A novel off-on fluorescent probe for sensitive imaging of mitochondria-specific nitroreductase activity in living tumor cells

Article abstract of DOI:10.1039/c7ob00781g

Sensitive and selective detection and imaging of nitroreductase (NTR) in cancer cells is of great importance for better understanding their biological functions. Since there are a few fluorescent probes concerning NTR activity specifically located in mito

Full text of DOI:10.1039/c7ob00781g

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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PAPER
A novel off-on fluorescent probe for sensitive imaging of
mitochondria-specific nitroreductase activity in living tumor
cells
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
＊
＊
Bo Huang, Wen Chen, Yong-Qing Kuang, Wei Liu, Xian-Jun Liu, Li-Juan Tang , Jian-Hui Jiang
www.rsc.org/
Sensitive and selective detection and imaging of nitroreductase (NTR) in cancer cells is of great importance for better
understanding their biological functions. Since there are few fluorescent probes concerning NTR activity specifically
located in mitochondria, we developed a novel fluorescent benzoindocyanine probe (BICP) for mitochondrial NTR activity
monitoring and imaging via extending benzoindole moiety into a benzoindocyanine based fluorophore (BICF) with strong
intramolecular charge transfer (ICT) effect and incorporating 4-nitrobenzyl as fluorescence-quenching and enzyme-
responsive moiety. Live cell imaging of HeLa and A549 demonstrates the developed BICP is able to realize sensitive and
selective mitochondrial NTR activity probing with high-contrast “off-on” fluorescence. These findings implied the great
potential of the develop probe for monitoring mitochondrial-speific NTR activities in living cells and related applications in
cell biology.
4
an amino group. There have been several successful designs
Introduction
of fluorescent probes by connecting aromatic nitro groups to
different fluorophores such as naphthalimide, resorufin,
Fluorescent probes allowing sensitive and high-contrast
imaging of molecules aberrant expressed in tumor tissues
represent crucial tools for molecular biology and theranostics
of cancer. Nitroreductase (NTR), a family of flavin-containing
enzymes which reduce nitro-containing compounds in the
presence of reduced nicotinamide adeninedinucleotide (NADH)
chemiluminescence, 3-hydroxyflavone and cyanine derivatives.
5
-9
Despite the fluorescent quenching property of the nitro
group, many of these probes still suffer the limitation of high
fluorescence background and limited fluorescence
enhancement ratio. Moreover, most of current probes can
only be used for detection and imaging of NTR in cytoplasm.
Because mitochondria are the main source of nitroradicals in
mammalian cells, the ability to monitor mitochondria-specific
NTR should be essential for elucidating the functions and
dynamics of NTR. Nevertheless, only one probe suitable for
imaging mitochondria-specific NTR activity has come into
1
as an electrondonor, has been consistently observed to be
abnormally up-regulated in cancer cells, especially is
2
accompanied with the hypoxia condition.
Moreover,
nitroreductase have also been reported to play a role in the
activation of nitro-compounds based prodrugs for cancer
3
therapeutics. Therefore, sensitive and selective detection and
1
0
reality very recently.
This probe shows that oxygen-
imaging of nitroreductase in cancer cells is of great importance,
either for better understanding their biological functions or for
better probes for cancer theranostics.
independent type I NTR is specifically localized within
mitochondria. However, there has no evidences from
fluorescent probes concerning whether there is oxygen-
dependent type II NTR in the mitochondria.
Recent years have witnessed significant progress in NTR-
responsive fluorescence probes. These probes are generally
based on the enzyme-catalyzed reaction of nitro group
substituted aromatic substrates, which can be reduced first to
a nitroso group, then a hydroxylamine group, and ultimately to
To elucidate the expression of type I or type II NTR in
mitochondria and probe mitochondria-specific NTR activity, we
develop a novel fluorescent probe of benzoindocyanine probe
(
BICP), which is able to target mitochondria and realize sensitive
and selective detection of NTR. The design of BICP is based on our
considerations that the benzoindole cation can act as
a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry
and Chemical Engineering, College of Biology, Hunan University, Changsha, Hunan
mitochondria-targeting moiety and an electron-withdrawing group.
