Rational Design of a Highly Selective Near-Infrared Two-Photon Fluorogenic Probe for Imaging Orthotopic Hepatocellular Carcinoma Chemotherapy

Article abstract of DOI:10.1002/anie.202101190

Selective fluorescence imaging of biomarkers in vivo and in situ for evaluating orthotopic hepatocellular carcinoma (HCC) chemotherapy remains a great challenge due to current imaging agents suffering from the potential interferences of other hydrolases. Herein, we observed that carbamate unit showed a high selectivity toward the HCC-related biomarker carboxylesterase (CE) for evaluation of treatment. A near-infrared two-photon fluorescent probe was developed to not only specially image CE activity in vivo and in situ but also target orthotopic liver tumor after systemic administration. The in vivo signals of the probe correlate well with tumor apoptosis, making it possible to evaluate the status of treatment. The probe enables the imaging of CE activity in situ with a high-resolution three-dimensional view for the first time. This study may promote advances in optical imaging approaches for precise imaging-guided diagnosis of HCC in situ and its evaluation of treatment.

Full text of DOI:10.1002/anie.202101190

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Rational Design of Highly Selective Near-Infrared Two-Photon
Fluorogenic Probe for Imaging Orthotopic Hepatocellular
Carcinoma Chemotherapy
Authors: Xiaofeng Wu, Rui Wang, Sujie Qi, Nahyun Kwon, Jingjing
Han, Heejeong Kim, Haidong Li, Fabiao Yu, and Juyoung
Yoon
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202101190
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10.1002/anie.202101190
Angewandte Chemie International Edition
RESEARCH ARTICLE
Rational Design of Highly Selective Near-Infrared Two-Photon
Fluorogenic Probe for Imaging Orthotopic Hepatocellular
Carcinoma Chemotherapy
Xiaofeng Wu,[a,+] Rui Wang,[b,+] Sujie Qi,[a] Nahyun Kwon,[a] Jingjing Han,[a] Heejeong Kim,[a] Haidong
Li,[a] Fabiao Yu,*[b] and Juyoung Yoon*[a]
[a]
Dr. X. Wu, S. Qi, Dr. N. Kwon, J. Han, H. Kim, Dr. H. Li, and Prof. Dr. J. Yoon
Department of Chemistry and Nanoscience, Ewha Womans University, Seoul 03706, Republic of Korea.
E-mail: jyoon@ewha.ac.kr.
[b]
Dr. R. Wang, and Prof. Dr. F. Yu
Key Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital of
Hainan Medical University, Institute of Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou
571199, China.
E-mail: yufabiao@hainmc.edu.cn
These authors contributed equally to this work.
[+]
Supporting information for this article is given via a link at the end of the document.
conditions.5 As a mainstream approach for biomarker detection,
Abstract: Selective fluorescence imaging of biomarker in vivo and in
situ for evaluating orthotopic hepatocellular carcinoma (HCC)
chemotherapy remains a great challenge due to current imaging
agents suffering from the potential interferences of other hydrolases.
Herein, we observed that carbamate unit showed a high selectivity
toward HCC-related biomarker (carboxylesterase, CE) for evaluation
of treatment. A near-infrared two-photon fluorescent probe was
developed to not only specially image CE activity in vivo and in situ
but also target orthotopic liver tumor after systemic administration. In
vivo signals of probe correlating well with tumor apoptosis make it
possible to evaluate the status of treatment. Excellent property of
probe enables the first imaging of CE activity in situ with high
resolution three-dimensional view. This study may promote
advancements in optical imaging approach for precise imaging-
guided diagnosis of HCC in situ and its evaluation of treatment.
