Aggregation-induced fluorescence probe for hypochlorite imaging in mitochondria of living cells and zebrafish

Article abstract of DOI:10.1039/d0tb01496f

Hypochlorite is an important active oxygen species formed in living organisms, and rapid and highly sensitive detection of trace hypochlorite is of great significance for understanding the mechanism of diseases caused by abnormal hypochlorite concentratio

Full text of DOI:10.1039/d0tb01496f

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Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
particles
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DOI: 10.1039/D0TB01496F
ARTICLE
Aggregation-induced fluorescence probe for hypochlorite imag-
ing in mitochondria of living cells and zebrafish
Received 00th January 20xx,
Accepted 00th January 20xx
Xiuli Zhong, Qing Yang, Yingshuang Chen, Yuliang Jiang,* and Zhihui Dai*
DOI: 10.1039/x0xx00000x
Hypochlorite is an important active oxygen species formed in living organisms, and rapid and highly sensitive detection of
trace hypochlorite is of great significance for understanding the mechanism of diseases caused by abnormal hypochlorite
concentrations at an early stage. Although aggregation-induced emission (AIE) probes are highly important for analyte de-
tection in living organisms, there is a lack of AIE probes for hypochlorite detection. In this study, two AIE probes based on
benzothiazole derivatives (BTD-1 and BTD-2) were designed and synthesized. Both the probes exhibited good AIE charac-
teristics and allowed different visual detection for hypochlorite. Additionally, the two probes could be used to detect
endogenous hypochlorite in mitochondria and were successfully applied for in vivo hypochlorite imaging in zebrafish.
to some extent by introducing water-soluble groups, but a complex
synthetic protocol and the unpredictable performance of the
Introduction
modified probe render this process somewhat impractical. Thus,
the development of appropriate parent materials to overcome the
above difficulties is still challenging.
Hypochlorite receives significant attraction owing to its crucial role
in living organisms.1-3 To elucidate the pathogenic mechanism
underlying the diseases caused by abnormal hypochlorite levels,
accurate, real-time, and high-sensitivity detection of trace
hypochlorite in vivo is extremely important. In recent years, many
research groups developed various detection methods, thereby
promoting the development of this field significantly.4-7
ACQ is known to affect the application of probes in the field of
life sciences, thereby presenting significant challenges for
researchers. In 2001, Tang et al. discovered a special phenomenon
that was opposite to ACQ; this was named aggregation-induced
emission (AIE).17 This important discovery could completely
circumvent the difficulties faced during the application of probes in
biological systems. Based on this important discovery, a large
number of parent fluorophores, such as tetraphenylethylene,
benzothiadiazole, naphthalene-diimide, siloles, and PEGylated
carbazole, exhibiting AIE have been developed18 and used for the
detection of proteins, DNA, amino acids, and other biomolecules,
thereby leading to considerable development in this field.19
However, AIE molecules have been rarely used for the designing
and construction of probes for hypochlorite detection, leading to a
lack of such probes. During the design and construction of
hypochlorite probe, the introduction of AIE parent unit, not only
enrich the types of AIE probes, moreover, it can be used to detect
hypochlorite in vivo owing to its excellent biocompatibility which
can further understand the pathogenesis of related diseases and
realize early diagnosis and cure.
Fluorimetry is relatively superior compared to many other
hypochlorite detection methods owing to its advantages such as
simple operation, non-destructive investigation, and high
sensitivity.8-10 Developing an ideal fluorescent probe is the core of
application of fluorescence-based techniques. The several different
types of hypochlorite probes developed so far have promoted an
extensive study on the pathogenic mechanism of hypochlorite.11-15
Yoon et.al systematically expounded the development of
hypochlorite probes in recent years and prospected the future
applications in vivo, encouraging further research in this field.16
However, the phenomenon of aggregation caused quenching (ACQ)
cannot be ignored, as it poses serious difficulties in the
development of effective probes. Fluorescent probe particles tend
to aggregate while transferring from organic phase to water phase,
which increases the collision probability between molecules,
leading to energy loss, and ultimately, fluorescence quenching.
