Observation of the Elevation of Cholinesterase Activity in Brain Glioma by a Near-Infrared Emission Chemsensor

Article abstract of DOI:10.1021/acs.analchem.0c02770

The excessive expression of cholinesterases (ChEs) directly disturbs the metabolism of acetylcholine (ACh), causing disordering neurotransmission in the brain or even Alzheimer's disease and cancer. However, the variation of ChEs including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in brain glioma has not yet been investigated. Therefore, the development of a suitable method for in situ imaging ChEs in brain tissues to understand the physiological functions of ChEs in depth is very important. Herein, a new near-infrared emission fluorescent probe (IPAN) for visualization of ChE activity was developed. IPAN exhibits ultrafast response to ChEs, low detection limit for AChE (0.127 U/mL) and BChE (0.0117 U/mL), and a large Stokes shift with near-infrared emission. Based on these excellent attributes, the IPAN was effectively utilized for imaging the fluctuations of ChE activity in the apoptosis cells and zebrafish. Notably, by utilizing the unique probe IPAN, we observed a significant enhancement of ChE activity in the tumor cells and brain glioma, for the first time. We believe that this interesting finding could provide a powerful guidance for tumor resection in the future.

Full text of DOI:10.1021/acs.analchem.0c02770

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Article
Observation of the elevation of cholinesterases activity
in brain glioma by a near-infrared emission chemsensor
Yanyan Ma, Wenjie Gao, Shihan Ma, Yongyuan Liu, and Weiying Lin
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.0c02770 • Publication Date (Web): 31 Aug 2020
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Observation of the elevation of cholinesterases activity in
brain glioma by a near-infrared emission chemsensor
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Yanyan Ma, Wenjie Gao, Shihan Ma, Yongyuan Liu, and Weiying Lin*
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials
Science and Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China
* E-mail: weiyinglin2013@163.com
ABSTRACT: The excessive expression of cholinesterases (ChEs) directly disturbs the metabolism of acetylcholine (ACh), causing
disordering neurotransmission in the brain or even Alzheimer's disease and cancer. However, the variation of ChEs including cetyl-
cholinesterase and butyrylcholinesterase in brain glioma has not yet been investigated. Therefore, the development of a suitable
method for in situ imaging ChEs in brain tissue to understand the physiological functions of ChEs in depth is very important. Here-
in, a new near-infrared emission fluorescent probe (IPAN) for visualization of ChEs activity was developed. IPAN exihibits ultra-
fast response to ChEs, low detection limit for AChE (0.127 U/mL) and BChE (0.0117 U/mL) as well as a large Stokes shift with
near-infrared emission. Based on these excellent attributes, the IPAN was effectively utilized for imaging the fluctuations of ChEs
activity in the apoptosis cells and zebrafish. Notably, by utilizing the unique probe IPAN, we observed a significant enhancement
of ChEs activity in the tumor cells and brain glioma, for the first time. We believe that this interesting finding could provide a pow-
erful guidance for tumor resection in the future.
20-25
Acetylcholine (ACh), the important neurotransmitter in the
ported.
However, additional excellent attributes of the
central nervous system, was significantly associated to recog-
probes for detection of ChEs are still needed to be improved to
study the internal relationship between ChEs and diseases in
vivo. For instance, large Stokes shifts with near infrared emis-
1-3
nition, learning, emotion and behavior.
It is reported that
the decrease of ACh in brain could cause neurotransmitter
4-6
metabolism dysfunction and several diseases.
Cholinester-
sion, which could achieve deep tissue penetration and avoid
26,27
ases (ChEs), which contains two major forms acetylcholines-
terase (AChE) and butyrylcholinesterase (BChE), could hy-
drolyze ACh to choline and acetic acid in cholinergic synapses.
The excessive expression of ChEs directly disturbs the me-
tabolism of ACh, thus disordering neurotransmission in the
self-absorption of fluorescence,
fast response for ChEs,
which could detect the fluctuation of ChEs rapidly in situ.