The positively charged, hydrophobic property of the benzoindole
cation is supposed to mediate localization of the probe inside the
mitochondrial matrix driven by the negative potential gradient
410082, P. R. China. E-mail: jianhuijiang@hnu.edu.cn; tanglijuan@hnu.edu.cn; Fax:
+86-731-88821916.
†Electronic Supplementary Informaꢀon (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
1
1
across the membrane. The strong electron-withdrawing property
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experiments. All these enzyme solutions were frozen immediately
1
2
at -80 °C for storage and allowed to thaw before use.
1
13
The compounds were characterized by H-NMR, C-NMR and
Mass spectrometry (MS). NMR spectra were obtained on a Bruker
1
Avance-III 400 instrument (400 MHz for H-NMR and 100 MHz for
1
3
C-NMR spectroscopy), tetramethylsilane (TMS) as internal
standard (0 ppm) substances, and DMSO-d as solvent. MS were
6
recorded on a LTQ Orbitrap Velos Rro mass spectrometer (Thermo
Fisher Scientific, Bremen, Germany). The UV-vis absorption spectra
were measured using a Shimadzu UV-2450 spectrophotometer. The
fluorescence spectra were recorded at room temperature in a
quartz cuvette on a FluoroMax-4 spectrofluorometer (HORIBA Jobin
Yvon, Inc., NJ, USA). Fluorescence images of HeLa cells and A549
cells were obtained using an OLYMPUSFV-1000 MPE inverted
fluorescence microscope.
Synthetic procedures
The BICP was efficiently synthesized following the synthetic
Scheme 1. (a) The design strategy of BICP; (b) The response mechanism of BICP to methodology shown in Scheme S1.
NTR.
Synthesis of Compound 1. 1,1,2-trimethyl-1H-benz[e]indole
1.31 g, 6.23 mmol, 1 equiv.) was dissolved in 5 mL toluene.
(
has the potential of using the mechanism of intramolecular charge
transfer (ICT) in activating the fluorophore. Motivated by the
hypothesis, we extend the benzoindole moiety into
benzoindocyanine based fluorophore (BICF) with strong ICT effect
and incorporate 4-nitrobenzyl as fluorescence-quenching and
enzyme-responsive moiety, as illustrated in Scheme 1a. The 4- solid without further purification (2.05 g, 93.6% yield). H-NMR (400
Iodomethane (1.33 g, 9.34 mmol, 1.5 equiv.) was added to the
solution, and the mixture was heated at 110 °C for 16 h under an
argon atmosphere. Then, the precipitate was collected by filtration,
a
and washed with diethylether to afford the Compound 1 as a gray
1
nitrobenzyl in the BICP also has very strong electron-withdrawing MHz, DMSO-d ) δ (ppm) 8.38 (d, J = 8.4 Hz, 1H ), 8.31 (d, J = 8.8 Hz,
6
ability, which interferes with the ICT effect in the BICF and thus 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 9.2 Hz, 1H), 7.80 (t, J = 7.2
largely quenches its fluorescence. According to this design, the BICP
merely delivers a low fluorescence background. Upon incubating
with cells, the probe is spontaneously localized in the mitochondria.
In the presence of NADH, the mitochondria-localized NTR, either
type I or type II, can catalyze removal of the 4-nitrobenzyl moiety
from the probe, resulting in a rearrangement-elimination reaction
that recovers the BICF fluorophore and activates strong
fluorescence signal, as depicted in Scheme 1b . Because of strong
fluorescence quenching efficiency of the 4-nitrobenzyl group, the
Hz, 1H), 7.73 (t, J = 7.6 Hz, 1H), 4.11 (s, 3H), 2.89 (s, 3H), 1.77 (s, 6H).
13
6
C-NMR (100 MHz, DMSO-d ) δ (ppm) 196.40, 139.96, 136.99,
1
3
33.51, 131.00, 130.23, 128.87, 127.61, 123.90, 113.65, 55.74,
-
5.67, 21.78, 14.58; MS (EI): calcd for C16
).
