An Activatable AIEgen Probe for High-Fidelity Monitoring of Overexpressed Tumor Enzyme Activity and Its Application to Surgical Tumor Excision

Article abstract of DOI:10.1002/anie.202001675

Monitoring fluctuations in enzyme overexpression facilitates early tumor detection and excision. An AIEgen probe (DQM-ALP) for the imaging of alkaline phosphatase (ALP) activity was synthesized. The probe consists of a quinoline-malononitrile (QM) core decorated with hydrophilic phosphate groups as ALP-recognition units. The rapid liberation of DQM-OH aggregates in the presence of ALP resulted in aggregation-induced fluorescence. The up-regulation of ALP expression in tumor cells was imaged using DQM-ALP. The probe permeated into 3D cervical and liver tumor spheroids for imaging spatially heterogeneous ALP activity with high spatial resolution on a two-photon microscopy platform, providing the fluorescence-guided recognition of sub-millimeter tumorigenesis. DQM-ALP enabled differentiation between tumor and normal tissue ex vivo and in vivo, suggesting that the probe may serve as a powerful tool to assist surgeons during tumor resection.

Full text of DOI:10.1002/anie.202001675

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Research Articles
Chemie
International Edition: DOI: 10.1002/anie.202001675
German Edition: DOI: 10.1002/ange.202001675
Fluorescent Probes
An Activatable AIEgen Probe for High-Fidelity Monitoring of
Overexpressed Tumor Enzyme Activity and Its Application to Surgical
Tumor Excision
+
+
+
Haidong Li , Qichao Yao , Feng Xu , Yueqing Li, Dayeh Kim, Jeewon Chung, Gain Baek,
Xiaofeng Wu, Prima Fitria Hillman, Eun Young Lee, Haoying Ge, Jiangli Fan, Jingyun Wang,
Sang-Jip Nam, Xiaojun Peng,* and Juyoung Yoon*
[
3]
[4]
Abstract: Monitoring fluctuations in enzyme overexpression
facilitates early tumor detection and excision. An AIEgen
probe (DQM-ALP) for the imaging of alkaline phosphatase
GGT), cyclooxygenase-2 (COX-2), and alkaline phospha-
[
5]
tase (ALP) are important clinical indicators that are closely
related to the occurrence, progression, and deterioration of
tumors. The current consensus is that the early diagnosis and
complete resection of cancerous tissue are particularly
important in reducing cancer mortality, further relieving the
heavy burden on families and society. The high selectivity,
sensitivity, spatial resolution, and non-invasive characteristics
of small-molecule fluorescent probes have made them indis-
(
ALP) activity was synthesized. The probe consists of
a quinoline-malononitrile (QM) core decorated with hydro-
philic phosphate groups as ALP-recognition units. The rapid
liberation of DQM-OH aggregates in the presence of ALP
resulted in aggregation-induced fluorescence. The up-regula-
tion of ALP expression in tumor cells was imaged using DQM-
ALP. The probe permeated into 3D cervical and liver tumor
spheroids for imaging spatially heterogeneous ALP activity
with high spatial resolution on a two-photon microscopy
platform, providing the fluorescence-guided recognition of
sub-millimeter tumorigenesis. DQM-ALP enabled differentia-
tion between tumor and normal tissue ex vivo and in vivo,
suggesting that the probe may serve as a powerful tool to assist
surgeons during tumor resection.
[
6]
pensable for biological imaging. There has been explosion in
the development of fluorescent imaging agents associated
with cancer diagnosis in recent decades, particularly for
activatable fluorescent probes with high signal-to-noise
[
3e,7]
ratios.
These probes can help surgeons visualize tiny
metastatic tumors to take preventive and therapeutic meas-
ures. Unfortunately, most traditional fluorescent probes
suffer from the aggregation-caused quenching (ACQ) effect
[8]
and readily leak out of cells during prolonged bio-imaging.
Introduction
This limits the fidelity of long-term dynamic observations in
biomedical and clinical research.
The overexpression of enzymes in tumor cells, such as
In 2001, Tang reported on the enhancement of fluores-
cence after molecular aggregation. This phenomenon is
[1]
[9]
membrane type 1-matrix metalloproteinase (MT1-MMP),
[
2]
aminopeptidase N (APN/CD13), g-glutamyltransferase (g-
referred to as aggregation-induced emission (AIE), which is
[
10]
directly opposite to the ACQ effect.
