Development of pH/Glutathione-Responsive Theranostic Agents Activated by Glutathione S-Transferase πfor Human Colon Cancer

Article abstract of DOI:10.1021/acs.jmedchem.0c00354

Two novel theranostic agents HJTA and HJTB have been designed and synthesized by covalently linking a β-carboline derivative, with antitumor activities and pH-responsive fluorescence, with a 2-exomethylenecyclohexanone moiety, which can be activated by the tumor-targeting glutathione (GSH)/glutathione S-transferase π(GSTπ). These agents showed pH- and GSH-dual-responsive fluorescence in tumor cells but not in normal cells. Importantly, HJTA selectively illuminated tumor tissue for up to 7 h and generated precise visualization of orthotopic colonic tumors through the blood circulation system in intraoperative mice. Furthermore, HJTA exhibited potent and selective antiproliferative activities and colonic tumor inhibition in mice. Finally, HJTA induced great cancer cell apoptosis and autophagy by regulating the expression of apoptotic and autophagic proteins. Therefore, this pH/GSH-dual-responsive fluorescent probe with cancer-targeting therapeutic activity provides a novel tool for precise diagnosis and tumor treatment, therefore broadening the impact of multifunctional agents as theranostic precision medicines.

Full text of DOI:10.1021/acs.jmedchem.0c00354

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Article
Development of pH/Glutathione Responsive Theranostic Agents
Activated by Glutathione S-transferase # for Human Colon Cancer
Ji Liu, Xin Liu, Jianqiang Qian, Chi Meng, Peng Zhu, Jiaying Hang, Yaling Wang,
biao xiong, Xiaodong Qiu, Weizhong Zhu, Yumin Yang, Yanan Zhang, and Yong Ling
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00354 • Publication Date (Web): 05 Aug 2020
Downloaded from pubs.acs.org on August 6, 2020
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Development of pH/Glutathione Responsive
Theranostic Agents Activated by Glutathione
S-transferase π for Human Colon Cancer
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a,#
a,#
a
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a,b
a
a,b
Ji Liu , Xin Liu , Jianqiang Qian , Chi Meng , Peng Zhu , Jiaying Hang , Yaling Wang ,
a
a
a
b
a,*
a,b,*
Biao Xiong , Xiaodong Qiu , Weizhong Zhu , Yumin Yang , Yanan Zhang Yong Ling
aSchool of Pharmacy and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug
Target, Nantong University, Nantong 226001, PR China.
bKey Laboratory of Neuroregeneration, Ministry of Education and Jiangsu Province,
Co-innovation Center of Neuroregeneration, Nantong University, Nantong 226001, PR China
Abstract: Two novel theranostic agents HJTA and HJTB have been designed and
synthesized by covalently linking a β-carboline derivative, with anti-tumor activities and pH
responsive fluorescence, with a 2-exomethylenecyclohexanone moiety which can be activated
by the tumor-targeting glutathione (GSH)/glutathione S-transferase  (GST). These agents
displayed pH- and GSH- dual responsive fluorescence in tumor cells but not in normal cells.
Importantly, HJTA selectively illuminated tumor tissue for up to 7h, and generated precise
visualization of orthotopic colonic tumors through the blood circulation system in
intraoperative mice. Further, HJTA exhibited potent and selective antiproliferative activities
and colonic tumor inhibition in mice. Finally, HJTA induced great cancer cell apoptosis and
autophagy by regulating expression of apoptotic and autophagic proteins. Therefore, this
pH/GSH dual responsive fluorescent probe with cancer-targeting therapeutic activity provides
a novel tool for precise diagnosis and tumor treatment, therefore broadening the impact of
multifunctional agents as theranostic precision medicines.
Keywords: β-Carbolines, Glutathione transferase π, pH/GSH dual responsive fluorescence,
Selective tumor suppression, Tumor microenvironment
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. Introduction
Tumor microenvironment (TME), the special environment formed by the interactions between
tumor cells and cell surroundings during the tumor cells growth process,1,2 plays a pivotal role
during tumor initiation, progression, and metastasis, and significantly influences therapeutic
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response and clinical outcome. The differences on the physical and chemical properties of tumor
microenvironment from normal tissue,4-6 have been widely targeted for the development of
targeted therapy in order to improve efficacy and reduce side effects. One of the main differences
is the up-regulated oxidative stress in TME, resulting from the over-activation of reactive oxygen
species (ROS) under anoxic conditions in different TME associated cells.7,8 The excessive ROS
9
formation in TME can cause cell damage and even death, and therefore, antioxidant systems are
concomitantly upregulated in order to maintain redox homeostasis.10 Glutathione (GSH) is the
main antioxidant involved in maintaining this redox balance through direct scavenging of
peroxides and free radicals,11,12 and elevated concentrations of intracellular GSH have been found
13
in many types of human tumor tissues. The human glutathione transferase π isozyme (GSTπ) is a
key member of the family of glutathione transferase (GST) proteins that catalyze the nucleophilic
attack and conjugation of GSH with reactive electrophiles. GSTπ is overexpressed in many cancer
cells,14 such as breast and colon cancer,15,16 and in drug-resistant tumors, and is associated with
cell carcinogenesis, tumor formation and drug resistance of human tissues.
17
The GSH-rich TME has been a target for anti-tumor medications development. Studies have
shown that (6-oxocyclohex-1-en-1-yl)methyl (E)-but-2-enoate (COMC-6) and its analogues, all
promising anticancer agents against human tumors,18 interact with the over-expressed GSTπ in
various tumor cells. When catalyzed by GSTπ, GSH conjugates with COMC-6 leading to a GSH
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addition
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which
subsequently
fragments
to
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-exomethylene-3-glutathionyl-cyclohexanone 3 and crotonic acid.19 The active intermediate 3
can combine with thiols on DNA or proteins via covalent bonds in many tumor cells, therefore
generating marked anticancer effect (Scheme 1).20
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Scheme 1. Reaction Mechanism of COMC-6 with GSH/GSTπ.