Notably, to the best of our knowledge, the detection of ChEs
activity in the brain glioma has not been reported yet. There-
fore, the construction of a near infrared emission fluorescent
probe with large Stoke shift for fast observation of the activity
of ChEs in the brain tumour is urgently required.
7,8
brain and even lead to depressive disorders, Alzheimer's dis-
9-11
ease and Parkinson's disease.
Furthermore, the anomalous
expression of ChEs may promote cell proliferation and partic-
ipate in tumorigenesis.12,13 However, the molecular mecha-
nisms of ChEs in cancer, especially in the complex brain, is
still unclear. Therefore, the development of a suitable method
for in situ imaging ChEs in brain tumor to understanding the
physiological functions of ChEs in depth is very highly signif-
icant.
Herein, we have developed a new near-infrared emission
fluorescent probe (IPAN) for detection of ChEs activity. The
new probe IPAN demonstrated ultrafast response to ChEs
with a large Stokes shift, excellent photo-stability and selectiv-
ity. IPAN was successfully utilized for visualization the
changes of ChEs activity in the apoptosis cells and zebrafish
which induced by H2O2. Significantly, an obvious enhance-
ment of ChEs activity in the brain glioma was revealed by
IPAN for the first time.
Currently, several methods have been used for the detection
of ChEs, including choline oxidase coupled multi-enzyme
assay, Ellman's colorimetric method and electrochemical anal-
14-16
Experimental Section
ysis.
However, these methods have several shortcomings,
such as laboring pretreatment, time-consuming procedures and
unsuitable for living biological systems detection. Fluores-
cence imaging, as a potential approach, has attracted signifi-
cant attention owing to their inherent advantages including
excellent compatibility, high spatiotemporal resolution and
real-time in-situ observation. 17-19 Very recently, some fluores-
cent probes for the detection of ChEs activity have been re-
Synthesis of the probe IPAN
Compound IPAH (149 mg, 0.5 mmol) and cesium car-
bonate (163 mg, 0.5 mmol) were added to dichloromethane,
and the mixture was stirred for 30 min under N2 atmosphere at
room temperature. Then, 100 μL dimethylcarbamoyl chloride
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was added to the above stirred mixture. Additional dimethyl-
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no fluorescence due to quenched group of carbamyl in the
IPAN. After addition of ChEs, the dimethyl carbamate moiety
was recognized, the ester bond was cleaved and then IPAN
was transformed to IPAH (Scheme 1). The characterizations
of IPAN were provided in the Supporting Information.
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carbamoyl chloride needed to be added every 12 h. After 72 h,
the mixture was purified by column chromatography (petrole-
um ether : CH2Cl2:MeOH=10:30:1) to afford a yellow product
1
(yield 80%). H NMR (400 MHz, DMSO) δ 7.72 (d, J = 8.4
Hz, 3H), 7.34 (dd, J1 = 37.4 Hz, J2 = 16.2 Hz, 2H), 7.16 (d, J =
8.8 Hz, 2H), 6.89 (s, 1H), 3.04 (s, 3H), 2.92 (s, 3H), 2.62 (s,
13
2H), 2.55 (s, 2H), 1.02 (s, 6H).
C (101 MHz, DMSO) δ
170.82, 156.32, 154.18, 152.69, 137.17, 133.44, 129.78,
129.25, 123.20, 122.78, 114.32, 113.49, 76.78, 42.79, 38.64,
36.78, 36.60, 34.36, 32.14, 27.90. HRMS (ESI): calcd. for
C22H23N3O2, [M+H]+, m/z, 362.1863, found: 362.1863.
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Cell imaging experiments
Scheme 1. Sensing mechanism of the probe IPAN for the detec-
tion of ChEs.
Before the imaging experiments, the NHA and U87 cells
were incubated into confocal plates for 24 h. Then the cells
were treated with 10 μM IPAN respectively for 30 min and
washed twice with PBS. The fluorescence images were ac-
quired with Nikon A1R confocal microscope.
