A turn-on optoacoustic probe for imaging metformin-induced upregulation of hepatic hydrogen sulfide and subsequent liver injury

Article abstract of DOI:10.7150/thno.30080

Metformin is currently the most prescribed oral agent for diabetes treatment; however the overdose or long-term use may cause some severe side effects such as liver injury. Researches indicate that metformin-induced liver injury is closely related to upre

Full text of DOI:10.7150/thno.30080

Theranostics 2019, Vol. 9, Issue 1
77
Ivyspring
International Publisher
Theranostics
2019; 9(1): 77-89. doi: 10.7150/thno.30080
Research Paper
A Turn-On Optoacoustic Probe for Imaging
Metformin-Induced Upregulation of Hepatic Hydrogen
Sulfide and Subsequent Liver Injury
Lihe Sun, Yinglong Wu, Junjie Chen, Jun Zhong, Fang Zeng, Shuizhu Wu
State Key Laboratory of Luminescent Materials & Devices, College of Materials Science & Engineering, South China University of Technology, Guangzhou
510640, China.
 Corresponding authors: Prof. F. Zeng & Prof. S. Z. Wu, South China University of Technology, 381 Wushan road, Guangzhou 510640, China. Email:
shzhwu@scut.edu.cn; mcfzeng@scut.edu.cn; Fax: +86 20 22236262; Tel: +86 20 22236262.
©
Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license
(https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Received: 2018.09.20; Accepted: 2018.11.19; Published: 2019.01.01
Abstract
Metformin is currently the most prescribed oral agent for diabetes treatment; however the
overdose or long-term use may cause some severe side effects such as liver injury. Researches
indicate that metformin-induced liver injury is closely related to upregulation of hepatic H2S. Hence,
monitoring hepatic H2S generation induced by metformin could be an effective approach for
evaluating hepatoxicity of the drug.
Methods: We present a novel turn-on and dual-mode probe for detecting and imaging
metformin-induced liver injury by specifically tracking the upregulation of hepatic H2S with
fluorescent and optoacoustic methods. After reaction with H2S, the strong electron-withdrawing
group dinitrophenyl ether (which acts as both the recognition moiety and the fluorescence
quencher) was cleaved and replaced by an electron-donating group hydroxyl. This correspondingly
leads to the changes of the probe’s electronic state and absorption red-shifting as well as the
subsequent turn-on fluorescent and optoacoustic signals.
Results: The probe was applied to the colon tumor-bearing mice model and the
metformin-induced liver injury mice model to achieve tumor imaging and liver injury assessment.
The biosafety of the probe was verified by histological analysis (hematoxylin and eosin staining) and
serum biochemical assays.
Conclusion: The probe responds quickly to H2S in tumors and the liver, and MSOT imaging with
the probe offers cross-secitonal and 3D spatial information of liver injury. This study may provide an
effective approach for accessing medication side effects by tracking drug-metabolism-related
products.
Key words: optoacoustic, imaging, metformin, hydrogen sulfide
Introduction
In vivo metabolism of many drugs, especially
those require continuous administration over
prolonged period of time, has been found to be
associated with some adverse side effects, including
hepatic injury [1-3]. Some drug-induced liver injury is
benign and could be improved after drug discontin-
uation but a lot more may progress to chronic liver
diseases or even acute liver failure [4, 5]. This is
probably the reason why drug-induced liver injury
has become one of the most challenging disorders for
gastroenterologists and usually is the most frequent
cause for withdrawing approved drugs. Hence,
evaluating liver injury via in-situ detecting the
elevation of drug-metabolism-related products at the
hepatic region would be helpful in drug development
and medication safety improvement.
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Metformin is a medicine commonly used in
diabetes treatment and has become the most
prescribed oral hypoglycemic agent which needs to be
taken over a long period of time [6]. It has also been
found to be functional in cardiovascular diseases
[7-8], cancer [9-11] and other diseases, and it can even
help to eliminate nicotine withdrawal syndrome [12].
But long-term use or overdose of metformin can cause
some side effects, such as heartburn, stomach pain,
and liver or kidney problems [13, 14]. Previous
researches indicate that metformin administration
may induce hepatotoxicity which is closely related to
the drug-induced upregulation of hydrogen sulfide
(H2S) in the hepatic region [15-18]. H2S is endogen-
ously synthesized during cysteine metabolism
primarily in liver and vasculature [19], involved in
various physiological and pathological processes [20,
21]. And its toxic effects are dose-related [20, 21]. As
the key regulatory site for H2S, the liver is uniquely
predisposed to be subject to high levels of H2S from
endogenous generation and exogenous source. The
oxidation of H2S by liver mitochondria exposes the
liver to periods of tissue hypoxia, which contributes to
hepatic injury via increased generation of reactive
oxygen species [15, 22, 23]. When the metformin-
induced H2S expression is excessive, severe hepatic
tissue hypoxia occurs, thus resulting in liver injury.
Therefore, monitoring hepatic H2S generation
induced by metformin could be an effective approach
for evaluating liver injury/hepatoxicity of the drug.
Compared with other conventional biological
detection methods, optical imaging has its advantages
of real-time noninvasive imaging and high resolution;
for example, fluorescence method has been widely
applied in the research of molecular biology and
metabolism of small molecules [24-28]; but
fluorescence method has unsatisfactory imaging
penetration depth due to low spatial resolution with
strong light scattering in tissue [29-34]. In contrast,
optoacoustic imaging technique avoids the inherent
limitations of fluorescent imaging [35-41] and in the
meantime retains other superior properties by
combining the advantages of both optical imaging
and acoustic imaging via detecting the ultrasonic
signal generated by local tissue expansion after
absorption of the external excitation laser [42-45]. In
this situation, the excitation light only needs to enter
the biological tissue in one direction instead of
traveling back and forth, thereby reducing light
scattering and deepening imaging penetration [46-54].
