Acid-Promoted D-A-D Type Far-Red Fluorescent Probe with High Photostability for Lysosomal Nitric Oxide Imaging

Article abstract of DOI:10.1021/acs.analchem.8b00612

To accurately monitor the variations of lysosomal nitric oxide (NO) under physiological condition remains a great challenge for understanding the biological function of NO. Herein, we developed a new chemotype probe, namely, MBTD, for acid-promoted and far-red fluorescence imaging of lysosomal NO in vitro and ex vivo. MBTD was rationally designed by incorporating o-phenylenediamino (OPD) moiety into the donor-acceptor-donor (D-A-D) type fluorophore based on a dual intramolecular charge transfer (ICT) mechanism. Compared to previously reported OPD-based NO probes, MBTD displays several distinct advantages including large stroke shift, huge on-off ratio with minimal autofluorescence, and high NO specificity. Particularly, MBTD exhibits an acid-promoted response to NO with high acid tolerance, which greatly improves the spatial resolution to lysosomal NO by excluding the background noise from other nonacidic organelles. Furthermore, MBTD displayed much longer-lived and more stable fluorescence emission in comparison with the commercialized NO probe. MBTD was employed for ratiometric examination of the exogenous or endogenous NO of macrophages. More importantly, MBTD was able to detect the variation of lysosomal NO level in an acute liver injury mouse model ex vivo, implying the potential of MBTD for real-time monitoring the therapeutic efficacy of drug candidates for the treatment of acute liver injury. MBTD as a novel type of NO probe might open a new avenue for precisely sensing lysosomal NO-related pathological and therapeutic process.

Full text of DOI:10.1021/acs.analchem.8b00612

Article
pubs.acs.org/ac
Cite This: Anal. Chem. XXXX, XXX, XXX−XXX
Acid-Promoted D‑A‑D Type Far-Red Fluorescent Probe with High
Photostability for Lysosomal Nitric Oxide Imaging
Fengyang Wang,†,‡ Shujuan Yu,†,§ Zhiai Xu,*,‡ Lingling Li,‡ Yijing Dang,‡ Xiaowei Xu,∥ Yi Luo,∥
Zhen Cheng,⊥ Haijun Yu,# Wen Zhang,‡ Ao Zhang,§,# and Chunyong Ding*,§,#
‡School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241, China
§CAS Key Laboratory of Receptor Research, Synthetic Organic and Medicinal Chemistry Laboratory, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203, China
∥State Key Laboratory of Fine Chemicals, School of Chemical Engineering, Dalian University of Technology, Dalian 116024, China
⊥Molecular Imaging Program at Stanford (MIPS), Bio-X Program, and Department of Radiology, Stanford University, Stanford,
California 94305-5344, United States
#State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai
201203, China
S
* Supporting Information
ABSTRACT: To accurately monitor the variations of
lysosomal nitric oxide (NO) under physiological condition
remains a great challenge for understanding the biological
function of NO. Herein, we developed a new chemotype
probe, namely, MBTD, for acid-promoted and far-red
fluorescence imaging of lysosomal NO in vitro and ex vivo.
MBTD was rationally designed by incorporating o-phenyl-
enediamino (OPD) moiety into the donor−acceptor−donor
(D-A-D) type fluorophore based on a dual intramolecular
charge transfer (ICT) mechanism. Compared to previously
reported OPD-based NO probes, MBTD displays several
distinct advantages including large stroke shift, huge on−off
ratio with minimal autofluorescence, and high NO specificity.
Particularly, MBTD exhibits an acid-promoted response to NO with high acid tolerance, which greatly improves the spatial
resolution to lysosomal NO by excluding the background noise from other nonacidic organelles. Furthermore, MBTD displayed
much longer-lived and more stable fluorescence emission in comparison with the commercialized NO probe. MBTD was
employed for ratiometric examination of the exogenous or endogenous NO of macrophages. More importantly, MBTD was able
to detect the variation of lysosomal NO level in an acute liver injury mouse model ex vivo, implying the potential of MBTD for
real-time monitoring the therapeutic efficacy of drug candidates for the treatment of acute liver injury. MBTD as a novel type of
NO probe might open a new avenue for precisely sensing lysosomal NO-related pathological and therapeutic process.
reactive characteristics of NO, as well as the acidic micro-
environment of lysosomal vesicles.