H18IN [M-I] m/z 224.3,
found 224.2. (Fig. S1-S3, ESI
†
Synthesis of BICF. To a mixture of Compound 1 (1.76 g, 5 mmol,
equiv.) and p-hydroxybenzaldehyde (0.73 g, 6 mmol, 1.2 equiv.) in
0 mL ethanol was added 2-3 drops of piperidine. Then the reaction
1
3
developed BCIP may provide a high-contrast “off-on” fluorescent solution was heated and reflux for 18 h under an argon
probe for sensitive and selective imaging of mitochondria-specific atmosphere. After cooled down to room temperature, the solvent
NTR activity in living cells.
was removed under reduced pressure, and the residue was
purification by flash column chromatography (silica gel,
CH
8% yield). H-NMR (400 MHz, DMSO-d
8.47-8.40 (m, 2H), 8.28 (d, J = 8.8 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H),
2 2 3
Cl :CH OH = 20:1, v/v) to get BICF as a black red solid (1.76 g,
Experimental section
1
7
6
) δ (ppm) 10.81 (s, 1H),
Reagents, materials and Instrumentations
8
1
.15 (d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.0 Hz, 1H), 7.80 (t, J = 9.2 Hz,
H), 7.71 (t, J = 8.0 Hz, 1H), 7.51 (d, J = 16.0 Hz, 1H), 6.98 (d, J = 8.0
All chemicals were obtained from commercial suppliers and used
without further purification, unless specifically stated otherwise.
The silica gel (200-300 mesh) was used for the flash
chromatography and thin layer chromatography (TLC) was carried
out with silica gel 60 F254. Both of them were obtained from
Qingdao Ocean Chemicals. Nitroreductase (NTR, ≥100 units) from
Escherichia coli, and NADH was purchased from TCI Co. Ltd. HeLa
cells and A549 cells were obtained from the cell bank of Central
Laboratory at Xiangya Hospital (Changsha, China). Ultrapure water
was obtained from a Milli-Q reference system (Billerica, MA, USA)
and had an electric resistance >18.25 MΩ. The lyophilized powder
of NTR was dissolved in ultrapure water and the aqueous solution
was divided into several parts as suitable amounts for daily
1
3
Hz, 2H), 4.22 (s, 3H), 2.00 (s, 6H). C-NMR (100 MHz, DMSO-d
6
) δ
(
ppm) 182.68, 163.54, 153.11, 139.95, 137.88, 133.92, 133.45,
1
1
31.22, 130.50, 128.83, 127.34, 127.18, 126.52, 123.51, 116.91,
13.66, 109.43, 53.85, 35.18, 25.92; HRMS (ESI): calcd for C23
H
22INO
-
[
M-I] m/z 328.1696, found 328.1692. (Fig. S4-S6, ESI
Synthesis of BICP. BICF (1.35 g, 3 mmol, 1 equiv.) and K
0.62 g, 4.5 mmol, 1.5 equiv.) were dissolved in N, N-
†).
2
CO
3
(
dimethylformamide (15 mL) at 45 °C and stirred for 15 min, 4-
nitrobenzylbromide (0.65 g, 3 mmol, 1 equiv.) in 3 mL N, N-
dimethylformamide was added dropwise into the reaction mixture.