Especially, multispectral optoacoustic tomography
(MSOT) [38, 39, 55-57] can distinguish ultrasound
signals generated by exogenous contrast agents from
the background signals of endogenous substances
such as hemoglobin, melanin and so on by irradiating
the sample with multiple wavelengths and detecting
ultrasound waves and through spectral unmixing and
deconvoluting the ultrasound waves emitted by
different absorbers. Hence, MSOT allows each
photoabsorber to be separately visualized in the target
tissue. Furthermore, by volumetric imaging technique
or by rendering stacks of 2D images as 3D images,
three-dimensional (3D) MSOT images can be
generated. Correspondingly, the roles of H2S in
physiological and pathological processes have evoked
interest among many researchers; and fluorescent
probes for imaging endogenous H2S have been
continuously
developed
[58-60].
However
optoacoustic probes for imaging H2S are still rare [61]
especially that using MSOT imaging [62]. Considering
the above and endeavoring toward developing a
molecular probe for detecting hepatic H2S level and
subsequent liver injury via optoacoustic imaging, in
this study, we synthesized a turn-on probe NR-NO2
for H2S detection and imaging. In this probe, a
benzothiazole-xanthene dyad serves as the reporting
unit, while a dinitrophenyl ether group acts as both
the recognition moiety for H2S and the fluorescence
quencher. In the absence of the H2S, the fluorescence
of the probe is quenched due to the existence of the
strong electron-withdrawing group dinitrophenyl
ether. The presence of H2S cleaves the strong
electron-withdrawing group dinitrophenyl ether and
generates an electron-donating group hydroxyl,
leading to the changes of the electronic state of the
probe molecule and red-shifting of absorption as well
as the subsequent turn-on fluorescent and
optoacoustic signals. We used the probe (NR-NO2) to
detect liver injury through its specific responding to
the elevated level of H2S in the liver induced by the
overdosed metformin in mice model. The schematic
illustration for the probe’s detection is displayed in
Figure 1C, and the experimental results indicate that,
the probe responds quickly to H2S in the liver, and the
MSOT imaging with the probe provides
cross-sectional and 3D spatial information of liver
injury via optoacoustic tomography (OA) imaging.
Material and methods
Preparation of the probe (NR-NO2)
Synthesis of Compound 1
Dimethylformamide (20 mL, 270 mmol) was
added to a 100 mL two-neck round flask under a
nitrogen atmosphere. Then, phosphorus oxychloride
(20 mL, 130 mmol) dissolved in 20 mL of
dichloromethane was slowly added at 0°C. The
reaction system was transferred to room temperature,
stirred for 30 min, and then cyclohexanone (5 g, 50
mmol) was slowly added. The temperature was raised
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to 80 °C and the mixture was refluxed for 2 hours.
Afterwards, the resulting mixture was cooled down
and poured into ice-cold water and allowed to stand
in a refrigerator overnight. The suspension was
filtered and precipitate was collected and dried in
vacuum as yellow solid (5.0 g, yield: 57%).
and the system was stirred at 75 °C for 1 h. The
solvent was removed by vacuum-rotary evaporation
procedure, and the resulting blue solid mixture was
purified by silica gel chromatography with
dichloromethane/methanol (20:1) as eluent to obtain
1
Compound 4 as a purple solid (0.3 g, yield: 90%). H
NMR (600 MHz, DMSO-d6, δ ppm): 8.94 (s, 1H), 8.50
(d, J = 9.3 Hz, 1H), 8.32 (d, J = 14.9 Hz, 1H), 8.21 (d, J =
7.9 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 7.77 (t, J = 7.8 Hz,
1H), 7.66 (t, J = 7.7 Hz, 1H), 7.57 (d, J = 8.5 Hz, 1H),
7.39 (d, J = 5.9 Hz, 2H), 7.22 (s, 1H), 7.14 (d, J = 8.4 Hz,
1H), 7.01 (d, J = 14.9 Hz, 1H), 4.79 (q, J = 7.1 Hz, 2H),
2.72 – 2.63 (m, 4H), 1.84 – 1.77 (m, 2H), 1.39 (t, J = 7.1
Hz, 3H). ESI-MS (m/z): 554.1 [M]+. 13C NMR (151
MHz, d6-DMSO) δ 170.60, 156.15, 155.69, 154.44,
153.65, 142.65, 142.56, 141.46, 140.28, 130.15, 129.65,
129.51, 129.28, 128.31, 128.13, 127.78, 124.29, 123.34,
122.44, 120.89, 120.06, 116.90, 116.34, 113.31, 107.95,
43.99, 29.22, 24.63, 20.35, 14.16.
