Precise imaging of mitochondria in cancer cells by real-time monitoring of nitroreductase activity with a targetable and activatable fluorescent probe

Article abstract of DOI:10.1039/d0cc00494d

An activatable and mitochondrial-targetable fluorescent probe was developed. This designed probe showed ratiometric fluorescence and light-up near-infrared emission responsiveness to nitroreductase, achieving precise imaging of mitochondria in cancer cells by real-time monitoring of nitroreductase activity.

Full text of DOI:10.1039/d0cc00494d

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DOI: 10.1039/D0CC00494D
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Precise imaging of mitochondria in cancer cells by real-time
monitoring nitroreductase activity with a targetable and
activatable fluorescent probe
Received 00th January 20xx,
Accepted 00th January 20xx
Ning Zhu,a,§ Ge Xu,a,§ Rongchen Wang, Tianli Zhu, Jiahui Tan, Xianfeng Gu,* Chunchang Zhao *
a
a
b
b
a
DOI: 10.1039/x0xx00000x
An activatable and mitochondrial-targetable fluorescent probe was conventional nonresponsive probes, activatable fluorescent
developed. This designed probe showed ratiometric fluorescence probes can switch on fluorescence that is distinguished from
and light-up near-infrared emission responsiveness to the original state only after specific activation by biological
9
nitroreductase, achieving precise imaging of mitochondria in targets. Therefore, activatable fluorescent probes usually have
cancer cells by real-time monitoring nitroreductase activity.
high selectivity and low background interference, which is
1
0
suitable for accurate detection of biological targets in vivo. In
this regard, we attended to design an activatable fluorescent
probe with a mitochondrial-targeted group, which can be
selectively activated by enzymes overexpressed in the cancer
cells, aiming at imaging of mitochondria in cancer cells with high
precision.
Mitochondrion, well-known energizing organelle in
a
mammalian cells, provides the energy required for cell
1
operation by generating ATP. Mitochondria are widely involved
in various cell processes, including signal transduction, energy
2
metabolism, autophagy and apoptosis. Accordingly, these
cellular organelles are of vital importance in maintaining the
Herein, we developed a mitochondria-targetable fluorescent
probe (BOD-TPP) that showed selective response to
nitroreductase. Such a probe was designed by linking p-
nitrobenzyl thioether and triphenylphosphine to the parent
3
normal physiological functions of organisms. Meanwhile,
mitochondrial function damage is also closely related to the
occurrence of various diseases such as cancer, cardiovascular
4
disease and dementia. Therefore, it’s of physiological and
11
boron dipyrromethene (BODIPY) platform (Scheme 1).
biological scientific interests for accurate imaging of
mitochondria and real-time monitoring of substances closely
related to mitochondrial function in cells. Until now,
Triphenylphosphine, a lipophilic cation, could specifically target
to the mitochondria. P-nitrobenzyl thioether could be reduced
12
5
to p-aminobenzyl thioether by nitroreductase (NTR), followed
by a 1,6-elimination reaction to release the required
fluorophore, showing advantages of dual-channel ratiometric
fluorescence and light-up near-infrared emission. As is well-
known, nitroreductase, a type of flavin-containing enzymes
overexpressed in cancer cells can be considered as a biomarker
fluorescence imaging has attracted wide attention to
mitochondrial imaging due to its advantages such as high
sensitivity, high spatio-temporal resolution and simple
6
operation. However, most of the available mitochondrial-
targeted fluorescent probes are always-on, endowing poor
specificity and thus incapable of specific imaging of
13
for cancers. Accordingly, this designed probe enabled highly
7
mitochondria in cancer cells. Therefore, the accurate imaging
accurate imaging of mitochondria in cancer cells by real-time
monitoring nitroreductase activity.
of mitochondrial in cancer cells remains a big challenge.
Activatable probes have been extensively explored in recent
8
years for targeting cancer cells imaging. Compared with
a. Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint
Research Center, Institute of Fine Chemicals, School of Chemistry and Molecular
Engineering, East China University of Science and Technology, Shanghai, 200237,
P. R. China. Email Address: zhaocchang@ecust.edu.cn
b.
Department of Medicinal Chemistry, School of Pharmacy, Fudan University,
Shanghai, 201203, P. R. China. Email Address: xfgu@fudan.edu.cn
These authors contributed equally.
Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
§
Scheme 1 Schematic illustration of BOD-TPP and the
mechanism for nitroreductase (NTR)-activated ratiometric and
NIR fluorescence light up.
†
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BOD-TPP was readily synthesized and purified by column
chromatography (Scheme 2). The detailed synthetic steps were
described in Scheme S1 (ESI†). Next the response of BOD-TPP
toward nitroreductase was evaluated under model conditions
DOI: 10.1039/D0CC00494D
(DMSO/Tris-HCl buffer, 1/3, v/v, 0.05 M, pH 7.4, 37 °C) by
monitoring changes in the absorption and emission spectra.
