Visualization of carboxylesterase 2 with a near-infrared two-photon fluorescent probe and potential evaluation of its anticancer drug effects in an orthotopic colon carcinoma mice model

Article abstract of DOI:10.1039/d0cc00297f

We establish a near-infrared two-photon fluorescent probe for the detection of CE2 with high selectivity and sensitivity. This probe exhibits low cytotoxicity and superior tissue penetration ability for evaluating the real-time activity of CE2 in living cells, in cancer tissues, and in a colon carcinoma mice model.

Full text of DOI:10.1039/d0cc00297f

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DOI: 10.1039/D0CC00297F
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Visualization of carboxylesterase 2 with a near-infrared two-
photon fluorescent probe and its potential evaluation of anticancer
drug effects in orthotopic colon carcinoma mice model
Received 00th January 20xx,
Accepted 00th January 20xx
Yan Wang,ab Feifei Yu,b Xianzhu Luo,ab Mingshun Li,ab Linlu Zhao,*b and Fabiao Yu*ab
DOI: 10.1039/x0xx00000x
We establish a near-infrared two-photon fluorescent probe for the competent for practical applications mainly due to their
detection of CE2 with high selectivity and sensitivity. This probe relatively short excitation/emission wavelength where has
exhibits low cytotoxicity and superior tissue penetration ability for strong intrinsic autofluorescence background from living
evaluating real-time activity of CE2 in living cells, in cancer tissues, tissues. This issue greatly restricts the deep-tissue penetration
and in colon carcinoma mice model.
and measurement accuracy under physiological conditions.
Compared to the single-photon confocal imaging, the
developed two-photon (TP) microscopy features the ability to
avoid biological autofluorescence and to reduce photodamage,
because the near-infrared (NIR) wavelength can achieve deeper
penetration depth and can exhibit higher 3D temporal spatial
resolution.17-20 Hence, a two-photon fluorescent probe displays
attracted extensive research interest in sensing of bioactive
macromolecule CE2 under sophisticated living systems.
However, there still exist obstacles of these reported probes,
which mainly lie in the deficiency with emission wavelength
since most of these TP probes only can emit fluorescence at
short wavelength (<550 nm).21 This issue may fail to the
practical bio-imaging especially in deep tissues as the collection
efficiency of fluorescence signal will be greatly decreased,
further affecting the sensing accuracy and sensitivity of TP
probes.22, 23 Therefore, it is of great significance to develop TP
probes with excitation/emission wavelength in NIR region21
which can not only minimize background fluorescence
interference owing to reducing absorption by bio-molecules,
but also achieve deeper penetration to facilitate precise
detection of CE2 in living systems.
Carboxylesterases (CEs), a component of α/β hydrolase fold
protein, plays pivotal roles in the hydrolysis of substances
containing esters, amides, carbamates and thioester.1-4 As a
major isoform of CEs, CE2 is found to be high expressed in many
cancer cells, and CE2 behaves in mediating the activation
process of many prodrugs such as Gemcitabine, Irinotecan (CPT-
11) and Capecitabine (CAPE).5, Therefore, the individual
6
differences in CE2 activity have been closely associated with the
cytotoxicity and efficacy of the relevant clinical anticancer
drugs.7 Furthermore, CE2 is considered to be the essential
determinant in intestinal first-pass metabolism, especially for
oral anticancer prodrugs.8 In view of the critical roles of CE2 in
metabolism of various anticancer prodrugs, it is quite necessary
to develop suitable techniques for accurate and sensitive
detection of CE2 in the complex physiological conditions.