Synthesis of Compound 2
Compound 1 (0.34 g, 2 mmol), 3-ethyl-2-methyl-
benzothiazolium iodide (1.22 g, 4 mmol) and sodium
acetate (0.33 g, 4 mmol) were dissolved in acetic
anhydride (20 mL). The mixture was refluxed at 130
°C for 1 h under nitrogen atmosphere. The solvent
was removed by vacuum-rotary evaporation proce-
dure, and the resulting green solid mixture was
purified by silica gel chromatography with dichloro-
methane/methanol (20:1) as eluent to obtain Comp-
1
ound 2 as a dark green solid (0.876 g, yield: 89%). H
NMR (600 MHz, DMSO-d6, δ ppm): 7.94 (d, J = 7.9 Hz,
2H), 7.71 (d, J = 8.3 Hz, 2H), 7.52 (t, J = 7.8 Hz, 2H),
7.33 (t, J = 7.6 Hz, 2H), 6.46 (d, J = 13.5 Hz, 2H), 6.17 (d,
J = 2.2 Hz, 2H), 4.44 (q, J = 7.1 Hz, 4H), 2.66 (t, J = 6.0
Hz, 4H), 1.83 (dd, J = 11.8, 6.0 Hz, 2H), 1.31 (t, J = 7.2
Hz, 6H). ESI-MS (m/z): 490.5 [M]+.
Optical characterization
Optical spectra were measured using Hitachi
U-3010 UV-vis spectrophotometer and Hitachi F-4600
fluorescence spectrophotometer. As for optical spectra
measurements, including absorption spectra and
fluorescence spectra, in the final test system, the probe
concentration was 10 μM, the volume percent of
DMSO was 10% and the experimental pH was
maintained at 7.4 by PBS. Fluorescence spectra were
recorded with excitation at 680 nm. For H2S response
measurement, NaHS was used as the H2S donor,
different concentrations of NaHS were added to the
solutions to track the response process for 20 min
(spectra were acquired every 2 min). The fluorescence
selectivity and limit of detection tests were also
performed in the same test conditions. Moreover, we
conducted optical response experiments at pH from
6.5 to 7.8.
Synthesis of Compound 3 (NR-OH)
Resorcinol (0.44 g, 4 mmol) and potassium
carbonate (0.55 g, 4 mmol) were dissolved into 50 mL
of dimethylformamide and the mixture was stirred
for 10 min. Afterwards, compound 2 (0.8 g, 1.6 mmol)
was added into the mixture and the system was
stirred at 60 °C for 5 h. The solvent was removed by
vacuum-rotary evaporation procedure, and the
resulting blue solid mixture was purified by silica gel
chromatography with dichloromethane/methanol
(20:1) as eluent to obtain Compound 3 as a dark blue
solid (0.58 g, yield: 93%). 1H NMR (600 MHz,
DMSO-d6, δ ppm): 9.14 (s, 1H), 8.31 (d, J = 14.6 Hz,
1H), 8.23 (d, J = 7.9 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H),
7.75 (t, J = 7.4 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.34 (d, J
= 8.4 Hz, 1H), 6.86 (d, J = 14.6 Hz, 1H), 6.79 (s, 1H),
6.21 – 6.15 (m, 2H), 4.74 (q, J = 7.0 Hz, 2H), 2.69 – 2.61
(m, 4H), 1.85 – 1.77 (m, 2H), 1.40 (t, J = 7.1 Hz, 3H).
ESI-MS (m/z): 387.3 [M]+. 13C NMR (151 MHz,
d6-DMSO) δ 161.41, 158.91, 158.36, 154.26, 142.59,
141.47, 131.58, 130.14, 129.38, 129.17, 127.52, 127.30,
126.04, 124.26, 115.74, 112.34, 106.64, 102.94, 102.33,
43.44, 28.95, 24.77, 20.54, 13.92.
Phantom optoacoustic imaging
Phantom optoacoustic imaging was conducted
with MSOT system (inVision 128, iThera Medical
GmbH). The test solutions containing 10 μM NR-NO2
(or NR-OH) and different concentrations of NaHS
were stirred for 20 min at room temperature, then
added into NMR sample tube for phantom
optoacoustic imaging.
Cell viability assay
Synthesis of Compound 4 (NR-NO2, the probe)
The cytotoxicity of the probe was evaluated by
MTT assay. Three cell lines (L929, HeLa and HCT-116)
were purchased from KeyGEN BioTECH. The cells
were cultured in medium (L929 & HeLa: DMEM;
HCT-116: McCoy’s 5A) added with 10% fetal bovine
serum (FBS), 100 U/mL penicillin and 100 μg/mL
streptomycin. Culture atmosphere was controlled at
Compound 3 (0.12 g, 0.3 mmol) and potassium
carbonate (0.21 g, 1.5 mmol) were dissolved into 10
mL of dimethylformamide and the mixture was
stirred for 10 min. Afterwards, 2,4-dinitro-1-fluoro-
benzene (75 µL, 0.6 mmol) was added into the mixture
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to accept standard care (including aseptic
environment, autoclaved rodent diet, sterile water
and light/dark cycle of 12 h) in Laboratory Animal
Center of South China Agricultural University. Mice
were randomly assigned to establish different animal
models and support subsequent experimental
investigations. Groups were randomly divided with 6
mice per group, ensuring sufficient replication of
results and a reduction in animal number. In the case
of lethal experimental procedures, mice would be
euthanized by exposure to carbon dioxide gas. All the
experimental protocols were approved by the Ethics
Committee of Laboratory Animal Center of South
China Agricultural University.
37 °C under 5% CO2 atmosphere. After a period of
culture, the cells were seeded in 96-well plates (about
5000 cells/well) and cultured for 24 h. The medium
was washed out with PBS (1X, pH=7.4) and replaced
with new medium containing NR-NO2 (concentra-
tion: 0, 1, 5, 10, 20, and 50 μM, containing 0.2% DMSO)
for one more 24 h. To reduce experimental error, we
prepared 8 parallel samples for each concentration.
