A rapid response near-infrared ratiometric fluorescent probe for the real-time tracking of peroxynitrite for pathological diagnosis and therapeutic assessment in a rheumatoid arthritis model

Article abstract of DOI:10.1039/d0tb01970d

Peroxynitrite (ONOO-) is a potent bio-oxidant involved in many physiological and pathological processes; however, most of the pathological effects associated with ONOO-in vivo are still ambiguous. Herein, we designed and synthesized two near-infrared ratiometric fluorescent probes, Ratio-A and Ratio-B, for the detection and biological evaluation of ONOO-. The recognition unit diene in the probes could be specifically cleaved by ONOO- with a 94-fold enhancement in the ratiometric fluorescence signal. By imaging ONOO- in immune stimulated cells and acute inflammation mice model using Ratio-A, we investigated the fluctuations of ONOO- levels in a rheumatoid arthritis (RA) model of mice. Ratio-A could be applied for the effective imaging of RA and could rapidly evaluate the response of the RA treatment with methotrexate (MTX). Thus, Ratio-A can be considered as a promising tool for pathological diagnosis and the therapeutic assessment of a wide range of diseases including RA.

Full text of DOI:10.1039/d0tb01970d

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Wei Wei, Guanghui Ma et al.
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J Po lue ran s ae l od fo Mn aot te rai ad l jsu Cs th em mai rs gt ri ny sB
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A Rapid Response Near-infrared Ratiometric Fluore
D
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Enabled In Real-Time Peroxynitrite Tracking for Pathological
Diagnosing and Therapeutic Assessment in Rheumatoid Arthritis
Model
Received 00th January 20xx,
Accepted 00th January 20xx
†
†*
†
†
†
†
†*
Jiao Lu , Zan Li , Xinrui Zheng , Jiangkun Tan , Zhongyin Ji , Zhiwei Sun , Jinmao You
DOI: 10.1039/x0xx00000x
−
Peroxynitrite (ONOO ) is a potent bio-oxidant involved in many physiological and pathological processes,
−
however, most of the pathological effects associated with ONOO in vivo are still ambiguous. Herein, we design
−
and synthesize two near-infrared ratiometric fluorescent probes Ratio-A and Ratio-B for ONOO detection and
−
biological evaluation. The recognition unit diene in the probes can be specifically cleaved by ONOO with a
−
ratiometric fluorescence signal enhancement of 94-fold. With the support of ONOO imaging in immune
−
stimulated cells and acute inflammation mice assisted by Ratio-A, we exploited to visualize ONOO fluctuations
in rheumatoid arthritis (RA) of mice. Ratio-A could be applied for effectively imaging of RA and rapidly evaluate
the treatment response of RA by methotrexate (MTX). It is envisioned that Ratio-A is a promising tool for
pathological diagnosing and therapeutic assessment in a wide range of diseases, including RA.
effective internal calibration can avoid these interferences.
−
Introduction
As a diffusion-controlled reaction product, peroxynitrite (ONOO−)
Therefore, ratiometric fluorescent probes for ONOO detection
are urgently required.
Rheumatoid arthritis (RA) is a chronic inflammatory disorder,
characterized by hyperplasia of synovium, inflamed joints, bone
damage and even disability. RA is an autoimmune disease of
unknown etiology, however, studies indicate that oxidative
damage induced by reactive oxygen species (ROS) is related to
As a reactive oxidant, ONOO is
utilized by phagocytes to kill invading microorganisms,
simultaneously inflicts damage on adjacent tissues, and is
is formed by fusion of nitric oxide radical (•NO) and superoxide
•
−
1-3
−
(O
2
). Being a strong oxidant and nucleophile, ONOO can damage
2
1
a wide range of critical molecular components in cells, including
4
,5
proteins, lipids, and DNA, and eventually causing cell death. There
are research evidences demonstrate that ONOO is the main cause
−
2
2,23
−
the pathophysiology of RA.