It was stirred at 45 °C for 4 h and then diluted with 20 mL
dichloromethane. The organic layer was separated, washed with
2
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-1
-1
water (50 mL × 3) and brine (50 mL × 3), and then dried over dry (10 μL well , 0.5 mg mL ), followed by further incubation for 4 h at
Na SO . The solvent was removed by evaporation and the residue 37 °C. Then the supernatant was abandoned, and 100 μL DMSO was
was purification by flash column chromatography (silica gel, added to each well to dissolve the formazan crystals. After shaking
CH Cl :CH OH = 20:1, v/v) to get BICP as an orange solid (0.96 g, the plates for 10 min, absorbance values of the wells were recorded
3% yield). H-NMR (400 MHz, DMSO-d6) δ (ppm) 8.50 (d, J = 16.4 with a microplate reader at 490 nm. The cell viability rate (VR) were
Hz, 1H), 8.43 (d, J = 8.8 Hz, 1H), 8.29 (t, J = 8.0 Hz, 5H), 8.23 (d, J = assessed by following equation: VR = A/A × 100%, where A and A
.4 Hz, 1H), 8.11 (d, J = 8.8 Hz, 1H), 7.84-7.78 (m, 3H), 7.73 (t, J = 7.2 are the absorbance of the experimental group (with BICP) and the
Hz, 1H), 7.63 (d, J = 16.4 Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 5.48 (s, 2H), control group (without BICP). The cell survival rate from the control
2
4
2
2
3
1
6
0
0
8
1
3
4
1
1
1
6
.27 (s, 3H), 2.02 (s, 6H). C-NMR (100 MHz, DMSO-d ) δ (ppm) group was considered as 100%.
82.83, 162.65, 152.29, 147.62, 144.73, 139.94, 138.23, 133.57,
33.37, 131.28, 130.50, 128.93, 128.87, 128.41, 127.51, 127.14, Results and discussion
24.16, 123.60, 116.22, 113.74, 110.92; HRMS (ESI): calcd for
Response of BICP to NTR
-
C
30
H
27IN
2
O
3
[M-I] m/z 463.2016, found 463.2016. (Fig. S7-S9, ESI
†
)
1
After confirming the synthetic intermediates and BICP by H NMR,
Spectrophotometric measurements
13
C NMR, and mass spectrometry (MS), we first investigated the
Both the fluorescence and UV-vis absorption measurements were absorption and fluorescence emission spectra of BICP in the
carried out in 10 mM PBS buffer solution (pH 7.4) containing 5% absence and presence of NTR. BICP displayed slight absorption in
DMSO. A volume of 10 µL BICP stock solution (0.2 mM), 10 µL NADH the long-wavelength region (Fig. S10, ESI
solution (10 mM), appropriate volume NTR sample solution, and NTR, its solution changed into rose red from chalky yellow (inset of
00 µL PBS buffer solution were added into a 200 µL tube. The final Fig. S10) with a new absorption peak at 523 nm which could be
†). Upon the addition of
1
solution volume was adjusted to 200 µL with PBS buffer, and the assigned to the characteristic absorption of BICF. This result
reaction solution was mixed up rapidly. The mixture solutions were demonstrated that BICP can serve as “naked-eye” probe for visual
incubated at 37 °C for 30 min in a thermostat and then the detection of NTR. We subsequently examined the fluorescence
fluorescence intensities were recorded in the range from 540 to 700 response of BICP to NTR (Fig. 1a). BICP (10 μM) merely displayed a
nm with λex = 523 nm from a Xe lamp, and both excitation and weak fluorescence signal, evidencing efficient fluorescence
emission slits of 5 nm.
quenching of BICF owing to the electron-withdrawing group of 4-
-1
nitrobenzyl. When 5 μg mL NTR added in the presence of 500 μM
NADH, the fluorescence intensity showed remarkable
Cell culture
a
HeLa cells and A549 cells were grown in RPMI-1640 medium
enhancement as high as ~83-fold. Such a high signal-to-background
(Thermo Scientific HyClone) supplemented with 10% fetal bovine
ratio indicated the BICP afforded high contrast for fluorescence
-
1
-1
serum (Invitrogen), 100
U
mL penicillin and 100
U
mL
2a, 9b, 9d, 9f, 14
imaging.
In the absence of 500 μM NADH, there was
streptomycin at 37 °C in a humidified atmosphere incubator
containing 5% wt/vol CO under normoxic condition (20% O ). The
little change in the signal, implying the NADH dependency of NTR.
