An Activity-Based Fluorogenic Probe Enables Cellular and in Vivo Profiling of Carboxylesterase Isozymes

Article abstract of DOI:10.1021/acs.analchem.0c01554

Carboxylesterases (CEs) exist as multiple types of isomers in humans, and two major types are CE1 and CE2. They are widely distributed in human tissues and well-known for their important roles in drug metabolism and pathology of various diseases. Thus, the detection of CEs in living systems could provide efficient proof in disease diagnostics, as well as important information regarding chemotherapeutic effects of antitumor drugs and prognosis. To develop a specific probe to discriminate CEs from other hydrolases, especially cholinesterases, is quite challenging due to their structural similarities and substrate specificity. To date, almost all of the fluorescent probes developed for CEs have been constructed with an acetyl group as the recognition unit. Herein we proposed a new design strategy of probe-cavity matching, which led to the identification of a new fluorogenic substrate (termed as HBT-CE) with high specificity toward both CE isomers and improved sensitivity, considering the higher binding affinity and catalysis efficiency. The promising capability of HBT-CE was further demonstrated for endogenous CEs imaging in living cells, zebrafish, and nude mice. In addition, HBT-CE was successfully applied in kinetically monitoring drug-induced CE regulation in cancer cells. All of these findings suggest that HBT-CE is a valuable tool for tracking and imaging endogenous CEs in complex biological systems.

Full text of DOI:10.1021/acs.analchem.0c01554

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Article
An Activity-based Fluorogenic Probe Enables Cellular
and In Vivo Profiling of Carboxylesterase Isozymes
Shi-Yu Liu, Ren-Yu Qu, Rong-Rong Li, Yao-Chao Yan, Yao Sun, Wen-Chao Yang, and Guang-Fu Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.0c01554 • Publication Date (Web): 08 Jun 2020
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An Activity-based Fluorogenic Probe Enables Cellular and
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In Vivo Profiling of Carboxylesterase Isozymes
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Shi-Yu Liu,†,§ Ren-Yu Qu,†,§ Rong-Rong Li,† Yao-Chao Yan,† Yao Sun,† Wen-Chao
Yang,*,† and Guang-Fu Yang*,†‡
†Key Laboratory of Pesticide & Chemical Biology of Ministry of Education,
International Joint Research Center for Intelligent Biosensor Technology and Health,
and Chemical Biology Center, College of Chemistry, Central China Normal
University, Wuhan 430079, P.R. China
‡Collaborative Innovation Center of Chemical Science and Engineering, Tianjin
30071, P.R. China.
—————————
§ These authors made equal contributions.
* Wen-Chao Yang, e-mail: tomyang@mail.ccnu.edu.cn; Tel: 86-27-67867706; Fax:
86-27-67867141; Guang-Fu Yang, e-mail: gfyang@mail.ccnu.edu.cn; Tel: 86-27-67867800;
Fax: 86-27-67867141.
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Abstract
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Carboxylesterases (CEs) exist as multiple types of isomers in humans, and two
major types are CE1 and CE2. They are widely distributed in human tissues and well
known for their important roles in drug metabolism and pathology of various diseases.
Thus, the detection of CEs in living systems could provide efficient proof in disease
diagnostics, as well as important information regarding chemotherapeutic effects of
anti-tumor drugs and prognosis. To develop a specific probe to discriminate CEs from
other hydrolases, especially cholinesterases, is quite challenging due to their structural
similarities and substrate specificity. To date, almost all of the fluorescent probes
developed for CEs have been constructed with an acetyl group as the recognition unit.
Herein, we proposed a new design strategy of probe-cavity matching, which led to the
identification of a new fluorogenic substrate (termed as HBT-CE) with high
specificity toward both CE isomers and improved sensitivity, considering the higher
binding affinity and catalysis efficiency. The promising capability of HBT-CE was
further demonstrated for endogenous CEs imaging in living cells, zebrafish, and nude
mice. In addition, HBT-CE was successfully applied in kinetically monitoring of
drug-induced CEs regulation in cancer cells. All of these findings suggest that
HBT-CE is a valuable tool for tracking and imaging endogenous CEs in complex
biological systems.