•
-
of cytotoxicity, which is previously attributed to the precursors O
2
6
,7
−
and •NO. Therefore, ONOO has been linked to a growing list of
diseases, such as neurological disorders, cancer, rheumatoid
2
4
8
,9
deemed to be of pathogenic significance in RA progression.
arthritis, inflammatory and autoimmune diseases. Emerging data
have shown that ONOO can be produced by immune cells such as
macrophages to kill invading microorganisms to play protective role
in immune system. Studies indicate that ONOO also plays crucial
roles in the regulation of pivotal signaling pathways by nitrosylation.
To deep investigate the pathophysiological roles of ONOO , there is
an imperious demand to develop a method to sensitively and
unambiguously detect ONOO both in vitro and in vivo. As ONOO
has short half-life (< 10 ms) and exhibits low steady-state
concentration (nM range),
efficiently detect ONOO in vitro and in vivo.
Fluorescence imaging, which features sensitivity, selectivity,
being noninvasive and real-time spatial imaging capacity, is an
attractive tool for ONOO detection.
−
However, the exact roles of ONOO in RA are still needed to be
further investigated. In addition, the assessment of RA
treatment effect in mice with antiarthritic drug, methotrexate
−
1
0
−
2
5,26
(MTX)
is lacking visual tool. Therefore, it is still desirable to
−
develop ratiometric probes for monitoring ONOO fluctuation
in RA progress, for directly reporting oxidative stress during
treatment and to get deep insights into the drug therapeutic
mechanism.
Ratio-A and Ratio-B were constructed based on a modified
which exhibited an emission
maximum at 564 nm. Zhang’s group have reported a
mitochondria targeted probe based on Rhodamine scaffold,
the ratiometric probe Mito-WQPhOH was successfully applied
in living cells to monitor ONOO . The differences of the previous
work is that we have changed the conjugating mode by
optimizing the sensing moietys. Therefore, the emission
wavelength of our probes are extended to the near infrared (≥
−
−
−
1
1,12
it is still a challenging issue to
2
7,28
Rhodamine scaffold QF,
−
4
0
−
13-15
During the past several
−
years, numerous fluorescent probes have been constructed for
−
ONOO detection, which enable us to understand the roles of
−
16-20
ONOO in specific biological progresses.
However, most of
these probes are intensity-based mode, the application is
susceptible by variations from performance of instruments,
intracellular microenvironment, local concentrations of the
probe and photobleaching. Ratiometric fluorescent probes with
7
00 nm) region, and the Stokes shift, response time and
detection limit of our probes have been considerably improved.
It is anticipated that our probes could be employed in living
cells, for diagnosis and therapeutic assessment in rheumatoid
arthritis model.
2
. Experiments Section
†The Key Laboratory of Life-Organic Analysis, Key Laboratory of Pharmaceutical
Intermediates and Analysis of Natural Medicine, School of Chemistry and Chemical
Engineering, Qufu Normal University, Qufu 273165, P.R. China.
E-mail: lilizanzan@163.com, jmyou6304@163.com
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
2
.1. Synthesis
2.1.1 Synthesis of QF
QF was synthesized according to the previous literature.
*
29
Cyclohexanone (441 mg, 4.5 mmol, 1.5 equivalents) was dropped
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Journal Name
into the ice-cooled concentrated H SO (15 mL) and then stirred for temperature. After completion of the reaction (aVsiewmAortinclietOonrleinde
2 4
3
0 min, 940 mg 2-(4-diethylamino-2-hydroxybenzoyl) benzoic acid (3 by TLC), the mixture was concentDrOatI:e1d0.1u0n39d/eDr0TrBe0d1u9c70eDd
mmol) was dissolved into the above solution in portions and then pressure and extracted with dichloromethane. The organic
heated to 95 °C for 3 h. The solution was cooled down to room layer was dried over Na SO and concentrated under vacuum.