The third day, the medium was washed out and
replaced with new medium containing 0.5 mg/mL
MTT, then incubated for another 2 h. Finally, the
medium in each well was washed out and replaced
with 150 μL DMSO to dissolve the precipitates. We
used Thermo MK3 ELISA reader to measure the
absorbance at 570 nm and estimate the viability of
cells in each well.
Mice model
The tumor-bearing mice model was established
by subcutaneous injection of HCT-116 cells (2×106
cells in 100 μL PBS per mouse) at the back of nude
mice. Tumors were then allowed to grow for 1-2
weeks before imaging. For the metformin-induced
liver injury mice model, the study protocol included:
metformin was dissolved in a saline solution, which
was then administered via intraperitoneal injection
with dosage of 1 mg or 2 mg metformin daily at 10:00
a.m. for 5 consecutive days (each administration of
200 μL). The control group received intraperitoneal
injection of physiological saline with the same volume
[15].
Flow cytometry
Cell uptake and activation of NR-NO2 was
measured by flow cytometry. Three cell lines (L929,
HeLa and HCT-116) were seeded into six-well plates
at 2 × 105 cells/mL and cultured for 24 h before
treatment. The second day, cells were incubated with
30 μM NR-NO2 and cultured for 0, 30 and 90 min
respectively. After culturing for the corresponding
period of time, the cells were washed, trypsinized,
centrifuged and dispersed into
a
single cell
suspension (in PBS). Then, around 10,000 cells were
used for analysis on a BD CS6 flow cytometer. The
signals in FL3 channel were collected.
Animal fluorescence imaging
Cell imaging
Animal fluorescence (in vivo and ex vivo) imaging
was performed using Ami small animal imaging
system (Spectral Instruments Imaging Co.). For the
tumor-bearing mice model, the probe (1.4 mg
probe/kg body weight, in PBS containing 4% DMSO)
was administered through subcutaneous intratumoral
injection. Then, the mice were anesthetized and
imaged for 60 min (images were acquired every 10
min) to track the response towards endogenous H2S in
tumor. In addition, we also studied the imaging of
exogenous H2S in tumor by intratumoral injection of
NaHS (1 mM, in PBS, 20 μL). The mice were imaged at
30 min after injection of probe; then NaHS was
injected into tumor and imaged again 5 min later.
Furthermore, we decreased the level of endogenous
H2S in HCT-116 tumors by intratumorally pretreating
mice with 100 nmol AOAA for 12 h to inhibit the
expression of CBS, and then injected NR-NO2 into
tumor at 30 min before in vivo fluorescence imaging.
For the metformin-induced liver injury mice model, 1
mg metformin-treated mice were intravenously
injected with the probe (2.8 mg probe/kg body
weight, in PBS containing 2% DMSO) for tracking the
probe’s metabolism in vivo. In addition, we also
Fluorescence images of living cells were
acquired through an Olympus IX71 inverted fluoresc-
ence microscope with a DP72 color CCD. Three cell
lines (L929, HeLa and HCT-116) were cultured in
medium containing different concentrations of
NR-NO2 for different lengths of time. Among them,
both L929 and HeLa cells were incubated with 30 μM
NR-NO2 for 90 min. For HCT-116 cells, the probe
concentration and incubation time were performed in
these following combinations: 0 μM & 90 min, 10 μM
& 90 min, 30 μM & 90 min, 50 μM & 90 min, 30 μM & 0
min, 30 μM & 30 min and 30 μM & 150 min. In
addition, aminooxyacetic acid (AOAA) was chosen as
an inhibition of cystathionine β synthase (CBS) to
reduce the generation of endogenous H2S in HCT-116.
HCT-116 cells were incubated with 200 µM AOAA for
30 min, washed by PBS twice, and then incubated
with NR-NO2 (30 µM) for 30 min before imaging.
Animal experiments
The BALB/c nude mice (female, 5- 6 weeks old)
were provided by Guangdong Medical Laboratory
Animal Center (GDMLAC). All animals were allowed
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studied the imaging of endogenous and exogenous
H2S in the peritoneal cavity of mice by intraperitoneal
injection. The groups were healthy mouse with probe
injection and healthy mouse with injection of both
probe and NaHS (the probe: 2.8 mg probe/kg body
weight, in PBS containing 2% DMSO; NaHS: 1 mM, in
PBS, 200 μL). In all the experiments mentioned in this
paragraph, excitation filter of 675 nm and emission
filter of 710 nm were used. All imaging parameters
were kept constant for the same mice model.
(ALT) was measured by Elisa kits. Mice were injected
with NR-NO2 solution (2.8 mg probe/kg body weight,
in PBS containing 2% DMSO) through the tail vein 24
h prior to serum testing. For histological study, mice
were treated with the probe 24 h in advance for the
treatment groups and PBS for the control groups;
organs (heart, liver, spleen, lung, and kidney) were
excised and tissue-fixed for hematoxylin and eosin
(H&E) staining.
Relative OA intensity
Animal optoacoustic imaging
Relative OA intensity was used to indicate
response of the probe to H2S (NaHS was used as the
H2S donor) of varied concentrations for the
optoacoustic method. It was calculated with the
equation:
Animal optoacoustic imaging was conducted
with MSOT system (inVision 128, iThera Medical
GmbH). For the tumor-bearing mice model, the probe
(1.4 mg probe/kg body weight, in PBS containing 4%
DMSO) was administered through subcutaneous
intratumoral injection; the mice were anesthetized
and placed in the prone position in animal holder for
imaging at 30 min post injection of NR-NO2.