There have been established several effective methods for
evaluating CE2 activity.9, 10 However, these technical methods
usually need time-consuming and complex procedures, high
cost, and skilled operators, which may largely restrict their
application in rapid detection. As attractive alternatives,
fluorescent probes have become indispensable tools toward
exploring bioactive molecules.11-13 Although small-molecule
based fluorescent probes have been synthesized and utilized for
the detection of CE2 in vitro and in vivo,14-16 few probes are
As illustrated in Scheme 1, the overall design strategy of the
probe DCM-CES2 was based on the attachment of an enzyme-
active moiety L-leucine to an excellent NIR TP chromophore
dicyanomethylene-4H-pyran (DCM), 11,13,24 because it held large
Stokes shifts and NIR emission (> 650 nm). Since the CE2 could
specifically hydrolyze the compound with large alcohol and
small acyl group, our probe DCM-CES2 was able to serve as a
highly selective substrate for CE2. The detailed synthetic route
a. The Key Laboratory of Life-Organic Analysis, Key Laboratory of Pharmaceutical
Intermediates and Analysis of Natural Medicine, College of Chemistry and Chemical
Engineering, Qufu Normal University, Qufu 273165, China.
b. Institute of Functional Materials and Molecular Imaging, Key Laboratory of
Emergency and Trauma, Ministry of Education, College of Pharmacy, Key
Laboratory of Hainan Trauma and Disaster Rescue, College of Clinical Medicine,
College of Emergency and Trauma, Hainan Medical University, Haikou 571199,
China.
1
was shown in ESI†. The compounds were characterized by H
NMR, 13C NMR and HR-MS.
We inspected the spectral characteristics of the probe DCM-
CES2 in simulated physiological conditions (PBS buffer pH = 7.4,
10 mM at 37 °C). As shown in Fig. 1a, the probe DCM-CES2
Electronic Supplementary Information (ESI) available: Probe synthesis and
characterization, cell imaging, mice imaging, and supplementary figures. See
DOI: 10.1039/x0xx00000x
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CE2 in living cells. The cytotoxicity of the probe was evaluated
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†
DOI: 10.1039/D0CC00297F
by Cell Counting Kit-8 assay (Fig. S4, ESI ) with human
hepatocellular liver carcinoma cell line (HepG2) and human
colorectal cancer cell line HT-29. The viability rates of the
selected model cell lines indicated the relatively low
cytotoxicity. To test the capability of DCM-CES2 responded to
CE2 activity in living cells, HepG2 cells were treated under
different treatment conditions. The addition of DCM-CES2
could generate strong red fluorescence, which revealed the
reactivity to the CE2-overexpressed system (Fig. S6h). Then two
representative inhibitors, BNPP for CEs and LPA for CE2, were
adopted to validate the specific detection of CE2 in cells. As
expected, there appeared severe fluorescence suppression in
Fig. S6i and S6j, respectively. The faint fluorescence was
attributed to the inhibition of intracellular CE2 activity, but the
CE2-selective inhibitor LPA could restrain almost all of
fluorescence emission, demonstrating the specific detecting
ability of our designed probe. We also pretreated the cells with
5-Fluorouracil (5-FU, 70 μM, a CE2 inducer26) for 48 h at 37 °C.
Interestingly, a stronger fluorescence increase was observed in
Fig. S6k, which conformed that the specifical response to
endogenous CE2 in living cells. The results obtained by flow
cytometry assays (Fig. S6m – S6q) were in well accordance with
the above imaging outcomes.
CE2 offered high activation effect on metabolism of
anticancer prodrug Irinotecan (CPT-11) in HT-29 cells.27 As the
same situation, anticancer prodrug Capecitabine (CAPE) held a
stronger tend to be firstly activated by CE2, and finally
metabolized to drug 5-FU that could inhibit the growth of
cancer cells.28 In view of the crucial role of CE2 involved in the
activation process of various anticancer prodrugs, it was quite
necessary to visualize CE2 for better understanding of its
metabolic mechanism in drug metabolism. We next attempted
to utilize the probe DCM-CES2 to detect the CE2 activity change
Scheme
1 Illustration of the molecular structure DCM-CES2 and its proposed
fluorescence response mechanism toward CE2.
provided characteristic absorption centred at 445 nm. With the
treatment of CE2, a new maximum absorption peak appeared
at 560 nm, which belonged to the released fluorophore DCM.
The color changed from light yellow to rose red allowing to the
colorimetric detection of CE2 via the naked eye, accompanied
by a large Stokes shift of 125 nm in the fluorescence spectra.