In addition, TME is also associated with lower extracellular pH than normal tissue environment
as well as lower intracellular lysosomal pH.21-24 This weak acidic extracellular environment
around tumor tissues results from the Warburg Effect, where the metabolism is altered with the
rate of glucose uptake increasing and lactate being produced, and could facilitate the invasion,
mutation, metastasis, and multi-drug resistance of the tumor.25-27 This characteristic of TME has
also been specifically targeted by many research teams. As a result, a variety of pH responsive
probes have been developed to study various tumor processes in cells and in animals,28 although
they are mainly based on nanoparticles thus far.29
Here we describe our efforts in the development of theranostics, agents that combine therapy
with diagnostics, that simultaneously target the over-expressed GSTπ and the acidic TME of the
tumors. Such rationally designed theranostic agents, in addition to providing targeted treatment,
would also allow imaging of the cancer site and determining biodistribution of the drug. Our
group has recently developed several series of promising antitumor agents based on the naturally
occurring β-carbolines,30-32 a class of compounds that possess anticancer activities and also have
fluorescence because of the highly conjugated planar polyaromatic structure. Interestingly, we
found for the first time that the fluorescence of these substituted β-carbolines derivatives was
highly pH-dependent, only present at acidic pH. Therefore, we examined the possibility of
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generating novel theranostic agents by replacing the crotonic acid by a β-carboline moiety with
pH-responsive fluorescence properties to provide a novel class of 2-exomethylenecyclohexanones
(HJTA and HJTB, Figure 1). Upon reaction with GSH and GSTπ, the active molecules 3 and
β-carbolines will be liberated (Figure 1). Given the antiproliferative properties of both COMC-6
and β-carbolines and their ability to detect and illuminate tissues with special pH’s as in the TME,
these novel agents may serve as excellent theranostic agents to provide in-vivo and real-time
diagnosis of tumor tissue and TME intraoperatively, as well as possessing chemotherapeutic
capability to prevent recurrence of residual tumor and improve patient outcome.
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HJTB
GSH
GSH, GST
pH of TME
COMC-6
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H2N
Protein-SH
Cancer cell imaging
6
GSH
O
Protein
S
Selective antitumor activity
4
Figure 1. Schematic illustration of constructing and activation the tumor-targeting and pH and
GSH/GSTπ dependent probes HJTA and HJTB.
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2
. Results and Discussion
.1 Synthesis of HJTA and HJTB
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The synthetic scheme of HJTA and HJTB is presented in Scheme 2. The starting
cyclohex-2-en-1-one
7
and
formaldehyde
were
reacted
to
provide
2
-(hydroxymethyl)cyclohex-2-en-1-one 8 by a Baylis–Hillman reaction. Intermediate 8 then
reacted with 4-nitrophenyl chloroformate to provide compound 9. Separately, the tricyclic system
of tetrahedron-β-carboline parent ring 12 was constructed via a Pictet-Spengler reaction between
L-tryptophane 10 and 3,4,5-trimetoxybenzaldhyde 11. Compound 12 was transformed to the ester
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3 under classical esterification conditions using SOCl and CH OH. The oxidization of ester 13
2 3
with KMnO in a DMF solution afforded compound 14, which was further treated with hydrazine
4
monohydrate to provide 15. Hydrazide compound 15 was then converted to acyl azide 16 using
sodium nitrite. Then the acyl azide group of 16 was transformed to an amino group in an aqueous
acetic acid solution by the Curtis rearrangement reaction to afford 3-amino-β-carboline 6. Finally,
HJTA and HJTB were constructed via an amidation reaction between 3-amino-β-carboline 6 and
intermediate 9 in the presence of DIPEA. The final compounds HJTA and HJTB were
characterized by H NMR, 13C NMR, and ESI mass spectrometry (Figure S7–S13, Supporting
Information).
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NO2
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NH
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OH
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N
O
N
NH2
NH2
N
N3
O
H
N
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f
g
h
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N
H
H
H
O
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O
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i
H
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+
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+
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O
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N
H
O
O
HJTA
HJTB
O
Scheme 2. Synthetic scheme for HJTA and HJTB. (a) HCHO, DMAP, 18 h, 86%; (b)
4
-nitrophenyl carbonochloridate, DIPEA, CH Cl , 0 °C, 2 h, 70%; (c) HAc, 100 °C, 4 h, 84%; (d)
2
2
SOCl , Methanol, -5 °C, 1 h, and then reflux 5 h, 89%; (e) KMnO , DMF, reflux, 4 h, 69%; (f)
2
4
NH −NH ·H O, Methanol, 0 °C, 1 h, 95%; (g) NaNO , HCl aqueous solution, 0 °C, 6 h; (h) HAc
2
2
2
2
aqueous solution, reflux, 6h, 71%; (i) DIPEA, CH Cl , 0 °C, 4 h, 40% and 28%.
2
2
2
.2 pH response of 6, HJTA, and HJTB
In order to confirm that the newly synthesized agents HJTA and HJTB have the same
pH-responsiveness that was observed with the 3-amino-β-carboline subunit 6, these compounds
were first evaluated using both UV-vis absorption and fluorescence emission spectroscopy. To our
delight, 3-amino-β-carboline 6 exhibited remarkable pH-responsiveness from pH 3 to 7 through an
internal charge transfer (ICT) process (Figure S1). We then probed the pH-responsive properties
of HJTA and HJTB. As shown in Figure 2A, as the pH decreased from 7.63 to 3.19, the
absorption band of HJTA gradually shifted from 398 to 445 nm, with a distinct isosbestic point
observed at 416 nm. The fluorescence intensity at 490 nm underwent a concomitant monotonic
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increase (Figure 2B). However, the fluorescence was fully quenched in an aqueous solution with
neutral pH. A quantitative analysis of the fluorescence intensity at 490 nm vs. pH (Figure 2D)
revealed a 65-fold (from 845.348 ± 12.47 to 13.035 ± 1.26) increase as the pH was lowered from
7
.67 to 3.17. The pKa of HJTA was calculated to be 5.2 (Figure S2) and the relative
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fluorescence-quantum yield is 0.63 at pH 4.0, compared with Quinine sulfate (Table S1).33
Similarly, the photophysical characteristics of HJTB were also measured under different pH’s.
A 50 nm red-shift with a distinct isosbestic point was observed at 418 nm and enhancement of
absorption at 447 nm occurred with the decrease of pH from 7.62 to 3.21. Similar to HJTA, a
6
0-fold enhancement of fluorescent intensity at 490 nm was also obtained with the decrease of pH
when excited at 447 nm. The pKa value of HJTB was 5.3 (Figure S3), which falls in the pH range
of TME over normal tissue. The relative fluorescence-quantum yield is 0.67 at pH 4.0, compared
with Quinine sulfate (Table S1).33 These results support that the pH-dependent fluorescent
properties are retained in these β-carboline based designed probes which are suitable for
visualizing TME.