Furthermore, we decreased the level of endogenous
H2S in HCT-116 tumors by intratumorally pretreating
mice with 100 nmol AOAA for 12 h to inhibit the
expression of CBS, and then injected NR-NO2 into
tumor 30 min prior to in vivo optoacoustic imaging.
Cross-sectional images were acquired through the
whole tumor with a step size of 0.1 mm. For the
metformin-induced liver injury mice model, the mice
were injected with the probe (2.8 mg probe/kg body
weight, in PBS containing 2% DMSO) via tail vein and
then imaged for 60 min (images were acquired every
15 min). Mice were anesthetized by 1% isoflurane and
placed in the prone position in a 34 °C water bath.
During the imaging process, continuous anesthesia
and oxygen were supplied through a mouse breathing
mask. Imaging wavelengths selected conforming to
the major turning points in NR-OH’s and
hemoglobin’s absorption spectra included 680 nm,
685 nm, 690 nm, 695 nm, 700 nm, 710 nm, 720 nm, 730
nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 800 nm
(background), 825 nm and 850 nm. We recorded 5
frames at every imaging wavelength. The region,
spanning through the liver and consisting of
transverse slices with a step size of 0.1 mm, was
selected by inspection of live MSOT images. After the
MSOT images were generated, the z-stack was
rendered as orthogonal-view three-dimensional
images (guided ICA spectral unmixing was utilized to
separate signals coming from the activated probe and
those from the photo-absorbing tissue elements in the
body (e.g. hemoglobin)).
Relative OA intensity = [(OA695 NaHS
)
- (OA695 control
)
] /
(OA695
)
control
Results and Discussion
Design and synthesis of the probe NR-NO2
The probe (NR-NO2) was designed by adopting
the strong electron-withdrawing group dinitrophenyl
ether as the recognition moiety for H2S and as the
fluorescence quencher as well (Figure 1A). The
1
structural characterizations (including H NMR, 13C
NMR and ESI-MS) of probe and intermediates are
shown in Figures S1-8. H2S can break the dinitro-
phenyl ether bond of NR-NO2 [63, 64], thereby
changing the chemical structure and leading to the
formation of the activated form of the probe (NR-OH);
and the structure of the product from the reaction of
NR-NO2 and NaHS (the reaction product was
purified by silica gel column chromatography) was
1
confirmed by H NMR and HR-MS spectra (Figures
S9-10). This response process strengthens the electron
push-pull
structure
(donor-π-acceptor),
and
eventually results in the enhanced fluorescent
emission for fluorescence imaging and red-shifted
absorption band (Figure 1B) for optoacoustic imaging.
Spectral response of the probe NR-NO2
toward H2S
Spectral properties of the probe NR-NO2 and the
probe’s response towards H2S were investigated
experimentally. First, absorption and fluorescence
spectra of the probe in the presence of NaHS (H2S
donor) were measured (excitation wavelength at 680
nm). Figure 2A and 2B show the time-dependent
changes in the absorption and fluorescence spectra.
The probe was incubated with NaHS (60 μM) for 20
min, during which the spectra were measured every 2
min to record the detailed spectral responses. In the
Serum biochemistry assessment and tissue
histological evaluation
Serum biochemistry indexes aminotransferase
absence of H2S, the probe exhibited
a strong
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absorption band centering around 560 nm and
negligible fluorescence emission at 725 nm. Whereas
in the presence of H2S, the absorption spectrum
red-shifted and the absorption peaked around 695 nm
and the fluorescence emission at 725 nm significantly
enhanced as the incubation time prolonged. These
prominent spectral changes were caused by the
H2S-mediated structural transformation from NR-
NO2 to NR-OH. Figure 2C shows the fluorescence
spectra of NR-NO2 incubated with different
concentrations of NaHS (0, 15, 30, 45 and 60 μM) for
20 min. From the spectra, we can see the response of
the probe towards H2S is concentration-dependent.
The absorbance at 695 nm and the fluorescent
intensity at 725 nm of the probe incubated with
different concentrations of NaHS as a function of time
are shown in Figure S11A-B, and the data at each
concentration for 20 min incubation were also
recorded as shown in Figure S11C-D; and the results
show a good NaHS concentration-dependent linear
relationship. In addition, the limit of detection (LOD)
towards NaHS was determined to be 40 nM (Figure
S12A-B), which is comparable to the reported probes
(Table S1). The fluorescence intensity reached plateau
at higher substrate concentration (Figure S12C-D).