Optical properties of the probe IPAN for ChEs
We evaluated the optical properties of IPAN toward ChEs
initially. The absorption of the free probe IPAN was approxi-
mately centered at 570 nm. In the presence of AChE or BChE,
a marked enhancement at this absorption peak can be observed
(Figure S1). The fluorescence titrimetric spectra were then
examined under the excitation at 545 nm. As shown in Figure
1A and B, IPAN emitted a faint emission band at 654 nm
(quantum yield, φ=0.038). Addition of increasing concentra-
tion of AChE or BChE resulted in obvious fluorescence in-
creases gradually, indicating that IPAN was transformed to
IPAH. Obviously, the IPAN possessed a large Stokes’ shift
which could avoid self-absorption of fluorescence. The probe
IPAN could determine AChE or BChE activity in the range
0−80 U/mL or 25-31 U/mL with a linear relationship (Figure
1C and D). The detection limit of IPAN for AChE or BChE
were calculated as 0.127 U/mL or 0.0117 U/mL. Moreover,
the fluorescence quantum yield of IPAN was obviously in-
creased after treated with AChE (φ=0.12) or BChE (φ=0.19).
In addition, the probe could detect AChE or BChE activity
quickly in less than 3s (Figure 2A and Figure S2) and its fluo-
rescence intensity remained stable within 1 h (Figure S3), sug-
gesting that the probe IPAN have a potential for real-time
detection the enzyme activity in living system. The sensing
mechanism of IPAN toward AChE or BChE was character-
ized by HRMS analysis. As shown in Figure S4, in the pres-
ence of AChE or BChE, the probe displayed a main peak at
m/z 291.1484 or 291.1580, which is consistent with IPAH
(calcd 291.1492 for C19H18N2O), demonstrating that the reac-
tion mechanism is similar as the depicted in Scheme 1. All
these data show that IPAN can detect AChE and BChE activi-
ty.
For the imaging of apoptotic cells, the NHA cells were pre-
treated with 1 mM H2O2 for 1 h. After that the NHA cells were
further incubated with 10 μM IPAN for another 30 min and
prepared for imaging. Emission was collected at 663-738 nm
(excited at 561 nm) for red channel. The mean fluorescence
intensity of each cell was measured (via “Measure” function)
and averaged across the three fields imaged.
Zebrafish imaging experiments
3-day-old zebrafish were purchased from the Nanjing Eze-
Rinka Biotechnology Co., Ltd.. Zebrafish were transferred
into confocal plates, and cultured in 1 mM H2O2 for 30 min.
Then the zebrafish were transferred to fresh water containing
10 μM IPAN for another 30 min. After that, the zebrafish
were anesthetized and imaged. The statistical analysis was
performed from three separate biological replicates. The exci-
tation wavelength was 561 nm and the emission wavelength
was collected at 663-738 nm.
Mouse brain slices fluorescence imaging
The 4-week old nude mice and nude mice were purchased
from Shandong University. All of the animal experiments
were carried out according to the Animal Management Rules
of the Ministry of Health in People's Republic of China (Doc-
ument NO. 55, 2001) and the guidelines for the Care and Use
of Animal Ethical Experimentation Committee of Shandong
University.
For the brain glioma imaging. U87 cells were injected into
nude mice by subcutaneously manner for the growth of xeno-
grafts tumor. After about 14 days, the glioma and brain were
excised from nude mice and cut into about 500 μm slices. The
slices were cultured with 20 μM IPAN for 30 min and imaged.
Results and Discussion
Molecular design and synthesis
The probe of IPAN was designed by introducing an N, N-
dimenthyl carbamyl moiety into IPAH through the acylation
reaction (Scheme S1). We speculated that the probe itself was
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we aimed to use the probe IPAN to detect ChEs activity in
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living cells during apoptosis induced by H2O2. As shown in
Figure 3, the NHA cells cultured with 1 mM H2O2 for 1h dis-
played stronger fluorescence signals than the untreated cells.
In order to further confirm the changes of fluorescence intensi-
ty was caused by the increase of ChEs activity, NHA cells
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were treated with donepezil, the inhibitor of ChEs. As ex-
pected, the fluorescent intensity of these cells was weak, indi-
cating that the enhancement of fluorescence signal is ascribed
to the over-activity of ChEs upon apoptosis and the probe can
be used for detection the changes of ChEs in living cells.