Figure 2. (A) Absorption spectra of HJTA (100 μM) at different pH (7.63 - 3.19) in an aqueous
solution containing 1% (v/v) DMSO. (B) Fluorescence emission spectrum of HJTA (1 μM) at
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different pH (7.67 – 3.17) in an aqueous solution containing 1% (v/v) DMSO, (λex = 445 nm). (C)
Absorbance-pH titration profiles of HJTA at absorption wavelength of 445 nm. (D) Fluorescence
intensity-pH titration profiles of HJTA at emission wavelength of 490 nm. Insert: fluorescence
images of HJTA (5 μM) at pH = 7.4 and pH = 4.0 under 365 nm UV light.
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Figure 3. (A) Absorption spectra of HJTB (100 μM) at different pH (7.62 - 3.21) in an aqueous
solution containing 1% (v/v) DMSO. (B) Fluorescence emission spectrum of HJTB (1 μM) at
different pH (7.69 – 3.19) in an aqueous solution containing 1% (v/v) DMSO, (λex = 447 nm). (C)
Absorbance-pH titration profiles of HJTB at absorption wavelength of 447 nm. (D) Fluorescence
intensity-pH titration profiles of HJTB at emission wavelength of 490 nm.
2
.3 GSH response of 6, HJTA and HJTB
The responses of HJTA and HJTB, along with 6, to GSH were measured in aqueous solutions
containing 1% DMSO (v/v) with the enzyme GSTπ at 37 °C. As shown in Figure S4, no
detectable change in fluorescence was observed with 6 after addition of GSH (0 – 360 μM).
However, the absorption of the HJTA (100 μM) solution rapidly increased to above 440 nm
(Figure 4A). Further, the solution exhibited a marked enhancement of fluorescence intensity at
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around 492 nm in a dose-dependent manner and showed obvious green fluorescence that could be
clearly observed under 365 nm ultraviolet lamp (Figure 4C). The fluorescent signal of HJTA after
addition of GSH was stable and lasted over 1 h at 37 °C (Figure 4D). These data support that
HJTA can serve as a prominent GSH responsive fluorescent on-off switch. From the fluorescence
spectrum in Figure S5b, it can be seen that both HJTA and HJTB displayed a typical
fluorescence enhancement in the range of 450-600 nm. Interestingly, while HJTB has two
GSH/GSTπ reactive sub-units and HJTA only has one such unit, HJTB had weaker fluorescence
than HJTA (Figure 4B vs. Figure S5B). Further, the time of fluorescence intensity reaching a
plateau (1.5h) after the addition of GSH was also longer for HJTB than that HJTA, suggesting
that HJTB is less sensitive for the rapid detection of GSH (Figure S5D).
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Figure 4. (A) Absorption spectra of HJTA (100 μM) in the presence of varying amount of GSH
(
0 ~ 10 equiv) with cat. GSTπ in an aqueous solution containing 1% (v/v) DMSO. The spectra
were recorded after incubation at 37 °C for 0.5 h. (B) Fluorescence emission spectrum of HJTA
1 μM) at 492 nm as a function of varying concentrations of GSH with cat. GSTπ in an aqueous
solution containing 1% DMSO (v/v), (λex = 445 nm). (C) Fluorescence intensity-concentration
GSH) titration profiles of HJTA at emission wavelength of 492 nm. Insert: fluorescence images
(
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(
of HJTA (5 μM) without and with the presence of GSH (50.0 μM) under 365 nm UV light. (D)
GSH concentration (0, 5, 15, 25, 50 μM)-dependent fluorescence activation rates of HJTA (1 μM).
(E) Fluorescence response of HJTA (1 μM) with cat. GSTπ after treatment with various
+
+
2+
2+
2+
3+
2+
2+
biological analytes: 1 mM of (K , Na , Ca , Mg , Zn , Al , Cu , Fe ); 100 μM of (GSH, lys,
his, ala, cys, glu, ser, gly, arg, VC, Na S); and 50 μM of NADH and H O . Bars represent
2
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2
comparative fluorescence changes at 492 nm, λ = 440 nm.
ex
The observed fluorescence change is likely the result of the formation of adduct 5, which
formed via the reaction of GSH with HJTA or HJTB (Figure 1). Thus, the aqueous solution of
the more GSH responsive HJTA and GSH were analyzed using HRMS. A mass peak of 809
+
(
exact mass calcd for M + H , 809.2825) was generated (Figure S14), confirming 5 formation.
This result indicates that GSH has the ability to covalently bind to the enone functionality of
HJTA.
To verify the selectivity of HJTA for GSTπ against other GST enzymes, the percentages of 6
release were analyzed by HPLC after incubation of HJTA (40 μM) in the presence of GSH (0.8
mM) and GSTπ or GSTα (0.03 μg/ml). Enzymatic reactions of HJTA with GSTπ, but not GSTα
(Figure S17), were observed under the same conditions, confirming the selectivity of HJTA for
GSTπ.
2
.4 Selective response of HJTA to GSH
To confirm that the response of HJTA toward GSH/GSTπ is selective, a variety of
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physiological environment relevant species, including inorganic metal ions, amino acids,
+
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reductants and oxidants, were analyzed at pH 7.4. As shown in Figure 4E, inorganic salts (K , Na ,
2+
2+
2+
3+
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2+
Ca , Mg , Zn , Al , Cu , Fe ), amino acids (lysine, histidine, alanine, cysteine, glutamic acid,
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serine, glycine, arginine), reductants (Vitamin C, Na S), H O , and NADH did not cause any
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apparent change of the fluorescence intensity. These results demonstrate that HJTA is highly
selective in specific response to GSH and pH.