Here, dimethyl sulfoxide was selected as a cosolvent to improve
the aqueous solubility of the probe. As expected, BOD-TPP
showed obvious responsiveness to nitroreductase. In the
absence of nitroreductase, BOD-TPP exhibited an intense
absorption at 495 nm and an emission band at 560 nm
(fluorescence quantum yield  of 17.8%). Upon addition of
NADH (500 μM) and nitroreductase (10 μg/mL), a new
absorption band appeared at 650 nm, and the original 495 nm
absorption band decreased concomitantly (Fig. 1a), showing a
big red-shift of 155 nm. Interestingly, the fluorescence titration
experiments showed that BOD-TPP can measure nitroreductase
in both dual-channel ratiometric and turn-on near-infrared
fluorescence modes. The presence of nitroreductase led to the
quenching of the fluorescence at 560 nm upon excitation at 495
nm, while a new near-infrared emission at 713 nm was
activated when the excitation wavelength was 650 nm (Fig. 1c).
These changes were ascribed to the successful conversion of
BOD-TPP to BOD-TPP-S ( of 5.3%) by nitroreductase that could
trigger a cascade reactions including the formation of the p-
aminobenzyl thioether linker and a subsequent 1,6-elimination
reaction to release the required fluorophore. This mechanism
could be confirmed by High-resolution mass spectrometry
Fig. 1 Time-dependent spectral changes of BOD-TPP (10 μM) in
the presence of nitroreductase (NTR) (10 μg/mL). (a) Absorption,
(
b) fluorescence quenching (λex = 495 nm) and (c) fluorescence
turn-on (λex = 650 nm) in buffer (DMSO/Tris-HCl buffer, 1/3, v/v,
.05 M, pH 7.4) at 37 °C. (d) Time-dependent fluorescence
0
intensity of I560nm and I713nm
.
BOD-TPP showed highly selective response to nitroreductase.
As shown in Figure 2a and Figure S5 (ESI†), interfering species
including other enzymes and thiols caused negligible
fluorescence changes, whereas the introduction of NADH and
nitroreductase caused a significant increase in fluorescence
intensity at 713 nm. It was worth noting that negligible
fluorescence enhancement was found upon pretreatment of
nitroreductase with an inhibitor dicoumarol (Fig. 2b),
suggesting that nitroreductase played a key role in activation of
BOD-TPP. This further illustrated that BOD-TPP unambiguously
exhibited high specificity for nitroreductase activity. In addition,
the probe exhibited good optical responsiveness to enzyme in
the physiological pH range (pH 8.5 to approximately 6; Fig. S6,
ESI†).
(HRMS) analysis (Fig. S1, ESI†). As shown in Fig. S1, HRMS
spectrum of BOD-TPP solution in the presence of NTR gave
+
peaks of 739.2641 (corresponding to [BOD-TPP-S - Br] ) and
+
8
74.2972 (corresponding to [BOD-TPP - Br] ). In the buffer
solution at pH 7.4, the kinetics values of BOD-TPP against
nitroreductase showed that Michaelis-Menten constant (K
m
)
-
1
was 33.70 μM and Vmax was 46.21 μM · min (Fig. S3, ESI†).
Importantly, a linear relationship between the fluorescence
intensity and nitroreductase concentration was noted in the
measured range of 0 μg/mL to 10 μg/mL, affording the
detection limit of 0.0168 μg/mL (Fig. S4, ESI†).
H
SH
N
NC
PPh3
Br
P+
Br
O2N
DMAP, CH3CN
O
NH CN
O
OHC
N
N
B
EtOH
OHC
S
F
F
N
N
B
F
N
N
B
N
H
S
F
Cl
F
F
O2N
O2N
BOD-TPP
Fig. 2 (a) The fluorescence intensity at 713 nm of BOD-TPP (10
μM) in the presence of nitroreductase (NTR) and other
biologically related analytes were incubated in buffer
Scheme 2 Synthetic route of BOD-TPP.
(DMSO/Tris-HCl buffer, 1/3, v/v, 0.05 M, pH 7.4) at 37 °C for 120
min: (0) Free; (1) aprotinin (30 μg/mL); (2) phosphatase (30
μg/mL); (3) collagenase (30 μg/mL); (4) lipase (30 μg/mL); (5)
BSA (10 mg/mL); (6) NQO1 (30 μg/mL); (7) NADH (500 μM); (8)
GSH (1 mM); (9) Cys (1 mM); (10) Hcy (1 mM ); (11) NADH (500
μM) + NTR (10 μg/mL). (b) NTR-induced the fluorescence
enhancement of BOD-TPP in the absence or presence of
inhibitor dicoumarol (0.2 mM).