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Figure 1. (A) The fluorescence spectra of IPAN in the presence
or absence of various concentration of AChE (0-80 U/mL) (B)
The linear relationship between the normalized fluorescence in-
tensity at 654 nm and the concentration of AChE. (C) Fluores-
cence titration spectra of IPAN for sensing different concentration
of BChE (0-30 U/mL). (D) The linear relationship between nor-
malized fluorescence intensity at 654 nm with 21 U/mL-31 U/mL
BChE.
Figure 3. (A) Fluorescence imaging of ChEs in living cells. First
row (IPAN): NHA cells incubated with 10 μM IPAN. Second
row (Donepezil + IPAN): NHA cells were pre-treated with 10 μM
donepezil for 1 h and then stained with 10 μM IPAN. Third row
(H2O2 + IPAN): NHA cells were pre-cultured with 1 mM H2O2
for 1 h and then incubated with 10 μM IPAN. (B) Fluorescence
quantitative analysis of A. Red channel: λex = 561 nm, λem
=
663−738 nm. Scale bar: 20 μm.
It is reported that the activity of ChEs is accompanied with
brain tumor. 31,32 Therefore, we selected U87 (human glioma)
cells as research object to investigate how ChEs activity varied
by using IPAN. As expected, the NHA cells cultured with
IPAN for 30 min exhibited weak fluorescence (Figure 4).
However, when U87 cells stained with IPAN, a brighter emis-
sion was obtained. To verify that the increase of fluorescence
signal was came from ChEs activity, an inhibitor of ChEs,
donepezil, incubated with NHA cells for 1 h. The cells treated
with donepezil for 1 h exhibited lower emission than cells
without donepezil, indicating that the activity of ChEs was
increased in U87 cells. These data displayed that the activity
of ChEs was enhanced in living glioma cells.
Figure 2. (A) Time-dependent fluorescence intensity of IPAN (10
μM) for response AChE activity. (B) Fluorescence responses of
IPAN (10 μM) to various representative analytes in PBS (pH 7.4,
20 mM, 50% DMSO) buffer: IPAN (10 μM), CaCl2 (1 mM),
CuCl2 (1 mM), DTBP (500 μM), GSH (2.5 mM), Hcy (1 mM),
Cys (1 mM), KCl (1 mM), KNO3 (1 mM), Na2S (1 mM), NaBr (1
mM), KF (1 mM), Na2HPO4 (1 mM), NH4OAc (1 mM), ZnCl2 (1
mM), CH3CHO (500 μM), MgCl2 (1 mM), FeSO4 (1 mM), H2O2
(500 μM), NaClO (500 μM), BSA (10 μg/mL), HSA (10 μg/mL),
DMEM, alkaline phosphatase (50 U/mL), phospholipase (50
U/mL), chymotrypsin (50 U/mL), thrombin (50 U/mL), lysozyme
(50 U/mL), trypsin (50 U/mL), AChE (80 U/mL) and BChE (27
U/mL). λex = 545 nm.
Fluorescence imaging of ChEs activity in living cells
The feasibility of IPAN for imaging ChEs activity in living
cells was then investigated. Before imaging, the cytotoxicity
of IPAN was firstly evaluated by MTT assay. As shown in
Figure S6, NHA and U87 cells treated with different concen-
trations of IPAN (0-50 μM) displayed high cell viability above
90%, suggesting that the probe possessed low toxicity for liv-
ing cells and was suitable for living organisms imaging.
We next moved on our attention to explore the ability of
IPAN for imaging the changes of ChEs in living cells. Previ-
ous studies reported that H2O2 could induce cells apoptosis
which is related to the change of ChEs activity. 28,29 Therefore,
Figure 4. (A) Fluorescence images of living cells. First row
(NHA): NHA cells were cultured with IPAN for 30 min. Second
row (NHA +donepezil): NHA cells pre-treated with donepezil for
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1 h and then stained with IPAN for 30 min. Third row (U87): U87
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cells incubated with IPAN for 30 min. (B) Fluorescence quantita-
tive analysis of A. Red channel: λex = 561 nm, λem = 663−738 nm.