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KEYWORDS carboxylesterases, fluorescent probe, probe-cavity matching,
bioimaging
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INTRODUCTION
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Carboxylesterases (EC 3.1.1.1, CEs), including, but not limited to, CE1 and CE2,(1)
are capable of hydrolyzing various endogenous and exogenous substrates, such as
short- and long-chain acylglycerols, drugs, pesticides and environmental toxicants.(2-4)
CEs are known to be classified into five groups(5) and commonly expressed in various
animal species.(6) In human, CEs are widely distributed in various tissues, especially
in the liver and intestine. Being regulated by various nuclear receptors,(7) CEs play
important roles in diseases diagnosis and drug discovery. It has been reported that CEs
are up-regulated in pancreatic cancer,(8) neuroblastoma,(9) non-small-cell lung
cancer(10) and colorectal cancer(11) tissues. Thus, CEs could serve as important
determinants for pharmacokinetics and pharmacodynamics of ester drugs or ester
prodrugs, such as irinotecan. Regulation of CEs is also involved in gambogic
acid-induced cell apoptosis in cancer cells,(12) CPT-11-related drug resistance,(13)
capecitabine-related toxicity(14), and inhibition of tumor growth.(15) Understanding the
behavior and regulation of CEs in various diseases is very beneficial for manipulating
the disease pathology and clinical drug usage. Interestingly, the abnormal secretion of
multiple forms of CEs into serum in diseases, such as acute liver injury(16-17) and acute
pancreatitis,(18-19) makes the total content of CE isomers a potential biomarker for the
development and/or perpetuation of the above diseases.(20) Therefore, it is essentially
important to develop a single method for simultaneous determination of CE isozymes
in complex biological systems and physiological events with temporal  spatial
resolution.
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Among various analysis techniques, fluorescent probes are attractive tools due to
their high temporal and spatial resolution.(21-24) Nevertheless, to acquire a highly
selective probe for real-time tracking particular target enzyme other than abundant
hydrolases and other biological molecules In Vivo is quite challenging.(25-27) To date,
almost all of the small-molecule-based probe targeting CEs have been constructed
with an acetyl group as the recognition group, and most of them incorporate a
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hydroxybenzyl moiety-based self-immolative linker.(28-34) In addition, interference
from other esterases, including acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE), presents a significant challenge due to their overlapping substrate
specificities with CEs. However, few of these works discussed the selectivity of the
probe for carboxylesterase and cholinesterase in detail.
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In this study, we selected 2-(2-hydroxyphenyl) benzothiazole (HBT), a typical
excited-state intramolecular proton transfer (ESIPT)-based fluorophore with a large
Stokes shift and good biocompatibility,(22) for the construction of a fluorogenic probe
(HBT-CE) that targets both CE isomers. In addition, the probe has a pivaloyl group,
with large steric hindrance, as the recognition unit rather the widely used acetyl group
(Scheme 1). This probe exhibits superior selectivity for two cholinesterases, and the
mechanism could be explained by analysis of the docking model and determination of
the kinetic parameters. Moreover, this new probe shows promising potential for the
detection of endogenous CEs in complicated biological systems due to successful
bioimaging in cellular and animal models.
Scheme 1. Recognition mechanism of HBT-CE toward CEs.
EXPERIMENTAL SECTION
Organic Synthesis
Reagents. Chemical compounds were of analytical grade and purchased from
Sigma-Aldrich (Shanghai, China), and they include bis-p-nitrophenyl phosphate
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(BNPP), 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), tacrine,
donepezil, sivelestat, fluoxetine, 5-fluorouracil, amino acids (Glu, Thr, Leu, Ser, Ala,
Arg, Tyr, Val, Asp, Cys, and Gly), some common inorganic salts (NaCl, CoCl2,
MnCl2, FeCl3, KCl, and CaCl2), organic compounds and solvents,
2-(2-hydroxyphenyl) benzothiazole (HBT), propionyl chloride, isobutyryl chloride,
pivaloyl chloride, Et3N, dichloromethane (DCM), petroleum ether, and ethyl acetate.