Consequently, the use of such probes in biological systems is almost
impossible. Certainly, the solubility of the probes can be improved
Benzothiazole derivatives (BTD) are important AIE matrices
and have been used for constructing fluorescent probes which can
be applied in the monitor of substances related to living systems.20-
24
Especially, the para- or ortho-position of phenol of BTD can be
further expanded and triggered excited-state intramolecular proton
transfer which allows dual channel signal detection, thereby
enhancing the detection sensitivity. Considering this, in this study,
benzothiazole was selected as a parent unit for constructing two
fluorescent probes (BTD-1 and BTD-2), using a simple synthetic
protocol. Both BTD-1 and BTD-2 exhibited ideal AIE characteristics,
Jiangsu Collaborative Innovation Center of Biomedical Functional Materials,
Jiangsu Key Laboratory of Bio-functional Materials, School of Chemistry and
Materials Science, Nanjing Normal University, Nanjing 210023, P. R. China.
E-mail address: 07205@njnu.edu.cn (Y.L. Jiang); Daizhihuii@njnu.edu.cn (Z.H.
Dai)
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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Synthesis of probe BTD-1
which ensured the feasibility of its biological application. Further
optical experiments showed that BTD-1 and BTD-2 displayed
View Article Online
DOI: 10.1039/D0TB01496F
The synthesis process of benzothiazolyl)benzaldehyde was
distinct specific recognition for hypochlorite. Unfortunately, while showed in ESI. To a solution of 4-(2-benzothiazolyl)benzaldehyde
(0.2409 g, 1 mmol), diaminomaleo nitrile (0.3246 g, 3 mmol) and
acetic acid (100 µL) in acetonitrile (20 mL) were added. The mixture
was stirred and refluxed in a flask for 8 h. The residual solvent was
removed immediately and washed with a small quantity of
we were still studying these molecules, BTD-2 was already reported
to be OCl- and CN− probe. However, the AIE performance, organelle
level, and living body were not discussed.25 Therefore, we
continued our investigations on the BTD-1 performance and
focussed on the AIE performance of BTD-2 and its application in
vivo and in organelles.
1
acetonitrile to yield BTD-1 as a pale green powder. Yield: 82.1% H
NMR (400 MHz, DMSO-d6) δ 8.34 (s, 1H), 8.23 (d, J = 8.4 Hz, 2H),
8.18 (d, J = 8.5 Hz, 5H), 8.13-8.09 (m, 1H), 7.58 (ddd, J = 8.3, 7.2, 1.3
Hz, 1H), 7.50 (td, J = 7.6, 7.1, 1.3 Hz, 1H); 13C NMR (100 MHz, DMSO)
δ(ppm) 39.55, 39.76, 39.97, 40.18, 40.39, 102.93, 114.16, 114.80,
122.95, 123.56, 126.34, 127.34, 127.90, 128.13, 130.25, 135.17,
135.38, 138.46, 154.07, 154.14, 166.94; MS (m/z): Calcd for
C18H12N5S+ [M]+, 330.0808; found, 330.0800.
Experimental section
Materials and Methods
All reagents were of analytical grade and used without further
purification unless otherwise specified. 1H NMR and 13C NMR
spectra of compounds were acquired on an AVANCE Ⅲ HD AN-400
MHz spectrophotometer (Bruker, Germany). Mass spectra (MS)
were acquired on a LCMS-2020 spectrophotometer (Shimadzu,
Japan). Absorption spectra were collected on a Lambda 1050+
spectrophotometer (PerkinElmer, USA), fluorescence data were
obtained from F-4700 fluorescence spectrophotometer (Hitachi,
Japan). High resolution mass spectroscopy data were collected from
a high resolution Orbitrap Fusion Lumos (Thermo Fisher Scientific,
USA) mass spectrometer. Fluorescence imaging of cells and
zebrafish was performed on an A1 confocal laser scanning
microscope (Nikon, Japan).
