Endoplasmic reticulum-targeted two-photon turn-on fluorescent probe for nitroreductase in tumor cells and tissues

Article abstract of DOI:10.1016/j.saa.2018.05.092

Hypoxia conditions could increase the activity of intracellular nitroreductase (NTR) and lead to many malignant diseases. Therefore, monitoring the activity of NTR is of great significance to study the related diseases. Organelles play crucial roles in the metabolism of living cells. In these organelles, the endoplasmic reticulum (ER) possesses single membrane structure, and it is the largest organelle in the cell. ER performs the synthesis, processing and modification of proteins and lipid, stabilizing the intracellular Ca²⁺ concentration and other physiological functions in living cells. Therefore, it is of great significance to develop ER-target probes in living system. Toward this goal, a new endoplasmic reticulum-targeted two-photon fluorescence turn-on NTR probe Na-NTR-ER is designed and synthesized. Probe Na-NTR-ER has been proved to display high sensitivity (36 ng/mL) and selectivity to NTR. Particularly, probe Na-NTR-ER has been successfully applied for the monitoring of NTR in ER with a high the Pearson's colocalization coefficient as 0.90 in HeLa cells and cancerous mouse tissues up to the depth of 100 μm with significant fluorescence signals.

Full text of DOI:10.1016/j.saa.2018.05.092

Accepted Manuscript
Endoplasmic reticulum-targeted two-photon turn-on fluorescent
probe for nitroreductase in tumor cells and tissues
An Xu, Yonghe Tang, Weiying Lin
PII:
S1386-1425(18)30493-1
doi:10.1016/j.saa.2018.05.092
SAA 16126
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 1 March 2018
Revised date:
Accepted
date:
18 May 2018
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7 May 2018
Please cite this article as: An Xu, Yonghe Tang, Weiying Lin , Endoplasmic reticulum-
targeted two-photon turn-on fluorescent probe for nitroreductase in tumor cells and tissues.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy(2017),
doi:10.1016/j.saa.2018.05.092
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ACCEPTED MANUSCRIPT
Endoplasmic reticulum-targeted two-photon turn-on
fluorescent probe for nitroreductase in tumor cells and
tissues
An Xu, Yonghe Tang, Weiying Lin*
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and
Chemical Engineering, School of Materials Science and Engineering, University of
Jinan, Jinan, Shandong 250022, P.R. China.
*
Corresponding author. Tel.: +86 53182769108; Fax: +86-531-82769031
E-mail address: weiyinglin2013@163.com.
1

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Abstract
Hypoxia conditions could increase the activity of intracellular nitroreductase
(
NTR) and lead to many malignant diseases. Therefore, monitoring the activity of
NTR is of great significance to study the related diseases. Organelles play crucial
roles in the metabolism of living cells. In these organelles, the endoplasmic reticulum
(ER) possesses single membrane structure, and it is the largest organelle in the cell.
ER performs the synthesis, processing and modification of proteins and lipid,
2
+
stabilizing the intracellular Ca concentration and other physiological functions in
living cells. Therefore, it is of great significance to develop ER-target probes in living
system. Toward this goal, a new endoplasmic reticulum-targeted two-photon
fluorescence turn-on NTR probe Na-NTR-ER is designed and synthesized. Probe
Na-NTR-ER has been proved to display high sensitivity (36 ng/mL) and selectivity to
NTR. Particularly, probe Na-NTR-ER has been successfully applied for the
monitoring of NTR in ER with a high the Pearson’s colocalization coefficient as 0.90
in HeLa cells and cancerous mouse tissues up to the depth of 100μm with significant
fluorescence signals.
Key Words: Fluorescence Probe; Two-photon Excited Fluorescence; Endoplasmic
Reticulum; Nitroreductase; Fluorescence Imaging
2