CE1 and CE2 were purchased from the Research Institute for Liver Disease (Shanghai,
China). Other proteins and enzymes, such as AChE, BChE, carboxypeptidase B
(CPB), human serum albumin (HSA), phosphatase, chymotrypsin, and trypsin, were
purchased from Sigma-Aldrich. Unless otherwise noted, all of the starting materials
were commercially available and treated with standard methods provided by the
manufactures’ instructions.
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Instrumentation. Fluorescence spectra were recorded with a microplate reader
(SpectraMax M5, Molecular Devices) using 96-well luminescent plates or quartz
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cuvette (Hellma, Germany). 1H NMR and C NMR spectra for the target compounds
were determined in CDCl3 on a Varian Mercury 500 MHz or 125 MHz spectrometer,
and resonances (δ) are given in ppm relative to tetramethylsilane (TMS).
High-resolution mass spectrometry (HRMS) was obtained on Agilent 6460 Triple
Quadrupole mass spectrometer.
Synthesis of probes 1-3 A mixture of the 2-(2-hydroxyphenyl) benzothiazole (0.44
mmol, 100 mg), propionyl chloride (0.54 mmol, 50 mg), and Et3N (0.88 mmol, 89 mg)
in THF (3 mL) was stirred at ambient temperature for 1.0 h. The progress of the
reaction was monitored by TLC. After the reaction was complete, the mixture was
poured into a separatory funnel containing 10 mL 1 M HCl and 10 mL CH2Cl2. The
layers were separated, and the aqueous layer was extracted with CH2Cl2 (2 × 10 mL).
The combined organic layers were dried over Na2SO4. The extract was concentrated
under vacuum, and the residue was purified by chromatography on silica gel using
petroleum ether: ethyl acetate = 40: 1 to afford probe 1 as a white solid. Synthesis of
probes 2-3 was conducted via the same procedure for probe 1, with isobutyryl
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chloride or pivaloyl chloride accordingly. The NMR and HRMS data are listed as
below.
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Probe 1 (yield 56.2%, 70.1 mg) H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 7.8 Hz,
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1H), 8.09 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.55 – 7.47 (m, 2H), 7.45 –
7.35 (m, 2H), 7.25 (d, J = 7.8 Hz, 1H), 2.81 (q, J = 7.8 Hz, 2H), 1.32 (t, J = 7.8 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 172.58, 162.59, 152.79, 148.34, 135.28, 131.43,
130.27, 129.35, 126.31, 126.00, 125.34, 123.68, 123.27, 121.32, 28.21, 8.84. HRMS
(MALDI): Calcd for C16H13NO2S [M+H]+ 284.0740. Found: 284.0741.
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1H), 8.08 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.55 – 7.48 (m, 2H), 7.44 –
7.35 (m, 2H), 7.21 (d, J = 8.0 Hz, 1H), 3.06 – 2.96 (m, 1H), 1.38 (d, J = 7.2 Hz, 6H).
13C NMR (101 MHz, CDCl3) δ 175.26, 162.72, 152.96, 148.53, 135.39, 131.37,
130.40, 126.28, 126.24, 125.29, 123.59, 123.30, 121.34, 34.52, 18.86. HRMS
(MALDI): Calcd for C17H15NO2S [M+H]+ 298.0896. Found: 298.0898.