2
4
temperature and poured into 100 g of ice. Afterwards, 70 % And then the products were purified by silica gel
perchloric acid (5 mL) was dropped to the above solution slowly to chromatography with DCM / methanol (100 : 1 to 30 : 1, and
obtain red precipitate. The precipitate was filtered off, and then 100 : 1 to 25:1 v/v) to obtain white solid P2 (1.97 mg, 26.1%)
1
washed with 100 mL cold water. Finally, the precipitate was and clay bank solid P1 (4.38 mg, 22.5%). P1: H NMR (400 MHz,
recrystallized with 5 mL of acetic acid to obtain purple solid (993.17 CDCl
3
) 7.97 (1 H, t, J 6.6), 7.69 (1 H, td, J 7.5, 1.1), 7.59 (1 H, dd,
1
3
mg, 83 %). H NMR (500 MHz, CDCl ) δ 8.27 (d, J = 7.7 Hz, 1H), 7.75 J 7.5, 0.8), 7.21 (1 H, t, J 6.6), 6.52 (1 H, d, J 2.6), 6.49 (1 H, d),
(
t, J = 7.3 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.4 Hz, 1H), 7.06 6.37 (1 H, dd, J 9.0, 2.6), 3.33 (4 H, q, J 7.1), 2.63 (2 H, m), 2.27
1
3
(dd, J = 16.5, 9.5 Hz, 2H), 6.85 (s, 1H), 3.61 (d, J = 7.0 Hz, 4H), 3.09 (s, (1 H, m), 1.97 (2 H, m),1.78 (1 H, m), 1.14 (6 H, t, J 7.1). C NMR
2
6
1
1
2
H), 2.33 – 2.16 (m, 2H), 1.96 (s, 2H), 1.76 (d, J = 5.1 Hz, 2H), 1.30 (s, (101 MHz, CDCl
3
) 192.29, 169.55, 151.93, 151.42, 149.58,
1
3
3
H). C NMR (126 MHz, CDCl ) δ 170.16, 166.59, 165.31, 159.53, 144.04, 135.04, 129.87, 128.34, 126.80, 125.22, 124.68, 123.43,
55.71, 134.30, 133.30, 131.84, 130.36, 130.27, 129.17, 128.43, 109.39, 103.32, 97.73, 84.39 , 44.45, 38.18, 22.90, 21.89, 12.47.
21.94, 118.03, 117.48, 95.48, 54.83, 46.23, 29.58, 25.10, 20.89, HR-MS: Calcd. P1, 390.1700, found: 390.1700. P2: H NMR (400
1
0.84. HR-MS: Calcd. QF, 376.1907, found: 376.1901.
.1.2 Synthesis of Ratio-A
Compound QF (951.4
MHz, DMSO) 12.61 (1 H, s), 7.93-7.87 (2 H, m), 7.04-7.00 (2 H,
m), 3.83 (3 H, s). C NMR (101 MHz, DMSO) 167.47, 163.30,
4- 131.80, 123.42, 114.27, 55.89. HR-MS: Calcd. P2, 151.0401,
1
3
2
mg,
2
mmol)
and
Methoxycinnamaldehyde (427.6 mg, 2.4 mmol, 1.2 equivalents) found: 151.0406.
were dissolved in 15 mL ethanol and stirred for 8 h under reflux. The 2.2. Evaluation of Ratio-A for ONOO imaging in live cells
−
ethanol was removed under reduced pressure, and the obtained
solid was purified by silica gel chromatography with DCM / methanol
RAW264.7 cells were incubated in DMEM medium with 10 %
FBS at 37 °C, the humidified CO
selectivity of Ratio-A for exogenous ONOO imaging in living
2
atmosphere was set as 5 %. The
1
(
100 : 1 to 20 : 1, v/v) to obtain green solid (906.88 mg, 87.2%). H
NMR (500 MHz, CDCl ) δ 8.09 (d, J = 6.3 Hz, 1H), 7.65 (d, J = 10.7 Hz,
H), 7.58 (t, J = 7.0 Hz, 1H), 7.48 (d, J = 8.2 Hz, 3H), 7.17 (d, J = 14.9
Hz, 1H), 7.00 (t, J = 11.2 Hz, 2H), 6.86 (d, J = 8.3 Hz, 2H), 6.80 (d, J =
−
3
cells was validated by employing ROS and RNS donors. 3-
1
morpholinosydnonimine hydrochloride (SIN-1) was used for
− 30
generating ONOO .