2
.5 Celluar uptake of HJTA
The intracellular fluorescence of HJTA was subsequently evaluated in cancer HT29 cells and
normal CCD841 cells to evaluate its cellular uptake. The time course of HJTA (1.0 μM) cellular
uptake was determined by quantifying intracellular fluorescent intensity of HT29 cells and
CCD841 cells within 60 min (Figure S6). The confocal fluorescence images indicate that the
uptake of HJTA was rapid and increased comparably for HT29 cells within the first 10 min
(Figure S6A), while it was minimal for CCD841 cells during the same time frame. In addition, the
final fluorescence intensity in HT29 cells was significantly higher than that of CCD841 cells,
suggesting more efficient accumulation of HJTA in HT29 cells. This is likely the result of the
targeting ability of HJTA for the overexpressed GSH and/or the lower pH in lysosomes in these
cancer cells.21,22
We then treated both HT29 and CCD841 cells with HJTA (1.0 μM) and co-incubated with a
commercial lysosomal probe (Lyso-Tracker), which labels and tracks acidic organelles in live
cells, for 60 min to examine intracellular fluorescence activation. For CCD841 cells which do not
overexpress GSH, the green emission upon treatment with HJTA was weak and little overlapped
with the red signal in the lysosomes (Figure 5B). However, the green emission was significantly
stronger for the GSH overexpressed HT29 cells than that of CCD841 cells upon treatment with
HJTA. Confocal images showed that the green fluorescent signal largely overlapped with the red
fluorescent signals from Lyso-Tracker (Figure 5A), consistent with its sensitivity to acidic
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environments and GSH. These studies clearly demonstrate that HJTA can target and monitor
cancer cells extensively overexpressing GSH and having lower pH with good sensitivity as
designed.
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Figure 5. Intracellular fluorescent activation of HJTA and cell viabilities. (A) Confocal
microscopy images of HT29 cells treated with HJTA and Lyso-Tracker. (B) Confocal microscopy
images of CCD841 cells incubated with HJTA and Lyso-Tracker. Scale bar for all images = 25
μm. For HJTA (green channel), an excitation wavelength of λex = 440 nm was used. For
Lyso-Tracker (red channel): λ = 561 nm.
ex
2
.6 In vitro antitumor activities and selectivity
Now that we have demonstrated the fluorescent and visualizing capability of HJTA in vitro,
we next determined the antiproliferative effect of HJTA and HJTB on HT29, HepG2 and 9L-2
3
4
cells by MTT assay. HT29, HepG2 cells as GSTπ activity “+” and 9L-2 cells as GSTπ activity “-”
were employed to examine the antiproliferative selectivity of HJTA and HJTB for
GSTπ-overexpressed cancer cells. Compound 6, COMC-6, and harmine (a naturally occurring
β-carboline) were used as the positive controls. Both HJTA and HJTB exhibited prominent
inhibitory activities against three tumor cell lines with IC50 values on single-digit micromole
levels. Furthermore, the inhibitory potencies of HJTA and HJTB on HT29 and HepG2 cells with
GSTπ activity as “+” (IC50 values of 0.92-2.69 μM) were greater than 9L-2 cells with GSTπ
activity as “-” (IC50 values of 4.32-4.85 μM), indicating a positive association between GSTπ
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activity and the antiproliferative activity of HJTA and HJTB. Noticeably, HJTA, the more potent
compound, exerted significant anticancer activities with IC50 value of 0.92 μM against colonic
cancer cells, 5-fold better than COMC-6 (IC = 4.37 μM), 14-fold better than β-carboline moiety
5
0
6
(IC50 = 13.5 μM), and even 3-fold better than the combination of COMC-6 and 6 (IC50 = 2.81
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μM). HJTA also exerted over 3-fold weaker toxicity on intestinal normal cells CCD841 than
COMC-6, demonstrating a greater therapeutic window for HJTA than COMC-6 (Table 1). HJTA
had greater anti-tumor activities than HJTB against all 3 cell lines. This result is consistent with
their GSH-responsiveness (HJTA > HJTB), likely leading to incomplete degradation of HJTB by
GSH/GSTπ and thus less amount of generated reactive species (compound 3). Based on these
considerations, HJTA was selected in subsequent investigations.
Table 1. The IC50 (μM) of HJTA and HJTB against three cancer cell lines and one intestinal
normal cell line a
a
IC (μM)
5
0
Compound
HT29b
9L-2 c
HepG2
CCD841
7.83 ± 0.55
ND
COMC-6
6
4.37 ± 0.39
13.5 ± 0.96
2.81 ± 0.43
0.92 ± 0.11
1.45 ± 0.24
41.8 ± 3.75
5.93 ± 0.67
17.1 ± 1.28
4.09 ± 0.56
1.57 ± 0.20
2.69 ± 0.31
49.6 ± 3.89
7.79 ± 0.48
ND
COMC-6 + 6
HJTA
ND
ND
4.32 ± 0.36
4.85 ± 0.61
47.8 ± 5.24
23.4 ± 2.72
28.1 ± 3.11
>50
HJTB
Harmine
aThe IC50 data (means ± SD) were from three separated experiments. GSTπ activity as “+”.
b
cGSTπ activity as “-”.
2.7 Imaging-guided surgery of colon tumors
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Considering its encouraging in vitro results in simultaneously targeting pH and GSH, HJTA
was investigated for its ability to visualize tumor tissues and to guide resection of orthotropic
HT29 colon tumors. Thus, luciferase-marked HT29 (HT29/Luc) cells were injected into the upper
part of the colons to construct an orthotopic colon tumor model in the mice. Two weeks later,
clear bioluminescence (BL) was seen, indicating successful formation of HT29/Luc tumors in the
colon of mice. Upon intravenous administration of HJTA, bright fluorescence was observed in the
colon position, overlapping well with the BL imaging position (Figure 6A). To further validate the
specificity of HJTA for tumor tissues versus normal tissues, the mice were sacrificed, and the
orthotropic colon tumor and main organs were analyzed by BL and fluorescence imaging. The
fluorescent signal was significantly greater in the colon tumor than the other organs, and also
overlapped well with the BL imaging (Figure 6B). These results confirm that HJTA, as a
promising molecular imaging tool, is capable of locating orthotopic colon tumor tissues in living
systems via noninvasive fluorescence imaging, which could assist fluorescence-guided surgical
removal of colon tumor.
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Figure 6. Fluorescence-guided location of orthotopic colon tumors. (A) Full-body
bioluminescence (BL, left) and fluorescence (FL, right) imaging of orthotopic HT29/Luc colon
tumor in intraoperative mice after 2 h post intravenous injection with HJTA (40 mg/kg). (B) Ex
vivo bioluminescence (up) and fluorescence (down) imaging of main organs removed from
orthotopic HT29/Luc colon tumor mice model (λex/em = 430/490 nm). (C) Ex vivo fluorescence
imaging of tumor and main organs removed from HT29 colon xenograft mice model after 2 h post
intravenous injection with HJTA (40 mg/kg). (D) Ex vivo fluorescence imaging of tumor
extirpated from subcutaneous tumor mice model at various time points post intravenously injected
with HJTA (40 mg/kg).