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Probe 3 (yield 59.9%, 82.1 mg) H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 7.2 Hz,
1H), 8.09 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.54 – 7.48 (m, 2H), 7.43 –
7.34 (m, 2H), 7.14 (d, J = 8.0 Hz, 1H), 1.42 (s, 9H). 13C NMR (101 MHz, CDCl3) δ
176.95, 163.03, 153.19, 148.86, 135.42, 131.40, 130.72, 126.67, 126.24, 126.22,
125.26, 123.57, 123.31, 121.39, 39.23, 27.28. HRMS (MALDI): Calcd for
C18H17NO2S [M+Na]+ 334.0872. Found: 334.0871.
Cell culture and live cell imaging Cancer cell lines were purchased from Procell
Life Science and Technology Co., Ltd. (Wuhan, China). Human liver hepatocellular
cells Hep G2, human ovarian carcinoma cells SKOV3 and human cervical carcinoma
cells HeLa were grown in DMEM (Gibco, USA) supplemented with 10% (V/V) fetal
bovine serum (Gibco). Human non-small cell lung carcinoma cells A549 were grown
in 1640 (Gibco) supplemented with 10% (V/V) fetal bovine serum (Gibco); both cell
lines were maintained at 37°C and 5% CO2.
Prior to the cellular imaging experiments, cells were seeded on glass-bottom cell
culture dishes (NEST, 15 mm) at a density of around 3 × 105 cells per well in 2.0 mL
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of culture medium. After the cells were washed with PBS, they were preincubated
with or without inhibitors (AEBSF, 1 mM or BNPP, 500 μM) for 30 min. Then,
HBT-CE (20 μM) in DMEM was added to the cells. Fluorescence images were
immediately acquired with an inverted fluorescence microscope (Olympus IX71,
Japan) every 10 min without any movement. Fluorescence image data were then
analyzed by quantifying the fluorescence intensity of each cell using ImageJ software
(National Institutes of Health, America). Each experiment included at least three
independent biological repeats, and at least 3 images from different fields of view
were captured for each well of cells.
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Live imaging in zebrafish. The zebrafish were provided by Nanjing EzeRinka
Biotechnology Co., Ltd. Before the imaging study, the zebrafish were placed in 35
mm confocal dishes and incubated with or without 50 μM tacrine, 500 μM BNPP or
500 μM AEBSF for 30 min. After the medium in each group was replaced with fresh
E3 medium, the cells were treated with 20 μM HBT-CE and further incubated for 20
min. Fluorescence images were acquired by an inverted fluorescence microscope with
a 4× objective lens.
Live imaging in nude mice. Nude mice (BALB/c-nu, 5 weeks) were intravenously
injected with 200 μL of 200 μM HBT-CE using a microinjector for 30 min.
Fluorescence images were then acquired with a Lago X In Vivo Imaging system
(Spectral Instruments Imaging, Arizona, USA) with an excitation laser at 365 nm and
an emission filter of 510 nm.
RESULTS AND DISCUSSIONS
Probe design and characterization
Broadly speaking, serine hydrolases, including CEs, utilize the same catalytic triad
(consisting of amino acid residues His, Asp or Glu and Ser) to carry out nucleophilic
attack of amide or ester bonds. Thus, there are two issues for designing a fluorescent
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probe for CE detection: the catalytic efficiency of both major CE isomers and the
discrimination between CEs and BChE activity. To address these challenges, we
analyzed the crystal structures of human BChE (PDB entry: 1P0M)(35) and CE (PDB
entry: 1MX5)(36) and proposed a “probe-cavity matching strategy” for the design of a
specific CEs probe. More specifically, the better the probe-cavity match, the higher
specificity it may achieve. Directed by this strategy, 2-(2’-hydroxyphenyl)
benzothiazole (HBT) was introduced as the fluorophore, and an alkanoyl moiety was
used to quench the fluorescence of HBT. An “off-on” fluorescence signal change
could occur when the ester bond was cleaved by the catalysis of CEs and, thus, could
illuminate CEs in living system. Three alkanoyl groups with different sizes were
introduced as recognition groups for the construction of the target probes, an ethyl
group for probe 1, isopropyl group for probe 2 and tertiary butyl group for probe 3.