itric oxide (NO) as a critical signaling molecular
Nparticipates in a large variety of physiological processes
including inflammation, blood pressure regulation, neuro-
transmission, and immunomodulation.1−7 The impairment of
intracellular NO homeostasis can induce a variety of
pathological conditions. Accumulating evidence indicates the
biological effects of NO are closely associated with its
concentration and diffusion dynamics in the subcellular
organelles (e.g., mitochondria and lysosome).8−12 It was
reported that lysosomal functions such as catabolic autophagy
process are affected by NO.11 To fully elucidate the
physiological roles of lysosomal NO, it is highly desirable but
challenging to develop an accurate method for in situ detection
of NO in complicated biological condition, particularly in
tissues and living organisms, due to the highly diffusible and
Of the reported techniques for detection of highly reactive
species,13−35 the fluorimetric strategy has attracted extensive
attention due to its high selectivity and sensitivity, as well as
real-time and noninvasive visualization of biological NO.13−33
Accordingly, a range of fluorescent NO probes have been
developed by coupling a NO-capturing moiety to organic
fluorophores such as rhodamine,23−28 naphthalimide,29 and
BODIPY derivatives.30−32 The o-phenylenediamino (OPD)
moiety is the most commonly used NO-capturing moiety,
which not only serves as a fluorescence quencher through
Received: February 5, 2018
Accepted: May 31, 2018
© XXXX American Chemical Society
A
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photoinduced electron transfer (PET), but also specifically
reacts with NO under aerobic conditions to form benzo-
triazoles, thus, restoring the fluorescence signals of fluorophore
by inhibiting the PET process.36 Despite being promising, the
application of the OPD-based probes for imaging lysosomal
NO were hindered by their intrinsic drawbacks such as
intolerance of the acidic pH, susceptibility to other reactive
species, low organelle specificity and poor photostability.
Moreover, most of these PET probes suffered from the short
emission wavelength (<600 nm), which restricts the in situ
tracking of NO in deep tissue and living organisms with strong
background autofluorescence. As such, various fluorescence
NO probes with improved sensing performance have been
developed recently.37−46 Nevertheless, few of them could be
utilized for accurately quantifying the variation of lysosomal
NO levels due to the interference of photobleaching,
subcellular microenvironment, and local probe concentration
variation.
The development of ratiometric fluorescent probe is a
practical solution to address the above concerns. Ratiometric
fluorescent imaging can synchronously record two signals at
different wavelengths, thus, providing a built-in reference to get
rid of the interference. Until now, ratiometric NO probes with
long wavelength emission (>600 nm) have barely been
reported.23,47 To this end, we herein present a novel OPD-
based fluorescent NO probe with far-red emission, namely,
MBTD (4,7-bis(4-(2-morpholinoethoxy)phenyl)benzo[c]-
[1,2,5] thiadiazole-5,6-diamine), for ratiometirc examination
of lysosomal NO in vitro and ex vivo. MBTD is designed based
on a dual intramolecular charge transfer (ICT) mechanism by
integrating the OPD moiety into a donor−acceptor−donor (D-
A-D) type fluorophore as the weak electron acceptor. To our
knowledge, D-A-D type fluorescent probes with far-red
emission have not been reported for in situ monitoring
lysosomal NO. MBTD not only possesses high acid-tolerance
and acid-promoted response to NO for fluorescent NO
imaging specifically in lysosomes, but also shows the intrinsic
potential for detecting NO of deep tissue. Moreover, MBTD
exhibits excellent photostability and high NO selectivity arising
from the unique D-A-D structure, suggesting a good potential
of MBTD as a new chemotype of fluorescent probe for accurate
sensing of lysosomal NO.
bovine serum (FBS) in an atmosphere of 5% CO2 at 37 °C.
The cell cytotoxicity of MBTD was evaluated by MTT assay.
One ×104 cells per well were seeded on 96-well plates and
incubated for 24 h at 37 °C under 5% CO2. MBTD at
concentrations of 0−15 μM was added to the wells and
incubated for 24 h. After that, the supernatant was removed and
cells were washed three times with 10 mM PBS buffer.
Subsequently, 0.5 mg/mL MTT was added and incubated for
additional 4 h. Finally, the formazan crystal was dissolved by
DMSO and the absorbance values were recorded at 570 nm
using a plate reader.
Fluorescence Imaging. For fluorescence imaging in vitro,
cells were seeded on 35 mm glass-bottom culture dishes with
8000 cells per well and incubated for 24 h at 37 °C. The cells
were treated with 5 μM MBTD for 1 h. After washing three
times with 10 mM PBS buffer, the cells were incubated with
various concentration of DEANO (NO donor) for 1 h. Finally,
the cells were washed four times with PBS buffer and observed
with confocal laser scanning microscopic (CLSM). For imaging
of endogenous NO in RAW 264.7 cells, the cells were
preincubated with 10 μg/mL LPS for 5 h, then stained with 5
μM MBTD for 1 h. To confirm the production of NO, the cells
were also treated with 10 μg/mL hemoglobin for 2 h for
comparison. After that, the cells were washed with PBS buffer
and imaged. In order to confirm the lysosome-targeting ability
of probe MBTD, cells were stained with 5 μM probe MBTD
for 1 h and incubated with 400 nM LysoTracker Red, 50 nM
MitoTracker Red separately for 1 h. The cells were also
incubated with 1 μM Baf-A1 for 2 h and incubated with 5 μM
MBTD for 1 h. For photostability study of different probes, the
cells were incubated with 5 μM probe MBTD or DAF-FM-DA,
and then treated with 100 μM DEANO for 1 h, finally the
fluorescence images were recorded. The mean fluorescence
intensity was analyzed with ImageJ Software.