the specificity of serum assays may be impaired due to
biomarkers or interference in the multiple organs or tissues in
addition to the disease lesions.6 Biomarker analysis at a specific
origin can more accurately reflect the real condition of the liver
under drug treatment. In addition, an ideal method for testing
biomarkers ought to have higher selectivity.4b
Thus, an activatable and highly selective NIR TP fluorescent
probe for localizing spatially and elevating CE levels in the
original hepatic organ is rather important and meaningful for
better accurate imaging-guided diagnosis of the orthotopic HCC
and its evaluation of treatments. As an indispensable element of
fluorescent probes, recognition moieties are extremely
responsible for selective and sensitive interactions with enzymes
of interest.7 However, currently, ester bonds such as acetyl units
are still employed as a main recognition moiety for constructing
fluorescent probes for CE activity besides butyrylcholinesterase
(BChE) and acetylcholinesterase (AChE), resulting in potential
mutual interferences.8 This may seriously compromise the
accurate measurement of CE activity in vivo and in situ. This
great challenge encourages us to engineer
a selective
Introduction
recognition moiety for constructing a NIR TP fluorescent probe
for imaging the CE. Fortunately, we noticed that rivastigmine
and physostigmine (commercial inhibitors of BChE and AChE)
contain carbamate moieties that can function to inhibit the
activity of BChE and AChE (Scheme 1A).9 However, irinotecan
(CPT-11) as a precursor of an anticancer drug containing a
carbamate unit could be a substrate catalyzed by CE to release
anticancer SN38 (Scheme 1A and Figure S1).10 This indicates
that compounds containing carbamate units may selectively
react with CE but simultaneously inhibit the activity of BChE and
Fluorescence imaging has been emerging as a powerful method
for realizing the selective imaging of enzyme activity in vivo for
accurate diagnosis, and therapy of cancer and evaluations of
corresponding drug treatments because of its unique
advantages, including good sensitivity and selectivity, in situ
and/or real-time detection, high spatiotemporal resolution and
noninvasive monitoring ability in living systems.1 However, due
to strong intrinsic light scattering in tissue, spatial resolution and
penetration are rapidly compromised in imaging in vivo with
depth. Near-infrared (NIR) two-photon (TP) fluorescence
imaging is a promising tool for in vivo imaging of enzyme activity
with decreased autofluorescence, high tissue penetration and
resolution.2
AChE, inspiring us to employ carbamate as
a specific
recognition unit for CE that can avoid potential interference from
AChE and BChE.
In this study, with these concerns and inspirations in mind, we
designed and synthesized a series of compounds containing
carbamate for screening the substrate with the best selectivity
for CE over BChE and AChE. To decrease the steric hindrance
between the signaling unit and active site of enzyme,11 these
compounds were prepared using substituted self-immolative
linker as a bridge between the signaling unit (resorufin) and
carbamate moiety (Schemes 1B and 1C).12 The detection
mechanism is based on hydrolysis by CE, followed by 1,6-
rearrangement elimination to afford free resorufin (Figure S2).
Based on the screened recognition moiety of substrate with the
best selectivity (P3 of Scheme 1c), we further developed a light
Hepatocellular carcinoma (HCC) is a highly aggressive liver
malignancy and a major cause of cancer-associated death.3 An
effective method is still urgently required for precisely diagnosing
the condition of liver and evaluating drug treatments. In medicine,
biomarkers can be tested in blood, tissue and body fluids as sign
reflecting the normal or abnormal conditions, allowing the
evaluating of individual treatment for cancer and detections of
cancer at early stages.4 Thus, given that carboxylesterase (CE)
is an important biomarker of HCC, a selective and sensitive
fluorescent probe for testing CE levels in vivo may be an
effective tool for indicating the severity or presence of liver
1
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10.1002/anie.202101190
Angewandte Chemie International Edition
RESEARCH ARTICLE
up NIR TP fluorescent probe (DCM-Cl-CE, Scheme 2).13 As
expected, this NIR TP fluorescent probe also displays excellent
selectivity for CE over BChE and AChE, and shows a new broad
emission band in the NIR region. Moreover, this probe
accurately enables monitoring the change in CE activity in cells
treated with anticancer drugs. Meanwhile, this conversely allows
for in vivo selective imaging of CE activity in orthotopic liver
tumor mice treated with anticancer drug, reflecting real
conditions of HCC. Importantly, to the best of our knowledge,
this is the first light up NIR TP fluorescent probe for in vivo and
in situ tracking of CE activity using high-resolution three-
dimensional (3D) fluorescence imaging, possibly allowing for
image-guided diagnosis, evaluation of treatment and surgical
resection of orthotopic hepatocellular tumors.
CE, we designed and prepared an NIR (λem > 650 nm) TP
fluorescent probe (DCM-Cl-CE, Scheme 2). All the detailed
synthetic steps, characterizations and reaction mechanisms are
shown in Schemes 1 and 2, and the Supporting Information.