The structural characterizations of probes 1-3 are provided in the supplementary
material (Figures S1S6). As shown in Figure S7, the fluorescence signal of probe 3
(termed as HBT-CE) was adequately quenched at 460 nm, with excitation at 325 nm.
All of the three probes exhibited similar responses against both CEs. More
importantly, although the relative activity for sensing CEs decreased with increasing
steric hindrance of the recognition group, the specificity of them over the BChE
improved and probe 3 showed the best specificity (Figure 1). The inteference of
various small molecules including amino acids and metal ions toward HBT-CE was
also investigated, as shown in Figure S8, the probe shows high selectivity among all
these analytes.
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Figure 1. Specificity of probe 1 (a), probe 2 (b) and probe 3 (c) (10 µM, 10 min)
toward CEs and other hydrolases or proteins in phosphate buffer (10 mM, pH 7.4) at
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30℃.The tested hydrolases or proteins include trypsin (10 µg/mL), chymotrypsin (10
µg/mL), phosphatase (10 µg/mL), HSA (10 µg/mL), CPB (10 µg/mL), AChE (10
µg/mL), BChE (10 µg/mL), CE1 (10 µg/mL), and CE2 (10 µg/mL).
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To understand the ability of probe HBT-CE to discriminate between CE and
BChE, we docked this probe into the active sites of both enzymes, respectively. As
depicted in Figures 2a and b, HBT-CE exhibited a better fit with the CE cavity than
that of BChE. In addition, it revealed that the proximity of catalytic Ser1221 to the
carbonyl group of HBT-CE was around 4.8 Å in CE, whereas the proximity of
catalytic Ser198 to the carbonyl group of HBT-CE was around 5.9 Å in BChE (Figure
2c and d). This suggests that HBT-CE in CE active center is much more susceptible
to Ser-triggered nucleophilic attack than that in the BChE active center. Further
experiments also confirmed its high selectivity for CEs over other small analytes,
such as amino acids and metal ions (Figure S8). The excellent selectivity of the probe
implies the efficacy of HBT-CE for evaluating CEs activity.
Figure 2. Simulated binding models of HBT-CE in human BChE (a in sphere type
and (c in cartoon type. The simulated binding models of HBT-CE in human CE (b in
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sphere type and (d in cartoon type. The probe HBT-CE is indicated in yellow, and the
key residues of the cavities for both enzymes are shown in cyan. Matching between
HBT-CE and the two enzymes is indicated by red dashed lines, respectively. The red
dash lines refer to the catalytic traid, and the distances between the hydroxyl of the
catalytic Ser residue and the carbonyl group of HBT-CE.
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Figure 3. Kinetic characterization of HBT-CE as a selective substrate for CEs. (a)
Time-dependent changes in the fluorescence intensity of HBT-CE (10 μM) in the
presence of PLE (1 μg/mL). (b) Dose-dependent changes in the fluorescence of
HBT-CE (10 μM) at various concentrations of PLE (0-0.04 U/mL). (c and d)
Michaelis  Menten curve for HBT-CE catalysis by CE1 (10 µg/mL) or CE2 (10
µg/mL), the assays were all conducted in phosphate buffer (10 mM, pH 7.4) at 30℃.