a great deal of attention in the last decade
AIE has attracted
[
11]
because
[
+]
[
*] Dr. H. Li, D. Kim, J. Chung, G. Baek, Dr. X. Wu, P. F. Hillman,
E. Y. Lee, Prof. Dr. S.-J. Nam, Prof. Dr. J. Yoon
Department of Chemistry and Nanoscience
Ewha Womans University, Seoul 03760 (Korea)
E-mail: jyoon@ewha.ac.kr
fluorescent dyes that are capable of AIE do not have the
inherent disadvantage of ACQ. AIE luminogens (AIEgens)
generally have twisted propeller-shaped conformations or
[
10b]
rotatable structures,
which effectively restrict intramolec-
[
+]
[+]
ular motion and render intermolecular p–p interactions in the
aggregation state to regulate the release of energy in the
excited state, resulting in brighter fluorescence. This property
makes AIEgens suitable for long-term, high-fidelity monitor-
ing of target molecules in biological systems. Many research-
ers have focused on “always-on” AIEgens based on tetra-
phenylethylene (TPE) and hexaphenylsilole (HPS) fluoro-
Q. Yao, F. Xu, H. Ge, Prof. Dr. J. Fan, Prof. Dr. X. Peng
State Key Laboratory of Fine Chemicals, Dalian University of
Technology, 2 Linggong Road, Dalian 116024 (P. R. China)
E-mail: pengxj@dlut.edu.cn
Prof. Dr. Y. Li
School of Pharmaceutical Science and Technology, Dalian University
of Technology, 2 Linggong Road, Dalian 116024 (P. R. China)
Prof. J. Wang
[12]
phore derivatives, which are characterized by low signal-to-
School of Life Science and Biotechnology, Dalian University of
Technology, 2 Linggong Road, Dalian 116024 (P. R. China)
noise ratios. Photobleaching and photo-induced damage are
[13]
less extensive in two-photon (TP) fluorescence microscopy.
Prof. Dr. J. Fan, Prof. Dr. X. Peng
TP fluorescence technology also provides higher 3D imaging
resolution due to the nonlinear optical process at the most
commonly used excitation wavelengths, which range from 700
to 1000 nm. Disappointingly, there are relatively few AIEgens
for TP fluorescence microscopy, despite their superiority in
biological imaging, to the best of our knowledge. 3D tumor
Research Institute of Dalian University of Technology in Shenzhen,
Gaoxin South fourth Road, Shenzhen 518057 (P. R. China)
+
[
] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202001675.
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spheroids, which are spherical aggregates of tumor cells,
bridge the distance between traditional 2D cell culture
cellular aggregates. The DQM-ALP nanoprobe was success-
fully employed to distinguish normal tissue from cancerous
tissue with high a signal-to-noise ratio, implying that DQM-
ALP AIEgen has the potential to assist surgeons in removing
tumors during surgery.
[
14]
models and complex real tissues.
Moreover, they can
provide more realistic and economical research models for
clinical and biological applications. In current research
models, the difference in scale between living cells and the
in vivo level may lead to an incomplete understanding of
tumor onset, progression, and deterioration mechanisms. Results and Discussion
Models at the sub-millimeter scale are lacking. In addition,
few activatable AIEgens that can differentiate between
cancer tissue from normal tissue to facilitate tumor resection
have been reported. These gaps motivated us to develop an
activatable two-photon AIEgen probe that would enable
fluctuations in tumor enzyme overexpression to be detected
and guide tumor surgery in clinical settings.
Synthesis and Spectroscopic Characteristics
The intermediates, DQM-ALP, and DCM-ALP (contrast
agent) were synthesized as shown in Scheme S2. Their full
1
13
31
characterization by H, C, and P NMR spectroscopy and
HRMS is described in the Supporting Information (Figur-
es S1–S27). To validate the initial design concept, DQM-ALP
nanoaggregates were obtained by dispersing a DQM-ALP
stock solution in DMSO into the aqueous solution. Nano-
aggregates of the amphiphilic structure were confirmed by
dynamic light scattering analysis (DLS, Figure 1a) and TEM
(Figure 1b).
The quinoline-malononitrile (QM) chromophore has
[
15]
recently emerged as a new AIE building block. It has the
characteristics of controllable excitation/emission wave-
[12]
length, facile modification, and good biocompatibility.
QM may be an alternative platform for the development of
activatable AIEgens. Alkaline phosphatase (ALP;
EC 3.1.3.1) is an enzyme containing one magnesium atom,
The effect of the volume fraction (f ) of distilled water
w
[
16]
two zinc atoms, and five cysteine residues.
In clinical
(DW) on UV/Vis absorption and fluorescence of DCM-OH,
DCM-ALP, DQM-OH, and DQM-ALP was evaluated using
DW/DMSO mixtures. As displayed in Figures S28–S30, the
DQM-ALP spectra were almost unaffected by increases the
fw, which were accompanied by low quantum yields (F)
ranging 0.4% to 0.1%. Fluorescence images of DQM-ALP
solutions confirmed that no AIE occurred (Figure S31). The
DCM-ALP exhibited similar spectral behavior (Figure S32–
S34). However, when the ratio of DW in the mixed solvent
exceeded 70%, the fluorescence intensity of DQM-OH
increased sharply due to restricted rotation of the N-ethyl
group (Figures S35 and S36). The quantum yield increased 13-
fold, which was indicative of AIE (Figure S37). The absorp-
tion spectra of DQM-OH decreased markedly with increasing
fw, which was due to the scattering effect of DQM-OH
aggregates (Figure S38). The DLS and TEM data confirmed
the presence of DQM-OH aggregates in aqueous solution
(Figures S39 and S40). In contrast, the ACQ behavior of
compound DCM-OH was observed in the DW/DMSO
mixtures, as shown in Figures S41–S43. As the pH value
decreased (10.01–4.73), the fluorescence intensity of DQM-
OH increased obviously (Figures S44 and S45). However, the
practice, ALP is recognized as a critical biomarker associated
[
17]
with signal transduction and tumors metabolism. Inspired
by the target-response strategy, we selected the hydrophobic
QM derivatives as the AIE core and hydrophilic phosphate
group as the ALP-recognition unit to design the activated
nanoAIEgen DQM-ALP (Scheme 1) for tumor diagnosis.