Synthesis of probe BTD-2
5-(2-Benzothiazolyl)-2-hydroxybenzaldehyde (0.2553 g, 1 mmol)
and diaminomaleonitrile (0.1189 g, 1.1 mmol) were stirred in
anhydrous ethanol (20 mL) at 80 °C for 12 h. A yellow solid
precipitate was obtained after the completion of reaction. The
resulting precipitate was filtered and washed three times with
ethanol. BTD-2 was obtained as a yellow solid. Yield: 85.3%. 1H NMR
(400 MHz, DMSO-d6) δ 11.21 (s, 1H), 8.70-8.62 (m, 2H), 8.12 (dt, J =
8.6, 2.0 Hz, 4H), 8.05-8.00 (m, 1H), 7.54 (d, J = 1.2 Hz, 1H), 7.47-7.41
(m, 1H), 7.14 (d, J = 8.7 Hz, 1H); 13C NMR (100 MHz, DMSO) δ(ppm)
39.75, 39.96, 40.17, 103.74, 114.58, 115.04, 117.86, 122.36, 122.65,
122.97, 125.30, 125.65, 127.02, 127.16, 127.99, 132.26, 134.95,
152.11, 154.07, 161.06, 167.45; MS (m/z): Calcd for C18H12N5OS+
[M]+, 346.0757; found, 346.0750.
Spectral measurements
All the spectral measurements were acquired in THF. The stock
solution of probe BTD-1 and BTD-2 were prepared with the
concentration of 1 mM. Other analytes (Ag+, Cu2+, Fe3+, Na+, Pb2+,
Cell culture and fluorescence imaging
HeLa cells and macrophage cells were received from Cell bank of
Chinese Academy of Sciences and the ability of the probe for
sensing OCl− in living cells was tested. HeLa cells were treated with
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
bovine serum at 37 °C in a humidified incubator with 5% CO2 for 24
h. Four groups of cells were investigated—three of them were
experimental groups containing different concentrations of OCl−
(10, 20, and 40 µM) and incubated for 30 min, while the other
group was a contrast group under the same conditions, incubated
only with the probe (10 µM), without OCl−.
-
-
-
Co2+, ONOO-, CO32-, H2PO4 , HSO3 , NO2 , Cys, Hcy, and H2O2) were
prepared in deionized water. Hypochlorite ions (OCl−) were
obtained from NaOCl. The detection limit (LOD) was calculated
based on the fluorescence titration experiment and the LOD was
obtained by using equation as follows:
LOD = 3σ/k,
where σ is the standard deviation of the measurement of the
blank solution (without the addition of OCl−), and k is the slope of
the intensity versus concentration (of OCl−) curve. σ was calculated
using the following equation:
Co-localization fluorescence imaging
2
To determine the capability of BTD-1 and BTD-2 inside the cells,
HeLa cells were co-stained with the probes (10 µM) and Mito-
tracker (200 nM) for 30 min at 37 °C respectively. Then, phosphate-
buffered saline (PBS) was used to washed the cells with three times
before imaging. Cells images were collected with 488/561 nm for
Mito-tracker red and 405/488 nm for Mito-tracker green.
∑
(푥 ― 푥푖)
σ =
n ― 1
Here, n is the number of measurements of the blank solution (n =
11); x is the mean of the blank measurements; xi is the
corresponding value of the blank measurement.
Fluorescence imaging of endogenous OCl− in living cells
Synthesis
The synthesis process of benzothiazolyl)benzaldehyde was
showed in ESI. The synthetic route of BTD-1 and BTD-2 were
showed in Scheme1 with the correlation NMR and MS spectra
exhibited in Fig.S1-Fig.S6.
Macrophages were grown in the required medium for 24 h and
then seeded on dishes. For imaging, the cells were incubated with
the probes (10 µM) for 30 min and then washed with PBS three
times. For the inhibition of OCl−, the second group was
subsequently incubated with N-acetylcysteine (NAC, OCl−
scavenger) for 30 min after being processed by the probe. In
another control group, we consulted the original group and added
exogenous OCl−.
Fluorescence imaging in zebrafish
Scheme 1. The synthetic route of BTD-1 and BTD-2
2 | J. Name., 2012, 00, 1-3
Zebrafish studies were approved by the Ethics Committee and
IACUC of Qilu Health Science Center, Nanjing Normal University,
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and were conducted in compliance with European guidelines for
Hypochlorite recognition by BTD-2 has been already reported.