We firstly tested the pH-dependent profile of HBT-CE catalyzed by CEs in
aqueous solution. As shown in Figure S9, the probe showed best performances at
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around physiological pH, and thus pH 7.4 was chosen as the in vitro test condition
prior to the application in biological system. Herein, we chose porcine liver esterase
(PLE) as the substitution of multiple CE isomers due to their high substrate
similarities.(29) As shown in Figure 3a, the initial fluorescence of HBT-CE (10 μM) at
460 nm was adequately quenched, and the FI increased over 52-fold within 20 min at
physiological conditions upon the addition of PLE. The enzyme-triggered reaction
mechanism was further validated by HPLC analysis and the data was shown in Figure
S10. Moreover, the fluorescence response of HBT-CE toward various concentrations
of PLE was linear, and the limit of detection (LOD) was 4.02 × 10-5 U/mL, which
indicates that HBT-CE is more sensitive than all of the former reported CEs probes
(Figures S11 and S12). To quantify the catalytic activity of both CEs against HBT-CE,
we characterized the hydrolysis of HBT-CE catalyzed by CE1 and CE2, respectively
(Figure 3c and d). Accordingly, the Michaelis constants (Km) and the catalytic
constants (kcat) were determined, and the data are listed in Table 1. Intriguingly,
although both kinetic parameters of HBT-CE catalyzed by CE1 are around one-fold
higher than those catalyzed by CE2, their overall catalytic efficiencies (kcat/Km) were
quite similar. This explains why HBT-CE exhibits a similar response toward both
CE1 and CE2. To compare the catalysis efficiency of HBT-CE among the existing
CEs probes, we further determined the kinetic parameters of HBT-CE toward PLE, as
shown in Figures S12 and S13. The kcat/Km value for HBT-CE is 6.4-fold higher than
the best performing probe reported recently.(29) The above analysis reveals that
HBT-CE should be a suitable substrate for both CE1 and CE2 detection in living
systems.
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Table. 1. Kinetics parameters of HBT-CE catalyzed by CE1 and CE2, respectively.
kcat/Km
Km (μM)
kcat (s-1)
(M-1·s-1)
CE1
CE2
14.69 ± 2.58
8.60 ± 0.58
3.07 ± 0.19
1.75 ± 0.07
2.09×105
2.03×105
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Cellular imaging and kinetic tracking
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To illustrate the capability of HBT-CE for the specific detection of endogenous
CEs in living cells, we first applied HBT-CE in Hep G2 cell lysates since the Hep G2
cell line is known to express CEs in high concentrations.(29) As shown in Figure S14,
preincubation with AEBSF (serine protease inhibitor) or BNPP (selective inhibitor for
carboxylesterases) resulted in fluorescence responses that had declined by over 90%,
compared with the cells treated with 0.1% DMSO. In addition, the preincubation with
other common esterase inhibitors, such as tacrine (inhibitor for cholinesterase),
donepezil (selective inhibitor for AChE) and sivelestat (particular inhibitor for
neutrophil elastase), did not change the fluorescence of HBT-CE, which primarily
demonstrated the potential of HBT-CE for bioimaging. Prior to cell image, we firstly
tested the cytotoxicity of HBT-CE against Hep G2 cells by MTT assay. As depicted
in Figure S15, the cell viability of Hep G2 cells treated with the probe after 24 h at
various concentration maintained over 80%, which demonstrated the good
biocompatibility of HBT-CE. Hence, we further adopted HBT-CE for kinetic
monitoring of CEs in living cells. As displayed in Figure 4a and b, the green
fluorescence of cells treated with HBT-CE quickly increased and became visible
within 10 min. The intracellular fluorescence signal enhanced in a time-dependent
manner and plateaued around 40 min. In contrast, no obvious green fluorescence of
cells pretreated with 1 mM AEBSF or 500 μM BNPP for 30 min before the addition
of HBT-CE was observed, which is in good agreement with the cell lysate-based
assay. These results suggest that HBT-CE could serve as a new tool for imaging of
endogenous CEs in living species, with high resolution and sensitivity.
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Figure 4. Real-time detection of CEs in living Hep G2 cells. (a, c, e) Time-lapse
imaging of endogenous CEs in Hep G2 cells using HBT-CE (20 µM) without (a) and
with pre-incubation of 1 mM AEBSF (c) or 500 µM BNPP (e) for 30 min. (b, d, f)
Quantification of the fluorescence intensity of the microscopy images.