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We have clearly demonstrated that HJTA successfully penetrated into cells/tissues and
selectively illuminated tumors as designed. Theranostic agents are also required to have good
stability and be retained at the targeted tissue for sufficiently long. Therefore, the fluorescence
biodistribution of HJTA was then investigated in tumor-bearing mice using ex-vivo imaging. Two
hours after intravenous injection of HJTA (40 mg/kg), the mice were sacrificed and the tumor and
major organs including the heart, lung, liver, kidney, colon, and spleen, were examined. As clearly
showin in Figure 6C, the fluorescent signal was significantly greater in the tumor than the major
organs (Figure 6C). Strong fluorescent signal was observed in tumors 1 hour after injection and
lasted for over 7 hours (Figure 6D). These results confirmed the efficient retention of HJTA in the
tumor with promising selectivity and visualization in vivo.
2
.8 In vivo anti-cancer efficacy of HJTA
To assess the in vivo safety and efficacy profile of HJTA, ICR mice were intraperitoneally
injected with different doses of HJTA. The percentage of survival was monitored for 14 days after
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administration. As depicted in Table 2, at the highest dose of HJTA (585.9 mg/kg), only one
mouse treated survived. In contrast, no death or abnormality in body weight, drinking, eating, or
activity was found after administration with HJTA at the lowest dose (240 mg/kg). Accordingly,
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the acute toxicity of HJTA was analyzed and the median lethal dose (LD ) value was determined
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to be 475 mg/kg.
Table 2. Acute toxicity of HJTA in mice
Dose
Number of Number of dead mice
Total
Survival (%)
(mg/kg)
mice
10
1h 4h
1d 2d 3d 4d 5-14d death
on day 14
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Figure 7. The antitumor effect of HJTA on HT29 xenograft in nude mice. (A) After establishing
xenograft mice model, tumor volume changes in tumor-bearing mice administrated with saline
control, COMC-6, or HJTA were surveyed during 21 days. *P < 0.05 vs control group. (B) Body
weight was monitored every three days. (C) Tumor weight was measured at the end of the
treatment . **P < 0.01 vs control group. (D) Histological sections of tumors stained with H&E.
Scale bar for all images = 100 μm.
In order to assess the in vivo antitumor efficacy of HJTA, four BALB/c nude mice groups were
subcutaneously injected with HT29 cells. After establishing xenograft mice model, these
tumor-bearing mice were intraperitoneally injected with saline, HJTA, and COMC-6 every day
for 21 days, respectively. The body mass and tumor size of nude mice were surveyed every three
days during the 21 days. As shown in Figure 7A, A sustainable tumor growth was observed with
the saline treated group. In contrast, the intraperitoneal treatment of HJTA apparently diminished
the volumes of xenograft tumors. HJTA also led to greater tumor reduction than COMC-6 at the
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end of the treatment period. However, no significant statistical difference in body mass changes
between the HJTA–treated groups and vehicle groups was observed (Figure 7B). The tumor
weight (0.48 ± 0.07 g) in mice dosed by HJTA at 40 mg/kg was diminished by 67.7% (w/w)
compared to the saline groups (1.49 ± 0.27 g), and was lower than the tumor weight (0.81 ± 0.12
g) of COMC-6 groups at 40 mg/kg (Figure 7C). Together, these data clearly confirm that HJTA
possessed remarkable inhibitory potency of the tumor growth in vivo.
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.9 Anticancer Mechanism Studies
Apoptosis is one of the critical mechanisms of cell death following exposure to
chemotherapy.35,36 Since HJTA exerted significant and selective tumor cell inhibitory activities, it
was evaluated for its potency to promote cancer cell apoptosis. Therefore, HT29 cells were
inoculated with COMC-6, harmine, or HJTA for 72 h, and the apoptotic cell percentages were
analyzed through FITC-Annexin V/PI staining and flow cytometry. As depicted in Figure 8, cells
incubated with increasing concentrations of HJTA exhibited potent apoptotic rates (36.8% for 1.0
μM and 52.3% for 2.5 μM) in annexin V + HT29 cells, which were significantly better than the
harmine (20.9% for 10 μM) and COMC-6 (28.8% for 2.5 μM) groups. Accordingly, HJTA
exerted HT29 cell apoptosis induction with greater efficacy than harmine and COMC-6.
To further verify the apoptosis induction effect of HJTA, expressions of apoptosis-related
proteins, including Bcl-2, Bax, and the cleavage of caspase-3 and PARP (apoptotic key markers)
were detected.37,38 HT29 cells were incubated with vehicle, HJTA, COMC-6, or harmine,
respectively, for 72 h and the cells were lysed and subjected to analysis in Western blot (WB).
According to Figure 8A, HJTA dose-dependently augmented the pro-apoptotic Bax levels but
diminished the anti-apoptotic Bcl-2 levels. In addition, HJTA also resulted in the accumulation of
cleaved-PARP and caspase-3, both more significantly than harmine- or COMC-6-treated group.
Overall, these analyses suggested that HJTA induced apoptosis by the regulation of apoptotic
related proteins in HT29 cells.
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Figure 8. Compound HJTA induced HT29 cell apoptosis. (A) HT29 cells were treated with the
different concentrations of HJTA, COMC-6, or harmine for 72 h, and stained with FITC-Annexin
V and PI through flow cytometry analysis. (B) The apoptotic rates were quantitatively analyzed,
and data (means ± SD) were collected from three separated experiments. *P< 0.01 vs control. (C)
The levels of apoptosis related proteins in HT29 cells upon with HJTA, COMC-6, or harmine in
WB assay. (D) The relative levels of each apoptosis protein were quantitatively analyzed
compared to the internal reference β-actin, and the related data (means ± SD) were from three
independent assays. *P < 0.01 vs vehicle control.
Autophagy, a natural and well-conserved cellular catabolic process regulating the cell in
disassembling dysfunctional or unnecessary components, is regarded as an effective strategy to
enhance antineoplastic effects of chemotherapeutic agents and improve their therapeutic outcome
in cancer patients.39-41 The potential and mechanism of HJTA inducing autophagy were assessed
by testing the levels of key autophagic related proteins. The conversion of LC3-I to LC3-II is a
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crucial point in autophagy. Thus, the changes of autophagic markers LC3-I/-II, Beclin-1, and p62
proteins in response to HJTA treatment were examined in HT29 cells. As depicted in Figure 9,
the levels of LC3-II and Beclin-1 proteins dose-dependently increased, while the expression of
p62 was downregulated upon treatment with HJTA of different concentrations. Noticeably, the
LC3-II/LC3-I ratio and the levels of LC3-II in HJTA-treated groups were higher than both the
harmine and COMC-6-treated groups (Figure 9B). These results indicated that HJTA could
markedly lead to an autophagic induction in HT29 cells.