Moreover, the probe is operable in the pH range of 6.5
– 7.8, as shown in Figure S13. To investigate the
sensing selectivity, the probe NR-NO2 was treated
with various potential biologically relevant species
(e.g., representative anions, reducing agents, reactive
oxygen species and small-molecule thiols), followed
by the fluorescence intensities measurement. As
shown in Figure 2D, addition of the representative
anions (Cl-, Br-, I-, NO3 , NO2 , CO3 , HCO3 , SO4 ) at 1
-
-
2-
-
2-
2-
2-
mM, reducing agents (SO3 , S2O3 ) at 100 μM,
reactive oxygen species (ClO-) at 50 μM, and
small-molecule thiols at the biologically relevant
concentrations (1 mM for HCy, 1 mM for cysteine and
10 mM for GSH) couldn’t induce marked fluorescence
emission at 725 nm. In contrast, upon treatment with
NaHS (60 μM) for 20 min, a remarkable 40-fold
fluorescence enhancement was observed. The above
results reveal that NR-NO2 has quite high selectivity
towards H2S over various species tested including
glutathione (GSH) and cysteine of the biologically-
relevant concentrations, confirming that H2S-
mediated thiolysis reaction of the dinitrophenyl ether
is highly selective and other free thiols in the
biological systems won’t affect the probe’s response
toward H2S. The results are in agreement with
literature reports in that the superior chemoselectivity
for H2S is based on the striking difference in size and
pKa values of H2S and other free thiols [65]. H2S has a
smaller molecular size, which makes the chemical
reaction with NR-NO2 easier. Additionally, the pKa
value of H2S (around 6.9 [66]) is lower than that of
typical cellular free thiols (around 8.5 [67]), all these
contribute to the sensing selectivity under physiolo-
gical conditions. Moreover, the extinction coefficient
for NR-OH (the activated form of the probe) at 695 nm
was calculated to be 2.2×104 M-1cm-1,
which is sufficient for optoacoustic
effect that converts the absorbed
optical energy into heat, accompanied
by generation of ultrasonic signal after
thermal expansion. We next evaluated
the probe’s optoacoustic response
toward H2S in phantom. The
optoacoustic intensities (%) of NR-OH
corresponding to different wave-
lengths measured by MSOT system
were given in Figure S14. And relative
optoacoustic intensity increases with
increasing concentration of NaHS
from Figure 2E. Moreover, the
optoacoustic response specificity
(selectivity) and anti-interference of
NR-NO2 are shown in Figure 2F. And
the experimental data indicate that the
probe shows excellent optoacoustic
response for H2S. These spectral
results confirmed that NR-NO2 could
be applied in imaging H2S by both
fluorescent and optoacoustic methods.
Figure 1. (A) Synthetic route for the probe NR-NO2. (B) Absorption spectra of the probe NR-NO2 and
its activated form NR-OH (10 μM). (C) A schematic representation of the probe’s diagnosis of liver
injury induced by metformin in mice model.
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Figure 2. (A) Time-dependent absorption spectra of NR-NO2 (10 μM) in the solution containing 60 μM NaHS. (B) Time-dependent fluorescence spectra of NR-NO2
(10 μM) in the solution containing 60 μM NaHS with an excitation wavelength at 680 nm. (C) Fluorescence spectra of NR-NO2 (10 μM) after 20 min incubation with
different concentrations of NaHS (0, 15, 30, 45 and 60 μM). Inset shows representative fluorescent images of the probes in the presence of different concentrations
of NaHS measured with Ami small animal imaging system (excitation filter of 675 nm and emission filter of 710 nm). (D) Fluorescent intensity at 725 nm of NR-NO2
-
-
(10 μM) in the presence of different species (Cl-, Br-, I-, NO3 , NO2 , CO32-, HCO3-, SO42-, HCy and Cysteine: 1 mM; SO32-, S2O32-: 100 μM; ClO-: 50 μM; GSH: 10 mM;
NaHS: 60 μM). (E) Optoacoustic response of NR-NO2 (10 μM) towards different concentrations of NaHS (n=3). Inset shows the representative optoacoustic images
of NR-NO2 (10 μM) in phantom with different concentrations of NaHS. (F) Relative optoacoustic intensity of NR-NO2 (10 μM) in the presence of different species
respectively (upper row) and in the presence of both NaHS with a potential interference substance (lower row) (n=3) (concentrations of all species were consistent
with D). Data were represented as mean ± SD.
time, the fluorescence of the cells was enhanced, and
the fluorescence of HCT-116 cells with the higher level
of endogenous H2S expression among the three cell
lines was the strongest when incubated with 30 μM
probe for 90 min, which was consistent with the
results of flow cytometry analysis. Furthermore, to
prove that the fluorescence signal was generated by
endogenous H2S in cells, we conducted relevant
experiments using AOAA (as an inhibitor of CBS) to
reduce H2S level in cells. As shown in Figure 3D,
fluorescence signal in HCT-116 pretreated with 200
μM AOAA for 30 min was negligible compared to
that in the group without AOAA pretreatment.
Imaging intracellular H2S in HCT-116 cells
Three cell lines were selected for the
experiments, including L929 cells (mouse fibroblastic
cells), HeLa cells (human cervical cancer cells), and
HCT-116 cells (human colorectal carcinoma cells). H2S
is expressed in all these three cell lines, but its level is
much higher in some cancer cells, especially in
HCT-116 cells [68]. As shown in Figure S15, the probe
NR-NO2 shows low cytotoxicity to all these three cell
lines. The viabilities of three cell lines can be
maintained above 80%, even after 24 h of incubation
with NR-NO2 at the concentration of 50 μM. The low
toxicity of the probe is advantageous for imaging
endogenous H2S in living cells. Then, we investigated
cellular uptake of the probe NR- NO2 and its
H2S-activated imaging in living cells. Figure S16
reveals that the cellular uptake and intracellular
fluorescence emission of the probe were time-
dependent by flow cytometry analysis. As the
incubation time prolonged, the intracellular fluores-
cence emission became stronger, which means that
more NR-NO2 molecules were activated into NR-OH
in the cells by endogenous H2S. We further confirmed
that the fluorescence response of the probe NR-NO2 to
endogenous H2S by fluorescence microscopy imaging
(Figure 3A-C). It is clear that, with the increase of the
probe concentration or the prolongation of the culture
Imaging endogenous and exogenous H2S at
tumor region or in peritoneal cavity in mouse
models
To evaluate the applicability of the probe in H2S
detection and imaging in vivo, we investigated the
probe’s performance in HCT-116 tumor-bearing mice
using both in vivo fluorescent and MSOT imaging. The
tumor-bearing mice model was established by
subcutaneous injection of HCT-116 cells at the back of
nude mice. As shown in Figure 4A, the fluorescent
signal in the tumor area was quite prominent only 10
min after the probe injection and gradually enhanced
over time, indicating the feasibility of NR-NO2 to
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rapidly respond to endogenous H2S in tumor for
real-time imaging. Mean fluorescent intensities at
region of interest (ROI) covering the tumor area were
given in Figure S17. Then, we pretreated tumor-
bearing mice with 100 nmol AOAA for 12 h, and then
injected NR-NO2 into tumor 30 min prior to in vivo
imaging. As can be seen from Figure 4B, fluorescence
signal was weak at 30 minutes after injection of
NR-NO2 into the back of healthy mice (the black circle
drawn with a marker pen on the mouse back shows
the injection position for control purpose), due to the
low level of endogenous H2S in this position.
