In vivo ratiometric tracking of endogenous β-galactosidase activity using an activatable near-infrared fluorescent probe

Article abstract of DOI:10.1039/c9cc06869d

Herein, we developed a dual-channel and light-up near-infrared fluorescent probe for ratiometric sensing of β-galactosidase (β-gal) activity. The well-designed probe, which shows ratiometric optical response with a significant red-shift (from 575 nm to 73

Full text of DOI:10.1039/c9cc06869d

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In vivo ratiometric tracking of endogenous
b-galactosidase activity using an activatable
near-infrared fluorescent probe†
Cite this: Chem. Commun., 2019,
55, 12308
Received 3rd September 2019,
Accepted 19th September 2019
Limin Shi,‡ Chenxu Yan,‡ Yiyu Ma, Ting Wang, Zhiqian Guo * and
Wei-Hong Zhu
DOI: 10.1039/c9cc06869d
rsc.li/chemcomm
Herein, we developed a dual-channel and light-up near-infrared good candidates for constructing fluorescent probes, such as
fluorescent probe for ratiometric sensing of b-galactosidase (b-gal) high quantum yields, excellent photostability and chemical
activity. The well-designed probe, which shows ratiometric optical stability under physiological conditions.28–30 Sparked by the
response with a significant red-shift (from 575 nm to 730 nm), was new tactics in the functionalization of the 1,3-dimethyl-BODIPY
successfully applied to detect endogenous b-gal activity in SKOV-3 core, due to its fine-tuned properties and easier purification
cells and tumor-bearing mice.
procedures, a veritable BODIPY renaissance has come into
being.31 However, most of the current BODIPY-based probes
Abnormal tumor microenvironmental factors such as hypoxia,1 focused on the OFF–ON response mode use the photon electron
low pH,2 and overexpressed tumor-related enzymes3–6 are transfer (PET) mechanism.32 In contrast, it would be an effective
widely accepted as the features of tumor tissue.7 In particular, approach to construct ratiometric BODIPY probes by a typical
b-galactosidase (b-gal) is known as an important biomarker intramolecular charge transfer (ICT) process.33 In particular,
involved in tumorigenesis and metastasis of ovarian cancers. when an electron-donating group (e.g. hydroxy) is covalently
For example, high levels of b-gal activity were detected in several conjugated with the 1,3-dimethyl-BODIPY core, a remarkable shift
metastatic ovarian cancer cells (including SHIN3, SKOV3, etc.) in the emission spectra can be obtained with effective ICT proper-
compared with that in a non-transformed human cell line.8 Thus, ties, making it an ideal NIR ratiometric fluorescent reporter for
the assessment of endogenous b-gal activity in vivo is a commonly in vivo application. Thus, we envision that a NIR ratiometric
accepted method for clinical diagnosis and therapeutic evaluation sensing of b-gal can be achieved by regulating the ICT process
of cancers.9 Apparently, fluorescence imaging offers great potential of a monostyryl-substituted BODIPY probe before and after the
for visualizing b-gal activity in preclinical diagnosis.10–14 Although a response with b-gal.
number of responsive fluorescent probes have been reported for
In this study, we report a NIR ratiometric probe (BODIPY-bgal)
tracking b-gal,15,16 the short emission extremely hinders their that can be used for accurately visualizing and analyzing the activity
in vivo applications due to the limited tissue penetration depth of b-gal in vivo. In our probe, monostyryl-substituted BODIPY
and inevitable auto-fluorescence interference from biosystems.17–20 (BODIPY-OH) is utilized as an NIR and wavelength-controllable
In addition, the OFF–ON detecting pattern makes it difficult to chromophore, and a b-gal cleavable unit (b-galactopyranoside) as
precisely acquire b-gal expression in tumors, which is obstructive an enzyme-active trigger (Scheme 1). Upon reaction between the
for evaluation of the cancer therapeutic efficacy.21,22 Therefore, a probe and b-gal, BODIPY-bgal is unmasked and converted into the
ratiometric near-infrared (NIR) fluorescent probe for tracking deprotected product (BODIPY-OH in the phenolate form) in a
endogenous b-gal is urgently required for in vivo accurate quanti- physiological context. As expected, a colorimetric change in the
fication analysis.23–27
absorption spectra and a remarkable red-shift to the NIR region in
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes the emission spectra were observed. Herein, we focus on regulating
have outstanding photophysical properties that make them the ICT process to achieve a ratiometric response which enables the
direct and accurate monitoring of intracellular b-gal distribution in
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of
Functional Materials Chemistry, Institute of Fine Chemicals, School of Chemistry
and Molecular Engineering, East China University of Science & Technology,
Shanghai 200237, China. E-mail: guozq@ecust.edu.cn
living cells and animals.