The reaction with CE2 would switch-on the fluorescence
emission of DCM-CES2 by restoring the intramolecular charge
transfer (ICT) process, thereby offered
a characteristic
fluorescence emission peak centred at 685 nm in NIR region
(Fig. 1b). The fluorescence intensity displayed a good linear
relationship. The regression equation was F560 nm = 10.27 [CE2]
+ 6.963 with a linear regression constant r = 0.9963 (Fig. 1c).
Based on the standard method of 3σ/k, the limit of detection
for CEs was 0.087 μg/mL, which indicated favourable sensitivity
for the CE2 detection in vivo.25 As indicated in Fig. 1d, there
appeared a quick response within 5 min. Continuous monitoring
demonstrated that the reaction completed within 10 min. The
maximal TP absorption cross section (δ) was measured as 26
GM at 830 nm (Fig. S2, ESI†), implying that DCM-CES2 was able
to track CE2 via TP microscope. These results validated that the
designed probe could serve as
fluorescent chemical tool for detecting CE2 under physiological
conditions.
a near-infrared off-on
A series of reconstructed human enzymes with hydrolytic
activity were selected to assess their possible interactions with
DCM-CES2 (Fig. 1e). DCM-CES2 was incubated with the selected
enzymes, respectively, only CE2 led to a remarkable increase in
fluorescence intensity at 685 nm, while the other hydrolases
exhibited negligible fluorescence changes. To further confirm
the CE2 dependent fluorescence changes, we chose two
representative esterase inhibitors to perform the chemical
inhibition assays. CE2 was pre-incubated with the non-covalent
inhibitor loperamide (LPA) and covalent inhibitor bis(4-
nitrophenyl) phosphate (BNPP) followed by the addition of the
probe DCM-CES2, respectively (Fig. 1e). Both inhibitors could
restrict fluorescence emission compared with the CE2 group,
which confirmed that the fluorescence change was dependent
on CE2. The results indicated that DCM-CES2 could serve as an
effective chemical tool to detect CE2 with high selectivity in
cells. We also determined the Michaelis constant (Km). The
value of Km was 33.43 μM, and the corresponding catalytic
constant (Kcat) and catalytic efficiency (Kcat/Km) were 3.75 s-1 and
Fig. 1 Spectral characterization of DCM-CES2 (10 μM). a) UV-Vis absorption spectrum
with CE2 (0 - 10 μg/mL) for 10 min. b) The fluorescence spectrum with CE2 (0 - 15 μg/mL)
for 10 min. c) Linear relationship between the fluorescence intensity and the activity of
CE2. d) Time-dependent kinetic measurement of the fluorescent response to CE2 (5
μg/mL). e) Selectivity towards various species. Blank, Paraoxonase (PON1 and PON2, 10
μg/mL), Human serum albumin (HSA, 0.5 mg/mL), Bovine serum albumin (BSA, 0.5
mg/mL), Acetylcholinesterase (AChE, 0.1 μg/L), Butyrylcholinesterase (BChE, 20 U/L),
CES1 (10 μg/mL), Loperamide (LAP, 100 μg/mL), Bis(4-nitrophenyl) phosphate (BNPP,
100 μg/mL), and CE2 (10 μg/mL). f) Lineweaver-Burk plot for the enzymatic reaction. The
Michaelis-Menten equation was expressed as: V = Vmax [probe]/(Km + [probe]), where V
was the reaction rate, [probe] referred to the probe concentration (substrate), and Km
was the Michaelis constant. λex = 560 nm.
-1
-1
0.17 μM
s
respectively, demonstrating that CE2-mediated
hydrolysis of DCM-CES2 possessed good kinetic property, high
catalytic activity, and powerful affinity (Fig. 1f).