The matched amphiphilic structure DQM-ALP enabled it to
self-assemble into a loosely packed nanoprobe in aqueous
solution. DQM-ALP molecules showed severe fluorescence
quenching in vitro. Activation of the probe by ALP, induced
the in situ aggregation of DQM-OH, which restricted free
molecular rotation and restored its intrinsic AIE fluores-
cence. When cancer cells were incubated with DQM-ALP,
overexpressed ALP in the cells cleaved its ALP-sensitive
moieties. Stronger AIE fluorescence enabled high-fidelity
tracking of fluctuations in ALP activity. DQM-ALP was
rapidly delivered into 3D tumor spheroids and enabled the
observation of ALP activity in the sub-millimeter multi-
weak ionization ability (pK = 8.75, Figures S46 and S47) of
a
DCM-OH phenolic hydroxy group significantly restricted
DCM-ALP for the detection of ALP in vitro (Figures S48 and
S49). Subsequently, the excitation and emission spectra of
DQM-ALP in the presence of ALP were investigated to
determine the optimal excitation wavelength (Figure S50).
The optical response of the DQM-ALP nanoprobe to
ALP was measured in a solution of Tris-buffer solution (TBS,
1
0 mm, pH 8.0, containing 1% DMSO). The fluorescence
profile of DQM-ALP (10 mm) changed significantly as the
À1
ALP concentration increased from 0 to 90 mUmL (Fig-
ure 1c) at 378C due to the scission of the phosphate
monoester bond. This converted DQM-ALP into DQM-OH
intermediate, and AIE was recovered in aqueous media.
These transformations were confirmed by variations in the
Scheme 1. Schematic illustration of the assembly of DQM-ALP and
process of AIE activation in vitro and in cell.
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Figure 1. Morphology and spectral characterization of DQM-ALP probe. a) DLS characterization of DQM-ALP (10 mm) in the aqueous solution.
b) TEM characterization of DQM-ALP (10 mm) in aqueous solution. c) Fluorescence spectra of DQM-ALP (10 mm) responding to ALP
À1
concentrations from 0 to 90 mUmL at 378C. d) Linear fitting of the fluorescence intensity at 550 nm towards the low concentrations of ALP,
incubation time 10 min. e) Response time of DQM-ALP (10 mm) in the absence (black) and presence (red) of ALP at 378C. f) ALP inhibition tests
with Na VO and DQM-ALP (10 mm) at 378C. g) Fluorescence spectra of DQM-ALP (10 mm) incubated with ALP and other analytes at 378C. The
3
4
+
+
À
À
À
À
À
À
2À
other analytes included Na (100 mm), K (100 mm), F (100 mm), Cl (100 mm), Br (100 mm), I (100 mm), NO (100 mm), CN (100 mm), S
100 mm), HSO4 (100 mm), SCN (100 mm), ClO4 (100 mm), NO2 (100 mm), CH COO (100 mm), HCO3 (100 mm), CO3 (100 mm), HPO4
100 mm), H PO4 (100 mm), SO4 (100 mm), ClO (100 mm), GSH (100 mm), Cys (100 mm), Hcy (100 mm), H O (100 mm), ascorbic acid
100 mm), DTT (100 mm), b-galactosidase (b-Gal, 100 mUmL ), xanthine oxidase (XO, 100 mUmL ), acetylcholinesterase (AChE, 100 mUmL ),
butyrylcholinesterase (BuChE), carboxylesterase (CE, 100 mUmL ), and alkaline phosphatase, (ALP, 75 mUmL ). h) Photostability of DQM-ALP
10 mm) and ICG dye (10 mm) under the same conditions. The experiments were repeated three times and the data were shown as mean (ÆS.D.).
3
À
À
À
À
À
À
2À
2À
(
(
(
3
À
2À
À
2
2
2
À1
À1
À1
À1
À1
(
absorption spectra (Figure S51), and characterization by DLS
and TEM (Figures S52 and S53). Meanwhile, the color
changes of probe solution indicated that DQM-ALP could
be used to recognize ALP with the naked eye (Figure S54).