Screening Recognition Moiety with the Optimal Selectivity
for CE. We first checked the selectivity of CP and P1-P8 toward
CE, BChE and AChE, respectively. As shown in Figures 1A and
S3, under the same conditions, CP has almost the same
response toward CE as AChE and BChE, indicating that this
traditional recognition moiety of acetyl unit is not selective for CE
and a fluorescent probe with acetyl unit for CE is interfered by
BChE and AChE. As shown in Figures 1B-1D, S3 and S4, P1-
P8 all produced better response toward CE but had almost no
any response to BChE and AchE. The ratios of fluorescence
changes from reactions of probes with CE, BChE and AChE
were termed R1 and R2, respectively, further clearly indicating
the selectivity of CE over BChE and AChE. Table 1 shows that
the values of R1 and R2 for CP are 1.03 and 1.0, respectively,
again clearly demonstrating that the acetyl unit has no difference
in response to CE, BChE and AChE. However, the values of R1
vary from 48.75 to 338.3, showing that P1-P8 all have better
selectivity for CE over BChE. P3 has the best selectivity (R1 =
338.3) while P6 displays the worst selectivity. In addition, for the
selectivity of CE over AChE, the R2 values for P1-P8 ranging
from 22.5 to 421 also indicated that P1-P8 all have better
selectivity toward CE than AChE, among of which P5 and P3
have relatively better selectivity. Altogether, P3 shows a better
recognition moiety for CE than BChE and AChE. All the above
data show that our design using carbamate to replace the acetyl
unit as a selective recognition moiety for CE is rational and
effective.
(A)
(B)
CP
1.0
0.8
0.6
0.4
0.2
0.0
CE
5000
4000
3000
2000
1000
0
P1 P2
AChE
P3
P4
P5
P6
P7
P8
CE
BChE
AChE
BChE
(C)5000
4000
(D)5000
4000
3000
2000
1000
3000
2000
600
300
0
300
0
P3 P4 P5 P6
P8
P7
P3 P4 P5 P6
P8
P1 P2
P7
P1 P2
Scheme 1. Illustration of the carbamate proposed as
a new specific
recognition moiety for CE. (A) Compounds with carbamate unit. (B)
a
Figure 1. (A) Normalized fluorescence intensity of CP (5 μM) reacting with CE,
BChE and AChE (1 U/mL) for 0.5 h at 37 oC in PBS buffer (pH 7.4),
respectively. Fluorescence response of P1-P8 (5 μM) toward (B) CE (1 U/mL),
(C) BChE (1 U/mL) and (D) AChE (1 U/mL) for 5 h at 37 oC in PBS buffer (pH
7.4), respectively. F is the fluorescence intensity difference after and before
Traditional acetyl unit for CE and the carbamate proposed as a new specific
recognition unit for CE. (C) Control probe (CP) and probes (P1-P8) designed
to screen substrates with the best selectivity for CE.
the reaction. The results are expressed as the mean  SD (n = 3). λex em
/
=
550/582 nm.
Results and Discussion
Design and Syntheses of CP, P1-P8 and DCM-Cl-CE. A
control probe (CP) containing an acetyl moiety was synthesized
for comparison. P1-P8 were prepared using the self-immolative
linker with different substituted groups (hydrogen, fluoride,
chloride, bromo, nitro, azide, amine and methoxyl groups) to
combine the carbamate unit with resorufin (Schemes 1B and
1C). The substituted units could also be used to tune the
selectivity toward CE over BChE and AChE. These compounds
were used as substrates to screen recognition moiety with the
best selectivity and reactivity for CE activity. To make the probe
suitable for imaging CE in vivo, based on the specific recognition
moiety of P3 presenting the best selectivity and reactivity toward
Selectivity of DCM-Cl-CE toward CE Over BChE and AChE.
As mentioned above, to further make it possible to image CE
activity in vivo, an NIR TP fluorescent probe (DCM-Cl-CE) was
prepared based on the best selective recognition unit of P3
(Scheme 2). The selectivity of DCM-Cl-CE with CE, BChE and
AChE was also first investigated. Upon titration with 1 U/mL of
CE, BChE and AChE, as shown in Figures 2A and S5, the
reaction of DCM-Cl-CE with CE produced
a significant
enhancement while the reactions of DCM-Cl-CE with BChE and
AChE did not generate any fluorescence. In addition, no
fluorescence peak was observed for DCM-Cl-CE reacting with
2
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10.1002/anie.202101190
Angewandte Chemie International Edition
RESEARCH ARTICLE
BChE and AChE, indicating that almost no reaction occurred.