Acute Liver Injury and Imaging of Frozen Tissue
Slices Ex Vivo. Male Balb/c mice (6−8 week old, 18−20 g)
were obtained from Animal Center of Shanghai Institute of
Material Medica, Chinese Academy of Sciences. All animal
procedures were carried out under the guidelines approved by
the Institutional Animal Care and Use Committee (IACUC) of
the Shanghai Institute of Material Medica, Chinese Academy of
Sciences. The mice were not given food and water for 12 h to
avoid the possible interferences of food. A total of 12 mice were
randomly divided into four groups. Control group includes
three mice. Test group includes nine mice. (a) Control group:
The mice were injected intraperitoneally with 200 μL of 0.9%
NaCl for 6 h. (b) LPS group: The mice were injected with 0.9%
NaCl containing 5 μg/kg LPS and 300 mg/kg D-GalN for 6 h.
(c) LPS+NAC group: The mice were injected with 5 μg/kg
LPS and 300 mg/kg D-GalN for 2 h, then with 300 mg/kg
NAC for 4 h. (d) L-NAME+LPS group: The mice were
injected with 50 μg/mL L-NAME for 6 h and then injected 5
μg/kg LPS and D-GalN for 6 h. After that, the mice were killed
using 10 mg/mL sodium pentobarital and the liver tissues were
embedded in OCT. Then the tissues were cut into 4-μm-thick
slices at −30 °C. The tissue slices were stained with 10 μM
MBTD for 1 h and incubated with 5 μg/mL DAPI for 5 min.
Finally, the treated tissue slices were observed with CLSM.
EXPERIMENTAL SECTION
■
Preparation of Reactive Oxygen/Nitrogen Species.
NO solution was prepared by bubbling NO gas to
deoxygenated water. The concentration of NO saturated
−
solution was measured by Griess Method. Nitrite (NO2 )
−
and nitrate (NO3 ) were prepared by dissolving sodium nitrite
and sodium nitrate in deionized water. Hydroxyl radical (•OH)
was generated by Fenton reaction (Fe2+/H2O2 = 1:6 mM).
Hypochloride anion (ClO−) was prepared by NaClO. O2− was
generated from KO2 solid diluted in DMSO. TBHP (tert-butyl
hydroperoxide) solution was prepared by diluting TBHP. H2O2
solution (1 mM) was provided by diluting 30% H2O2.
Peroxynitrite (ONOO−) solution was generated by H2O2
solution (1 mM) reacting with NaNO2 (1 mM). The single
oxygen (1O2) was produced by reaction between NaClO (1
mM) and H2O2 (1 mM).
RESULTS AND DISCUSSION
Cell Culture and Cytotoxicity Assay. RAW 264.7 cells
were obtained from cell bank of Chinese Academy of Sciences
(Shanghai, China). RAW 264.7 cells were cultured in DMEM
medium with 1% penicinllin/streptomycin and 10% fetal
■
Design and Synthesis of the D-A-D Type NO Probe.
The D-A-D architecture employed for fluorescent probe design
is featured with a series of distinct optical properties including
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Scheme 1. Strategies of the OPD-Based Fluorescent Probe for NO Based on Different Mechanisms: (A) Previous Probes Based
on PET; (B) D-A-D Type Probe MBTD of This Work Based on ICT
a
Scheme 2. Synthesis of Compounds MOBTD, MBTD, and MBTT
a
Conditions and reagents: (a) (4-methoxyphenyl)boronic acid, Pd(PPh3)4, K2CO3, toluene, 110 °C; (b) BBr3, DCM, −78 °C; (c) 4-(2-
bromoethyl)morpholine, NaH, DMF; (d) Fe/ACOH; (e) NaNO2/HOAc.
significant absorption and emission in the red or near-infrared
(NIR) region, large Stokes’ shift, and high photostability.48−50
The benzo(1,2-c:4,5-c′)bis([1,2,5]thiadiazole) core has com-
monly been used as the electron acceptor of D-A-D compound
due to its substantial quinoid character and great electron
delocalization.51−54 Inspired by this, we employed thiadiazole-
fused OPD (TOPD) scaffold as both the NO-capturing moiety
and the weak electron acceptor for MBTD design. Moreover,
two alkyoxyl groups severing as the electron donors were linked
to the 4- and 8-positions of TOPD via a benzene unit as a π
spacer, and followed by two morpholinyl groups for specifically
targeting lysosomes (Scheme 1). It is envisioned that, upon
reaction with NO, TOPD is transferred into the tricyclic
thiadiazole-fused benzotriazole as another electron acceptor
with stronger electron-withdrawing ability,49 and the resulting
product MBTT possesses an enhanced ICT ability, leading to a
large red-shift of the excition/emission wavelengths.