Scale bar: 20 μm.
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Fluorescence imaging of ChEs activity in living zebrafish
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Inspired by the successful application of IPAN in cellular
imaging, we intended to further explore the performance of
IPAN for detection of ChEs activity in living small-animal.
Figure 6. (A) Fluorescence images normal brain (first row) and
brain glioma by using the probe IPAN for 30 min incubation. (B)
Fluorescence quantitative analysis of A.
Zebrafish were used as living organism model due to their
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genes are similar to human beings.
As shown in Figure 5,
Conclusion
when the zebrafish stained with only probe IPAN for 30 min,
a dark fluorescence signal in the red channel was detected. By
contrast, the zebrafish pre-incubated with H2O2 for 1 h, a sig-
nificant enhancement of fluorescence signal was observed,
suggesting that the probe IPAN is capable of imaging the
changes of ChEs activity in living zebrafish.
In summary, we have presented a new near-infrared emis-
sion fluorescence probe for real time imaging ChEs activity in
vitro and in vivo, particularly in brain glioma for the first time.
The powerful probe IPAN possesses ultrafast response, high
sensitivity, low detection limit and large Stokes shift. Based
on these excellent attributes, the IPAN was successfully used
to detect the changes of ChEs activity in the living cells and
zebrafish during apoptosis. Notably, the in situ imaging stud-
ies by the probe IPAN revealed, for the first time, that the
activity of ChEs in the tumor cells and brain glioma was high-
er than that of the normal cells and brain. We believe that this
novel NIR probe could serve as a powerful tool for exploring
the roles of ChEs activity in biological and pathological pro-
cesses and provide a guide for further diagnosis and treatment
of ChEs related diseases.
Figure 5. (A) Fluorescence imaging of ChEs activity in zebrafish
First row (IPAN): zebrafish were incubated with 10 μM IPAN for
30 min. Second row (IPAN + H2O2): Zebrafish were pre-treated
with 1 mM H2O2 for 1 h and then stained with 10 μM IPAN for
30 min. (B) Fluorescence quantitative analysis of A. Red channel:
λex = 561 nm, λem = 663−738 nm. Scale bar: 20 μm.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website. Synthesis of the probes, absorption
and fluorescence spectra, imaging assays, 1H NMR and 13 C NMR
spectra. (PDF)
Fluorescence imaging of ChEs activity in living glioma
Motivated by the excellent attributes of IPAN for distin-
guishing cancer form normal cells, we further attempted to
verify the suitable of IPAN for imaging the changes of ChEs
in brain tumor and normal brain. The nude mice were pre-
inoculated U87 cells before 14 days for imaging, then the
brain of normal mice and glioma tissue of tumour mice were
excised and cut into 500 μM for imaging. As presented in Fig-
ure 6, the normal brain exhibited a dark emission intensity,
however, the fluorescence intensity of glioma tissue enhanced
about 2-fold than the normal brain tissue, indicating that the
activity of ChEs was elevated in brain tumor. Importantly, it is
the first time that the enhancement activity of ChEs in brain
glioma tissue was monitored by using a unique fluorescence
probe. In addition, the activity of ChEs in whole blood of liv-
ing tumor-loaded mice and living normal mice were deter-
mined. The whole blood were obtained from normal and tu-
mor-bore living mice respectively, then diluted 500-fold with
PBS buffer and measured finally with fluorescence spectrome-
ter. The blood of the tumor mice indicated a distinct fluores-
cence elevation (about 2-fold) than that of the blood of normal
mice (Figure S7). All of these data show that the probe was
successfully detected the fluctuation of ChEs activity in tumor
mice and normal mice for the first time.
AUTHOR INFORMATION
Corresponding Author
*Fax: + 86-531-82769031. E-mail: weiyinglin2013@163.com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was financially supported by NSFC (21472067,
21672083,
(TS201511041), and the startup fund of University of Jinan (309-
10004).
21877048),
Taishan
Scholar
Foundation
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