View Article Online
the care and use of laboratory animals. Zebrafish (3-5 days old) Thus, we only studied the optical performance of BTD-1. The
DOI: 10.1039/D0TB01496F
were first incubated with the probe (10 µM) for 30 min and then spectral properties of BTD-1 was investigated in THF solution. Free
further coexistence with OCl− (5, 20, and 40 µM) for 30 min. BTD-1 showed an obvious absorption peak at 396 nm. Upon the
Subsequently, the zebrafish were washed with culture water to addition of 300 μM OCl−, a remarkable increase in the absorption
remove any remaining probe molecule. Fluorescence imaging of intensity was observed (Fig. S8). Free BTD-1 exhibited weak
zebrafish was performed on Nikon A1 confocal fluorescence fluorescence at about 448 nm. Upon the addition of OCl−, significant
microscope and imaging channel including green channel (500–550 enhancement in the fluorescence intensity and solution color was
nm) and blue channel (425-475 nm).
observed. Fluorescence titration was then conducted for
investigating the relationship between the probe concentration and
fluorescence intensity (Fig. 2a).
A good linear relationship,
represented by the equation Y = 18.190X + 2868.1 (R2 = 0.9903),
with the OCl− concentration in the range 0-260 μM (Fig.2b) was
observed. Based on the linear calibration, the detection limit (LOD =
3σ/k) of BTD-1 for OCl− was calculated to be 0.014 μM.
Results and discussion
The AIE characteristics of BTD-1 was first examined in THF/water
mixtures containing various water fractions. Fluorescence from
BTD-1 was almost negligible when water in the mixture was less
than 50 vol% (Fig.1a). The fluorescence intensity increased
gradually with the water fraction increasing from 50% to 90%, and a
sharp increase in the fluorescence intensity was observed at 98
vol% water. Plot of relative peak intensity (I/I0) and ratio of water
was showed in (Fig.1b). Thus, increasing the water content in the
THF/water mixture reduced the solubility of BTD-1 and induced the
formation of aggregates. This could be further confirmed by
scanning electron microscopy (SEM). The SEM image was captured
by dissolving BTD-1 in THF and its binary solution of H2O and THF
containing 90% water respectively (Fig.S7). This indicated that BTD-
1 agglomerated as globular species, with a particle size of about 100
nm. Such a particle size can be well related for applications in
biological systems. Thus, the occurrence of AIE in BTD-1 was
evident.
Fig. 2 (a) Changes of fluorescence spectra of BTD-1 (10 mM) with
titration of different concentration of OCl- (0-300 μM); (b) The
relationship of fluorescence intensity and concentration of OCl-
from 0 to 260 μM.
The selectivity of BTD-1 for OCl− was further assessed in the
presence of different interfering ions such as Ag+, Cu2+, Fe3+, Na+,
-
-
-
Pb2+, Co2+, ONOO-,CO32-, H2PO4 , HSO3 , NO2 , Cys, Hcy,H2O2, and OCl-
under similar conditions. In contrast to the interfering ions, OCl−
could significantly enhance the fluorescence (Fig.3). Competition
experiments further confirmed the high selectivity of BTD-1 (Fig.S9).
Fig. 1 (a) Fluorescence spectra of probe BTD-1 in THF/water
solutions with different water fractions;(b) Plot of relative
peak intensity (I/I0) and ratio of water.
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the probe (Fig.S11). Thus, the two probes were suitable for
Fig. 3 The change of fluorescence spectrum of BTD-1 in the
existence of various species (300 μM).
View Article Online
applications in biosystems. Further, almosDt OnIo: 10fl.1u0o3r9e/sDc0eTnBc0e14w96aFs
observed in any channel from the HeLa cells incubated with 10 μM
BTD-1 (30 min at 37 °C). Addition of various concentrations of OCl−
(10, 20, and 40 μM) to the cells, followed by incubation for another
30 min, led to a gradual increase in the fluorescence intensity in the
blue channel (Fig.5). Under the same experimental conditions, BTD-
2 showed similar fluorescence enhancement in both blue and green
channels (Fig. 6). Thus, BTD-1 and BTD-2 can be used as effective
tools for the specific sensing and imaging of OCl− at the cellular
level.
OCl− has a short half-life inside organisms, and thus, a rapid
response during real time in vivo hypochlorite detection must be
ensured.26 Herein, time-dependent fluorescence response
experiments were developed, and the result is illustrated in Fig.4.