•NO was generated from 3, 3-
1
3
1.1 Hz, 1H), 6.77 (d, J = 8.9 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 3.80 (s,
H), 3.50 (d, J = 6.2 Hz, 4H), 2.64 (s, 2H), 2.23 (s, 1H), 1.99 (d, J = 14.1
•−
bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18), O
2
was
from menadione sodium bisulfite (MSB), and FeTMPyP was
1
3
Hz, 1H), 1.66 (d, J = 24.3 Hz, 2H), 1.23 (dd, J = 13.6, 7.0 Hz, 6H).
C
− 18,31
used for consuming ONOO .
Ratio-A were co-incubated
3
NMR (126 MHz, CDCl ) δ 169.68, 160.58, 156.44, 153.28, 141.51,
with RAW264.7 cells for 30 min, after that the cells were stained
with 4, 6-diamidino-2-phenylindole (DAPI, 100 ng/mL, 1 μL) and
then washed with phosphate buffered saline (PBS), 50 μM SIN-
1
1
1
35.16, 132.16, 132.01, 129.73, 129.67, 129.52, 129.16, 126.69,
22.08, 114.27, 114.19, 96.23, 55.42, 45.55, 29.67, 25.15, 21.17,
2.63. HR-MS: Calcd. Ratio-A, 520.2482, found: 520.2483.
1
, 500 μM NOC-18, 100 μM MSB, 50 μM SIN-1 and 50 μM
FeTMPyP were co-incubated for 1 h, respectively. Under the
confocal fluorescence microscope, the excitation was set as 488
nm and 594 nm, corresponding emission were set as 520-600
nm (green channel) and 670 nm-750 nm (red channel), blue
channel: 430-480 nm (λex=405 nm).
The evaluation of intracellular retention of Ratio-A and the
stability of fluorescent product. RAW264.7 cells were stained
with Ratio-A (10 μM) for 30 min. the control group was treated
with PBS solution, and the experimental group were treated
with 50 μM SIN-1 for 30 min. The medium was removed and
cells were washed with PBS for three times, and then images
were acquired at indicated time points.
2
.1.3 Synthesis of Ratio-B
Compound QF (951.4 mg, 2 mmol) and 4-(dimethylamino)
cinnamaldehyde (420.5 mg, 2.4 mmol, 1.2 equivalents) were
dissolved in 15 mL ethanol and stirred for 8 h under reflux. The
ethanol was removed under reduced pressure and the obtained solid
was purified by silica gel chromatography with DCM / methanol (100
1
:
1 to 15 : 1, v/v) to obtain green solid (891.17 mg, 83.6 %). H NMR
(500 MHz, CDCl
3
) δ 8.16 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 11.4 Hz, 1H),
7
7
6
=
.57 (t, J = 7.3 Hz, 1H), 7.50 (t, J = 7.4 Hz, 1H), 7.44 (d, J = 8.3 Hz, 2H),
.13 (d, J = 14.9 Hz, 1H), 7.02 (d, J = 7.3 Hz, 1H), 6.99 – 6.93 (m, 1H),
.79 (d, J = 9.1 Hz, 1H), 6.71 (s, 1H), 6.67 (d, J = 8.3 Hz, 2H), 6.61 (d, J
9.1 Hz, 1H), 3.49 (d, J = 6.9 Hz, 4H), 3.02 (s, 6H), 2.65 (s, 2H), 2.30
The assessment of Ratio-A to visualize the production of
(
s, 1H), 2.04 (d, J = 16.2 Hz, 1H), 1.71 (d, J = 34.9 Hz, 2H), 1.24 (s, 6H).