15
2
2
In the presence of 0.1 mM NTR inhibitor dicoumarin,
the
cells were plated on a 35 mm Petri dish with 10 mm bottom well in
RPMI-1640 medium for 24 h, and then the dishes were washed with
PBS buffer (2 mL × 3 times).
fluorescence intensity became much lower than that obtained after
NTR reaction. A higher concentration of dicoumarin (0.2 mM)
resulted in a further decrease in the fluorescence intensity. These
Confocal fluorescence imaging for living cells
The cells were treated with 10 µM BICP in 1 mL RPMI-1640 medium
for 30 min at 37 °C firstly and then washed with PBS buffer (2 mL ×
3
2
times), then cultured under normoxic (20% O ) and different
hypoxic status (15%, 10%, 5%, 1%) for 4 h. All fluorescence images
were acquired using a confocal laser scanning fluorescence
microscope setup consisting of an Olympus IX81 inverted
microscope with an Olympus FV1000-MPE confocal scanning
system.
Fig. 1. (a) Fluorescence spectral responses of BICP. BICP (black); BICP plus NADH
Cytotoxicity assay
without NTR (red); BICP plus NTR without NADH (blue); BICP plus NADH and NTR
Cell viability was used as an index to assess the cytotoxicity of
fluorescence dyes for cells. The cytotoxicity of BICP to HeLa cells
(
green); BICP plus NADH, NTR and 0.1 mM dicoumarin (pink); BICP plus NADH,
NTR and 0.2 mM dicoumarin (purple). (b) Fluorescence responses of BICP to
various species: (1) control, (2) glutathione (GSH, 5 mM), (3) cysteine (Cys, 1
mM), (4) homocysteine (Hcy, 1mM), (5) NaClO (10 μM), (6) dithiothreitol (DTT, 1
1
3
were evaluated following an approach reported previously.
In
4
brief, HeLa cells (1 × 10 cells per well) were seeded into 96-well U-
bottom plates in 100 μL RPMI-1640 medium and incubated in 5%
mM), (7) KCl (50 mM), (8) NaCl (10 mM), (9) MgCl
11) NaHS (10 μM), (12) glycine (Gly, 1 mM), (13) tryptophan (Try, 1 mM), (14)
vitamin C (1 mM), (15) Na SO (10 mM), (16) H (1 mM), (17) tyrosine (Tyr, 1
2 2
(2.5 mM), (10) CaCl (2.5 mM),
2
CO at 37 °C for 24 h. Then, the cells were treated with BICP at
(
varied concentrations (0-30 μM) for additional 24 h, respectively.
After that, the culture media was discarded, and 100 μL 3-(4,5-
2
3
2 2
O
mM), (18) glucose (10 mM), (19) arginine (Arg, 1 mM), and (20) NTR (5 μg/mL).
Fluorescence response of probe BICP (10 μM) toward various species in 10 mM
PBS (pH 7.4, containing 5% DMSO) at 37 °C for 30 min. λex/em = 523 nm/570 nm.
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT)
solution (0.5 mg/mL, RPMI-1640 medium) was added to each well
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broad range of pH values or temperatures and the optimal activity
was achieved at physiological conditions of pH 7.4 and 37 °C,
suggesting the utility of BICP for live cell imaging applications (Fig.
S16 and Fig. S17, ESI†).
The potential cytotoxicity of BICP was assessed by MTT assays
with HeLa cells. The results showed that the cell viability was not
significantly changed upon treatment. The cell viability decreased
by ~7% after 24 h incubation at a concentration up to 30 μM BICP
(
Fig. S18, ESI†), indicating low cytotoxicity and good
Fig. 2. (a) Fluorescence spectral responses of BICP to NTR of varying biocompatibility of this probe.
concentrations. (b) Calibration curve of BICP to NTR, Inset: the linear correlation
Fluorescence imaging of NTR in living cells
between emission intensities and NTR concentrations. The reaction was
conducted in the presence of 500 μM NADH in 10 mM PBS (pH 7.4, containing 5%
DMSO) at 37 °C for 30 min. λex/em = 523 nm/570 nm.