Moreover, AOAA treatment did significantly reduce
intratumoral fluorescence signal, indicating that the
expression of endogenous H2S in the tumor was
reduced and that the fluorescence signal was
produced in response to H2S. To verify the probe’s
imaging towards exogenous H2S, we then injected
NaHS into the tumor. As expected, a stronger
fluorescence emission was observed after injection of
NaHS (Figure 4C).
Figure 3. (A) Bright-field (top) and fluorescent (bottom) images of HCT-116 cells after incubation with different concentrations (0, 10, 30, 50 μM) of the probe for
90 min. (B) Bright-field (top) and fluorescent (bottom) images of HCT-116 cells after incubation with the probe (30 μM) for different period of time (0, 30, 90, 150
min). (C) Bright-field and fluorescent images of three cell lines after incubation with the probe (30 μM) for 90 min. (D) HCT-116 cells were incubated with the
inhibitor AOAA (200 μM) for 30 min and then probe (30 μM) for 30 min (left), or with probe (30 μM) only for 30 min (right).
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Figure 4. (A) Representative photographs for time-dependent response of the probe towards endogenous H2S in mice tumor through intratumoral injection. (B) In
vivo fluorescence imaging for healthy mice (left), AOAA-pretreated tumor-bearing mice (middle) and no-pretreatment tumor-bearing mice (right). (the black circle
drawn with a marker pen on the mouse back shows the injection position for control purpose). (C) Representative photographs of tumor-bearing mice (left), in vivo
fluorescence imaging towards endogenous (middle, 30 min after intratumoral injection of the probe) and exogenous (right, 30 min after intratumoral injection of the
probe and 5 min after intratumoral injection of NaHS) H2S in tumor-bearing mice. (D) Typical cross-sectional MSOT images (scale bar: 3 mm) of AOAA-pretreated
tumor-bearing mice (upper row) or the tumor-bearing mice without AOAA pretreatment (lower row) at 30 min post intratumoral injection of the probe. (E) Typical
orthogonal-view 3D MSOT images (scale bar: 3 mm) of AOAA-pretreated tumor-bearing mice (upper row) or tumor-bearing mice without AOAA pretreatment
(lower row). Left: overlay of NR-OH (activated probe) signal with the grayscale single-wavelength (800 nm) background image; Right: NR-OH (activated probe) signal.
On the other hand, the real-time MSOT imaging
provided further evidence that NR-NO2 has excellent
tumor imaging capability. It is worth mentioning that
the orthogonal-view 3D MSOT images (z-stack
orthogonal maximal intensity projection images)
covering the whole tumor allows us to determine the
relative position and the volume of solid tumors on
the body of mice, more veritably and directly (Figure
4D-E). As expected, AOAA in tumors reduces the
generation of endogenous H2S, thereby attenuating
H2S-activated optoacoustic signal in tumors. In
addition, we also evaluated the probe’s capability to
image H2S in abdomina. As shown in Figure S18,
through intraperitoneal injection, the probe can image
H2S of both endogenous and exogenous sources in the
peritoneal cavity of mice. These results demonstrate
that the probe NR-NO2 can respond to both H2S in
tumor region and abdominal, and it can serve as a
fluorescent/optoacoustic dual-mode molecular probe
in vivo.
Diagnosing metformin-induced liver injury
Metformin overdose can cause increased H2S
level in mouse liver [17], which in turn induces liver
injury. In this experiment, we established the
metformin-induced liver injury mice model (metfor-
min dose: 1 mg and 2 mg) to explore whether NR-NO2
is capable of imaging the upregulation of hepatic H2S
and reflecting liver injury caused by metformin. First,
by in vivo fluorescence imaging, we evaluated the
metabolism of the probe NR-NO2 injected through tail
vein of the mice, and the results are shown in Figure
S19. For the mice pretreated with 1 mg of metformin,
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Theranostics 2019, Vol. 9, Issue 1
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at one hour upon i.v. injection of the probe NR-NO2,
fluorescence in liver region emerged and reached the
maximum at about 60 min post injection, then
gradually faded due to the metabolism. This result is
consistent with fluorescence imaging of the excised
organs as displayed in Figure S20.