This shows that carbamate is an excellent recognition moiety for
CE, and DCM-Cl-CE is a highly selective NIR TP fluorescent
probe for the detection of CE activity in vivo.
buffer solution incubated at 37 oC (Figure S10). The plot
between the concentration of DCM-Cl-CE and the emission
intensity is displayed, and good linearity was obtained at
concentrations of 0-6 U/mL. The linear regression equation is F
= 3381 × [activity of CE] + 55.2, with a correlation coefficient of
0.998. The limit of detection (3/slope) is 0.013 U/mL,12 also
showing sensitive detection of CE (Figures 2C and S11). The
Table 1. Selectivity of CE over BChE and AChE.
Michaelis-Menten constant (Km) and the catalytic constant (kcat
)
Compd.
CE[a]
BChE[b]
AChE[c]
R1[d]
R2[e]
of CE toward DCM-Cl-CE were also measured to be 0.21 M
and 0.91 s−1, respectively (Figure S12). The catalytic efficiency
(kcat/Km) of CE for DCM-Cl-CE was calculated to be 4.52 × 106
M-1 s-1. These data indicated that CE displays high affinity and
effective cleavage toward DCM-Cl-CE. The maximum TP
absorption cross section (δ) was also determined to be 24.1 GM
at 830 nm (Figure S13), implying that CE could be imaged by
DCM-Cl-CE with a TP microscope.
CP
P1
P2
P3
P4
P5
P6
P7
P8
4833
4371
4716
4831
3520
4126
3442
4346
4299
4708
28.1
4835
118.9
52.41
32.7
44.06
9.8
1.03
1.0
153.6
177.6
338.3
90.6
36.6
90.0
148.0
79.9
421.0
37.0
62.1
22.5
26.56
14.28
38.85
21.53
70.6
(B)8000
(A) 4000
DCM-Cl-CE
DCM-Cl-CE + CE
6000
3000
191.6
48.75
57.6
4000
2000
0
2000
1000
0
92.97
70
75.44
52.3
600
700
800
900
191.1
82.2
CE
BChE
AChE
Wavelength (nm)
(C)25000
(D)
Equation y =
Adj. R-Sq
a
+
b*x
0.99774
4000
3000
2000
1000
0
Valu Standar
55.234 2.40407
3810.8 77.8182
[a] [b] [c] Fluorescence intensity difference (F) of probes (5 M) with CE,
BChE and AChE (1 U/mL), respectively, after and before reaction. [d] R1 =
(F of CE)/(F of BChE), and [e] R2 = (F of CE)/(F of AChE), indicating the
selectivity of CE over BChE and AChE. All were measured in PBS solution
(7.4).
20000
15000
10000
5000
0
B
B
Intercept
Slope
0
1
2
3
4
5
6
1 2 3 4 5 6 7 8 9 1011
Species
Conc. of CE (U/mL)
(E)
6000
5000
4000
3000
2000
1000
0
DCM-Cl-CE
DCM-Cl-CE + CE
DCM-Cl-CE + BNPP (0.1 M)
DCM-Cl-CE + BNPP (1 M)
(F)
1.0
0.8
0.6
0.4
ICG
DCM-Cl-OH
DCM-Cl-CE
0
10 20 30 40 50 60
Time (min)
600
700
800
900
Wavelength (nm)
Figure 2. In vitro properties of DCM-Cl-CE. (A) Fluorescence response of
DCM-Cl-CE toward CE, BChE and AChE (1 U/mL). (B) Fluorescence spectra
of DCM-Cl-CE (5 M) in the absence and presence of CE (1 U/mL). (C)
Linear relationship between the fluorescence intensity and CE concentration
(0-6 U/mL) for 5 h at 37 oC. (D) Fluorescence response of DCM-Cl-CE (5 μM)
to various enzymes, including (1) the blank; (2) -glucosidases (10 U/mL); (3)
xanthine oxidase (10 mU/mL); (4) tyrosinase (10 U/mL); (5) ALP (10 U/mL);
(6) MAO-A (10 μg/mL); (7) MAO-B (10 μg/mL); (8) trypsin (10 μg/mL); (9) HSA
(10 μM); (10) BSA (10 μM) and (11) CE (1 U/mL). (E) Inhibitory activity of
DCM-Cl-CE against BNPP under different conditions. (F) Photostability of
DCM-Cl-CE, DCM-Cl-OH and ICG in PBS detected via absorbance spectra.
The samples (all at 5 μM) were continuously irradiated by a light source (25
mW/cm2).
Scheme 2. Synthesis of the NIR TP fluorescent probe (DCM-Cl-CE) for CE
and its reaction mechanism with CE.