with BBr3 followed by nucleophilic substitution reaction with 4-
(2-bromoethyl)morpholine provided the precursor 3, which
then underwent Fe/AcOH-mediated reduction reaction to
furnish MBTD. Further reaction of MBTD with NaNO2/
HOAc gave rise to the fluorescent product MBTT. To
determine if the morpholinyl groups account for the
lysosome-targeting ability of MBTD, another probe MOBTD
was synthesized as a comparison according to the similar
procedure. All synthesized final compounds were characterized
1
by H NMR, 13C NMR spectroscopy, and LR/HR-ESI-MS,
respectively (Figures S1, S2, S4, and S5). HPLC chromatogram
showed MBTD has a high purity of 96.9% (Figure S3).
Photophysical Properties of MBTD. The photophysical
properties of MBTD were first investigated by using UV−vis
and fluorescence spectroscopic examinations, respectively.
MBTD showed UV−vis absorbance peaked at 365 nm and
moderate fluorescence emission at 565 nm (λex = 365 nm) due
to the weak ICT process induced by TOPD (Figures 1A and
S9A). Negligible fluorescence emission was detected in aqueous
solution of MBTD when excited at 488 nm (Figure 1B),
The MBTD probe was synthesized by Suzuki coupling of
4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole with (4-
methoxyphenyl)boronic acid (Scheme 2). Demethylation of 1
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Figure 1. (A) UV−vis absorbance and (B) fluorescence spectra of 50 μM MBTT and MBTD in the absence and presence of NO (100 μM) under
the aerobic condition, λex = 488 nm. Inset: the photographs of MBTD probe (a), MBTD in the presence of NO (b), and the product MBTT (c).
(C) Absorbance spectra of MBTD probe (50 μM) upon the addition of different concentrations of NO. (D) Fluorescence response of probe MBTD
in the presence of various concentration of NO, λex = 421 nm. Inset: Plot of F625nm/F550nm ratio of NO-incubated MBTD against NO concentration
(5−20 μM). Buffer: 10 mM PB (pH = 5.0, 10% DMAC).
−
Figure 2. (A) Fluorescence responses (F625nm/F550nm) of MBTD (50 μM) incubated with 100 μM NO, 1 mM ROS/RNS 2−11 (blank, NO2 ,
−
NO3 , •OH, ClO−, TBHP, H2O2, O2•−, 1O2, ONOO−), 1 mM active biomolecules 12−16 (GSH, Cys, Hcy, AA, DHA), and 1 mM metal ions 17−
23 (K+, Na+, Ca2+, Fe2+, Fe3+, Zn2+, Mg2+). Reaction time: 30 min, λex = 421 nm. (B) Photostability of 50 μM MBTD (λex = 365 nm, red line) and 50
μM MBTT (λex = 488 nm, black line). Buffer: 10 mM PB (pH = 5.0, 10% DMAC). (C) Effects of pH on F625nm/F550nm ratio of MBTD in the
absence or presence of 100 μM NO, λex = 421 nm.
verifying minimal basal level autofluorescence of MBTD. In
contrast, upon addition of 100 μM NO under aerobic
condition, the maximum absorption was considerably red-
shifted to 488 nm together with obvious color change from
yellow-green to red under the ultraviolet lamp (Figure 1A,B).
The corresponding fluorescence emission peak dramatically
shifted to 625 nm (λex = 488 nm) with a 446-fold off−on ratio
(Figure 1B), which could be attributed to an enhanced ICT
process by the resulting thiadiazole-fused benzotriazole as
stronger electron acceptor. The effect of O2 on the fluorescence
responses was investigated. As shown in Figure S6, the
absorbance and fluorescence responses are significantly sup-
pressed at low concentration of O2 by bubbling pure N2 gas. In
the case of bubbling pure O2 gas, the response decreases
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Scheme 3. Proposed Mechanism for Acid-Promoted Fluorescence Property of MBTT
Table 1. Structures and Energies of the HOMO and LUMO of MBTD, MBTT, and MBTT-H+ at the B3LYP/6-31+G (d) Level
a
in the Gas Phase
a
Egap = ELUMO − EHOMO
.
slightly, and this was probably ascribed to oxidization of minor
NO into NO2. These results demonstrated that O2 is crucial for
the reaction. To validate the reaction mechanism we proposed,
MBTT was synthesized by reaction of MBTD with NaNO2 in
glacial acetic acid (Scheme 2), and we found its absorption and
emission maxima profiles were essentially identical to those of
MBTD treated with NO (Figure 1A,B). Besides, HPLC-MS
analysis showed that treating MBTD with NO quantitatively
produced a new product with the same mass peak and retention
time as those of MBTT (Figures S7B and S8A). Obviously,
these results suggested that reaction of MBTD with NO in pH
5.0 PB buffer produced the benzotriazole product MBTT,
which was in good agreement with the mechanism proposed in
Scheme 1.
detection by both colorimetry and fluorescence ratiometry in
far-red emission region.
