A sensitive fluorescent probe for β-galactosidase activity detection and application in ovarian tumor imaging

Article abstract of DOI:10.1039/d0tb02269a

The development of non-invasive and sensitive optical probes for in vivo bioimaging of cancer-related enzymes is desirable for early diagnosis and effective cancer therapy. β-galactosidase (β-gal) is regarded as a key ovarian cancer biomarker, owing to its overexpression in primary ovarian cancer. Herein, we designed a sensitive near-infrared (NIR) probe (DCMCA-βgal) for the detection and real-time imaging of β-gal activity in ovarian tumors, thereby achieving the visualization of ovarian tumors by β-gal activity detection. DCMCA-β-gal could be triggered by β-gal, resulting in the release of a NIR chromophore, DCM-NH2; the linear range of fluorescent response to β-gal concentration was 0-1.2 U with a low detection limit of 1.26 × 10-3 U mL-1. We used DCMCA-β-gal to detect and visualize β-gal activity in SKOV3 human ovarian cancer cells, as well as for real-time imaging of β-gal activity in ovarian cancer mouse models. DCMCA-β-gal possessed high sensitivity, "turn-on"NIR emission, a large spectral shift, and high photostability in a dynamic living system and thus could serve as a highly sensitive sensor for real-time tracking of β-gal activity in vivo and ovarian tumor imaging.

Full text of DOI:10.1039/d0tb02269a

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Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
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DOI: 10.1039/D0TB02269A
ARTICLE
A sensitive fluorescent probe for β-galactosidase activity
detection and application in ovarian tumors imaging
Fang Fan, Li Zhang, Xinguang Zhou, Fangya Mu and Guoyue Shi*
Received 00th January 20xx,
Accepted 00th January 20xx
The development of non-invasive and sensitive optical probes for in vivo bioimaging of cancer-related enzymes is desirable
for early diagnosis and effective cancer therapy. β-galactosidase (β-gal) is regarded as a key ovarian cancer biomarker, owing
to its overexpression in primary ovarian cancer. Herein, we designed a sensitive near-infrared (NIR) probe (DCMCA-βgal) for
the detection and real-time imaging of β-gal activity in ovarian tumors, thereby achieving the visualization of ovarian tumors
by β-gal activity detection. DCMCA-βgal could be triggered by β-gal, resulting in the release of a NIR chromophore DCM-
DOI: 10.1039/x0xx00000x
2
NH U
; the linear range of fluorescent response to β-gal concentration was 0-1.2 U with a low detection limit of 1.26×10-3
-1
mL . We used DCMCA-βgal to detect and visualize β-gal activity in SKOV3 human ovarian cancer cells, as well as for real-
time imaging of β-gal activity in ovarian cancer mouse models. DCMCA-βgal possessed high sensitivity, “turn-on” NIR
emission, a large spectral shift, and high photostability in a dynamic living system and thus could serve as a more sensitive
sensor for real-time tracking of β-gal activity in vivo and ovarian tumors imaging.
auto-fluorescence and deep penetration in tissues, which have
proven beneficial for in vivo bioimaging [30-32]; however,
highly sensitive NIR probes for imaging ovarian tumors by
Introduction
Early accurate diagnostics and effective therapy have great
significance in improving cancer survival rates. Ovarian cancer
is the leading cause of cancer-associated death in women, with
a five-year survival rate < 50%. This high mortality rate is often
attributed to delayed diagnosis; approximately 75% of patients
with ovarian cancer are diagnosed in later stages [1, 2].
Therefore, early diagnosis is crucial for improving survival;
however, the early diagnosis of ovarian cancer is hindered by
the lack of obvious symptoms.
Biomarkers are synthesized or released from cells during
cancer onset; hence, accurate biomarker detection and activity
monitoring in living systems can be utilized for early stage
cancer diagnosis, further promoting effective treatment [3]. β-
galactosidase (β-gal), with overexpression in primary ovarian
cancer, is considered an important biomarker of ovarian
cancers [4-6]. Therefore, developing an accurate and fast
method for in situ detection and real-time monitoring of β-gal
activity is desirable for early diagnosis of ovarian cancer [7, 8].
Non-invasive optical imaging has recently developed to be
an ideal tool for tracking enzyme activity in situ [9-14]. Up to
now, several optical probes for detecting and visualizing β-gal
activity in living systems have been designed and produced [15-
tracking β-gal activity in vivo are still rare.