Spectroscopic Properties of DCM-Cl-CE and Its Responses
to CE. To test the validity of this probe, the spectroscopic
properties of DCM-Cl-CE were investigated in physiological
buffer. DCM-Cl-CE itself shows a peak at 400 nm while upon
reaction with CE, the absorbance shifts to approximately 540 nm
(Figure S6), which is roughly consistent with the absorbance
peak of DCM-Cl-OH (Figure S7). The color ranges from
yellowish to rose red, making it favorable for colorimetric
detection using the naked eye (Figure S6). As expected, when
excited at 540 nm, an NIR fluorescence signal at 685 nm was
observed fully matching the emission wavelength of DCM-Cl-OH
(Figures 2B and S8). Time-dependent experiments revealed that
after 5 h, 83-fold fluorescence enhancement could be found at
685 nm (Figure S9), benefiting the sensitive detection of CE.
The fluorescence intensity almost reached a maximum with
To investigate the interference, the reaction of DCM-Cl-CE
with various species was performed, including enzymes [-
glucosidases,
xanthine
oxidase,
tyrosinase,
alkaline
phosphatase (ALP), monoamine oxidase A/B (MAO-A/B), and
trypsin], human serum albumin (HSA), bovine serum albumin
(BSA), several inorganic salts, glucose, vitamin, some amino
acids, glutathione, urea and various reactive oxygen species
(Figures 2D, S14 and S15). However, only when treated with CE
was the strongest and most distinct fluorescence enhancement
observed. These results indicate the super excellent selectivity
of DCM-Cl-CE toward other competitive species, which is
3
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10.1002/anie.202101190
Angewandte Chemie International Edition
RESEARCH ARTICLE
required for selective and accurate detection under complex
biosystems.
photon fluorescence for imaging CE activity in HepG2 cells
could also be successfully observed (Figure S24).
To further investigate the applicability of DCM-Cl-CE in the
field of drug development, an inhibitory experiment was
implemented in HepG2 and LO2 cells. Compared to that in
HepG2 cells and LO2 pretreated with only DCM-Cl-CE, the
fluorescence intensity in HepG2 and LO2 cells pre-treated with
BNPP and then DCM-Cl-CE significantly decreased, indicating
that the fluorescence intensity indeed arose from the reaction of
DCM-Cl-CE and CE (Figures S25 and S26).
Moreover, an inhibitory experiment was performed to
demonstrate that the reaction site was cleaved by CE. The effect
of an inhibitor of CE, bis(4-nitrophenyl)phosphate (BNPP) was
examined.21 As shown in Figure 2E and S16, compared to the
group (DCM-Cl-CE + CE), with increasing concentrations of
BNPP, the fluorescence intensity decreased dramatically,
indicating that the fluorescence change of the reaction indeed
arose from CE-catalytic hydrolysis.
Stabilities of DCM-Cl-CE and DCM-Cl-OH. Their high stability
benefits DCM-Cl-CE and DCM-Cl-OH in performing in vivo long-
term tracking of enzyme activity in preclinical applications. The
photostability of DCM-Cl-CE and DCM-Cl-OH was estimated
based on time-course absorbance measurements under
continuous irradiation by a light source with a power of 25
mW/cm2 in aqueous solution. The clinically approved NIR dye
indocyanine green (ICG) was chosen as the control
compound.15 As shown in Figures 2F and S17, after continuous
exposure to light for 1 h, an almost 61% decrease in absorbance
indicated that 39% of ICG was left. However, almost 100%
DCM-Cl-OH and 90% DCM-Cl-CE remained under the same
conditions. The photostability of both DCM-Cl-OH and DCM-Cl-
CE is better than that of ICG. In addition, the effect of the
temperature on the thermal stability was also investigated.
Different temperatures (25, 30, 37 and 42 oC) and long-term
o
incubation at 37 C also had almost no effect on the stability of
DCM-Cl-CE and DCM-Cl-OH (Figures S10, S18 and S19).
These data show that high photo- and thermal stabilities are
highly desirable for long-term tracking and bioimaging of CE
activity in vivo.
pH Profiles of DCM-Cl-CE and DCM-Cl-OH. The pH is an
important factor that has great effect on the photophysical
properties of probes. Consequently, the pH-dependent
fluorescence emission profiles were evaluated. There was
negligible change in fluorescence emission at 685 nm for DCM-
Cl-CE in pH range from 4.0-11.0. Upon titration with CE,
fluorescence intensity enhancement was generated, and the
strongest fluorescence intensity could be observed at pH 7.4,
making DCM-Cl-CE desirable for the detection of CE activity
under physiological conditions (Figure S20). Besides, the
hydrolytic product of DCM-Cl-CE, that is DCM-Cl-OH was found
its pKa is calculated to be 7.18 (Figures S21 and S22), also
allowing DCM-Cl-CE to working better under the physiological
conditions of living systems.