The general approach for the synthesis of b-gal probes
involves a deacetyl step in a strong alkaline solution (by using
NaOMe/MeOH) to attach the b-galactopyranoside group as an
activatable unit.34 However, owing to the poor chemical stabi-
lity of BODIPY dyes in strong alkaline solution, it is extremely
† Electronic supplementary information (ESI) available: Experimental details and
additional figures and table. See DOI: 10.1039/c9cc06869d
‡ These authors contributed equally to this work.
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Scheme 1 Ratiometric tracking of endogenous b-gal activity using an
activatable near-infrared probe BODIPY-bgal.
difficult to synthesize a BODIPY-based b-gal probe. In order to
overcome the hurdles, we innovatively described an alternative
synthetic route (Scheme 2): initially a de-protected key inter-
mediate compound 2 was successfully synthesized, and then
the target product BODIPY-bgal was effectively obtained via a
typical Knoevenagel reaction within the neutral pH range.
To test the validity of our designed probe, the spectral
properties of BODIPY-bgal were investigated with b-gal in an
optimal physiological buffer solution. Upon titration with b-gal
(5 U), the absorption peak sharply decreased at 560 nm, and a
concomitant new absorption peak appeared at 620 nm (Fig. 1a),
which is in perfect accordance with the absorption of BODIPY-
OÀ (Fig. S1, ESI†). In addition, an obvious color change from
pink to dark blue allows the colorimetric detection of b-gal
using the naked eye, which is invoked by a red-shift of
ca. 60 nm in the absorption spectra.
As expected, a clear bathochromic shift in the emission
spectra was observed. Initially, BODIPY-bgal possessed a strong
fluorescence signal at 575 nm (Table S1, ESI†), but a very weak
fluorescence signal at 730 nm. In the presence of b-gal, there
was a significant new enhanced emission peak at 730 nm upon
excitation at 660 nm (Fig. 1c), whereas the initial fluorescence
at 575 nm was decreased (Fig. 1b). These aforementioned observa-
tions demonstrated that upon a specific reaction of BODIPY-bgal
with b-gal, there was a light-up NIR fluorescent signal with a
significant red-shift (ca. 155 nm). Clearly, the distinct large red-
shifted fluorescence response confirms that b-gal controlled hydro-
lysis liberates the oxygen atom of BODIPY-bgal as a strong electron
donor, thereby shifting the emission maximum to the NIR region. In
addition, the ratiometric fluorescent signal (I730nm/I575nm) depends
Fig. 1 Spectral profiles of BODIPY-bgal (10 mM) incubation with b-gal
(5 U) in a mixture solution (phosphate-buffered saline (PBS)/dimethyl
sulfoxide (DMSO) = 1 : 1 v : v, pH = 7.4). Absorption (a) and fluorescence
spectra, lex = 530 nm (b) and lex = 660 nm (c); and (d) time-dependent
fluorescence intensity I730nm and I575nm
.
linearly as a function of the b-gal concentration (from 0 U to 3 U) at a
fixed incubation time with a detection limit of 4.6 Â 10À3 U mLÀ1
(Fig. S2 and S3, ESI†). Fig. 1d depicts the fluorescent ratio signal
(I730nm and I575nm) as a function of time. Dual-channel fluorescent
signal both take around 30 min to reach a plateau. We also evaluate
the sensing performance of BODIPY-bgal in different pH solutions.
Notably, BODIPY-bgal displayed similar dual-channel response in
pH 6.0 (Fig. S4, ESI†), suggestive of its application in an acidic
tumor microenvironment. Thus, these results offer a possibility to
quantitatively and rapidly detect b-gal using the intensity ratios of
I730nm/I575nm
.
To further confirm that the leaving group could be specifi-
cally cleaved by b-gal and accompanied by in situ release of the
fluorophore BODIPY-OH, HPLC and HRMS analyses were systemi-
cally performed. As shown in Fig. S5 (ESI†), the retention time of
free BODIPY-bgal and BODIPY-OH is 4.0 and 6.7 min, respectively.