Thus, we develop a very sensitive NIR probe for detecting
and real-time imaging of β-gal activity in ovarian tumors, and
further for visualizing ovarian tumors. Our probe, DCMCA-βgal,
2
utilizes DCM-NH as the NIR fluorescence reporter [33, 34] and
β-galactose as the enzyme-activating trigger; these two
components are conjugated by a bridge of 4-hydroxy-3-
nitrobenzyl alcohol (Scheme 1A). DCMCA-βgal exhibits “turn-
on” NIR emission, a large spectral shift, and high photostability
under normal physiological conditions; these characteristics
make it more suitable for tracking β-gal activity in vivo.
Particularly noteworthy is its high sensitivity for β-gal
detection, suggesting that the use of DCMCA-βgal could enable
clearer imaging of ovarian tumors and the detection of much
smaller metastases. Early reports indicate that the detection
and removal of ovarian cancer peritoneal metastases < 1 mm in
diameter could improve the five-year survival rate [35, 36].
Therefore, DCMCA-βgal has great potential value for
fluorescence-guided diagnosis and therapy of ovarian cancer in
preclinical applications.
2
9]. Compared with traditional probes, near infrared (NIR)
Results and discussion
probes demonstrate unique characteristics, such as decreased
Design of DCMCA-βgal
2
In our probe, DCM-NH was used as the NIR fluorescent
a. School of Chemistry and Molecular Engineering, East China Normal University,
Dongchuan Road 500, Shanghai 200241, China E-mail: gyshi@chem.ecnu.edu.cn
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
reporter in terms of its controllable emission, and a β-gal
cleavable unit (β-galactose) was used to be the enzyme-
activating trigger. These two units were connected by a 4-
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hydroxy-3-nitrobenzyl bridge, which could enhance the pull- sensitivity and accuracy (Fig. S9). As shown in Figs.V1ieCw Aarnticdle1ODnl,inae
electron effect in the molecule, thus facilitating the cleavage of good linear relationship was observed b
D
e
O
t
I
w
: 1
e
0
e
.1
676 nm 550
n I /I
039/D0TB0226 9n mA
the C-O bond between the bridge and trigger site by β-gal, and β-gal concentration, ranging from 0 to 1.2 U, and the I676
thereby improving the response rate and sensitivity of the nm/I550 nm ratio demonstrated an approximate 19-fold increase.
probe. This feature was confirmed in subsequent experiments. These results demonstrated the high sensitivity of DCMCA-βgal,
1
-3
-1
L
DCMCA-βgal and other intermediates were characterized by H- a low detection limit (D ) of 1.26×10 U mL was achieved (S/N
NMR and HR-MS (Figs. S1–S8).
= 3).
The selectivity of DCMCA-βgal for β-gal was evaluated using
common biomolecules and several enzymes. As illustrated in
Fig. 1F, no obvious changes in FL intensity (I676 nm) were detected
following the addition of 100 equiv. of the interferents. Only
after β-gal (1.2 U) was added, was a distinct enhancement in FL
intensity (I676 nm) detected, indicating that DCMCA-βgal could be
selectively hydrolyzed by β-gal. Consequently, DCMCA-βgal
could be employed as a β-gal detection sensor with high
sensitivity and selectivity.
In addition, the pH dependence of the DCMCA-βgal (5 μM)
was also investigated (Fig. S10). DCMCA-βgal was stable within
a pH range of 5 to 10, suggesting that β-gal could be detected in
dynamic living systems using DCMCA-βgal without pH
interference.
Scheme 1 (A) The DCMCA-βgal fluorescent probe for β-gal detection. (B) DCMCA-βgal
synthesis process.
DCMCA-βgal response to β-gal in vitro
To assess whether DCMCA-βgal can actually detect β-gal, we
studied the optical characteristics of DCMCA-βgal in reaction
with β-gal in a physiological buffer solution. Fig. 1A showed that
DCMCA-βgal exhibited robust absorption at 452 nm. Following
the addition of β-gal, the absorption at 452 nm decreased, while
a new red-shifted band at approximately 526 nm appeared. This
new absorption pattern was identical to the absorption of DCM-
2 2
NH , demonstrating DCM-NH production following cleavage of
the β-galactose unit by β-gal. Additionally, an alteration in color
from pale yellow to light red was detected in the test solution.