We next utilized the probe DCM-CES2 to detect the level of
2 | J. Name., 2012, 00, 1-3
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subcutaneous intestinal cancer slices (100 μm) to validate the
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deep imaging capability of our probe.DTOhI:e10t.1i0ss3u9/eD0sCliCce002w9a7Fs
incubated with DCM-CES2 (20 μM) for 60 min, then washed
with PBS for three times before confocal fluorescence imaging
(Fig. 3a). The cancer tissues displayed strong fluorescence
responded with collection window from 650 nm to 750 nm (λex
= 830 nm). We utilized the z-scan mode of the TP microscope to
visualize the fluorescence signals at different depth of mice
colon cancer slices (0 – 160 μm), and then reconstructed its 3D
fluorescence image (Fig. 3b). These assays indicated that the
probe DCM-CES2 could measure CE2 at penetration depth of up
to 160 μm in tissues by TP microscopy, which could be
attributed to its superior NIR-emissive TP fluorescence
properties for in vivo sensing of CE2. Many of the reported
fluorescent probes for imaging of CE2 activity were obstructed
to achieve applications for in vivo experiments since their
emissions were not in NIR region.30 Motivated by the
predominant NIR-emissive properties of the designed probe,
we then explored its ability to real-time in vivo visualization of
CE2 activity in tumour lesions. To optimize the imaging time in
mice, the probe DCM-CES2 (100 μM, 30 μL in PBS) was
administered to human colorectal xenograft tumour mice
model by intra-tumoral injection and then scanned at time
points 10, 20, 30, 60, 90, 120, 150 and 180 min using an IVIS
Lumina Kinetic Series III imaging system. Images were taken
with an excitation filter of 560 nm and emission windows from
650 nm to 750 nm. As displayed in Fig. S7, the control group was
injected with PBS. As expected, the in situ injection of DCM-
CES2 resulted in gradually fluorescence increase, indicating its
Fig. 2 Two-photon confocal fluorescence imaging (λex = 830 nm, λem = 650 - 750 nm) and
flow cytometry assay (λex = 560 nm, λem = 650 - 750 nm) for detection of CE2 in HT-29
cells. a - b) HT-29 cells pretreated with CPT-11 and CAPE, respectively, and then
incubated with DCM-CES2. c - d) Corresponding flow-cytometric analysis to a and b,
respectively. e - f) PE Annexin V/7-AAD assays of HT-29 cells in a and b, respectively, using
flow cytometry, Q1: necrosis cells, Q2: late apoptotic cells, Q3: early apoptotic cells, Q4:
survival cells. g - h) Mean fluorescence intensities of the images in a and b, respectively.
i - j) Mean values of c and d, respectively. k - l) Apoptosis percentages of e and f,
respectively. The data were shown as mean (± s.d.).
during the activation procedure in living cells. HT-29 cells were
parallelly divided into seven groups and treated with CPT-11 (20
μM) for 0, 2, 4, 8, 12, 24, 48 h, respectively, then incubated with
the probe for 30 min (Fig. 2a and 2b). Upon treatment with CPT-
11 for the initial 4 h, there appeared a fluorescence
enhancement in the testing system, while continuous effect of
CPT-11 gradually weakened the fluorescence signal as
incubation time extended to 24 h. When the examination was
continuous for 48 h, we noticed that the activity of CE2
gradually returned to the original level, which was in well
agreement with the metabolic cycle of CPT-11.29 We speculated
that the cells were suffering from a stress response to
metabolize the prodrug, which would promote CE2 activate
stronger, but occupied by amount of prodrug molecules. Once
the prodrug was decomposed, it would lead to the decreased
vitality of CE2, thus reducing the obtained fluorescence
intensity as the control group. Further evaluation was carried
out to detect CE2 activation response to CAPE in HT-29 cells. As
shown in Fig. 2b, the overall fluorescence trend was almost as
same as that in Fig. 2a, except for acquiring a stronger
fluorescence increase at 48 h. This phenomenon probably
caused by the participant of 5-FU which not only served as a
metabolite from CAPE through multiple enzymes including CE2,
but also could be an inducer to stimulate the activity increase of
CE2.26 The results demonstrated that the different anticancer
drugs offered the distinct activation process of CE2, which might
instruct various cancer cells resistance mechanisms to different
drugs. Flow analysis was shown in Fig. 2c and 2d. In addition, PE
Annexin V/7-AAD assays of HT-29 cells was also studied to
illustrate cell apoptosis caused by both anticancer drugs (Fig. 2e
and 2f). All of these results suggested that DCM-CES2 could be
utilized to detect the crucial role of CE2 during anti-cancer drugs
activation process.