Activation by ALP converted DQM-ALP to tightly
aggregated DQM-OH, indicating that the DQM-ALP nanop-
robe formed loosely packed aggregates in the aqueous
medium without fluorescence. The excellent linear relation-
biomedical applications, particularly the long-term monitor-
ing of target activity for disease diagnosis and visual therapy.
The commercial dye indocyanine green (ICG) has been
[19]
approved for clinical use by the FDA. Under irradiation
À2
with light at 350 mWcm (Scheme S1), the absorbance
spectra of commercial dye ICG distinctly decreased over
time (Figure 1h and Figure S60). In contrast, irradiation had
no effect on the absorbance of DQM-ALP and DQM-OH
(Figure 1h and Figure S61 and S62), which clearly demon-
strated that they could be used for long-term biological
imaging. As illustrated in Figures S63 and S64, DQM-ALP
showed good stability at pH values from 4.54 to 10.02 in
aqueous solutions and optimum value of increased fluores-
cence intensity for the recognition of ALP at 378C. Our
experimental results suggested that DQM-ALP would effi-
ciently identify ALP activity under physiological conditions.
2
ship (R = 0.9942) between the fluorescence intensity cen-
tered at 550 nm and various ALP concentrations (0–
À1
6
0 mUmL ) was obtained, as shown in Figure 1d. Addition-
ally, the detection limit of the DQM-ALP for ALP based on
À1
3
s/slope formula was determined to be 0.15 mUmL , which
suggested that the sensitivity of DQM-ALP detection of ALP
in vitro was high (Table S1). The Michaelis constant (K ) and
m
maximum initial reaction rate (Vmax) were calculated to be
À1
5
.16 mm and 4.83 mm min by Lineweaver–Burk analysis
(
Figure S55). Compared to without ALP group (Figure S56),
the fluorescence intensity of DQM-ALP gradually increased
to an equilibrium in the presence of ALP after 10 min
Docking Calculations and Verification of the Response
Mechanism
(
Figure 1e and Figure S57), indicating that loose aggregation
of amphiphilic structure of the DQM-ALP favored the ALP-
mediated enzymatic reaction. When sodium orthovanadate
To validate the relationship between DQM-ALP and
ALP, docking calculations were performed using the Discov-
ery Studio platform. We chose the crystal structure of ALP as
[
18]
(
Na VO ), an ALP inhibitor was added to the solution, the
3 4
[
20]
fluorescence intensity of DQM-ALP was almost completely
suppressed (Figure 1 f and Figure S58). To our satisfaction,
only ALP induced a dramatic change in fluorescence
behavior under identical conditions (Figure 1g and Fig-
ure S59). This demonstrated that DQM-ALP could serve as
an ALP-specific tool for monitoring ALP activity in complex
biological samples. Photostability is another vital parameter
used to evaluate the performance of fluorescent probes for
the docking model. As depicted in Figure 2a,b, the DQM-
ALP probe tended to approach the double zinc ion (named
2
+
2+ [21]
Zn1 and Zn2
)
coordination center in the ALP cavity.
The lengths of the coordinate bonds (Figure 2c and Fig-
2
+
ure S65) between probe and ALP were 2.015 ꢀ (OÀZn ),
1
2
+
2+
2.041 ꢀ (OÀZn ), and 2.829 ꢀ (OÀZn ). These were
1
2
extremely close to the inherent distances of the coordinate
bonds between zinc ion and amino acid residues in ALP,
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MTT Assays and Cell Imaging
Prior to cell fluorescence imaging, we examined the
cytotoxicity of DCM-ALP and DQM-ALP using a standard
[3f]
MTT assay.
As shown in Figures S67–S70, cell viability
remained high after treatment with the probes for 24 h. The
probes caused no noticeable cytotoxicity. Thus, the ability of
DQM-ALP for imaging the activity of endogenous ALP was
further investigated in living cells. Human cervical carcinoma
cells (HeLa cells) and hepatoma carcinoma cells (HepG-2
cells) were chosen as model cells owing to their overexpres-
[22]
sion of ALP. Compared to the control group (Figure 3b),
obvious fluorescence signal (Figure 3e) was observed in
HeLa cells incubated with DQM-ALP for 0.5 h. This was
caused by in situ enzymatic dephosphorylation, which re-
leased the nanoaggregates DQM-OH. Similarly, this was also
consistently observed in HepG-2 cells treated with DQM-
ALP for 0.5 h (Figure S71). We also performed time-depen-
dent imaging of DQM-ALP in live HeLa cells. As shown in
Figure S72, fluorescence intensity increased sharply over
Figure 2. Docking of DQM-ALP on ALP based on Discovery Studio
platform. a) Structure of ALP. The binding site is indicated by the red
transparent sphere. b) Partial enlargement of (a). c) Lengths of coor-
dinate bonds between DQM-ALP and ALP. d) Lengths of coordinate
2+
bonds between Zn and amino acid residues in ALP.
1
0 min, which was consistent with our in vitro characteriza-
tion.