In terms of the fluorescence response, an obvious
ratiometric fluorescent signal (I676 nm/I550 nm) was observed
following β-gal addition (Fig. 1B). The fluorescence intensity (FL
intensity) decreased at 550 nm, but drastically increased in the
6
00 – 800 nm range, especially at 676 nm. This new and unique
red-shifted response was attributed to the liberation of
amidogen following β-gal-catalyzed DCMCA-βgal hydrolysis.
The formed amino group was a stronger electron donor, which
could enhance the intramolecular charge transfer (ICT) and
create the NIR emission of DCM-NH
2
.
Then the FL intensity (I676 nm) as a function of time was
Fig. 1 (A – B) The optical spectra of DCMCA-βgal with or without the addition of β-gal. (C)
measured. As presented in Fig. 1E, the FL intensity at I676 nm Fluorescence emission of DCMCA-βgal (5 μM) incubated with 0 – 1.2 U of β-gal for 30
min at 37 °C (λex = 464 nm). (D) I676 nm/I550 nm vs. β-gal concentration plot. (E) Fluorescence
time-course study of DCMCA-βgal in reaction with β-gal (1.2 U). (F) FL intensity response
of DCMCA-βgal (5 μM) to various interferents (100 equiv.): a) reductase; b) phosphatase;
plateaued in 30 min after β-gal (1.2 U) was added to the
DCMCA-βgal solution, with an approximate 12-fold
enhancement detected. These results indicated that this rapid
response was available for rapid detection of β-gal, and that 30
min was selected as the optimal detection time in subsequent
tests.
2 2 2
c) cellulase; d) lysozyme; e) GSH; f) H O ; g) cysteine; h) H S; i) α-gal; j) β-gal.
Sensing mechanism
Following fluorescence titration with β-gal, 5 μM of DCMCA- To get a further insight into the specific sensing mechanism
βgal was chosen as the optimal concentration in terms of underlying the response of DCMCA-βgal to β-gal, HPLC
2
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experiments were conducted. As shown in Fig. 2, following the
incubation of DCMCA-βgal with β-gal, a new peak appeared
with a retention time consistent with the fluorophore DCM-
View Article Online
DOI: 10.1039/D0TB02269A
NH
2
. These results confirmed that an enzyme-triggered
had formed;
cleavage reaction had occurred and that DCM-NH
2
the β-glycoside bond was cleaved by β-gal, removing the 4-
hydroxy-3-nitrobenzyl bridge, then generating an electron-rich
amino group. The enzymatic reaction product DCM-NH
produced a clear fluorescent signal in the NIR region.
2
Fig. 3 Fluorescent imaging of DCMCA-βgal incubated with (A – C) 293T/lacZ cells highly
expressing β-gal, (D – F) SKOV3 cells, and (G – I) SKOV3 cells following pre-treatment with
an inhibitor Scale bar: 50 μm.
Visualizing β-gal activity in vivo and ovarian tumors imaging
NIR probe possesses unique advantages for in vivo bioimaging,
the NIR emission enables to penetrate deep into tissues and
avoid interference of auto-fluorescence in vivo. Thus, we
designed DCMCA-βgal with “turn-on” NIR emission, and
evaluated its ability to visualize β-gal activity in vivo. First, we
constructed an ovarian cancer mouse model by subcutaneous
inoculation of control 293T and SKOV3 cells into the left and righ
2
Fig.2 HPLC traces of DCMCA-βgal, DCMCA-βgal reacted with β-gal, and DCM-NH ,
respectively. HPLC conditions: detection wavelength, 450 nm; eluent solvent, 15%
methanol in water with 0.3 mL min-1 flow rate.
Sensing endogenous β-gal in live cells
Subsequently, we evaluated the ability to utilize DCMCA-βgal to
monitor and image β-gal activity in live cells using 293T cells as
the model. Initially, the lacZ gene was transferred into the 293T
cells via facile transfection, resulting in cells (293T/lacZ cells)
harboring endogenous β-gal [37, 38]. Low fluorescence was
detected in three specific channels (blue, green, and red
channels), when the 293T cells were treated with DCMCA-βgal
(5 μM) at 37 ºC for 30 min. In contrast, strong red fluorescence
(670 – 800 nm), corresponding to the emission band of DCM-
2
NH , was clearly observed in 293T/lacZ cells overexpressing β-
gal (Fig. 3B). The mean FL intensity in 293T/lacZ cells was three
times greater than that in untreated 293T cells (Fig. S12). Thus,
these observations suggested that DCMCA-βgal could image β-
gal activity in live cells.