To examine the NO specificity of MBTD, we inspected the
F
625nm/F550nm ratios of MBTD with the addition of a wide array
of the potentially interfering substances, which were categorized
−
−
as reactive oxygen/nitrogen species (ROS/RNS: NO2 , NO3 ,
•OH, ClO−, TBHP, H2O2, O2•−, 1O2 and ONOO−), metal ions
(K+, Na+, Ca2+, Mg2+, Fe2+, Fe3+, and Zn2+), and active
biomolecules (GSH, Cys, Hcy, ascorbic acid (AA) and
dehydroascorbic acid (DHA)). All the interfering substances
showed negligible influence on MBTD fluorescence, while only
NO remarkably increased the F625nm/F550nm fluorescence ratio
(Figure 2A), suggesting high NO selectivity of MBTD, which
could be most likely explained by the relative low reactivity of
electron-withdrawing TOPD functionality.
NO-dependent changes of the MBTD absorption and
emission spectra were further investigated by adjusting the
concentration of NO. Along with the increase of NO
concentration up to 100 μM, the intensity of the 488 nm
absorbance (ε = 11600 M−1 cm−1) increased gradually while
the 365 nm absorption decreased with an obvious isoabsorp-
tion point at 421 nm (Figure 1C). Meanwhile, two character-
istic fluorescence bands appeared at 550 and 625 nm in the
emission spectra (λex = 421 nm). Interestingly, the 625 nm
emission enhanced gradually while the 550 nm emission kept
constant (Figure 1D), indicating a ratiometric fluorescence
response of MBTD probe. The maximal UV−vis absorbance at
488 nm and its emission at 625 nm linearly increased in a
concentration-dependent manner in a wide NO concentration
range, with detection limits of 1.2 and 1.1 μM (3σ/slope),
respectively (Figure S9B−D). In particularly, the 625 to 550
nm fluorescence ratios (F625nm/F550nm) increased linearly as a
function of NO concentration with an improved detection limit
of 0.71 μM (Figure 1D). To keep consistence with that of
imaging application, the F625nm/F565nm ratio upon excitation at
488 and 405 nm, respectively, were also shown (Figure S10).
These results suggested the potential of MBTD for NO
The real-time fluorescence intensity variations of MBTD
probe toward NO was tested by recording the fluorescent
intensity at two emission wavelengths of 550 and 625 nm,
respectively. Upon addition of 100 μM NO, the fluorescence
intensity at 550 nm was weak, and kept intact during 180 min
(Figure S11A). In contrast, the emission intensity at 625 nm
remarkably increased and quickly reached a plateau within 20
min, with a 33-fold enhancement in comparison with the basal
level. The fluorescence signal kept steady for several hours (>2
h), verifying the potential of MBTD for real-time fluorescence
imaging of biological NO (Figure 2B). The fluorescence
lifetime measurement revealed that the average lifetime of
MBTD determined by multiexponential fitting was 2.68 ns,
while MBTT displayed a longer lifetime up to 7.91 ns, which
could avoid the interference of background fluorescence and
photobleaching (Figure S11B). These results showed that
MBTT possessed a persistent fluorescent emission for a long-
term fluorescence imaging.
In the assessment of pH responses of MBTD toward NO, it
was found that the F625nm/F550nm fluorescence ratios of MBTD
were nearly 1.0 with negligible variation throughout the tested
pH range of 4.5−9.0 (Figure 2C), indicating its high tolerance
E
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Figure 3. (A, C) Colocalization of MBTD with the lysosomal (A) and (C) mitochondrial vesicles in RAW 264.7 cells. (B, D) The corresponding
intensity profiles. (A−D, probe channel: λex = 405 nm λem = 520−570 nm. Red channel: λex = 552 nm; λem = 590−700 nm). (E) MBTD-loaded cells
incubated with 200 μM DEANO for 1 h. (F) 1 μM Baf-A1-pretreated cells treated with 5 μM MBTD followed by incubation with 200 μM DEANO.