2
| J. Name., 2012, 00, 1-3
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confirmed that the probe we designed could bVeiewspAreticcliefiOcnallinlye
The low cytotoxicity of BOD-TPP to living cells was localized in the mitochondria of living AD5O4I9: 1c0e.1l0ls3.9N/Do0tCaCb0ly0,49th4De
demonstrated through CCK-8 experiment (Fig. S7, ESI†). Then common nitroreductase probes are known to imaging
we testified its ability to selectively identify cancer cells by nitroreductase activity in hypoxic tumor cells, while few probe
tracking enzyme activity in real time. In order to testify the show selective monitoring of nitroreductase under normoxic
1
3
feasibility of visualization of cancer cells based on conditions. The selective responsiveness of this probe to
nitroreductase activity, imaging experiments were performed nitroreductase in normoxic tumor cells might enable the
1
3a
on A549 cells (high nitroreductase activity). As shown in Fig. accurate imaging of the mitochondria in cancer cells.
a-3c, the fluorescence ratio (Ired/Igreen) of BOD-TPP-stained
3
A549 cells showed a time-dependent enhancement. Obviously,
the fluorescence signals in the green channel was continuously
attenuated as prolonging the incubation time, while the signals
in red channel progressively increased, resulting in the change
of Ired/Igreen from 0.08 to 5.5. On the contrary, pretreatment of
the cells with the enzyme inhibitor dicoumarol significantly
lowered the ratio of Ired/Igreen due to the suppression of the
nitroreductase activity (Fig. 3d), which further indicated that
fluorescence imaging signals were indeed initiated by
nitroreductase activity.
Fig. 4 Fluorescence emission from BOD-TPP colocalized to
mitochondria in live A549 cells. (a) A549 cells were incubated
with Mito-Tracker Green (50 nM) for 30 min. (b) A549 cells were
incubated with BOD-TPP (10 μM) for 60 min. (c) Bright field. (d)
’
Overlay of images a−c. (e) Pearson s colocalization coefficient. (f)
The intensity profile showed the fluorescence intensity along
the yellow line across the cell. Scale bar = 20 μm.
In summary, we have developed a novel activatable BODIPY-
based NIR fluorescent probe for monitoring of nitroreductase
activity and imaging of mitochondria in cancer cells. This
activatable probe was designed by appending p-nitrobenzyl
thioether and triphenylphosphine function to the parent
BODIPY platform. Nitroreductase could trigger the formation of
the p-aminobenzyl thioether linker that was capable of
undergoing a 1,6-elimination reaction to release a NIR
fluorophore, enabling selective detection of nitroreductase. In
light of the mitochondria targetable feature, ratiometric
fluorescence and NIR emission light-up responsiveness to
nitroreductase, our probe achieved high-precision imaging of
mitochondria in cancer cells by real-time monitoring
nitroreductase activity. We expect that our design approach
could have more applications in nitroreductase-based biology
or mitochondrial biology.
Fig. 3 Visualization of A549 cells by confocal microscopy imaging.
A549 cells incubated with BOD-TPP (10 μM) for (a) 15 min; (b)
3
0 min and (c) 60 min. (d) A549 cells pretreated with dicoumarol
(0.5 mM) for 60 min, followed by treatment with BOD-TPP (10
μM) for 60 min. Green channel at 525–600 nm with λex = 488
nm excitation, red channel at 675–750 nm using λex = 561 nm;
ratio image was Ired /Igreen. Scale bar = 10 μm.
We gratefully acknowledge the financial support by the
National Natural Science Foundation of China (21874043,
To further investigate the subcellular localization of the
probe, a widely used commercial mitochondrial targeting dye
Mito-Tracker Green was used for colocalization study. As shown
in Fig. 4, the results clearly showed the red signal was generated
by the probe, and the green channel illustrated visualization of
mitochondria by Mito-Tracker Green. Superposition of panels a-
c showed complete overlap of the red and green signals.
2
1672062, 21977018), the Shanghai Municipal Science and
Technology Major Project (Grant No. 2018SHZDZX03) and the
Programme of Introducing Talents of Discipline to Universities
(B16017).
Conflicts of interest
There are no conflicts to declare.
’
Pearson s colocalization coefficient, which used to describe the
correlation of the intensity distribution between the two
channels, was caculated to be 0.96 (Fig. 4). All these results
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Gu, G. Xu, R. Wang, T. Zhu, Y. Zhu, C. Fan, C. ZhaVoieawnAdrtiHcle. OTinalinne,
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TOC
A probe with the mitochondria targetable feature and ratiometric fluorescence as well as NIR emission light-up responsiveness to
nitroreductase is presented for precise imaging of mitochondria in cancer cells by real-time monitoring nitroreductase activity.
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