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1
. Introduction
Hypoxia could lead to many malignant diseases [1-5]. Recent researches have
shown that cells under hypoxia conditions may produce elevated levels of specific
bioreductase, such as nitroreductase (NTR), azoreductase, quinonereductase, and
DT-diaphorase[6-10]. Amongst, NTRs belong to a kind of flavin-containing enzymes
[11], which can reduce the nitro group to the correspounding amino moiety with the
help of nicotinamide adenine dinucleotide (NADH) through a series of one-electron
reduction processes [12]. In recent years, NTRs, as prodrug-activating enzyme, are
also used for cancer gene therapy [13-15]. Therefore, it is of great significance to
monitor the bioactivity of NTR in living system.
Most eukaryotic cells contain certain organelles, such as endoplasmic reticulum
(ER), mitochondria, lysosomes, and Golgi apparatus. In these organelles, the ER is
the largest organelle in the cell with single membrane structure, which plays a
significant role in living systems and involves a great deal of essential biological
processes [16], including synthesis, processing and modification of proteins and lipid,
2
+
maintaining the intracellular Ca concentration, and so on [17]. Furthermore, ER is
also closely related to other organelles in living cells [18-20]. On the other hand, the
hypoxia condition may affect ER homeostasis and causing an imbalance between
protein folding load and capacity, which is known as ‘ER stress’, and this celluar
process is frequently found in tumor cells [21-22]. Therefore, it is very important to
develop a ER-targeted NTR fluorescent probe for further disentangling
physiopathological function of ER.
Fluorescence imaging technique has received wide attention because of its high
sensitivity and selectivity, fast response, and technical simplicity [23-25]. Particularly,
the two-photon (TP) fluorescence imaging shows many advantages, such as higher
spacial resolution, deep penetration, and low background fluorescence [26-29]. In
recent years, several fluorescence probes have been reported to detect NTR [30-42].
However, the organelle-targeted two-photon fluorescent NTR probes are still lacking.
In particular, there is no endoplasmic reticulum-targeted NTR fluorescent probe.
3

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Therefore, the development of organelle-targeted (especially endoplasmic
reticulum-targeted) two-photon NTR fluorescence probes has important value.
In this work, we reported a novel two-photon fluorescent probe Na-NTR-ER for
detection of ER NTR in a turn-on mode. Na-NTR-ER was able to preferentially
locate in the ER of the cells due to the methyl sulphonamide moiety [43-45].
Meanwhile, the probe showed good selectivity and sensitivity for NTR and was
suitable for the detection of ER NTR in living HeLa cells by two-photon fluorescence
microscope and confocal microscope. Significantly, we have successfully used the
new probe to monitor ER NTR in both living cells and live tumor tissues of mice.
2
. Experimental
2
.1. Materials and instrumentation
Unless otherwise stated, all solvents and reagents were commercially available
and used without further purification. Solvents used were purified by standard
methods prior to use. Doubly distilled water was used throughout all experiments.
Thin-layer chromatography (TLC) analysis was performed on silica gel plates and
column chromatography was conducted over silica gel (mesh 200-300), both of which
were obtained
from the Qingdao Ocean Chemicals. MTT (3-(4,
5
-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and Escherichia coli
nitroreductase (NTR) were from Sigma-Aldrich. The NTR was dissolved into
ultrapure water, and the enzyme solution was stored immediately at -80 °C to keep the
enzyme activity, and thawed quickly at 37 °C before use. β-Nicotinamide adenine
dinucleotide disodium salt (NADH) was purchased from J&K Scientific Ltd.
ER-Tracker Red was purchased from Beyotime Biotechnology co. Ltd.
High-resolution mass spectra (HRMS) were recorded on a Bruker Apex Ultra 7.0 T
1
FTMS mass spectrometer in electrospray ionization (ESI) mode. The H NMR and
1
3
C NMR spectra were recorded on a Bruker AVANCE III 400 MHz Digital NMR
Spectrometer, and using DMSO-d6 as solvent and tetramethylsilane (TMS) as internal
reference respectively. UV-vis absorption spectra were recorded on a Shimadzu
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UV-2600 UV-vis spectrophotometer and fluorescent spectra were measured on a
Hitachi F-4600 luminescence spectrophotometer with a 1 cm standard quartz cell. The
fluorescence imaging of cells was performed using a Nikon A1MP confocal
microscope. The pH measurements were carried out on a Mettler-Toledo Delta 320
pH meter.
2
.2. Synthesis of Na-NTR-ER
The synthetic routes of the probe Na-NTR-ER and compound Na-ER were shown
in Scheme S1. Compound 2, compound 7, compound 8, and compound Na-ER were
synthesized according to the previous literature [43]. Compound 4 and compound 5
were also synthesized according to the previous reports [46].
Compound 2 (320 mg, 1.5 mmol) and compound 5 (243 mg, 1 mmol) were added
to ethanol (20 mL), triethylamine (0.1 mL) was dropwise added to the above
suspension with stirring at room temperature. After refluxing overnight, the reaction
was cooled down to room temperature. The solvent was removed by evaporation
under reduced pressure, and the resulting residue was further purified by column
chromatography to obtain a brown yellow solid product Na-NTR-ER 224 mg with a
1
yield of 51%. H NMR (400 MHz, DMSO-d6) δ 8.71 (dd, J1=8.7 Hz, J2=0.9 Hz, 1H),
8
1
.60 (dd, J1=7.2 Hz, J2=0.8 Hz, 1H), 8.56 (m, 2H), 8.09 (td, J1=7.6 Hz, J2=1.3 Hz,
H), 7.80 (t, J=5.8 Hz, 1H), 7.57 (d, J=8.2 Hz, 2H), 7.22 (d, J=8.2 Hz, 2H), 4.11 (t,
1
3
J=6.4 Hz, 2H), 3.11 (q, J=6.4 Hz, 2H), 2.25 (s, 3H); C NMR (100 MHz, DMSO-d6)
δ 163.48, 162.69, 149.60, 142.92, 138.16, 132.09, 130.55, 129.95, 129.17, 128.84,
1
27.12, 126.77, 124.65, 123.27, 123.14, 40.11, 21.30; HR-MS calculated for
+
C21H17N3O6S [M+H] m/z 440.0911, found 440.0913.
2
.3. Spectrum measurement
All the spectrum measurements were carried out as following. The stock solution
of Na-NTR-ER was prepared with the concentration of 1.0 mM in DMSO. NADH
5