Next, to further confirm the utility of DCMCA-βgal to
monitor β-gal activity in live cells, SKOV3 human ovarian cancer
Fig. 4 (A) Fluorescence imaging of DCMCA-βgal in ovarian tumors. DCMCA-βgal was
intratumorally injected into tumor-bearing mice; the 293T control tumor is on the left
cells was proposed. SKOV3 cells have been shown to highly and the SKOV3 tumor is on the right (λex = 465 nm). (B) Comparison of fluorescence
signals following intratumoral injection of DCMCA-βgal in both flanks. Quantitative
analysis was conducted using the IVIS imaging system. (C) Fluorescent images of the
dissected tumor tissues.
express endogenous β-gal. As presented in Fig. 3E, strong red
fluorescence was detected in SKOV3 cells, while no
fluorescence was observed in negative control SKOV3 cells
pretreated with an inhibitor (Fig. 3H). The mean FL intensity in
positive SKOV3 cells increased 25-fold compared with the
negative control (Fig. S13). Thus, the results demonstrated that
DCMCA-βgal could be triggered by β-gal in cells to release the
-
t flanks of mice, respectively. As illustrated in Fig. 4A and Fig.
S14, 10 min post injection of DCMCA-βgal into the tumors,
obvious NIR fluorescence signals ranging from 680 to 720 nm
were detected on the right flanks, which gradually increased to
DCM-NH
2
fluorophore, which provided “turn-on” NIR emission
(λ = 676 nm) for monitoring β-gal activity.
This journal is © The Royal Society of Chemistry 20xx
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a maximum at 1 h; however, no fluorescence signals were Malononitrile, 2'-hydroxyacetophenone, sodium hVyidewriAdretic(leNOanHlin)e,
detected on the left flanks, even at 1 h post injection. IVIS acetic anhydride, 4-acetamidobenzald ip
imaging system analysis demonstrated that the FL intensity at acetobromo-α-D-galactose, 4-hydroxy-3-nitro benzaldehyde,
80 – 720 nm in tumors on the right flanks was much higher silver oxide, sodium borohydride, triphosgene, Hünig’s reagent
D
O
e
I
h
: 1
y
0
d
.1
e
0
,
39/D
p
0
T
e
B0ri
2
d
2
i
6
ne,
9
A
6
than in the control and a clear description was achieved by (DIEA), citric acid, sodium methoxide (MeONa), and Amberlite
+
quantitative study of the fluorescence images (Fig. 4B). These IR-120 plus (H ) were provided by Tokyo Chemical Industry Co.,
results showed that DCMCA-βgal could be trigged by β-gal in Ltd. (Shanghai, China). Hydrochloric acid (HCl), acetic acid
tumors, releasing the NIR fluorophore to image β-gal activity. (AcOH), anhydrous sodium sulfate (Na
Additionally, fluorescence imaging of tumor tissues showed a phosphate (NaH PO ·2H O), sodium bicarbonate (NaHCO
large increase in NIR fluorescence signals in the ovarian tumors dibasic sodium phosphate (Na HPO ·12H O), tetrahydrofuran
2
SO
4
), sodium dihydrogen
2
4
2
3
),
2
4
2
compared with the control (Fig. 4C). The result further (THF), hexane, ethyl acetate (EtOAc), toluene, methanol
confirmed that DCMCA-βgal could be utilized to visualize and (MeOH), acetonitrile, dichloromethane (DCM), ethanol (EtOH),
3
track β-gal activity in ovarian tumors, and in turn to image trichloro-methane (CHCl ), isopropanol (i-PrOH), and
ovarian tumors by β-gal activity detection.
deuterated chloroform (CDCl ) were supplied by Aladdin
3
Chemistry Co. Ltd. (Shanghai, China).
2 2
β-gal, glutathione (GSH), hydrogen peroxide (H O ), α-gal,
reductase, phosphatase, cellulase, lysozyme, cysteine,
hydrogen sulfide (H S), and dimethyl sulfoxide (DMSO,
2
molecular biology grade) were bought from Sangon Biotech Co.,
Ltd. (Shanghai, China).
The absorption spectrum was measured using a Shimadzu
UV-1800 spectrometer (Kyoto, Japan). Fluorescence emission
spectrum was collected using a microplate reader (Infinite
M200 PRO; TECAN, Switzerland).