(E, F) Green channel: λex = 405 nm, λem = 520−570 nm. Red channel: λex = 488 nm, λem = 590−650 nm. Scale bar = 10 μm.
in a wide pH range with low background fluorescence. In the
presence of excessive NO (2 equiv), the F625nm/F550nm ratios
were significantly enhanced under acidic conditions in a pH-
dependent manner, and reached to the maxima in the pH range
of 4.5−5.0, which is in good accordance with the lysosomal
acidity (e.g., pH < 6.0), while it dropped to a comparable
baseline-level to that of free MBTD under alkaline conditions
(pH > 8.0). HPLC-MS analytic results showed that MBTD
react with NO in pH 5.0 buffer solution to generate MBTT
(Figures S8A and S7), while the reaction failed in pH 9.0 buffer
solution, and most of MBTD still remained (Figures S8B and
S12). Given the fact that MOBTD without the morpholinyl
group also displayed a similar pH-dependent NO response
(Figure S11D), this unique pH-dependent reactivity of MBTD
with NO is probably attributed to the blunt nucleophilic ability
of TOPD, which reacts only with NO in acidic conditions, but
not with alkaline NO. These results revealed that MBTD is an
acid-promoted and acid-tolerant fluorescent probe for intra-
cellular NO determination in the acidic lysosome. Besides, the
triazole product MBTT also displayed a similar pH-dependent
fluorescence emission curve with the calculated pKa value of 5.6
(Figure S11C). The remarkable reduction in the fluorescence
intensity under nonacidic condition was probably ascribed to
the formation of the triazolate MBTT-H+ as a weak electron
acceptor (Scheme 3).
MBTD and MBTT were obtained from density functional
theory (DFT) calculations in the gas phase. As shown in Table
1, the HOMOs of these two compounds were essentially
delocalized on the whole molecular backbone, and the LOMOs
were mainly distributed on the electron accepting unit
(thiadiazole-fused benzotriazole). Moreover, MBTT presented
higher-lying calculated HOMO and LUMO levels, and
displayed lower bandgap than MBTD, which may account for
the relatively stronger ICT effect that led to the large red-shift
of the exciting/emission wavelengths. It was also found that the
HOMO−LUMO bandgap of MBTT-H+ was larger than that of
MBTT, indicating the attenuation of ICT process induced by
the deprotonation of the triazole in the electron accepting unit.
Fluorescence Imaging of NO in Living Cells. Both
MBTD and MBTT showed negligible cytotoxicity in RAW
264.7 cells at the concentrations from 1 to 15 μM as revealed
by MTT assay (Figure S13). We began to investigate the
imaging ability of the probe at different times by CLSM
examination (Figure S14). After 40 min, a strong red
fluorescence was observed. We next evaluated the lysosome-
targeting ability of the MBTD probe. The intracellular
lysosomal vesicles and mitochondria of RAW 264.7 cells were
stained with LysoTracker Red or MitoTracker Red, respec-
tively. Figure 3A−D showed that in the absence of exogenous
NO, the cells displayed noticeable green fluorescence post 1 h
incubation with MBTD at 37 °C. The green fluorescence
emission of MBTD colocalized well with the red fluorescence
signal of LysoTracker Red while not MitoTracker Red. The
To validate the reaction mechanism we proposed in Scheme
1, the highest occupied molecular orbitals (HOMO), lowest
unoccupied molecular orbitals (LUMO), and band gap levels of
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Figure 4. (A−D) CLSM imaging of RAW 264.7 cells treated with MBTD at various DEANO concentration (0, 50, 150, 300 μM) for 1 h. (E) The
ratiometric intensity of red/green fluorescence as a function of DEANO concentration. Green channel: λex = 405 nm, λem = 520−570 nm. Red
channel: λex = 488 nm, λem = 590−650 nm. (F) Flow cytometric analysis results. λex = 355 nm, λem = 515−545 nm; Red channel: λex = 488 nm, λem
564−606 nm. Scale bar = 10 μm.
=
intensity profiles of the linear regions of interest across the cells
that stained with MBTD and LysoTracker Red varied in very
close synchrony with Pearson’s colocalization coefficient and
overlap coefficient of 0.83 and 0.99, respectively, while those
stained with MBTD and MitoTracker Red varied in remarkable
inconsistency (Figure 3B,D). In contrast, MOBTD without the
morpholinyl group poorly colocalized with the lysosomal
vesicles, indicating poor lysosome specificity (Figure S15), and
the morpholinyl group accounts for the excellent lysosome-
targeting ability of MBTD.
To investigate the ability of MBTD to image NO in the
lysosomes, the MBTD-loaded cells were incubated with
DEANO, an exogenous NO donor, for 1 h. As shown in
Figure 3E, remarkable green and red fluorescence were
observed in the lysosome, respectively. Upon pretreatment
with NO scavenger hemoglobin (Hb), the cells showed barely
detectable red fluorescence, verifying the increased fluorescence
emission could be attributed to the presence of lysosomal NO
(Figure S16). To further demonstrate if it could specifically
response to the coexistence of H+ and NO in the lysosome,
RAW 264.7 cells were pretreated with Baf-A1, a H+-ATPase
inhibitor to neutralize the acidic surrounding of the lysosome.