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was dissolved in ultrapure water to obtain the stock solution with a concentration of
1
00 mM. The other analytes were dissolved in ultrapure water to obtain appropriate
concentration. For the spectrum test, the probe Na-NTR-ER (5.0 μM) and NADH
500 μM) were dissolved in 10 mM PBS buffer (pH 7.4, with 5 % DMSO as
(
co-solvent). Before recording the spectra, the test solution was incubated with
appropriate testing species for 60 min at 37 °C. The excitation wavelength of test
solution was 440 nm. The test solution was shaken well and incubated with certain
analytes for 60 min before recording the spectra.
2
.4. MTT assays
The HeLa cells were cultured in Dulbecco’s Modified Eagle Medium media
(DMEM, Hyclone) supplemented with 10 % heat-inactivated fetal bovine serum (FBS,
Sijiqing) in an atmosphere of 5% CO2 and 95% air at 37 °C up to appropriate density.
4
Before the experiments, the HeLa cells (1×10 /well) were placed in a 96-well plate
and further cultured for another 24 h. Then the cells were incubated with varying
concentrations of Na-NTR-ER (0, 1.0, 2.0, 5.0, 10.0 μM) at 37 °C for 24 h, followed
by the MTT assays. Untreated groups were also conducted under the same conditions.
2
.5. HeLa cells culture and hypoxic imaging in HeLa cells
The HeLa cells were inoculated into 35 mm glass-bottom culture dishes (Nest).
The cells were incubated under the normoxic condition (21 % O2) and the different
hypoxic conditions (15-1 % O2) for 12 h. The cells were added the probe
Na-NTR-ER (5.0 μM) and further incubated under the same conditions for another
6
0 min at 37 °C, and then the HeLa cells were imaged with a two-photon (TP)
fluorescence confocal microscope. The cells were excited with 488/760 nm
one-photon (OP)/two-photon (TP) mode), and the emission wavelengths were
(
collected from 500 nm to 550 nm.
The co-localization experiment: the HeLa cells were incubated under the hypoxic
conditions (1 % O2) for 12 h, then added the probe Na-NTR-ER (5.0 μM), and
further incubated under the same condition for another 60 min. After removing the
6