We investigated the applicability of BICP to imaging NTR activities in
living cells. Confocal fluorescence microscopy was used to visualize
HeLa cells and A549 cells, which are known to increase expression
2
a, 2b, 6c, 8b,9d, 9f, 14a-d, 16
of NTR under hypoxic conditions.
observed that HeLa cells, after incubated under the hypoxic
condition of 1% O for 4 h, did not display appreciable fluorescence
Fig. S19, ESI ). In contrast, when HeLa cells incubated with 10 μM
BICP under the hypoxic condition, a bright fluorescence image was
observed in the cells (Fig. S19, ESI ), which suggested fluorescence
It was
results revealed that the fluorescent response of BICP was specific
to the activity of NTR. Furthermore, we found that other
biologically related species induced no obvious colour changes (Fig.
2
(
†
S11, ESI
b), suggesting the specificity for BICP in response to NTR.
We further evaluated the performance of the BICP for
quantitative detection of NTR. With increasing concentrations of
†) and little enhancement of fluorescence intensity (Fig.
1
†
activation for the BICP in the NTR-overexpressed cells. The
fluorescence from HeLa cells pretreated with 0.1 mM dicoumarin,
an known inhibitor for NTR, under the hypoxic condition, became
much weaker, indicating that the fluorescence signal was specific
for the activity of NTR in the cells. A closer interrogation was
performed with HeLa cells and A549 cells incubated with 10 μM
-1
NTR in the range of 0 to 10 μg mL , the fluorescence signals
exhibited a dynamic increase (Fig. 2a) with a saturated response
-1
reached at 5 μg mL (Fig. 2b). This saturated signal seemed
-1
attributed to complete depletion of BICP by 5 μg mL NTR. The
fluorescence intensities at 570 nm were found to be linearly
correlated to NTR in the concentration range from 0.025 to 0.75 μg
BICP for 30 min firstly and then treated with normoxic (20% O
2
) and
hypoxic (1% O ) conditions, respectively, for 4 h. Interestingly, HeLa
2
-
1
2
mL with a regression equation y = 723585x + 17627 (R = 0.9984)
cells treated with BICP under normoxic conditions were found to
show appreciable fluorescence contrast (Fig. 3a), and the
fluorescence contrast became much more higher under the hypoxic
conditions (Fig. 3b). For A549 cells, similar results were obtained
(
inset of Fig. 2b). The limit of detection was determined to be 1.9 ng
-1
8b, 9b,
mL , which was better or comparable than existing methods,
9
d, 9e, 9f, 14a, 14d, 16
suggesting that BICP held potential as a highly
sensitive probe for NTR detection.
(
Fig. 3c-d). These findings revealed that oxygen-independent type I
Real-time kinetic was also measured for NTR-catalyzed cleavage
NTR was expressed in both cell lines even in situations when the
cells were incubated under normoxic conditions, while under
hypoxic conditions, the expression of NTR was increased
reaction of BICP. In real-time fluorescence assays (Fig. S12, ESI†),
-1
with the NTR concentration over 0.25 μg mL , the fluorescent
signal of the reaction system was up to a plateau within ~30 min.
Meanwhile, free probe showed no detectable change with time,
implying that free BICP was stable in the reaction system. By using
1
7
the Michaelis−Menten equaꢀon, the Michaelis constant (K
m
) and
maximum of initial reaction rate (Vmax) were estimated to be 34.99
-1
μM and 0.06 μM s (Fig. S13, ESI†), respectively, comparable to
5
, 6a, 6b, 9c, 9e, 14a-d, 18
previous studies.
Further experiments were performed to interrogate the
mechanism and performance for BICP. The HRMS (ESI) spectra of
the reaction solution of BICP with NTR shows a peak at m/z =
-
3
28.1629 [M-I] , which is characterized as BICF; the peak at m/z =
-
4
63.2005 [M-I] is the residue of BICP (Fig. S14, ESI†). Moreover,
HPLC analyses further verified the generation of BICF as a major
final reaction product. As shown in Fig. S15 of ESI , after reacting
†
with NTR the peak at 7.27 min suggested BICP decreased
significantly, concomitant with the increased peak intensity at 2.05
min for BICF (curve c). All these results supported the fact that the
fluorescence enhancement of the BICP for NTR was resulted from
the release of BICF. Investigation of the effects of pH and
temperature indicated that BICP was chemically stable under a
Fig. 3. Cells were incubated with 10 μM BICP for 30 min, then kept under
normoxic (20% O
images of HeLa cells under normoxic (20% O
d) Fluorescence images of A549 cells under normoxic (20 % O
) conditions. (e-h) The merged fluorescence imaging and corresponding
2
) and hypoxic (1% O
2
) conditions for 4 h. (a-b) Fluorescence
) and hypoxic (1% O ) conditions; (c-
) and hypoxic (1%
2
2
2
O
2
differential interference contrast (DIC) images. ( λex = 559 nm, λem = 565-600 nm)
4
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substantially. However, alike to existing NTR probes, our probe also
could not discriminate between two types of NTR, so we were
unable to identify which type of NTR was dominately expressed
under the hypoxic conditions.