DQM-ALP inhibition experiments were performed to
which were 2.002 ꢀ, 2.029 ꢀ, 2.011 ꢀ, 2.099 ꢀ, 2.083 ꢀ, and
1
.979 ꢀ (Figure 2d). With the help of the catalytic system
demonstrate that increased fluorescence intensity in living
centered on double zinc ion, the probe was transformed into
the DQM-OH intermediate. This restored the AIE and strong
fluorescence of the probe was measured, which was consistent
with the process depicted in Scheme 1.
cells was triggered by ALP. When Na VO4 were pre-
3
incubated with HeLa cells for 1 h, followed by the addition
of DQM-ALP for incubation another 0.5 h, the fluorescence
intensity in the cells decreased (Figure 3h) until it eventually
disappeared (Figure 3k), manifesting the activity of endoge-
nous ALP inhibited by Na VO . Levamisole hydrochloride,
In addition, high resolution mass spectra were collected to
confirm the generation of DQM-OH in vitro. A distinct peak
at m/z 340.1449 was observed in the HRMS spectrum. This
was in accordance with the calculated m/z of DQM-OH in
3
4
23]
[
another phosphatase inhibitor, was less effective in inhibit-
ing cellular ALP activity (Figure 3n,q). These results sug-
gested that DQM-ALP probe could be activated by the
overexpression of ALP in tumor cells for in situ monitoring of
ALP activity, and that it could be employed for visual high-
throughput screening ALP inhibitors. The advantages of two-
photon fluorescence imaging include relatively low photo-
+
positive mode, which was 340.1444 for [M+H] (Figure S66),
which further validated that DQM-OH resulted from ALP
catalytic activity towards the DQM-ALP probe.
Figure 3. Confocal fluorescence images of HeLa cells incubated with DQM-ALP (10 mm) at 378C. a–c) Control group, d–f) cells treated with DQM-
ALP for 0.5 h; Na VO group: Preincubated with g–i) 0.1 mm and j–l) 1 mm Na VO , followed by the addition of DQM-ALP for 0.5 h. Levamisole
3
4
3
4
group: Preincubated with m–o) 0.1 mm and p–r) 1 mm levamisole, followed by the addition of DQM-ALP for 0.5 h. l =473 nm, l =490–
ex
em
5
90 nm. Scale bar=30 mm.
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induced damage, photo-bleaching, deeper penetration, and
higher spatial and temporal resolution. We thus evaluated the
imaging performance of the probe using a two-photon
platform. As expected, fluorescence was observed from
HepG-2 cells treated with DQM-ALP under a Mai Tai
femtosecond laser at 810 nm (Figure S73).
intensity of their channels (Figure 4s,t), which indicated that
cortisol was a more efficient inducer than NaBu. These
observations demonstrated that DQM-ALP could serve as an
AIEgen probe with high spatiotemporal resolution (HSR) to
monitor the upregulation of ALP activity.
ALP plays a vital role in catalyzing the dephosphorylation
[24]
of small molecules, proteins, and nucleic acids. Thus, the
visualization of ALP activity provides a simple and effective
tool for chemical biology and clinical medicine. Previous
studies have shown that sodium butyrate (NaBu) as an
inducer increases the activity of ALP by stimulating de novo
Long-Term Cell Imaging Assessment
Long-term, high-fidelity bioimaging with AIEgens is of
great importance in the field of biomedicine. We further
investigated its ability to monitor the overexpression of ALP
in cells with HSR over long periods of time. To demonstrate
the inherent advantages of the AIE probe, the DCM-ALP
(ACQ probe) based on the DCM-fluorophore modified with
the same identification unit was used for the control group.
Distinct fluorescence images (Figure 5b,n) were observed
from the HeLa cells after incubation with DQM-ALP and
DCM-ALP for 0.5 h, which indicated that both probes could
be used to monitor ALP activity in the tumor cells.
Fluorescence in the ACQ group (Figure 5q,z) nearly vanished
within 3 h. However, the DQM-ALP group exhibited obvious
intracellular fluorescence (Figure 5e), and the fluorescence
intensity in the cells had only decreased to 66.98% of the
original value (Figure 5y). These results suggested that
DQM-ALP was activated by ALP to generate AIEgen
aggregation in situ, which reduced the rate of exocytosis due
to a size effect. The sharp drop in fluorescence in the control
group was attributed to the ACQ effect and extracellular
[25]
synthesis of the enzyme. Therefore, HeLa cells were pre-
incubated with NaBu to induce the synthesis of ALP
[
26]
mRNA.