To further investigate the subcellular localization of CEs, HBT-CE was
employed to stain cells, with commercial organelle dyes as references. In general, Hep
G2 cells were co-stained with HBT-CE and three separate red dyes: ER-tracker red,
Mito-tracker red and Lyso-tracker red (Figure 5). Thus, the Pearson correlation
coefficient (Rr) was determined to calculate the linear correlation between two color
staining methods. From Figure 5, CEs mainly distributed in the endoplasmic
reticulum and mitochondria, rather than in lysosomes.
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Figure 5. Determination of subcellular localization of CEs with HBT-CE. (a  c)
Co-incubated with HBT-CE (20 µM) and commercial organelle trafficking dye
ER-tracker red (a, 1 µM), Mito-tracker red (b, 1 µM), and Lyso-tracker red (c, 1 µM).
From left to right: Bright-field images; commercial dye images; HBT-CE images;
merged images of commercial dye and HBT-CE images; and pseudo color images
indicating the extent of correlation between the intensities of HBT-CE and organelle
trackers.
The high selectivity of this newly identified probe for CEs monitoring
inspired us to extend its application in understanding biological events. As
mentioned before, CEs have important roles in drug metabolism and disease
pathology;(12, 37-38) thus, identifying the relative content of CEs in different cell
lines (especially cancer cell lines) could be useful in predicting the
chemotherapeutic effect of anti-cancer drugs and prognosis. First, we employed
the HBT-CE probe to compare endogenous CEs among various cell lines,
including human liver carcinoma cells Hep G2, non-small-cell lung carcinoma
cells A549, ovarian carcinoma cells SKOV3, and cervical carcinoma cells HeLa.
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As shown in Figures 6a and 6b, the green fluorescence intensity of Hep G2 cells
treated with HBT-CE is much higher than that of other cancer cell lines. Thus, it
was speculated that the CEs content in Hep G2 cells is about 4.1-fold and 9.6-fold
that of SKOV3 cells and HeLa cells, respectively. The above result suggests that
the metabolism of ester-bond-bearing drugs in HeLa cells may be less efficient,
compared with the other three tested cell lines.
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Figure 6. Determination of the relative content of endogenous CEs in different cell
lines. (a) Fluorescence images taken from different cells (Hep G2, A549, SKOV3,
HeLa) incubated with HBT-CE (20 µM) for 30 min. (b) Quantification of relative
fluorescence intensities from the microscopy images.
Enzyme prodrug approaches are widely adopted in cancer therapy, and the
activity of CEs is crucial for the conversion of many prodrugs to their active
ingredient, such as irinotecan(37) and capecitabine;(14) thus, an activity-based
evaluation of CEs could be more reliable than protein expression level analysis in
this aspect. Since extensive research has proved the combination of
chemotherapeutic agents could improve anti-tumor drug efficacy,(39-41) the ability
to predict the interaction between drugs and CEs could help to instruct the usage
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of CEs catalyzed prodrugs. Next, we utilized the HBT-CE probe to monitor the
kinetic change in CEs in the presence of drug-induced regulation. 5-fluorouracil
(5-FU) is a widely used anti-tumor drug that is frequently applied in
combinational therapy(42-43) with other chemotherapy agents. Therefore, the
interaction between CEs and 5-FU can influence the efficacy of CEs-dependent
pro-drugs, such as irinotecan. As shown in Figure 7, treatment with 20 µM
5-fluorouracil significantly induced the up-regulation of CEs activity in Hep G2
cells, whereas the activity of CEs decreased significantly when the cells were
treated with fluoxetine, an antidepressant drug that could down-regulate the
expression of CEs.(44) These results indicate that 5-FU might promote the
conversion of irinotecan to the active metabolite, due to the up-regulation of CEs
activity. Thus, the combination of 5-FU and irinotecan could be quite beneficial
in cancer therapy.
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Figure 7. Monitoring of drug-induced regulation of CEs in Hep G2 cells by HBT-CE.