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culture medium and washing the cells with PBS for three times, the cells were added
the ER-Tracker Red (1.0 μM) and incubation for another 15 min.
2
.6. Rat tissue slices preparation and fluorescent imaging
Four weeks old Balb/c mice were kindly kept during the experiments. 4T-1 cells
were injected to the mice. When the tumor grows to certain size, the mice were killed.
Then the tumor tissues were removed from the mouse. After washing off the blood
with PBS, the tumor tissues were cut into suitable size. After incubated with the probe
Na-NTR-ER (30 μM) for 60 min, the tumor tissues were washed three times with
PBS. Then the tumor tissue slices were imaged with a TP fluorescence microscope.
The tissues were excited at 760 nm (TP mode), and the emission wavelengths were
collected from 500 nm to 550 nm.
3
. Results and discussion
3
.1. Design and synthesis of Na-NTR-ER
The naphthylamide dye has outstanding TP fluorescent properties and is widely
used in preparing various fluorescent probes [43, 47, 48]. The nitroaromatics are
frequently used during the development of pro-drugs for hypoxia [6, 49, 50].
Consequently, we used naphthylamide as the fluorescent platform and nitro group as
the NTR identification site, and introduced methyl sulphonamide moiety, the
ER-target group, to construct a fluorescent NTR probe Na-NTR-ER. Due to the
strong electron-withdrawing effect of the nitro group, the electron structure of the
probe Na-NTR-ER behaved as “A-π-A”. The fluorescent probe had almost no
fluorescence due to the specific electronic structure. Under the catalytic reduction of
NTR, the nitro group was reduced to hydroxylamine, and then to amino moiety via a
one-electron reduction pathway. The nitro moiety was eventually reduced to amine
moiety, the strong electron-donating group, changes the electron structure of
fluorophore from “A-π-A” to “D-π-A”. Consequently, the intramolecular charge
transfer (ICT) effect of the molecule resulted in strong fluorescence of Na-ER
(Scheme 1 and Scheme S2).
7

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Scheme 1. The structures of the probe Na-NTR-ER and compound Na-ER, and the
proposed recognition mechanism of the probe Na-NTR-ER for NTR.
3
.2. Photophysical properties of Na-NTR-ER
After the fluorescence probe Na-NTR-ER was obtained, we first tested the
absorption spectra in PBS buffer. As shown in the Fig. S1, the maximum absorption
-
1
-1
wavelength of Na-NTR-ER was about 356 nm (ε=19,200 M cm ). In contrast, the
-
1
-1
maximum absorption wavelength of Na-ER was about 440 nm (ε=19,600 M cm ).
Similar to the absorption spectrum, the emission spectrum also had a significant
change. As designed, due to the specific electronic structures of “A-π-A”,
Na-NTR-ER showed almost non-fluorescent. Under the catalytic reduction of NTR,
the nitro group was eventually reduced to amine group, an intense fluorescence peak
around 543 nm was observed (Fig. 1). According to titration test, the detection limit of
Na-NTR-ER for NTR was calculated as 36 ng/mL (Fig. S2). These data indicated
that Na-NTR-ER has a high sensitivity to detect NTR, and the sensitivity of the
already described methods could detect the NTR values reported for living systems.
8