compared to that treated with 1 mg of metformin,
which verified the dose dependency of metformin to
induce liver injury. Furthermore, to verify the
biosafety and non-toxic nature of the probe, we
performed some experiments, which include mice
body weight tracking, H&E staining of liver sections,
and serum liver function index measurement by using
Elisa kits (Figure 5F and Figure S21-22). From Figure
5G, it can be seen that metformin administration
leading to the increase of hepatic H2S is dose
dependent; and in combination with the results of
serum tests as shown in Figure 5F, it is clear that the
severity of liver injury is dependent on the extent of
H2S generation caused by the drug metformin. Figure
S21 shows there was no abnormal change in mice
weight within one week after i.v. injection of the
probe, which indicated the low toxicity of the probe in
vivo. Next, histological analysis of the main organs by
using H&E staining (Figure S22) displayed that, there
was no obvious difference between the control group
and the mice group with i.v. injection of the probe,
confirming the insignificant toxicity of the probe
NR-NO2 in vivo. While for the mice treated with
metformin (Figure 5H), liver injury was evident for
We then conducted optoacoustic imaging
experiments. The mice were randomly assigned into
three groups: the control group was injected (i. v.)
with 200 μL of physiological saline at 10:00 a.m. every
day for five consecutive days; and the two treatment
groups were injected with 1 mg or 2 mg of metformin
instead. On the fifth day, the mice were intravenously
injected with the probe through tail vein and then
subject to optoacoustic imaging. The MSOT system
was used to monitor the severity of metformin-
induced liver injury by imaging the elevated level of
hepatic H2S, and the results are presented in Figure 5.
Figure 5A-C show the representative cross-sectional
MSOT images from the mice at different time points
in each group upon i.v. injection of the probe
NR-NO2. The upper row shows the overlay of NR-OH
(activated probe) image with a single wavelength (800
nm) background image as the anatomical reference,
while the lower row presents the MSOT signal from
the activated probe NR-OH. For the control group
treated with saline, only weak signal was detected in
the liver (Figure 5A). In contrast, for the mice treated
with metformin, the multispectrally resolved signal of
NR-OH was significantly stronger and gradually
increased over a period of 60 min (Figure 5B-C).
Overdose of metformin caused upregulation of
hepatic H2S, and the probe molecules accumulated in
the liver via metabolism were activated to produce
optoacoustic signals. It can also be seen in the figure
that, the mice exposed to 2 mg of metformin exhibited
stronger MSOT signals. Moreover, the mean optoaco-
ustic intensities in region of interest (ROI) over time
were given in Figure 5D. By referring to a cryosection
image of a mouse (Figure 5E), we can find that the
signal of NR-OH is mainly located in the liver region,
indicating an increase in the level of hepatic H2S.
MSOT technique is capable of rendering
multiple two-dimensional tomographic images as
orthogonal-view 3D images (orthogonal maximal
intensity projection images), which would aid in
spatially positioning disease focus. In this study, we
obtained orthogonal-view 3D images for the control
mice as well as the mice groups treated with
metformin, as shown in Figure 5G (at 30 min after i.v.
injection of NR-NO2). Upon treatment with metfor-
min, hepatic H2S levels were remarkably higher
compared to that of the control group. Figure 5G also
indicates that, the optoacoustic signal from the mouse
treated with 2 mg of metformin was more evident
the mouse treated with
2 mg of metformin.
Additionally, we measured the activities of serum
alanine aminotransferase (ALT) by using Elisa kits
and the results were given in Figure 5F. The data
reveal that metformin overdose does cause liver
injury to mice, and our probe has negligible effect on
liver function. All the above experiments prove that,
NR-NO2 is of good biosafety and can be applied to
detect the upregulation of hepatic H2S and assess the
severity of metformin-induced liver injury in mice
model.
Conclusions
Taken all together, we have developed a novel
molecular probe that specifically responds to H2S, and
it can be applied to imaging endogenous H2S in
HCT-116 tumor-bearing mice model. More importan-
tly, it can also be used to monitor metformin-induced
liver injury in mice model via detecting hepatic H2S.
With the aid of MSOT imaging method, the probe can
be successfully used to distinguish and precisely
position liver injury, and in the meantime visualize
the volume of liver injury. Moreover, the probe
NR-NO2 exhibits quite good response specificity and
sensitivity towards H2S and good biosafety. The work
herein could offer an optoacoustic probe for H2S and
an approach for designing chromophores for turn-on
optoacoustic/fluorescent imaging, as well as provides
helpful insights for devising an effective strategy for
assessing the side effects of the drug by tracking its
metabolism-related products.
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Figure 5. Typical cross-sectional MSOT images (scale bar: 3 mm) for a mouse treated with physiological saline (A), 1 mg (B) or 2 mg (C) of metformin at varied time
post i.v. injection of the probe. Upper row: merged signals of NR-OH and the grayscale-wavelength (800 nm) background. Lower row: signal of NR-OH. Organ
labeling: 1. spinal cord; 2. aorta; 3. liver. The mouse lay on its chest with a certain tilt as indicated by the position of spinal cord. (D) Mean optoacoustic intensities in
liver area (ROI) for control mice and metformin-treated mice (n=6) at corresponding time post i.v. injection of the probe. (E) A cryosection image of a female mouse
corresponding to the cross section shown in A-C. (F) Serum levels of ALT for the mice (n=6) with different treatment methods (+ means treatment, - means no
treatment). (G) Orthogonal-view 3D MSOT images (scale bar: 3 mm) for the mice shown in A-C at 30 min post i.v. injection of the probe. (H) Representative H&E
staining of liver sections from mice with different treatment methods: control mice (left) and 2 mg metformin-treated mice with probe injection at the fifth day. (Scale
bar: 100 μm).
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Supplementary Material
Supplementary figures and tables.
http://www.thno.org/v09p0077s1.pdf
Acknowledgment
This work was supported by NSFC (21788102,
21875069, 51673066 and 21574044) and the Natural
Science Foundation of Guangdong Province (2016A03
0312002).
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