BTD-1 exhibited weak fluorescence, and the addition of 300 μM
OCl− enhanced the fluorescence, which rapidly reached saturation
within 10 s. Compared to previous reports (table 1),27-31 BTD-1
showed excellent response time and lower detection limit which
can be realized real-time, sensitive monitor OCl− in biological
systems .
Fig. 4 Time response of BTD-1 and BTD-1+OCl-.
Table 1. The comparision of BTD-1 with published probes for OCl-
Fig.5 Confocal fluorescence images of Hela cells. Flurescence image
with blue channel (A1-D1) bright field (A2-D2) and merge (A3-D3)
of Hela cells pretreated with 10 μM probe BTD-1 for 30 min and
then followed by incubation with 10, 20, 40 μM OCl- for another 30
min, respectively. The cells were excited with a light laser at 405
nm. Scale bar: 10 mm.
Response
time (s)
50
＜10
60
Detection
limit (nM)
705
Ref.
Probe
BL
B6S
RCP
27
28
29
200
70
94
30
CSN
within 60
RT-1
Zcp-Me
180
900
2.18
89
31
32
This work
BTD-1
about 8s
14
Always, C=N as a classical hypochlorite targeting functional
groups have been used for constructing hypochlorite probes widely,
and there were also relevant reports in our early work.33-35 The
recognition mechanism of BTD-2 involves the breaking and
hydrolysis of C=N to an aldehyde group. In this study, the
recognition mechanism of BTD-1 for hypochlorite was also
confirmed by mass spectrometry (Fig.S10, m/z 240.0476). The
specific mechanism is shown in Scheme 2
H2N
H2N
R
CN
CN
HN
S
N
S
N
H2O
N
S
N
CHO
CN
CN
OCl-
OCl
O
R
R
H
H
BTD
R=H/OH
Scheme 2. Proposed mechanism of probe BTD for highly selective
sensing of OCl-.
Considering the excellent optical properties of probes BTD-1 and
BTD-2, we further explored their application inside HeLa cells. First,
the toxicities of BTD-1 and BTD-2 against HeLa cells were
determined using the MTT assay. Both BTD-1 and BTD-2 exhibited
low cytotoxicity against HeLa cells, with over 88% and 89% cell
viability, respectively, after 24 h of incubation with 100 μg/mL of
Fig.6 Confocal fluorescence images of Hela cells. Fluorescence
image with blue channel (A1-D1) green channel(A2-D2) bright field
(A3-D3) and merge (A4-D4) of Hela cells pretreated with 10 μM
probe BTD-2 for 30 min and then followed by incubation with 10,
4 | J. Name., 2012, 00, 1-3
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20, 40 μM OCl- for another 30 min, respectively. The cells were
excited with a light laser at 405 and 488 nm. Scale bar: 10 μm.
View Article Online
DOI: 10.1039/D0TB01496F
Although most of the existing probes can image hypochlorite in
cells, the real-time detection of endogenous hypochlorite is the key
to practical applications of the probe. In this study, we further
explored the detection ability of the probe for endogenous
hypochlorite in cells. Macrophage cells were selected as the
bioassay model and used for the detection of endogenous
hypochlorite. The living macrophage cells were loaded with BTD-1
and BTD-2 (10 µM), which emitted obvious fluorescence. Addition
of inhibitor NAC (10 µM), followed by incubation with BTD-1 and
BTD-2 for another 30 min, almost quenched the fluorescence,
proving that the two prepared probes can be used for the detection
of endogenous hypochlorite.
Fig.8 Intracellular co-localization fluorescence imaging of BTD-1
and BTD-2; A1) bright field image; B1) from green channel
(mitochondria staining); C1) confocal images of BTD-1 on the blue
channel (λex = 405 nm); D1) overlay of brigh field, green, and blue
channels; E1) Intensity profile of linear region of interest across the
HeLa cell costained with Mito Tracker green channel of BTD-1. A2)
bright field image; B2) from red channel (mitochondria staining);
C2) confocal images of BTD-2 on the green channel (λex = 488 nm);
D2) overlay of brigh field, red, and green channels; E2) Intensity
profile of linear region of interest across the HeLa cell costained
with Mito Tracker green channel of BTD-2. Scale bar = 10 μm.