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Fig. 1. (A) Fluorescence spectra of the probe Na-NTR-ER (5.0 μM) in 10 mM PBS
buffer (pH 7.4, 5 % DMSO) with various concentrations of NTR (0-5.0 μg/mL) and
o
NADH (500 μM) for 60 min at 37 C. (B) Fluorescence intensity ratio (F/F0) changes
at 543 nm of Na-NTR-ER (5.0 μM) with the amount of NTR (0-5.0 μg/mL) in the
presence of NADH (500 μM). λex = 440 nm. n = 3, RSD ≤ 10.2 % (B).
The response rate is also one of the basic parameters to evaluate the identification
ability of the probe. The dynamic tests of the probe Na-NTR-ER (5.0 μM) treated
with varying concentration of NTR (0-5.0 μg/mL) and NADH (500 μM) were carried
out (Fig. S3). The fluorescent intensity of the probe (5.0 μM) only treated with NADH
(500 μM) was almost unchanged. In comparison, after the probe incubated with NTR
and NADH, strong changes of fluorescent signals were observed at about 543 nm.
And the fluorescence signal could reach a plateau state after about 60 min. In spite of
the response rate for NTR is not very fast, but the fluorescence enhancement ratio is
enough to apply to cell imaging after 60 min.
The activity of biological enzymes is closely related to the change of temperature.
We conducted experiment to investigate the fluorescent signal changes of
Na-NTR-ER (5.0 μM) treated or untreated with NTR (2.0 μg/mL) and NADH (500
μM) at different temperatures (20-40 °C). As shown in the Fig. S4, the fluorescence
intensities of Na-NTR-ER had almost no change over a wide temperature range of
2
0-40 °C without NTR, suggesting that Na-NTR-ER is stable in this temperature
9

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range. By contrast, after Na-NTR-ER was incubated with NTR (2.0 μg/mL) and
NADH (500 μM) for 60 min, an intense fluorescence signal was observed. Especially
under 37 °C (the physiological temperature condition), the fluorescence enhancement
phenomenon was the most significant. Solution pH also has major influence on the
activity of biological enzymes. The probe (5.0 μM) with NADH (500 μM) showed
few fluorescence intensity changes in a wide pH range (from 4.0 to 10.0), while a
remarkable fluorescent enhancement was observed when the NTR (2.0 μg/mL) was
introduced especially at pH 7.4 (the physiological) (Fig. S5). These results reveal that
Na-NTR-ER gives intense signals under about pH 7.4 and 37 °C (the physiological
conditions) and is suitable for biological applications, which should be ascribed to
rigorous reaction conditions of biological enzyme.
High selectivity is one of the basic characteristics of fluorescent probes for
applications in complex biological system. Therefore, the probe Na-NTR-ER was
incubated with various biologically relevant analytes, including biothiols (Cys, Hcy,
2
+
2+
2+
2+
2+
+
+
-
-
-
-
-
GSH), cations/anions (Ca , Cu , Fe , Mg , Zn , K , Na , F , Cl , Br , I , SCN ,
-
-
-
-
2-
NO2 , NO3 , OAc , HS , SO4 ), reactive oxygen species (ROS) (H2O2, HClO,
tert-butyl hydroperoxide (TBHP), •OH), and NTR, in PBS buffer. As shown in the Fig.
S6, in the absence of NADH, all the analytes almost had hardly change the
fluorescence emission intensity of the probe, suggesting that These results indicated
those relevant substance do not interfere with the fluorescence of probe directly and
NADH plays a key role in the process of enzymatic reduction. At the same condition,
with the introduction of NADH, the other analytes (except NTR) also did not change
the fluorescence intensity of the probe. By contrast, a strong turn-on signal was
observed after Na-NTR-ER treated with NTR for 1 h (Fig. 2). The selection test
results indicated that Na-NTR-ER is highly selective for NTR over other tested
analytes, indicating that the probe is favorable for investigation of NTR in biological
systems.
10