Figure 3. Comparison of CE levels in human liver cancer cells (HepG2 cells)
and human normal liver cells (LO2 cells). (A) One-photon fluorescence
imaging of DCM-Cl-CE in HepG2 and LO2 cells at different time points (0, 1,
2.5 and 5 h). The control group: fluorescence imaging of cells without probe.
NIR: near-infrared emission channel; BF: bright field channel. (B) Relative
fluorescence intensity in panel (A). (C) Analyses of CE levels by commercial
kit in HepG2 and LO2 cells. (λex = 540 nm and λem = 650-750 nm). Scale bar:
20 μm.
Comparison of CE Activity within Human Liver Cancer Cells
and Normal Liver Cells. The high selectivity, sensitivity and
stability of DCM-Cl-CE make it possible to image CE activity in
living cells. The HepG2 cell line (human liver cancer cell) and
LO2 cell line (human normal liver cell) were chosen as model
cell lines. Prior to the study, the potential toxicity of DCM-Cl-CE
Discovery of the Effect of Anti-liver-cancer Drug on CE
Activity in Hepatocellular Carcinoma Cells. Encouraged by
the excellent performance of DCM-Cl-CE imaging the
overexpression of CE activity in HepG2 cells, we will next
evaluate the effect of the anti-liver-cancer drug on CE activity in
HepG2 cells with DCM-Cl-CE. An anticancer drug (sorafenib)
approved for the treatment of liver cancer was employed.17 As
shown in Figure 4, HepG2 cells were divided into seven groups
treated with sorafenib for different time points (0, 2, 4, 8, 12, 24
and 48 h), and then incubated with DCM-Cl-CE for 5 h. The
fluorescence intensity in the cells without sorafenib treatment
was used as a control (0 h). With prolonged time, we found that
the fluorescence intensity dramatically decreased (Figures 4B,
4C and S27), indicating that the CE level also decreased, which
is further evidenced by analysis of the relative CE level (Figure
4D). These results proved that DCM-Cl-CE has the capacity to
selectively monitor the change of CE activity and image the
different conditions of liver cancer cells during the anticancer
drug activation process.
was evaluated by
a standard counting kit-8, and high
biocompatibility could be observed for HepG2 and LO2 cells
even as the concentration of DCM-Cl-CE reached 100 μM
(Figure S23). The activity of CE in HepG2 and LO2 cells was
compared under the same conditions for demonstrating whether
CE activity is overexpressed in the human liver cancer cells. As
shown in Figure 3A, time-dependent experiments showed that
after incubation of DCM-Cl-CE with cells, the fluorescence
intensity in HepG2 cells gradually increased, while almost no
fluorescence could be observed in LO2 cells (Figure 3B),
indicating that the CE level in HepG2 cells may be higher than
that in LO2 cells. The analysis of CE level by a commercial kit
further demonstrated that the CE level was higher in HepG2
cells than in LO2 cells (Figure 3C), implying that CE is
overexpressed in HepG2 cells,16 and may be a potential
biomarker for indicating the condition of liver cancer.5c,5d Two-
4
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10.1002/anie.202101190
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RESEARCH ARTICLE
liver region, and thus fluorescence regions may be tumor site in
the two-dimensional (2D) ex vivo imaging of the separated
organs, indicating that probe bears targeting ability to liver tumor
(Figures 5E and 5F). To further confirm tumor-targeting ability of
probe and treating effect of sorafenib, various staining methods
have been performed to illustrate the pathological morphology of
the tissues from the fluorescent regions in the liver. As shown in
Figures 5G and S28, the tissue slices in the control group (0
day) were mainly composed of tumor cells. The tumor tissues
were less interstitial, and the tumor cells were uniform and tightly
distributed. Compared to the control group, the tumor tissue in
the treatment group was relatively loose with flaky cell necrosis
or apoptosis, and as the time increased, the area of necrosis
increased, demonstrating that the tumor-targeting ability of
DCM-Cl-CE and anticancer drug has some therapeutic effect on
the liver cancer. Importantly, from the analysis of above data, we
can find that with drug post-treated days (i.e., mice under
different conditions), the fluorescence intensity of DCM-Cl-CE
imaging CE at hepatic organ decreased, accompanied by
continuous tumor apoptosis, and a positive correlation between
fluorescence change and tumor apoptosis may be established.