After reaction with 5U b-gal, BODIPY-bgal exhibited an intense
peak with a retention time of 6.7 min, suggesting that BODIPY-
bgal is a substrate of b-gal and the sensing product is exclusively
BODIPY-OH. Moreover, the cleavage product was further unam-
biguously confirmed by HRMS analysis. In the ensemble system of
BODIPY-bgal and b-gal, the peaks of BODIPY-bgal and its cleavage
product were found to be at m/z 485.18 and 323.16, respectively
(Fig. S6, ESI†). Collectively, all of these results substantiate that the
enzyme-catalyzed reaction in situ produces BODIPY-OH with NIR
fluorescence.
We next investigated the photostability, pH stability and
selectivity of probe BODIPY-bgal. Time-dependent photo-
bleaching measurements show that BODIPY-OH still main-
tained 60% absorbance after sustained irradiation for 10 min,
while the absorbance of ICG (an FDA approved NIR dye) as a
control sharply decreased and reached a minimum level under
the same irradiation conditions (Fig. S7, ESI†). These data
demonstrate that the BODIPY-OH dye is more photostable than
ICG. To gain insight into the influence of the hydroxyl group
Scheme 2 Synthetic route of BODIPY-bgal.
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and the b-galactopyranoside group in the BODIPY fluorophore, D-galactose (an inhibitor of b-gal) for 0.5 h and then treated with
we examined the pH effect on the photophysical properties of BODIPY-bgal for 0.5 h, the NIR fluorescence signal was clearly
BODIPY-OH and BODIPY-bgal. As the pH increased from 1.7 blocked, but a strong fluorescence from the green channel was
to 10.0, the emission peak of BODIPY-OH around 575 nm observed, illustrating that these distinct signal changes were
decreased significantly, while a new red-shifted emission band triggered by intracellular endogenous b-gal. In comparison, upon
was observed around 730 nm (Fig. S8 and S9, ESI†). This large incubation with cancer cells HepG2, a bright fluorescence signal
red-shift (155 nm) in the emission spectra with increased pH can was observed in the green channel, whereas weak NIR fluorescence
be explained by the deprotonation of BODIPY-OH. Interestingly, was observed in the red channel (Fig. 2c), suggesting that the
the dual-channel emission of BODIPY-bgal shows stable photo- intracellular b-gal activity in HepG2 cells is very low. Furthermore,
physical properties in the pH range of 1.7–10.0 (Fig. S9, ESI†). the emission ratio value Fred/Fgreen of BODIPY-bgal in living cells
These results show that the b-galactopyranoside group stabilizes was in accordance with the aforementioned dual-channel cell
the form of BODIPY-bgal but not that of BODIPY-OH. Besides, imaging (Fig. 2d). These results confirmed that BODIPY-bgal could
BODIPY-bgal also displayed excellent stability in fresh human react with endogenous b-gal and emit strong NIR fluorescence,
serum (Fig. S10, ESI†). Furthermore, BODIPY-bgal showed excel- which is consistent with the in vitro spectral experiments.
lent selectivity towards b-gal over other competitive bioanalytes
On the basis of the BODIPY-bgal optical properties and the
because of the b-galactopyranoside unit as a specific enzyme- above intracellular imaging of b-gal, we next examined its
active trigger moiety, suggesting its potential as a bioprobe for capability for real-time in vivo visualization of b-gal activity in
tracking b-gal in living cells (Fig. S11, ESI†).
tumors. Human lung xenograft tumor cells (A549 cells, without
Encouraged by the desirable fluorescence response of overexpressed b-gal) of the mice model were utilized. Living
BODIPY-bgal with b-gal, the probe for ratiometric tracking of mice were pre-injected with b-gal to establish a mouse model
endogenous b-gal activity was evaluated in living cells. Initially, with overexpressed b-gal at the tumor site and then injected
the cytotoxicity of the probes was determined by MTT assay orthotopically with BODIPY-bgal (Fig. 3c and d). On the other
(Fig. S12, ESI†). The results indicated that the cell viability was hand, the nude mice were pre-injected with PBS (and then
still as high as 95% even after incubation with 20 mM BODIPY- injected with BODIPY-bgal) as a control (Fig. 3a and b). Notably,
bgal for 24 h, suggesting low cytotoxicity towards the cells. the control group displays a distinct fluorescence emission at
Based on the excellent biocompatibility, the dual-channel cell the 600 nm channel, while a non-fluorescent signal at the
imaging was investigated by confocal laser scanning microscopy 730 nm channel (Fig. 3a and b). In comparison, for the pre-
(CLSM). Herein, human ovarian cancer cells SKOV-3 were chosen treated-b-gal tumor-bearing mouse, after merely 5 min post-
as cell models because of their high levels of endogenous b-gal, injection, the fluorescence signal at 730 nm was already clearly
whereas human hepatic cancer cells HepG2 as a contrast.