As expected, no obvious red fluorescence was observed and
only weak green fluorescence remained after incubation with
MBTD in the presence of NO (Figure 3F), indicating that the
intracellular response behavior of the MBTD probe toward NO
happened specifically in the acidic lysosomes rather than in
other nonacidic organelles. The ability of MBTD to monitor
the dynamic pH changes of lysosome in RAW 264.7 cells in the
presence of DEANO was investigated (Figure S17). It was
found that red fluorescence gradually decreased with the
increase of the lysosomal pH value, indicating the potential of
MBTD for monitoring lysosomal pH changes.
CLSM examination was further performed to investigate the
feasibility of MBTD for the ratiometric measurement of the
lysosomal NO. In absence of NO, the probe-loaded cells
showed noticeable green fluorescence without the appearance
of red fluorescence (Figure 4A). Intracellular red fluorescence
was observed post 1 h incubation of the cells with 50 μM
DEANO as exogenous NO donor (Figure 4B). Moreover, the
red fluorescence increased gradually with the increase of
DEANO concentrations (Figure 4C,D). The relative fluores-
G
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Figure 5. (A) MBTD and (B) DAF-FM-DA-mediated dynamic CLSM examination of exogenous NO in RAW 264.7 cells (Green channel: λex = 405
nm, λem = 520−570 nm; Red channel: λem = 488 nm, λem = 590−650 nm; Scale bar = 10 μm). (C, D) Relative fluorescence intensities of MBTD (C)
and DAF-FM-DA (D) along with time, respectively.
cent intensity ratio (IR/IG) displayed a positive correlation with
DEANO concentration ranging from 0 to 300 μM (Figure 4E).
Flow cytometric measurement was performed to demon-
strate the potential of MBTD probe for quantitative detection
of exogenous NO. Compared to the control cells without
staining, cells incubated with MBTD alone show marginally
detectable fluorescence intensity. The red fluorescence
increased upon DEANO incubation at 100 or 450 μM, without
affecting the intensity of green fluorescence (Figures 4F and
S18). Accordingly, the CLSM and flow cytometric data
consistently suggested the good potential of MBTD for
accurately monitoring the variation of exogenous NO. As
shown in Figures S19 and S20, MCF-7 and HepG2 cells were
also used to image NO in living cells. The results confirm the
imaging capability of MBTD probe in other cells lines.
The photostability of MBTD after incubation with DEANO
in RAW 264.7 cells was assessed under laser irradiation at 405
and 488 nm, respectively. A commercially available NO probe
DAF-FM-DA was selected as a parallel control.55Figure 5A,C
showed negligible fluctuation in both red and green
fluorescence intensity upon laser irradiation for 6 min. In
contrast, when the cells were treated with DAF-FM-DA and
DEANO, the green fluorescence decayed gradually at 30 s, and
completely disappeared within 3 min (Figure 5B,D). These
results indicate that MBTD possesses much better fluorescence
stability than DAF-FM-DA probe, allowing for continuously
monitoring the dynamic change of NO in living cells for a long
period.
We next sought to testify the feasibility of MBTD for
measuring endogenous NO in vitro. Cells were stimulated with
lipopolysaccharide (LPS) and then treated with MBTD. Figure
S21A showed LPS stimulation followed by MBTD incubation
led to a marked enhancement in red fluorescence (Figure
S21B). To verify whether the enhanced fluorescence could be
attributed to LPS-induced massive endogenous NO, the cells
were coincubated with LPS and Hb, and subsequently treated
with MBTD probe. The CLSM image exhibited a barely
detectable red fluorescence (Figure S21C), as indicated by 2.5-
D image and IR/IG ratio histogram, respectively (Figure
S21D,E). These results clarify the potential of MBTD probe
for ratiometric detection of the endogenous NO in living cells.
Ex Vivo Imaging of NO in an Acute Liver Injury Mouse
Model. After confirming the ability of MBTD to track both
exogenous and endogenous NO in living cells, we further
explored its potential for in situ detection of the lysosomal NO
in an acute liver injury mouse model (Figure 6A). The Balb/c
mice were intraperitoneally (i.p.) injected with LPS and D-
galactosamine (D-GalN) at a dose of 5 μg/kg and 300 mg/kg,
respectively. The mice were sacrificed at 4 h post-injection.
Haematoxylin-eosin (H&E) staining of the liver sections
showed significant vacuolization of the hepatic parenchymal
cells, verifying LPS-induced inflammation of the liver tissue
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DOI: 10.1021/acs.analchem.8b00612
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Analytical Chemistry
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drug-induced acute liver injury. We thus attempted to explore
the potential application of MBTD probe for real-time
monitoring the therapeutic efficacy of NAC. As shown in
Figure 6E, treatment of the liver injured mice with NAC led to
a markedly decreased red fluorescence with a lower IR/IG value
of 0.2, illustrating that NAC could effectively alleviate the liver
injury of the inflamed mice leading to a significantly decreased
NO level. Both the 2.5D CLSM images and IR/IG ratio
histogram vividly displayed the variation of the relative
fluorescence intensity related with the NO levels in the
inflammed mice with different treatment (Figure 6F,G). The
above results demonstrate the promising potential of MBTD
probe for in situ monitoring the therapeutic efficacy by
detecting the variation of pathological NO.