(a) Live imaging fluorescence image taken from Hep G2 cells preincubated with
DMSO (0.5%), fluoxetine (25 µM) or 5-fluorouracil (20 µM) for 24 h, followed by
incubation with HBT-CE (20 µM) for 30 min. (b) Quantification of relative
fluorescence intensities from microscopy images.
Specific detection of CEs in living zebrafish
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To further assess the potential of HBT-CE for visualizing CEs In Vivo, we
performed CEs sensing in an aquatic model organism. Zebrafish are frequently
adopted as a model system for fluorescent probe-based bioimaging.(45-47) We
applied HBT-CE into live image with zebrafish, and strong green fluorescence
appeared throughout the zebrafish bodies, including eyes (Figure 8). As a control
experiment, zebrafish treated with tacrine prior to the treatment of HBT-CE had
no obvious effect on the FI signal. However, the fluorescence signal in zebrafish
preincubated with CEs specific inhibitor BNPP or serine protease inhibitor
AEBSF exhibited a significantly diminished fluorescence signal. The above
results demonstrate that HBT-CE is a valuable visualizing tool for monitoring
CEs activity in living organisms.
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Figure 8. In Vivo detection of CEs in zebrafish by HBT-CE. (a) Live imaging
fluorescence image taken from zebrafish preincubated with DMSO (0.5%), Tacrine
(50 µM), BNPP (500 µM) or AEBSF (500 µM) for 30 min, followed by incubation
with HBT-CE (20 µM) for 20 min. (b) Quantification of relative fluorescence
intensity from microscopy images.
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Determination of the biodistribution of CEs in tissue of nude mice
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To further investigate the feasibility of HBT-CE in detecting CEs in living
animals, we applied HBT-CE in nude mice via tail vein injection. Although the tissue
penetration of HBT-CE in live imaging is not sufficient, the remarkable fluorescence
signal, however, was clearly observed in the abdomen of a nude mouse, and this is
consistent with a previous report (Figure 9a).(48) To obtain more precise
biodistribution data of CEs in various organisms, we dissected major organs for
in-situ imaging. As shown in Figure 9b, the fluorescence signal was mainly localized
in the small intestine, liver and lung, and no signal was detected in the heart, kidney
and spleen. Although the tissue distribution of CEs has already been well studied via
the Northern blot assay,(49) the HBT-CE probe provides a fast and efficient method for
evaluating CEs biodistribution in living samples.
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Figure 9. Determination of the biodistribution of CEs in nude mice (BALB/c-nu, 5
weeks) with HBT-CE. (a) Live fluorescence image taken from nude mice
intravenously injected with 350 µL HBT-CE (200 µM). (b) Fluorescence image of ex
vivo-dissected organs: (1) heart, (2) kidney, (3) spleen, (4) small intestine, (5) liver
and (6) lung. All of the images were taken with an excitation laser at 365 nm and an
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emission filter of 510 nm.
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Conclusion
A new strategy of probe-cavity matching was proposed to design a new
fluorescent probe (HBT-CE) targeting CE isomers. HBT-CE showed a similar overall
catalytic efficiency toward both CE isomers, with superior sensitivity and high
specificity. It was also successfully applied to real-time monitoring of endogenous
CEs in various cancer cell lines and live tracking of drug-induced CEs regulation.
Furthermore, the In Vivo bioimaging ability of HBT-CE was demonstrated by sensing
CE activity in living zebrafish and nude mice. The results reveal that HBT-CE could
serve as a potential tool in CE-related drug discovery and disease diagnostics, as well
as the functional study of drug metabolism and disease pathology.
Acknowledgment
This work was funded by the National Key Research and Development Program of
China (No. 2017YFA0505200), the National Natural Science Foundation of China
(Nos. 21837001 and 21977036), and Hubei Province Natural Science Foundation (No.
2018CFA072).
Supporting information
Probe characterization, HPLC analysis and additional data. This material is available
free of charge via the Internet.
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