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Figure 9. Compound HJTA promoted autophagic induction. (A) Autophagy-associated proteins
(LC3-I/-II, p62, and beclin-1) were detected by WB assay in HT29 cells treated with vehicle,
HJTA, COMC-6, or harmine. (B) The LC3-II/LC3-I ratio was quantitatively analyzed. (C) The
comparative levels of LC3-I/-II, p62, and beclin-1 to the internal reference β-actin were
quantitatively analyzed. The related data (means ± SD) were from three independent assays. *P <
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.01 vs respective vehicle control.
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. Conclusions
In summary, we have for the first time developed excellent theranostics HJTA and HJTB as
intraoperative fluorescence diagnostic and chemotherapeutic agents for colonic cancer in vivo. The
results showed that HJTA and HJTB not only displayed dual pH/GSH responsive fluorescence
features, but also selectively inhibited tumor cell proliferation. Importantly, HJTA selectively and
precisely illuminated cancer cells and tumor tissues, and accurately located orthotopic colonic
tumor foci through the blood circulation system, providing an image-guided tool for surgical
resection of tumor tissues in intraoperative mice. Meanwhile, the positive tumor inhibition of
HJTA and its significant apoptotic and autophagic induction may provide simultaneous
chemotherapy, in addition to real-time fluorescence diagnosis, to prevent invasion and metastasis
of cancer cells. Thus, we believe that this theranostic probe designed for intraoperatively
monitoring of TME combined with selective antitumor activity would add a much needed tool to
the current cancer diagnosis strategies and may improve patient prognosis and broaden the impact
of small multifunctional molecules in enzyme mapping, tumor theranostic and image-guided
surgery.
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. MATERIALS AND METHODS
4.1 Materials
All chemical reagents were purchased from Aladdin or Sigma-Aldrich and used without further
1
purifications. All reactions were carried out under air atmosphere if no additional indication. H
and 13C NMR spectra were obtained on a Bruker AV 400 M spectrometer with tetramethylsilane
as the internal standard. Mass spectra was recorded on a Mariner mass spectrometer (ESI).
UV-visible spectra were obtained on a spectrometer (UV1800PC, Jinghua, China). Fluorescence
spectroscopic studies were performed on a fluorescence spectrophotometer (RF-5301PC,
Shimadzu, Japan). HRMS data were recorded on a JMS-SX102A (FAB) or LC/MSD TOF.
Compounds 7, 10, 11 were commercially available. Compounds 6 and 12-16 were synthesized
according literatures.42 Purities of target compounds reached at least >95% pure by analytical
HPLC (Figure S15, S16).
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4.2 Synthesis of HJTA and HJTB
-Hydroxymethyl-2-cyclohexen-1-one (8)
To a solution of 2-cyclohexen-1-one 7 (4.8 g, 50 mmol) in 30 mL THF were added
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formaldehyde (4.2 g, 60 mmol) and DMAP (0.63 g, 5 mmol)and stirred for 18 hours. After TLC
monitored the total consumption of starting material, the reaction mixture was quenched with
water 120 mL, followed by extracted with CH Cl (40 ml × 3). The combined organic extracts
2
2
were dried over anhydrous Na SO , filtered, concentrated in vacuo, and finally purified by flash
2
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column chromatography to give the product 8 as a yellow oil (5.4 g, 85.7%). Spectral data are in
agreement with literature values.43 1H NMR (DMSO-d , 400 MHz): δ 6.95 (s, 1H), 4.77 (t, 1H, J =
6
5
.5 Hz), 4.12 – 3.94 (m, 2H), 2.00 – 1.78 (m, 4H), 2.00 – 1.78 (m, 2H).
(6-Oxocyclohex-1-en-1-yl)methyl (1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indol
-3-yl)
carbamate (HJTA) and di(6-oxocyclohex-1-en-1-yl)methyl(1-(3,4,5-trimethoxy phenyl)-9H-
pyrido[3,4- b]indol-3-yl)carbamate (HJTB)
Compound 8 (252 mg, 2.0 mmol) and 4-nitrophenyl carbonochloridate (605 mg, 3.0 mmol)
were dissolved in 15 mL dry CH Cl cooled to 0 °C. DIPEA (505 mg, 5.0 mmol) was added and
2
2
the mixture was stirred for 2 h at 0 °C, at which point reaction was completed. The mixture was
diluted with CH Cl (40 mL), washed with saturated aqueous NaHCO (15 mL), and brine. Then
2
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organic layer was dried with Na SO and concentrated under reduced pressure. The crude product
2
4
was purified by silica gel column chromatography using Petroleum ether/EtOAc (8:1, v : v) as
eluent to afford 9 as a yellow solid (410 mg, 70.4%).
Compound 9 (436 mg, 1.5 mmol) and compound 6 (349 mg, 1.0 mmol) were dissolved in 5 mL
dry CH Cl at 0 °C, and DIPEA (505 mg, 5.0 mmol) was added to the above solution and the
2
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mixture stirred at room temperature for 4 hours. The solvent was evaporated under vacuum. The
crude solid waspurified by a silica gel column using Petroleum ether/EtOAc (2:1, v : v) as eluent
to obtain HJTA (202 mg, 40.3%) and HJTB (140 mg, 27.9%).
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Analytical data for HJTA: H NMR (DMSO-d , 400 MHz): δ 10.83 (s, 1H), 8.07 (d, 1H, J =
6
7
.6 Hz), 7.49 – 7.45 (m, 2H), 7.21 (s, 2H), 7.13 – 7.09 (m, 2H), 6.99 (s, 1H), 6.13 (s, 1H), 4.11 –
4.10 (m, 2H), 3.91 (s, 6H), 3.76 (s, 3H), 2.43 – 2.36 (m, 2H), 2.34 – 2.33 (m, 2H), 1.95 – 1.87 (m,
2
1
H). 13C NMR (DMSO-d , 101 MHz): δ199.3, 153.4, 152.5, 145.8, 143.0, 138.2, 137.0, 128.4,
6
27.9, 126.6, 122.0, 121.3, 118.8, 116.3, 112.5, 106.1, 95.6, 60.5, 56.3, 38.6, 25.7, 23.2. HRMS:
+
(
ESI, m/z) Calcd for C H N NaO [M+Na] : 524.1792, found 524.1785.