CONCLUSIONS
■
In summary, we have developed a novel fluorescent NO probe
MBTD with far-red emission based on a dual ICT mechanism
by merging of the OPD moiety into the D-A-D type
fluorophore. MBTD possesses several distinct merits including
large fluorescence on−off ratio, long-lived fluorescence with
better stability, and high specificity for NO over other
disturbing species. Moreover, MBTD is featured by lysosomal
acid-compatible properties for specific imaging of lysosomal
NO. All these distinct advantages collectively enable it to
accurately detect the variation of both exogenous and
endogenous NO levels in cells. More importantly, the probe
is able to monitor the variation of lysosomal NO level in the
inflamed mouse model, which not only reveals iNOS is
critically responsible for NO generation in the inflamed liver,
but also suggests that NO is one of important therapeutic
indicators of acute liver injury. Taken together, MBTD as a
novel type of NO probe might open a new avenue for precisely
detecting lysosomal NO-related biological process.
Figure 6. (A) Experimental schedule for MBTD-mediated fluo-
rescence examination of endogenous NO in an inflamed mouse model.
(B) The saline group. (C) LPS/D-GalN-induced inflammed mice
group. (D) L-NAME-treated infection mice group. (E) NAC-treated
infection mice group. Scale bar = 100 μm in B−E. (F) 2.5D images of
B, C, D, and E groups, respectively. (G) The IR/IG fluorescence
intensity ratio of B−E groups. Blue channel: λex = 377 nm, λem = 410−
480 nm; Green channel: λex = 469 nm, λem = 495−570 nm; Red
channel: λex = 531 nm, λem = 550−650 nm.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.8b00612.
(Figure S22). As vividly illustrated in Figure 6B, the MBTD-
stained liver slices of unstimulated mice displayed measurable
green fluorescence in the saline-injected control group, and
negligible red fluorescence was observed in red channel. The
image of the liver slice of the inflamed mice exhibited a notably
enhanced red fluorescence with an IR/IG value of 2.4 (Figure
6C), indicating the generation of the massive endogenous NO.
It was reported that the overactivation of iNOS during the
infection and inflammation was mainly responsible for the
production of NO and other reactive nitrogen species.56,57 To
determine if the aforementioned red fluorescence enhancement
was induced by in vivo generated NO, the mice were pretreated
with an iNOS inhibitor N-nitro-arginine methyl ester (L-
NAME) for 6 h, and then with 5 μg/kg LPS and 300 mg/kg D-
GalN. CLSM examination in Figure 6D showed a dramatic
decrease of red fluorescence with an IR/IG ratio of 0.3 due to
the suppressed NO generation by L-NAME, further suggesting
the great potential of MBTD to investigate the regulation
mechanism of endogenous NO by iNOS in the inflammation.
NO has been defined as one of indicators reflecting the
degree of inflammation.58,59 Meanwhile, N-acetylcysteine
(NAC) is a FDA-approved drug for clinical treatment of the
Synthesis, flow cytometric examination, theoretical
calculation, and supplementary figures (PDF).
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: zaxu@chem.ecnu.edu.cn. Tel./Fax: +86-21-54340053.
*E-mail: chding@simm.ac.cn. Tel./Fax: +86-21-50806600.
ORCID
Zhiai Xu: 0000-0002-4391-2507
Yi Luo: 0000-0001-6390-8639
Zhen Cheng: 0000-0001-8177-9463
Haijun Yu: 0000-0002-3398-0880
Ao Zhang: 0000-0001-7205-9202
Chunyong Ding: 0000-0003-2379-8740
Author Contributions
†These authors contributed equally to this work (F.W. and
S.Y.).
Notes
The authors declare no competing financial interest.
I
DOI: 10.1021/acs.analchem.8b00612
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(29) Sun, X.; Xu, Y.; Zhu, W.; He, C.; Xu, L.; Yang, Y.; Qian, X. Anal.
Methods 2012, 4, 919−922.
ACKNOWLEDGMENTS
■
The authors thank the assistance from Prof. Guang Liang at
Wenzhou Medical University for in vivo liver infection model,
and Dr. Chengyuan Peng and Prof. Ruimin Huang at SIMM for
cell imaging. Financial supports by the National Natural
Science Foundation of China (Nos. 21675055, 81402790,
21775046, 21635003) are greatly appreciated. Financial
supports from the International Cooperative Program of the
Chinese Academy of Sciences (GJHZ1622) and the Key
Program of the Frontier Science of the Chinese Academy of
Sciences (160621) were also appreciated.
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