These data fully indicated that probe has an excellent capacity
to target orthotopic liver tumors, and imaging different conditions
of liver cancer and elevating the drug treatment of HCC.
Moreover, 3D imaging is a powerful tool for clearly accurate
disease diagnosis, especially for uncertain lesions with high
spatiotemporal precision.18 To improve the observation of the
CE distribution in deep hepatoma tissue, we carried out a 3D
fluorescence molecular tomography (3D FMT) imaging
technique based on accurate reconstruction of the in vivo
fluorescence distribution. The 3D FMT fluorescence imaging of
the orthotopic hepatoma mice was reconstructed through diffuse
tomographic algorithms and the CE locations and lesions in the
hepatoma were also clearly shown from coronal, sagittal, and
transverse views with NIR light-up fluorescence signals (Figures
5H and S29-S32). For the first time, we exemplify in situ 3D
fluorescence imaging to clearly visualize the CE activity in
tumors. This could be provided as an important tool for the
imaging-guided diagnosis and surgical resection of HCC.
Figure 4. Visualization of CE activity in HepG2 cells treated with an anti-liver-
cancer drug (sorafenib) using TP fluorescence imaging. (A) Schematic
illustration of DCM-Cl-CE imaging CE activity in HepG2 cells treated with
sorafenib and lysate extraction for analysis of CE levels. (B) TP confocal
fluorescence imaging for CE activity in HepG2 cells after treatment with
sorafenib (10 μM) for different times (0, 2, 4, 8, 12, 24 and 48 h) and then
incubation with DCM-Cl-CE. (C) Relative fluorescence intensity in panel (B).
(D) Analyses of CE levels in HepG2 cells with (a) sorafenib for 12 h or without
(b) sorafenib via a commercial kit. λex = 830 nm and λem = 650-750 nm. Scale
bar: 20 μm.
Discovery of DCM-Cl-CE Imaging CE Activity in the
Orthotopic Liver Tumor During Treatment of Anticancer
Drug. Motivated by the performance of DCM-Cl-CE in liver
cancer cells, we used this probe to explore the therapeutic effect
of an anti-liver-cancer drug (sorafenib) on orthotopic HCC and
whether DCM-Cl-CE could indicate the in vivo therapeutic
condition of mice. We first observed the fluorescence change of
DCM-Cl-CE in tumor over time. DCM-Cl-CE was administered to
the subcutaneous HepG2-xenografted tumor-bearing mouse
model by intratumoral injection and scanned at different time
points (0, 0.5, 1.5, 2.5 and 5 h) using in vivo imaging system. As
shown in Figure 5A, obvious fluorescence enhancement could
Conclusion
In summary, we have proposed dimethyl carbamate as a
specific recognition moiety for CE activity, which was combined
with a substituted self-immolative linker to develop a series of
compounds (P1-P8) for screening the best substrate for CE
activity. Based on the best recognition moiety of P3, a NIR TP
fluorogenic probe (DCM-Cl-CE) was prepared for in vivo
imaging of CE activity that can also effectively eliminate the
interference from other hydrolyses. This probe displayed high
sensitivity, selectivity, stability, and pH independence.
Importantly, this probe has proven to image the difference in CE
activity between HepG2 and LO2 cells, and successfully monitor
changes in CE activity in HepG2 cells treated with anticancer
drugs, making it applicable to indicate for the evaluation of drug
treatment of HCC in vivo. With condition changing at different
time points post-treatment in the mice model bearing orthotopic
liver tumor (i.e., mice under different conditions), a positive
correlation may be established between the fluorescence
intensity of DCM-Cl-CE in vivo and continuous tumor apoptosis,
indicating DCM-Cl-CE can be used to evaluate the condition of
HCC chemotherapy. Moreover, for the first time, the CE activity
in the tumor site with a high-resolution 3D view was visualized,
allowing for possible image-guided diagnosis and surgical
resection of HCC in the future. Considering the high selectivity
and sensitivity, stabilities, and biocompatibility, we believe this
enzyme-activated NIR TP fluorescent probe will be provided as
an effective tool for in vivo and in situ monitoring the condition of
the liver cancer and evaluations of drug treatments.