DCMCA-βgal synthesis
Fig. 5 Fluorescence imaging of DCMCA-βgal in ovarian tumors. DCMCA-βgal was
intraperitoneally injected into tumor-bearing mice (λex = 465 nm).
DCMCA-βgal and other related intermediates were synthesized
according to Scheme 1B and Scheme S1, specific details of the
synthesis processes were provided in the supporting materials.
Incubation and determination system
Then, to further confirm the suitability of DCMCA-βgal for The optical spectra of DCMCA-β-gal in reaction with β-gal were
ovarian tumors imaging, we built another tumor model of obtained at 37 °C in phosphate buffer saline (PBS, 0.1 M,
ovarian cancer by intraperitoneal injection of SKOV3 cells into pH=7.4, 30% DMSO). The fluorescence spectra were collected
the nude mice. After raised for 10 days, the non-tumor- and over a wavelength range of 500 – 800 nm after excitation at 464
tumor-bearing mice were both intraperitoneally injected with nm. The selectivity of DCMCA-βgal for β-gal was evaluated with
the probe DCMCA-βgal. At 40 min post injection, the mice were various interferents (100 equiv.) in our incubation system
2 2 2
killed and the abdominal cavities imaging were performed. As including GSH, H S, H O , cysteine, lysozyme, reductase,
displayed in Fig. 5, the NIR fluorescence signals were observed phosphatase, α-gal, and cellulase.
in the tumor-bearing mice and using the signals the ovarian Cell culture and imaging
tumors could be clearly visualized, which suggested that
Human embryonic kidney cells (293T cells) and SKOV3 cells
DCMCA-βgal had been hydrolyzed by β-gal in the tumors and
the NIR fluorophore DCM-NH was released; while no NIR
were provided by Shanghai Institutes of Biological Science, CAS
Shanghai, China). The 293T and SKOV3 cells were grown at 37
C, 5% CO in Dulbecco’s modified Eagle’s medium (DMEM) and
2
(
signals was captured in non-tumor-bearing mice. Thus, these
results demonstrated the suitability of DCMCA-βgal for ovarian
tumors imaging. Additionally, the high sensitivity of DCMCA-
βgal enabled the acquisition of much clearer images, which was
conducive for the detection of smaller tumor metastases with
great significance for improving the the five-year survival rate
of patients.
°
2
McCoy’s 5A Medium, respectively. The cytotoxicity of DCMCA-
βgal towards 293T and SKOV3 cells was assessed by a
colorimetric assay (MTT based). The cells were plated in a 96-
well plate containing DMEM, and then different concentrations
of DCMCA-βgal (containing 0.1% DMSO) were added. The
absorbance of each well was recorded after 24 h of incubation.
For fluorescent imaging, the cells were incubated at 37 °C
with DMEM and DCMCA-βgal (5 μM) for 30 min. As a negative
control, SKOV3 cells were also pretreated with the β-gal
competitive inhibitor (D-galactose, 1 mM) for 1 h. Fluorescence
Experimental methods
Reagents and instruments
2
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imaging and image collection were performed using a
fluorescent microscope (BX53, Olympus, Japan).
In vivo imaging of β-gal activity
View Article Online
DOI: 10.1039/D0TB02269A
We created two ovarian cancer mouse models with SKOV3 cells.
One was that, 1 × 10 SKOV3 and control 293T cells were
Notes and references
7
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subcutaneously inoculated into the right and left flanks of 7– 8-
week-old female nude mice, respectively; and the other was
that SKOV3 cells were intraperitoneally injected into mice.
Tumor-bearing mice of two models were both raised for
approximately 10 days. In vivo fluorescence imaging was
performed using the IVIS imaging system (PerkinElmer, USA),
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possessed the advantages of high sensitivity, good
photostability in dynamic living systems, and a larger spectral
shift, which performed well in visualizing β-gal activity in SKOV3
human ovarian cancer cells and real-time imaging of β-gal
activity in ovarian cancer mouse models. This study confirmed
that DCMCA-βgal could provide an accessible tool for tracking
β-gal activity in vivo and thereby imaging of ovarian tumors,
which provides great preclinical potential for early diagnosis
and therapy of ovarian cancer.
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Conflicts of interest
There are no conflicts to declare.
2
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Acknowledgements
This work was supported by the Key Project of Science and
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Journal of Materials Chemistry B
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Graphical abstract
A sensitive fluorescent probe for β-galactosidase activity detection in vivo and near-
infrared imaging ovarian tumors by detecting β-gal.