28 27
3
6
1
Analytical data for HJTB: H NMR (DMSO-d , 400 MHz): δ 10.89 (s, 1H), 8.15 (d, J = 8.1 Hz,
6
1H), 7.50 – 7.43 (m, 2H), 7.23 (s, 2H), 7.18 (s, 1H), 7.13 – 7.09 (m, 1H), 6.73 (s, 2H), 4.35 (s, 4H),
.89 (s, 6H), 3.76 (s, 3H), 2.42 – 2.37 (m, 4H), 2.31 (s, 4H), 1.92 – 1.90 (m, 4H). 13C NMR
DMSO-d , 101 MHz): δ199.8, 153.4, 151.5, 145.4, 142.9, 138.4, 138.0, 134.8, 134.7, 134.1,
3
(
6
1
28.5, 127.5, 122.3, 105.8, 56.1, 47.6, 38.6, 25.7, 23.2. HRMS: (ESI, m/z) Calcd for C H N O
36 36 3 9
+
[
M+H] : 654.2452, found 654.2455.
4.3 GSH dependent absorption spectra
Solutions of HJTA (100 μM) or HJTB (200 μM) were prepared with deionized water
containing 1% (v/v) DMSO with GSH (0 ~ 20 equiv) and cat. GSTπ. All absorption spectra were
recorded at 37 °C for 0.5 h and scanning wavelength range was 350–700 nm with scanning speed
is 1.0 nm/s.
4.4 GSH dependent emission spectra
Solutions (1.0 μM) of HJTA or HJTB were prepared in deionized water containing 1% (v/v)
DMSO with GSH (0 – 100 μM) and cat. GSTπ. All emission spectra were performed at 37 °C for
0.5 h and excited at 440 nm and recorded from 450 nm to 650 nm.
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.5 pH dependent absorption spectra
Solutions (100 μM) of HJTA, HJTB or 6 were prepared in deionized water containing 1% (v/v)
DMSO with pH ranging from 7 to 3. All absorption spectra were recorded at room temperature
r.t.) and scanning wavelength range was 350–700 nm with scanning speed is 1.0 nm/s.
(
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.6 pH dependent emission spectra
Solutions (1.0 μM) of HJTA, HJTB or 6 were prepared in deionized water containing 1% (v/v)
DMSO with pH ranging from 7 to 3. All emission spectra were performed at r.t. and excited at
4
50 nm and recorded from 460 nm to 650 nm.
4
.7 Fluorescence selectivity evaluation
Solutions (1 μM) of HJTA with cat. GSTπ and various biological analytes (Lys, His, Ala, Cys,
2+
+
2+
+
2+
2+
2+
3+
Glu, Ser, Gly, Arg, H O , Ca , K , Mg , Na , Zn , Fe , Cu , Na S, Al , Vc, NADH, GSH, 20
2
2
2
μM) were prepared at pH = 4.0 with deionized water containing 1% (v/v) DMSO at 37 °C for 0.5
h. All emission spectra were excited at 440 nm and recorded from 450 nm to 650 nm.
4
.8 Quantum efficiencies of fluorescence
The quantum yield of HJTA + GSH, HJTA at pH = 4.0 or HJTB at pH = 4.0 were determined
in deionized water containing 1% (v/v) DMSO, using Quinine sulfate (Φ = 0.55 in 1 N H SO ) as
S
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a reference. The quantum yield was calculated according to Equation 1:
2
Φ = Φ (A F /A F ) (n /n ) (1)
X
S
S
X
X
S
X
S
Φ: quantum yield; A: absorbance at the excitation wavelength; F: integral area of fluorescence
spectra at the same excitation wavelength; n: the refractive index of solvents; S and X represent
the reference standard and unknown sample, respectively.
4
.9 Cell Culture
Human cancer cell lines (HT29 and HepG2) or normal cell lines (CCD841) were purchased
from Shanghai Institute of Cell Biology (China), and murine gliosarcoma cell line (9L-2) was
from department of neurological surgery tissue bank at University of California, San Francisco.
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These cell lines were routinely put in DMEM/RMPI 1640 medium possessing 10% FBS
(Invitrogen), 100 units/ml penicillin, and 100 units/ml streptomycin at 37 °C under an atmosphere
of 5% CO2.
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4.10 In vitro cellular uptake studies
5
For time dependent-cellular uptake: HT29 (2 × 10 cells/well) were seeded into 35 mm glass
bottom cell culture dishes respectively in 2.0 mL culture medium. After cell attachment, the
culture medium was replaced by the DMEM containing HJTA (1 μM) and the fluorescent images
were obtained every one minute for 60 minutes by Leica TCS SP5 LSM confocal microscope
using a 40X objective water lenses. The green fluorescence of HJTA was obtained by using a 405
nm laser. Filter set: 480-530 nm.
4
.11 MTT assay
The in vitro antiproliferative effects were assessed on HT29, HepG2, 9L-2, and CCD841 by the
MTT method according to previous literature.44 After washing the cells with PBS buffer, 0.5
mg/mL MTT solution was added and the cells were kept at 37 °C for 3 h. After MTT medium
removal, the formazan crystals were dissolved in DMSO and the absorbance was measured at 570
nm using a BioTek Microplate Absorbance Reader according to the protocol. The inhibitory effect
was expressed as percentage. Corresponding IC50 values were then calculated through
corresponding software (GraphPadPrism Version 4.03).
4
.12 Intracellular fluorescence activation
5
HT29 and CCD841 cells (2 × 10 cells/well) were seeded into 35 mm glass bottom cell culture
dishes in 2.0 mL culture medium respectively. The cells were incubated in DMEM containing
HJTA (1 μM) with LysoTracker red DND-99 (1 μM) for 30 minutes. The fluorescent images
were obtained by Leica TCS SP5 LSM confocal microscope using a 40X objective water lenses.
The green fluorescence of HJTA was obtained by using a 405 nm laser. Filter set: 480-530 nm.
The red fluorescence of LysoTracker red was obtained by using a 561 nm laser. Filter set: 580-630
nm.