After 4 h, there was no significant difference
between the fluorescence intensities of the cells (Figure 4b)
and the control group (Figure S74). Remarkable fluorescence
was observed from the treated cells after 28 h and 48 h
(
Figure 4e,s), suggesting that ALP overactivity induced by
NaBu had a time lag. As expected, the DQM-ALP was
triggered by high ALP activity. Plenty of DQM-OH was
generated, leading to the formation of aggregates in the cells
and AIE fluorescence (Figure 4h). Cortisol was also used to
increase the activity of HeLa ALP by modifying the enzyme
during or shortly after synthesis, transforming the conforma-
tion of the enzyme protein, resulting in improved the catalytic
[
27]
efficiency. Under 3 mm cortisol pretreatment, HeLa cells
exhibited brighter yellow fluorescence (Figure 4n) than
NaBu-stimulated cells after 28 h, in accordance with the pixel
Figure 4. Confocal fluorescence images of endogenous ALP activity in the living HeLa cells induced by sodium butyrate (NaBu, 1 mm) and
cortisol (3 mm) at 378C for 4 h (a–c and j–l), 28 h (d–f and m–o) and 48 h (g–i and p–r), followed by treatment with DQM-ALP for 0.5 h. s,t) The
normalized fluorescence intensity of cells treated with sodium butyrate and cortisol, respectively. Note: the pixel intensity of each channel in
images (h) and (q) was defined as 1.0. Error bar=RSD (n=9), l =473 nm, lem =490–590 nm. Scale bar=30 mm.
ex
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Figure 5. Long-term fluorescence imaging of HeLa cells treated with DQM-ALP. a–l) l =473 nm, l =490–590 nm; m–x) l =561 nm,
ex
em
ex
lem =655–755 nm. Normalized fluorescence intensities of cells incubated with DQM-ALP (y) and DCM-ALP (z). Note: The pixel intensity of each
channel from the image (b) and (n) was defined as 1.0, respectively. Error bar=RSD (n=9). Scale bar=30 mm.
leakage. Even after 13 h, the intracellular fluorescence
intensity remained high level of 51.07% of the original
intensity (Figure 5k,y).
ALP were successfully obtained by scanning along the z-axis
with two-photon excitation at 810 nm (Figure 6m1–6m3).
With the advantage of two-photon longitudinal imaging,
fluorescence was observed in each 8.48 mm layer of a HeLa
tumor spheroid (Figure 6C and Figure S76). Similar fluores-
cence was also observed in the HepG-2 tumor spheroids
Sub-organelle Localization Experiment
(Figures S77). This implied that ALP overexpression could
Co-localization experiments were carried out to further
investigate the distribution of DQM-ALP in the cells after
responding to ALP. HeLa cells were pre-treated with the
DQM-ALP probe at 378C for 20 min. The commercial
indicators (Hoechst 33342, LysoTracker Deep Red, and
MitoTracker Deep Red FM) were then successively added
into confocal dishes and incubated for another 20 min,
respectively. As shown in Figure S75, the apparent fluores-
cence signal from the AIEgen nanoaggregates (Figure S75 f,j)
overlapped well with the deep red fluorescence signal labeling
by Lyso-Tracker (Figure S75g) and Mito-Tracker dye (Fig-
ure S75k) with high Pearsonꢁs correlation coefficients of 0.92
and 0.82. These were higher than that of the nucleus (P =
activate more of DQM-ALP probes in the HepG-2 tumor
spheroids in situ to generate the obvious AIE fluorescence
signal. Our observations suggested that DQM-ALP was
rapidly delivered into the 3D-structured spheroids and
activated by ALP overexpression. This filled the gap between
simple two-dimensional cell culture models and complex real
tissues, resulting in a more vivid and economical research
model for clinical and biological applications.
Imaging ALP Upregulation in a Drug-Induced Acute Liver Injury
Model
0
.32). These results demonstrated that the product of probe
The activation of DQW-ALP by ALP in vitro, in cells, and
in tumor spheroids were assessed. The AIEgen probe was
used to monitor the fluctuations in ALP activity in the drug-
induced acute liver injury (DIALI) model. Overdoses of
acetaminophen (APAP) can cause acute liver injury. This is
generally accompanied by an increase in the ALP level.
Compared to PBS-treated mice (Figure 7b), the surfaces of
livers in mice administered with APAP were covered with
many hepatic parenchymal injury areas (Figure 7g). The
livers became lighter in color and nuclei of the tissue sections
were wrinkled (Figure 7h), which indicated the DIALI model
was smoothly established. 20 mm sections of normal liver and
DIALI liver tissue were prepared using a freezing microtome
and incubated with the DQM-ALP probe. Isolated liver tissue
from the mice pre-treated with APAP for 12 h, followed by
incubation with DQM-ALP probe showed distinct AIE with
increasing time (Figure 7i and Figure S78). This indicated
that the ALP content increased in the liver due to APAP-
induced liver injury. In contrast, the fluorescence intensity in
liver tissues from Balb/c mice pre-treated with PBS did not
increase significantly after incubation with DQM-ALP (Fig-
ure 7d and Figure S79). These results suggested that the
DQM-ALP responding to ALP mainly accumulated in
lysosomes and mitochondria.