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Fig. 2. Fluorescence responses of the probe Na-NTR-ER with various relevant
analytes. The data were obtained after the incubation of the probe Na-NTR-ER (5.0
o
μΜ) with the analytes and NADH (500 μΜ) for 60 min at 37 C in PBS buffer (pH 7.4,
5
% DMSO). Legend: (1) free probe; (2) CaCl2 (2.5 mM); (3) CuCl2 (2.5 mM); (4)
FeSO4 (2.5 mM); (5) MgCl2 (100 μM); (6) ZnCl2 (2.5 mM); (7) KF (2.5 mM); (8) KI
2.5 mM); (9) KSCN (2.5 mM); (10) NaBr (2.5 mM); (11) NaNO2 (2.5 mM); (12)
(
NaNO3 (2.5 mM); (13) NaOAc (2.5 mM); (14) Na2SO4 (2.5 mM); (15) NaHS (2.5
mM); (16) Cys (2.5 mM); (17) GSH (2.5 mM); (18) Hcy (2.5 mM); (19) •OH (100
μM); (20) HClO (100 μM); (21) H2O2 (100 μM); (22) TBHP (100 μM); (23) NTR (2.0
μg/mL). λex = 440 nm. n = 3, RSD ≤ 7.5 %.
Moreover, the photostability of the probe was also tested. The irradiation of short
wavelength (365 nm) UV-light did not change the fluorescence intensity of
Na-NTR-ER (Fig. S7), suggesting that Na-NTR-ER has high photo-stability. We
also used spectral experiments and HR-MS analyses to verify the identification
mechanism of Na-NTR-ER for NTR. From the absorption spectrum, the maximum
absorption wavelength of Na-NTR-ER was at about 356 nm. When NADH was
introduced, a strong absorption peak was produced at 345 nm, which was the
absorption peak of NADH, and the absorption peak was no change at about 440 nm.
When NTR was added to the test solution, a new absorption peak had been generated
at 440 nm, and the absorption peak is similar to that of compound Na-ER (Fig. S8).
For the fluorescence emission spectra, when the probe treated with NTR and NADH,
11

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a maximum fluorescence emission wavelength was found at 543 nm, which was also
similar to the maximum emission wavelength of compound Na-ER (Fig. S9). The
HR-MS analyses test result confirms that the reduction product of Na-NTR-ER and
NTR is the compound Na-ER. (Fig. S10). These results agreed with the proposed
mechanism in Scheme 1 and Scheme S2.
3
.3. Fluorescence imaging of NTR in the living HeLa cells
As an imaging reagent, Na-NTR-ER should have low cytotoxicity. The data of
cell cytotoxicity assay showed that the survival rate of HeLa cells remained above
0% (treated with 0-10 μM Na-NTR-ER) (Fig. S11), indicating that Na-NTR-ER
9
has low cytotoxicity to the HeLa cells and Na-NTR-ER is suitable for imaging
experiments.
Due to the activity of NTR is closely related to the degree of hypoxia in cells, we
conducted NTR imaging experiments under different oxygen content conditions. As
naphthylamide is a classic TP dye, we applied Na-NTR-ER for imaging NTR in the
HeLa cells by the TP fluorescence and confocal microscope with the OP and TP
modes. The HeLa cells displayed almost no fluorescent signal when the cell treated
with Na-NTR-ER (5.0 μM) at normal oxygen content (21 % O2) (Fig. 3A f, p). By
contrast, when HeLa cells were cultured under hypoxic conditions (15–1% O2) with
Na-NTR-ER (5.0 μM), the different fluorescent signals were observed (Fig. 3A g-j
and q-t). As the oxygen content decreases, the fluorescence intensity of the HeLa cells
increased gradually (Fig. 3B and 3C). These studies demonstrated that Na-NTR-ER
is suitable for monitoring the activity of endogenous NTR at different oxygen content
in living HeLa cells by using OP/TP microscopical technique.
12

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Fig. 3. A) Fluorescence imaging of NTR in the living HeLa cells. a-e) Bright-field
image of the HeLa cells; f-j) One-photon image of HeLa cells; k-o) The merge of the
bright-field image and one-photon image; p-t) Two-photon fluorescence images of the
Hela cells. Scale bar: 20 μm. B) The one-photon fluorescence intensity of the Hela
cells images, n=3, RSD≤9.8%. C) The two-photon fluorescence intensity of the Hela
cells images, n=3, RSD≤14.6%.
13