Fig.7 Confocal fluorescence images of endogenous hypochlorite in
Macrophage cells. Fluorescence image with blue channel (A) BTD-1
and green channel (B) Macrophage cells pretreated with 10 μM
probe BTD-2 for 30 min. The cells were excited with a light laser at
405 and 488 nm. Scale bar: 10 μm.
Mitochondria, an important organelle is the main sources of cell
energy which is also a pivotal production place of hypochlorite.
Imaging study on the level of cell mitochondria could bring more
accurate expression of the physiological and pathological processes
related to mitochondrial functions. So, whether it can be applied to
the study of mitochondrial imaging is an important criterion to
evaluate the value of probes. Herein, conventional co-localization
experiments were performed with commercially available probes
Mito Tracker green and Mito Tracker red. Briefly, HeLa cells were
co-incubated with BTD-1 (10 μM) and Mito-Tracker Green FM (200
nM) at 37 °C for 30 min. Comparative experiments were further
developed by adding OCl− (40 μM) to the above solution. Figure 8
shows that the fluorescence images of BTD-1 and Mito-Tracker
green overlaid well with each other. Mito-Tracker red was used
similarly for assessing BTD-2, and BTD-2 was also found to localize
in the mitochondria of the HeLa cells. This indicated that probes
BTD-1 and BTD-2 could selectively localize and accumulate in
mitochondria, thus enabling the imaging of exogenous OCl− in HeLa
cells and endogenous OCl− in living macrophage cells. Thus, our
original aim of designing the probe is realized.
Fig.9 (a) Fluorescence imaging of BTD-1 responding to OCl- in
zebrafish with various concentrations of OCl- (0, 5, 20 and 40 μM);
blue channe (A1-D1); bright field image (A2-D2); the merged image
of blue channel and bright field image (A3-D3);(b) Fluorescence
imaging of BTD-2 responding to OCl- in zebrafish with same
concentration as above; blue channe (A4-D4); green channe (A5-
D5);bright field image (A6-D6); the merged image of blue and green
channel, bright field image (A7-D7); All scale bars = 100 μm.
Based on the successful application of BTD in cells, we further
explored the applicability of BTD-1 and BTD-2 as an imaging tool in
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vivo. Zebrafish embryos (three days old) were chosen as the animal
model. Figure 9a and 9b showed that when the zebrafish were
pretreated with BTD-1 and BTD-2 (10 μM) for 30 min only a faint
fluorescence can be found. Subsequently, OCl− were added (5, 20,
and 40 μM), and the system was incubated for another 30 min. An
obvious enhancement in fluorescence was observed in the blue
channel for BTD-1 and in the blue and green channels for BTD-2.
This indicates that these two probes could enter zebrafish and
could be used to monitor OCl- in actual time with a turn-on
fluorescence response. Thus, BTD-1 and BTD-2 could be a useful
probe for monitoring OCl− distribution in living organisms.
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Yuan, F.; Han, Z. X.; Gong, A. H.; Wang, K. Simultaneous
discrimination of hypochlorite and single oxygen during sSepsis by a
dual-functional fluorescent probe. Anal. Chem. 2020, 92, 6072-6080.
15 Gong, J.; Yu, M. X.; Wang, C. F.; Tan, J. Y.; Wang, S. C.; Zhao, S. Y.;
Zhao, Z. J.; Qin, A. J.; Tang, B. Z.; Zhang, X. J. Reaction-based
chiroptical sensing of ClO− using circularly polarized luminescence
via self-assembly organogel. Chem. Commun. 2019, 55, 10768 -
10771.
In summary, two aggregation-induced fluorescence probes, BTD-
1 and BTD-2 were designed based on benzothiazole derivatives for
the detection and imaging of OCl−. BTD-1 exhibited rapid and highly
selective and sensitive detection for hypochlorite. To our delight,
BTD-1 and BTD-2 were successfully applied for monitoring OCl− in
HeLa cells as well as for tracking endogenous OCl− in macrophage
cells. In addition, the two probes could also be applied for
hypochlorite imaging in zebrafish. We believe that researchers
engaged in studying the effects of OCl− on biological systems will
significantly benefit from these new probes.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the Natural Science Foundation of
China for the project (Nos. 21625502 and 21974070) and the China
Postdoctoral Fund Surface Project (2017M610335) and a Project
Funded by the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD).
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