In order to evaluate the subcellular localization, colocalization
experiments were performed by co-staining HeLa cells with
®
MitoTracker Green FM, DAPI and BICP (Fig. 4). The cells were
treated with 10 μM BICP for 30 min, then kept under hypoxic
2
conditions (1% O ) for another 4 h followed by addition of 1.0 μM
®
-1
MitoTracker Green FM (30 min incubation) or 2.0 μg mL DAPI (10
min incubation). It was observed that the fluorescence signal from
Fig. 5. (a-e) The fluorescence images of HeLa cells under (20% O
2
) and different
hypoxic (15%, 10%, 5% and 1% O ) conditions for 4h (λex = 559 nm, λem = 565-600
2
®
BICP showed substantial overlap with that from MitoTracker Green
nm); (f-j) The merged fluorescence imaging and corresponding differential
interference contrast (DIC) images.
FM but not with that from DAPI (Fig. S20, ESI†). Moreover, the
variations in intensity profile of linear regions of interest (ROI) were
across well in the two channels and showed tendency to synchrony
with a Pearson’s colocalization coefficient of 0.92. The above Conclusions
investigations suggested that BICP could specifically accumulate in
In summary, we have developed a novel mitochondria-
the mitochondria rather than in other organelles, implying our
design of using the positively charged and hydrophobic benzoindole
cation enabled selective targeting of mitochondria in cells.
targeting fluorescent probe BICP for mitochondrial NTR activity
monitoring and imaging. By virtual of the modulated ICT effect
for the benzoindocyanine fluorophore, the developed BICP
affords specific mitochondria-targeting ability and very low
fluorescence background. Highly selective and sensitive NTR
detection was realized both in aqueous solution and in living
tumour cells. BICP has been successfully applied to detect the
mitochondria-specific activities of type I and II NTR in tumor
cells. Low cytotoxicity and good biocompatibility of this probe
has also been demonstrated. The results show oxygen-
independent type I NTR was expressed in tumor cells even in
situations when the cells were incubated under normoxic
conditions, while under hypoxic conditions, the expression of
NTR was increased substantially. Moreover, BICP is able to
indicate the NTR activities in dynamic correlation to the
concentrations of NTR. These findings reveal BICP can provide
a promising molecular tool for elucidating mitochondria-
specific NTR activity for varying applications in NTR-based or
mitochondrial biology.
Mitochondrial localization feature of BICP allowed us to further
investigate the applicability of BICP for monitoring mitochondria-
specific NTR activity under different hypoxic conditions. In this
experiment, HeLa cells were treated with 10 μM BICP for 30 min at
3
7 °C and then kept under normoxic (20% O
15%, 10%, 5% and 1% O ) for 4 h. As depicted in Fig. 5, HeLa cells,
when cultured with decreased O concentrations, showed increased
fluorescence intensities (Fig. 5a-e), implying cells expressed
increasing amounts of NTR at decreasing O concentrations.
2
) or hypoxic conditions
(
2
2
2
Moreover, further inspection using flow cytometry showed the
same variations of NTR activity under these conditions (Fig. S21,
ESI†). This result confirmed that BICP could be catalytically
activated by active NTR under different hypoxic conditions,
delivering off-on fluorescence signals in dynamic correlation to the
concentrations of NTR.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (21527810, 21575036,
2
1521063), the National Key Basic Research Program
(
2017CB911000).
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