Fig. 3 a) The confocal z-scan TP imaging at different depths. b) 3D fluorescence image in
a. scale bar: 100 μm. c - g) Evaluation of CE2 in orthotopic tumor-bearing mice model
treated with CPT-11 by tail intravenous injection (20 mg/kg) for 0 - 4 weeks and
fluorescence images of CE2 with DCM-CES2. h) H&E stained colonic cancer tissues
histopathology images. i) Regional TUNEL staining in the colonic cancer tissues. k)
Masson's stained slice of colonic cancer tissues.
We further evaluated the in vivo applicability of our NIR-
emissive TP probe. We performed TP imaging of CE2 in
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J. Name., 2013, 00, 1-3 | 3
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1
2
S. C. Laizure, V. Herring, Z. Hu, K. Witbrodt and R. B. Parker,
rapid activation by CE2 in vivo. The intensity reached maximum
at 150 min after injection and remained stable up to 180 min.
After successfully validating the availability of DCM-CES2 to
achieve real-time monitoring of CE2 in mice, we further
investigated the therapeutic effect of the CE2-realated
anticancer prodrugs. Herein, human colonic cancer cells MC38
was inoculated in colon to construct the orthotopic tumour-
bearing mice model, which was then treated with CPT-11 by tail
intravenous injection for 0, 1, 2, 3 and 4 weeks. We recorded
fluorescence images of these mice in five parallel groups after
150 min post abdominal intra-tumoral injection of DCM-CES2
(100 μM, 30 μL in PBS), respectively. It was worth noting that
the “probe light-up” region of tumour lesion gradually
narrowed, and in the 4th week there exhibited pronounced
improvement of tumour (Fig. 3c – 3g). The experimental results
demonstrated that the endogenous CE2 in tumour lesions was
always activated during the treatment of colon cancer with CPT-
11. The results in H&E, TUNEL and massion slices (Fig. 3h – 3j)
illustrated the degree of tumour treatment, which provided the
satisfactory treatment outcomes with anticancer prodrug CPT-
11. Hence, this promising probe could give convincing
assessment to the anticancer efficacy of CES-2-related
prodrugs.
In conclusion, we have successfully designed and synthesized
a NIR-emissive two-photon fluorescent probe DCM-CES2 for
endogenous CE2 detection in living cells and in vivo. The probe
offers a switch-on behavior for the detection of CE2 activity
under simulated physiological systems with a large Stokes shift
of 125 nm and a low LOD as 0.087 μg/mL. Cell experiments
illustrate that DCM-CES2 can realize specific detection of
endogenous CE2 with low cytotoxicity and satisfactory cell
membrane permeability. Tissue fluorescence imaging exhibits
high resolution at penetration depth up to 160 μm due to its
excellent NIR-emissive TP properties. DCM-CES2 has been
applied for real-time monitoring of CE2 activity during the
activation process of anticancer prodrugs CPT-11 and CAPE in
cells. The probe also achieves in situ and in vivo evaluation of
therapeutic efficacy of anticancer prodrug CPT-11 at tumour
lesion in orthotopic colonic mice model. The results of DCM-
CES2 reveal that this NIR two-photon probe holds potential
advantages for the further study of the biological function of
CE2 in complex systems.
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Pharmacotherapy, 2013, 33, 210-222. DOI: 10.1039/D0CC00297F
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This work was supported by Hainan Provincial Natural Science
Foundation of China (819QN225 and 2019RC210), National
Nature Science Foundation of China (No.21775162), Hainan
High Education Research Project (Hnky2019ZD-30 and
Hnky2019ZD-52), Talent Program of Hainan Medical University
(HYPY201905, XRC180006, XRC180010), and Hundred-Talent
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Conflicts of interest
The authors declare no conflicts of interest.
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Notes and references
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