Multicellular Tumor Spheroid Imaging
3
D-structured spheroids are used to better simulate the
microenvironment of cells and reflects multicellular aggrega-
tion and avascular metastasis in vivo. Consequently, we
evaluated the capability of enzyme-triggered DQM-ALP to
be turned on inside cells of sub-millimeter 3D-structured
spheroids. Exposure of the HeLa tumor spheroids to the
DQM-ALP probe led to a significant increase in fluorescence
under one-photon irradiation (Figure 6a2–6 f2) accompanied
by bright dots of aggregates, implying that the AIE probe was
activated by the overexpression of ALP in the tumor
spheroids. We also used DQM-ALP to monitor ALP activity
in the HeLa spheroids on the two-photon microscope plat-
form. Significant fluorescence enhancement was also ob-
served (Figure 6g2–6l2), as time went on. Reconstructed 3D
fluorescence images of HeLa spheroids treated with DQM-
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Figure 6. One- and two-photon microscope fluorescence imaging of HeLa multicellular tumor spheroids treated with DQM-ALP. A) One-photon
model: a1–a3) 30 min; b1–b3) 60 min; c1–c3) 90 min; d1–d3) 120 min; e1–e3) 150 min; and f1–f3) 180 min. Two-photon model: g1–g3) 30 min;
h1–h3) 60 min; i1–i3) 90 min; j1–j3) 120 min; k1–k3) 150 min; and l1–l3) 180 min. B) 3D tumor spheroid images (m1–m3) obtained along the z-
axis direction at depths of 15300–15800 mm by each interval of 8.48 mm as a step (C). One-photon mode: l =488 nm, l =520–620 nm; two-
ex
em
photon mode: l =810 nm, l =575–630 nm. Scale bar=200 mm.
ex
em
could distinguish between tumor tissue and normal tissue.
After the addition of probe DQM-ALP, hepatocellular
carcinoma tissues exhibited the evident granular fluorescent
spots just 10 min later (Figure 8a2) owing to the generation of
nanoaggregates due to ALP overexpression. The fluorescence
intensity of DQM-ALP increased with prolonged incubation
(Figure 8a2–8e2). No obvious fluorescence was observed in
normal liver tissue (Figure 8 f2–8j2), which suggested that the
DQM-ALP probe could be used to differentiate between
tumor tissue and normal tissue. In addition, cervical cancer
tissues were also used to as a research model owing to
overexpression of ALP. Strong fluorescence appeared in the
tumor tissues (Figure 8l2 and Figure S80). Fluorescence in
normal tissue was negligible (Figure 8k2). Interestingly, 3D
tissue imaging performed with two-photon platform, provid-
ed images that showed deep permeation of the probe into
tissue and its activation by ALP overexpression in 100 mm
hepatocellular carcinoma tissue (Figure 8m1–8m3 and Fig-
ures S81 and S82) and cervical cancer tissue (Figure 8n1–8n3)
pre-loaded with DQM-ALP for 30 min. These results showed
that the DQM-ALP could efficiently permeate tumor tissues
and be selectively activated by ALP overexpression. Aggre-
gation resulted in strong AIE fluorescence, which would
allow tumors to be distinguished from normal tissue.
Figure 7. Imaging ALP up-regulation in the drug-induced acute liver
injury model. A) Schematic illustration of APAP administered to Balb/c
mice and fluorescence imaging of tissue sections. B) Mice treated with
À1
PBS (a–e) or APAP (500 mgkg , f–j) for 12 h, followed by incubation
with DQM-ALP (10 mm). l =488 nm, l =520–620 nm. Scale
ex
em
bar=40 mm.
probe could be used to monitor fluctuations in ALP activity
induced by APAP in mouse liver, which was favorable for
early DIALI predictions.
Differentiating Tumor Tissue from Normal Tissue
Accurate identification of tumor tissue and normal tissue
is very important for the surgical resection of tumors to
prevent recurrence, reduce cutting, and to avoid damaging
normal tissue. We investigated whether the DQM-ALP probe
Visualization ALP Activity in Mice Xenograft Tumor Model
We then evaluated the performance of DQM-ALP for
monitoring ALP activity in mice bearing tumor xenografts.
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Figure 8. 2D and 3D imaging of tissues treated with DQM-ALP to monitor ALP activity. A) Time-dependent fluorescence imaging of 10 mm
hepatocellular carcinoma tissue (a–e) and normal liver tissue (f–j). B) 2D fluorescence imaging of 10 mm normal tissue tissue (k1–k3) and cervical
cancer tissue (l1–l3) were treated with DQM-ALP for 30 min. C) 3D fluorescence imaging of 100 mm hepatocellular carcinoma tissue (m1–m3)
and cervical cancer tissue (n1–n3) under two-photon excitation. One-photon mode: l =488 nm, l =520–620 nm; two-photon mode:
ex
em
lex =810 nm, lem =575–630 nm. Scale bar=50 mm.