be observed after
5 h, indicating that DCM-Cl-CE could
successfully image CE activity in the liver tumor in vivo. To
further reflect the condition of real liver cancer and evaluate the
drug treatment, orthotopic liver cancer model was established at
the original hepatic organ. The mice with orthotopic liver cancer
were separated into five groups, and then pretreated with an
anticancer drug (sorafenib) by intravenous injection for different
periods (0, 7, 14, 21 and 28 days). Afterwards, DCM-Cl-CE by
intravenous injection was incubated with each group for 5 h and
then imaged with the imaging system (Figures 5B and 5C). As
shown in Figure 5D, compared to the control group (0 day), the
fluorescence intensity gradually decreased with prolonged time,
indicating that CE activity also decreased. This phenomenon
was fully consistent with the fluorescence change in HepG2 cells
treated with sorafenib and the analysis of the relative CE activity
(Figures 4B-4D). Meanwhile, it also indicated that DCM-Cl-CE
could selectively image the biomarker (i.e., CE) of liver cancer at
the original liver. Besides, based on the higher CE level and
selective imaging capacity of DCM-Cl-CE in HepG2 cells in
Figure 3, it is reasonable to speculate that the fluorescence
intensity in the liver tumor may be higher than that in the normal
5
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10.1002/anie.202101190
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RESEARCH ARTICLE
(C)
(A)
(B)
5 h
0.5
1.5
2.5
0
0
14
21
7
28 (d)
(D)
(E)
0
(F)
9
8
7
6
5
4
3
2
1
0
7
0
7
14
21
28 (d)
14
21
28
(d)
Heart
SpleenLung
Liver
Kidney
21
0
7
14
28 (d)
(G)
H&E
(H)
Masson
TUNEL
Figure 5. Visualization of imaging the conditions at the orthotopic liver cancer mouse model treated with the anti-liver cancer drug. (A) Time-dependent
fluorescence imaging of CE activity in the subcutaneous HepG2-xenografted tumor-bearing mouse model was obtained at different time points (0, 0.5, 1.5, 2.5
and 5 h) by intratumoral injection of DCM-Cl-CE (10 μM). Red dotted circles indicate the tumor site. (B) Schematic illustration of DCM-Cl-CE imaging CE activity
in the mouse treated with anticancer drug (sorafenib). (C) Timeline for the development of mouse model treated with sorafenib and bimodal imaging. (D) Imaging
CE activity in orthotopic tumor-bearing mice treated with sorafenib by intravenous injection for different times (0, 7, 14, 21 and 28 days), followed by intravenous
injection of DCM-Cl-CE (10 μM, 100 μL). (E) 2D ex vivo imaging of CE in separated organs (heart, liver, spleen, lung, and kidney) sacrificed from mice in panel
(D). Red dotted circles indicate the tumor site. (F) Fluorescence intensity of the images in the panel (E). (G) Hematoxylin and eosin (H&E) staining of orthotopic
liver tumor tissue; Masson’s staining of orthotopic liver tumor tissues; regional terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of
orthotopic liver tumor tissues. (H) 3D in vivo imaging of CE activity in the orthotopic liver cancer mouse model pretreated with sorafenib for 0 day, and then treated
with DCM-Cl-CE (10 μM, 100 μL) for 5 h by intravenous injection. 3D reconstruction of fluorescence molecular tomographic (FMT) imaging of CE activity from
coronal, sagittal, and transverse views. (λex = 540 nm and λem = 650-750 nm).
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J.Y. thanks the National Research Foundation of Korea (NRF),
which was funded by the Korea government (MSIP) (No.
2012R1A3A2048814). F.Y. thanks CAMS Innovation Fund for
Medical Sciences (2019-I2M-5-023).
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Keywords: activatable probes• analytical methods • specific
substrate • fluorogenic imaging• orthotopic hepatocellular
carcinoma
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RESEARCH ARTICLE
Entry for the Table of Contents
3D
Orthotopic
liver cancer
3D imaging
NIR imaging
2. i.v. injection
of probe
1. i.v. injection of drug
⚫
⚫
⚫
⚫
NIR and TP fluorescence emission
High selectivity and sensitivity
Monitoring the real condition of HCC
3D imaging for clear lesion location
NIR&TP
emission
cell imaging
Herein, we proposed that the carbamate as recognition unit showed an excellent selectivity for imaging HCC-related biomarker
(carboxylesterase, CE) without the interferences from other hydrolases. An engineered near-infrared two-photon fluorescent probe
shows high selectivity and strong fluorescence signals towards CE activity, enabling it to successfully monitor CE activity for
evaluating HCC chemotherapy in vivo and in situ.
8
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