appreciated in tumor cells, indicative of the rapid activation of
After incubating SKOV-3 with BODIPY-bgal (10 mM) for 30 min, BODIPY-bgal with b-gal in situ. The NIR signal reached a
we observed weak fluorescence in the green channel (570–620 nm)
while significant NIR fluorescence in the red channel (670–800 nm)
(Fig. 2a), indicating that b-gal is overexpressed in the cancer cells
SKOV-3. To verify that the obtained fluorescence signal is indeed
derived from endogenous b-gal activity, an inhibitor assay was
performed (Fig. 2b). When SKOV-3 cells were exposed to 1 mM
Fig. 2 CLSM images of SKOV-3 and HepG2 cells incubated with BODIPY-
bgal (10 mM) for 0.5 h: (a) SKOV-3 cells and (b) SKOV-3 cells pretreated with
1 mM inhibitor for 0.5 h and (c) HepG2 cells. Note: the green channel
obtained from 570 to 620 nm, lex = 560 nm; the red channel obtained
from 670 to 800 nm, lex = 660 nm; ratiometric images generated from
the red and green channel. (d) The emission ratio value Fred/Fgreen of
BODIPY-bgal with b-gal in living cells in the absence and presence of the
inhibitor. Error bars represent the standard deviation (S.D.) with n = 3.
Fig. 3 Tumor-bearing mice were pre-treated with PBS (a, b, e and f) or
b-gal at the tumor site (c, d, g and h) and then injected orthotopically with
BODIPY-bgal (0.048 mg kgÀ1). (a–d) In vivo dual-channel fluorescence
imaging of the b-gal activity in xenograft tumor bearing mice at various
times (5 min, 1 h and 3 h) after tumor injection. (e and f) Ex vivo
fluorescence imaging of the excised organs (tumor, heart, liver, spleen,
lung and kidney) at 3 h tumor injection of BODIPY-bgal. Note: fluores-
cence signals at 600 nm (rainbow scale) and 730 nm (yellow-red scale).
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almost disappeared. To further confirm that the dual-channel
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b-gal in the tumor cells, we also recorded the ex vivo fluores-
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(Fig. 3e–h). These results confirm that the dual-channel NIR
probe BODIPY-bgal has the capability to selectively detect and
image b-gal in vivo.
In summary, we have developed a ratiometric and light-up NIR
probe BODIPY-bgal, which is composed of monostyryl-substituted
BODIPY-OH as a wavelength controllable fluorescent reporter with
effective ICT properties, and a b-gal cleavable unit as an enzyme-
specific trigger. There was a significant new enhanced emission
peak at 730 nm, whereas the initial fluorescence at 575 nm of
BODIPY-bgal was decreased in the presence of b-gal. Importantly,
this probe was successfully applied for real-time trapping of
intracellular endogenous b-gal distribution in overexpressed living
SKOV-3 cells, as well as in vivo visualization of b-gal activity in a
mice model. In light of its simplicity, sensitivity, and resistance to
photobleaching, this enzyme-activatable ratiometric and light-up
NIR fluorescent probe provides an accessible tool for in vivo targeted
visualization and a therapeutic evaluation approach.
This work was supported by the NSFC/China (21622602,
21788102, 21421004, 21636002, 21878087 and 21908060), the
National Key Research and Development Program (2017YFC-
0906902), the Innovation Program of Shanghai Municipal
Education Commission, the Scientific Committee of Shanghai
(15XD1501400), the Shuguang Program (18SG27) and the China
Postdoctoral Science Foundation (Grant 2019M651417). This
study was performed in strict accordance with the NIH guide-
lines for the care and use of laboratory animals and was
approved by the Institutional Animal Care and Use Committee
of the National Tissue Engineering Center (Shanghai, China).
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There are no conflicts to declare.
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