Upon tumor formation, ex vivo imaging of the dissected
organs and tumors after immersion in DQM-ALP was carried
out using small-animal imaging equipment. As shown in
Figure S83, HepG-2 tumor exhibited brighter fluorescence
than other organs including the heart, liver, spleen, lung, and
kidney after soaking for 15 min, which reached equilibrium in
rapid and highly sensitive. The recognition of DQM-ALP by
ALP was verified with docking calculations and HRMS
analysis. DQM-ALP exhibited its inherent AIE fluorescence
in response to ALP overexpression in tumor cells. The probe
was firstly used to monitor the down-regulation of ALP
activity with sodium orthovanadate and levamisole hydro-
chloride, and the up-regulation of ALP activity by sodium
butyrate and cortisol stimulation. Unlike DCM-ALP, the
DQM-ALP probe was visualized for up to 13 h in high-fidelity
cell imaging. Under two-photon platform, DQM-ALP probe
was delivered into 3D-structured spheroids to monitor
spatially heterogenous ALP activity with high spatial reso-
lution. Moreover, relying on abnormal ALP activity, DQM-
ALP was successfully used to differentiate between tumors
tissues and normal tissues in nude BABL/c mice bearing
HepG-2 and HeLa xenograft tumors. Thus, we expect that the
enzyme-activated AIEgen DQM-ALP can be a valuable tool
for the early diagnosis and resection of cancerous tissues.
1
h (Figure 9a). HeLa tumor also exhibited significant signal
(
Figure 9c) compared to normal liver (Figure 9b,d), which
was caused by overexpression ALP in the tumor. A rapid
fluorescence increase of the DQM-ALP-treated group was
observed in the tumor region (Figure 9e–h) of tumor-bearing
mice, demonstrating that DQM-ALP probe could rapidly
monitor the ALP activity in mice. Fluorescence-guided
surgery has attracted much attention in clinical research to
achieve accurate resection of tumors. The biggest bottleneck
for this technology is the lack of suitable clinical contrast
agents, particularly activatable agents for high-fidelity imag-
ing. We explored the use of probe for fluorescence-mediated
surgical resection. As shown in Figure 9C, by direct spraying
DQM-ALP probe in situ, fluorescence was remarkable in the
cancerous tissue and there was a clear boundary between Acknowledgements
tumor tissue and normal tissue (Figure 9j). With fluorescence
guidance, the tumor tissue was successfully removed intact
using a scalpel (Figure 9k).
This work was supported by grants from the National
Research Foundation of Korea (NRF) funded by the Korean
government (MSIP) (No. 2012R1A3A2048814 for J. Y.), and
National Science Foundation of China (No. U1608222 for X.
P.). Mass spectral data were obtained from the Korea Basic
Science Institute (Daegu) on a Jeol JMS 700 high-resolution
mass spectrometer.
Conclusion
In summary, we rationally designed and synthesized the
activatable AIEgen DQM-ALP for the specific monitoring of
ALP activity. Owing to its matched amphiphilic structure,
molecules DQM-ALP could form the loose nanoprobe by Conflict of interest
self-assembly in aqueous solution. Their formation was
confirmed through the characterization of DLS and TEM.
Detection of ALP activity with nanoprobe DQM-ALP was
The authors declare no conflict of interest.
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Figure 9. Fluorescence images of endogenous ALP activity in nude
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tumor and normal liver immersion in DQM-ALP solution (50 mm):
a) HepG-2 tumor, b) normal liver, c) HeLa tumor, and d) normal liver.
B) Fluorescence imaging of HeLa tumor-bearing Balb/c mice at
various times (e, 0 min; f, 15 min; g, 30 min; h, 35 min) after injection
with DQM-ALP, and the green areas represent the tumor. C) Schematic
illustration of tumor resection by spraying DQM-ALP and fluorescence-
mediated tumor resection: i) fluorescence image (control group),
j) pre-treated by spraying DQM-ALP (50 mm), followed by fluorescence
image, k) surgical removal of tumor, and the green areas represent the
tumor. Fluorescence imaging of mice were recorded through small-
animal imaging equipment (PerkinElmer IVIS Spectrum).
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Keywords: aggregation-induced emission · AIEgens ·
enzyme activity · nanoprobes · tumor detection
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Manuscript received: February 1, 2020
Version of record online: && &&
,
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Research Articles
Fluorescent Probes
The cutting edge: An enzyme-activated
two-photon AIEgen, DQM-ALP, for
imaging the alkaline phosphatase (ALP)
activity in tumor cells and 3D tumor
spheroids with high spatial resolution
was synthesized. DQM-ALP enabled
accurate differentiation between tumor
and normal tissue ex vivo and in vivo,
suggesting that the probe may serve as
a powerful tool to assist surgeons during
tumor resection.
H. Li, Q. Yao, F. Xu, Y. Li, D. Kim,
J. Chung, G. Baek, X. Wu, P. F. Hillman,
E. Y. Lee, H. Ge, J. Fan, J. Wang, S.-J. Nam,
X. Peng,* J. Yoon*
&&&& — &&&&
An Activatable AIEgen Probe for High-
Fidelity Monitoring of Overexpressed
Tumor Enzyme Activity and Its
Application to Surgical Tumor Excision
&
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