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3
.4. Colocalization experiments of the probe in the living cells
We also conducted co-localization experiment to detect the targeted-feasibility of
Na-NTR-ER in living cells. The HeLa cells were incubated with Na-NTR-ER and
ER-Tracker Red (Beyotime Institute of Biotechnology) under hypoxic conditions (1%
O2). The HeLa cells showed significant green fluorescence as Na-NTR-ER reacted
with the NTR (Fig. 4a). At the same time, strong red fluorescence was observed in the
HeLa cells due to the presence of ER-Tracker Red (Fig. 4b). The merged image
showed that the green fluorescence and the red fluorescence can be highly overlapped
(Fig. 4c). Two fluorescence channels show high co-localization coefficients, the
Pearson’s colocalization coefficient was 0.90, and the Mander's overlap coefficient
was 0.91 (Fig. 4d). In addition, the spatial distribution of the two fluorescent probes in
HeLa cells also tended to be consistent (Fig. 4e). These results demonstrated that, as
designed, Na-NTR-ER can be highly enriched in the ER of cells and can be used to
imaging the NTR in the ER.
Fig. 4. The images of the living HeLa cells co-incubated with the probe Na-NTR-ER
TM
(5 μM), and ER-Tracker
Red under hypoxic condition (1% O ). a) The
2
fluorescence image of the green channel; b) The fluorescence image of the red
channel; c) The merged image of a and b; d) Intensity scatter plot of the green and red
channels. e) Intensity profile of linear region of interest across in the HeLa cells
14

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co-stained with ER-Tracker Red and green channel of Na-NTR-ER. Scale bar: 20
μm.
3
.5. Two-photon fluorescence imaging of NTR in living mouse tumor tissues
We also applied Na-NTR-ER to carry out the tissue imaging experiment to detect
NTR in the living mouse tumor tissues by using TP fluorescence microscope. For a
control experiment, the tumor tissue slides showed almost no fluorescence without the
incubation of fluorescent probes (Fig. 5a). By contrast, the tumor tissue slides treated
with Na-NTR-ER displayed green fluorescent emission up to a depth of 100 μm (Fig.
5
b). The sharp experiment contrast demonstrated that Na-NTR-ER is suitable for
monitoring the activity of endogenous NTR in living tissues by using TP
microscopical technique.
Fig. 5. Two-photon fluorescence imaging of NTR in the living tumor tissue slices.
Fluorescence images of the tumor tissue slices incubated with the probe Na-NTR-ER
(30 μM) for 60 min. Excitation was at 760 nm by femtosecond laser, and the emission
15

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collection was from 500-550 nm. Scale bar: 20 μm.
4
. Conclusions
In this work, we reported a novel two-photon fluorescent probe, Na-NTR-ER for
the detection of ER NTR. Na-NTR-ER can be highly enriched in the ER of cells, and
it is the first ER-targeted NTR fluorescent probe. The identification site, nitro group,
is directly connected to the fluorescence platform 1,8-naphthalimide and causes the
fluorescence quenching. After the catalytic reduction by NTR, a strong fluorescence
signal was observed at 543 nm. Overall, Na-NTR-ER has several advantages in
bioimaging experiments, such as high sensitivity and selectivity to NTR, low
cytotoxicity, optimal responses in a physiological environment (physiological pH 7.4
and temperature 37 °C). Especially, Na-NTR-ER can be used to detect the activity of
endogenous NTR at different oxygen content in living HeLa cells. Na-NTR-ER was
successfully applied to detect endogenous NTR in the living mouse tumor tissues, and
the penetration depth was up to 100 μm. Importantly, base on the excellent
ER-targeted feature of Na-NTR-ER, we believe that Na-NTR-ER as a powerful
molecular tool for studying the physiological function of NTR in ER of living cells
and tissues.
Acknowledgements
This work was financially supported by NSFC (21472067, 21672083), Taishan
Scholar Foundation (TS201511041), and the startup fund of University of Jinan
(309-10004).
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Graphical Abstract
A new turn-on endoplasmic reticulum-targeted two-photon fluorescent probe
(Na-NTR-ER) for nitroreductase was engineered for monitoring nitroreductase in the
living cells and tissues.
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Highlights
1) The probe Na-NTR-ER is a novel two-photon fluorescent
nitroreductase (NTR) probe.
2) The probe Na-NTR-ER is the first fluorescent ER-targeted probe for
NTR.
3) The probe Na-NTR-ER shows high selectivity and sensitivity to
NTR.
4) The probe Na-NTR-ER could respond for the degree of hypoxia in
the living cells.
5) The probe Na-NTR-ER could monitor NTR in tumor tissues up to
depth of 100 μm.
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