4
.13 Animals and tumor model
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All animal experimental protocols were approved by the Animal Research and Care Committee
of Nantong University. To set up the subcutaneous tumor mouse model, female nude mice (5-6
weeks old) were purchased from the Model Animal Research Center Affiliated to Nanjing
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University (Nanjing, China). 1×10 HT29 or HT29/Luc cells were subcutaneously injected into
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the BALB/c mice. Animals bearing tumor were established (approximately 3 to 4 weeks) before
used for fluorescence imaging.
6
To establish an orthotropic colon tumor model, 1×10 luciferase-transfected HT29 cells
(
HT29/Luc) were directly injected into the upper part of the colons after anaesthesia in nude mice
5-6 weeks old). The tumor growth and development in mice were monitored by bioluminescent
(
imaging. Two weeks later, the orthotropic colon tumors were successfully established.
4
.14 Ex vivo fluorescence imaging and tissue biodistribution
HT29 tumor-bearing mice were used for fluorescence imaging amd before imaging, the
tumor-bearing mice were intravenously (i.v.) injected with HJTA (40 mg/kg). First, the mice were
placed onto the warmed stage inside of an IVIS light-tight chamber and anesthesia was maintained
with 1% pentobarbital. All the image acquisitions were performed with Caliper IVIS Lumina II in
vivo optical imaging system equipped with excitation filter: 465 nm and emission filter: GFP
when the mice were anesthetized at 0 h, 1 h, 2 h, 4 h, 8 h, and 24 h postinjection. Then, the mice
were sacrificed after imaging. The major organs including the heart, lung, liver, kidney, spleen,
colon, and tumor were collected and imaged with the fluorescence imaging system as described
above.
4
.15 Acute toxicity determination
The female ICR mice were randomly treated by intraperitoneal injection of a single dose of
HJTA at 240.0, 300.0, 375.0, 468.8, and 585.9 mg/kg or vehicle control (n = 10 per group),
respectively. The mice were monitored for their survival, body weights, behaviors, and movement
abilities up to 14 days post treatment. The experimental protocols were evaluated and approved by
the Nantong University Animal Care and Use Committee.
4
.16 In vivo tumor growth inhibition
All animal experimental protocols were approved by the Animal Research and Care Committee
of Nantong University. Female BALB/c nude mice at the age of 5 to 6-week-old were inoculated
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subcutaneously with 1×10 HT29 cells. When tumor volumes reached 100 mm , the mice were
randomly administered with HJTA, COMC-6, and vehicle, respectively. The body weight of all
animals was monitored throughout the study and animals were euthanized if they incurred 20 %
weight loss between observations. Two axes (mm) of a tumor (L, longest axis; W, shortest axis)
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‘
tumor volume = ½ (L×W )’. Progression of tumors was monitored every 2 days up to 21 days
posttreatment. At the end of the experiment, the mice were sacrificed, and their tumors were
dissected out and weighed.
4
.17 Cell apoptosis assay
After 12h culture of HT29 cells, HJTA (0.4, 1.0, 2.5 μM), COMC-6 (2.5 μM), harmine (10
μM), or vehicle were incubated with the cells for 72 h. The cells were harvested and then stained
with APC-Annexin V and 7-AAD at 37 °C for 15 min. Flow cytometry analysis (Calibur, BD,
USA) was used to determine the percentage of apoptotic cells, using APC signal (FL4) (excitation
¼
¼
633 nm; emission ¼ 660 nm) and 7-AAD staining signal (FL3) (excitation ¼ 488 nm; emission
647 nm). The data were analyzed using WinList 3D (7.1) and the histogram was plotted using
Excel 2016.
4
.18 Western blot analysis
The activities of apoptosis- and autophagy-related proteins were analyzed by the Western blot
5
assay. HT29 cells (1.5×10 /mL) were treated with or without vehicle, HJTA, COMC-6, or
harmine at indicated dosages for 72h. The cell lysates (0.05 mg/lane) were separated by
SDS-PAGE (12% gel) and transferred onto nitrocellulose membranes. When the reaction was
blocked using 5% non-fat milk, the target proteins were then detected by different antibodies, such
as anti-Bax, anti-Bcl-2, anti-cleaved-caspase 3, anti-cleaved-PARP, anti-p62, anti-LC3, anti-p62,
anti-beclin-1, and anti-β-actin antibodies (Cell Signaling Technology, MA, USA), respectively.
The bound antibodies were detected by HRP-conjugated second antibodies and then visualized
using the chemiluminescent reagent.
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ASSOCIATED CONTENT
Supporting Information
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Supporting Information is available free of charge via the Internet at http://pubs.acs.org.
Molecular formula strings (CSV)
The pH-responsive spectroscopy properties, Calculations of pKa and pKa*, GSH-responsive
1
13
properties, time-dependent cellular uptake, relative quantum yield, H & C NMR spectra, HRMS
and HPLC analysis.
AUTHOR INFORMATION
Corresponding Author
*Tel.: +86-513-85051892; E-mail for Y. Ling: Lyyy111@sina.com, e-mail for Y.A. Zhang:
zhangyanan@gmail.com.
AUTHOR CONTRIBUTIONS
#
J. Liu and X. Liu contributed equally to this work.
NOTES
The authors declare no competing financial interest.
ABBREVIATIONS USED
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Ala, alanine; Arg, arginine; Cys, cysteine; DMAP, 4-(N,N-dimethylamino)pyridine; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; DNA, deoxyribonucleic acid; Glu, glutamic acid;
Gly, glycine; GST, glutathione S-transferase; His, histidine; HRMS, high-resolution mass
spectrometry; Lys, lysine; MS, mass spectrometry; NADH, reduced nicotinamide adenine
dinucleotide; Ser, serine; ROS, reactive oxygen species; THF, tetrahydrofuran; TLC, thin-layer
chromatography.
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ACKNOWLEDGEMENTS
This work was financially supported by the Natural Science Foundation of China (Nos.
3
1830028 & 21977058), China and Jiangsu Province Postdoctoral Science Foundation
(
2018T110533, 2016M590488, and 1601136B), the Project of “Jiangsu Six Peaks of Talent”
2016-SWYY-CXTD-008 and 2014-SWYY-044), the Project of “Jiangsu 333 high-level talents”,
(
Jiangsu Province Innovation Project of Postgraduate Training (KYCX19 2086), and Applied
Research Projects of Nantong City (MS12018079 and JC2018125).
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