−
endogenous ONOO . RAW264.7 cells were stimulated with 1
1
3
3
C NMR (126 MHz, CDCl ) δ 169.08, 158.19, 156.24, 152.83, 151.30,
μg/mL lipopolysaccharides (LPS), 100 ng/mL of interferon-
gamma (IFN-γ) and 10 nM phorbol 12-myristate 13-acetate
1
1
1
43.07, 136.11, 132.36, 132.00, 129.66, 129.52, 129.32, 126.51,
24.99, 124.87, 119.84, 118.03, 118.00, 113.46, 113.13, 112.21,
05.01, 96.06, 53.37, 45.28, 40.26, 25.18, 21.16, 12.77. HR-MS:
3
2
(
(
PMA). Aminoguanidine (AG, iNOS inhibitor) and apocynin
3
3
NOX inhibitor) were used to conduct enzyme inhibition assay.
Calcd. Ratio-B, 533.2799, found: 533.2792.
RAW264.7 cells were divided into four groups. The first group
was set as control group; the second group was pre-incubated
with LPS and IFN-γ for 15 h, followed by PMA for 1 h; the third
group was incubated with 5 mM AG for 4 h before LPS and IFN-γ
2
.1.4 Preparation of P1 and P2
−
A mixture of Ratio-A (31 mg, 0.05mmol) and ONOO (2.82
mmol/L, 35 mL) in 10 mL PBS solution was stirred at room
4
| J. Name., 2012, 00, 1-3
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for 15 h, and 10 nM PMA for 1 h; the fourth group was pre- P2 in Ratio-A (calculated value: 151.0401) (Figure S14). Zhang’s
View Article Online
DOI: 10.1039/D0TB01970D
treated with 100 µM apocynin for 1 h, LPS and IFN-γ for 15 h group has reported the formation of compound P1 and
4
0
and then 10 nM PMA for 1 h. Ratio-A was incubated for 30 min confirmed the exact mass. And then we isolated the products
−
before imaging.
2
after reaction with ONOO , P1 and P2 were purified and
−
1
13
.3. ONOO imaging in acute inflammation model and CIA charactered by HNMR and CNMR to further confirm the
mice model.
proposed sensing mechanism. The corresponding products of
All animal experiments were performed according to the Ratio-B were also studied and confirmed by HR-MS (Figure
guidelines issued by The Ethical Committee of Qufu Normal S15). HPLC analysis was further used to verify the response
University (approval No. QF2018-015L). The evaluation of mechanism during detection process of Ratio-A towards
−
−
endogenous ONOO levels in acute inflammation mouse ONOO . As shown in Figure S16, the peaks at 8.07 min and 10.13
model.34 Three groups of Kunming mice were employed, the min were belonged to the carbonyl fluorophore and carboxylic
first group mice were treated with saline; the experimental phenyl compound, and the two peaks were appeared after
−
group mice were administered with intraperitoneal (i.p.) addition of ONOO , while the peak at 4.82 min (Ratio-A) was
injection of 100 μL LPS (200 μM in saline) to induce peritonitis; diminished. The results were consistent with the HR-MS data,
the enzyme activity blocking group was treated with 4 mM which was suggested that the products of Ratio-A were
−
apocynin followed by 100 μL LPS (200 μM in saline), 100 μL generated after the reaction with ONOO .
Ratio-A (200 μM in saline) was injected in the same position 4 h
later. In vivo images were acquired at 30 min.
Collagen-induced arthritis (CIA) model was established in
male Kunming mice (8-10 weeks) according to the published
approach. To establish CIA model, all Kunming mice were
randomly divided into four groups. Three groups of mice were
injected with 2.0 mg/mL Bovine type II collagen (BCol II)
intradermally, which was emulsified in equal volume of
complete 2.0 mg/mL of Freund's Adjuvant (CFA) in tail. A
booster immunization was operated at 21 days after the
primary immunization, which was treated with BCol II, equally
3
5
emulsified in incomplete Freund's Adjuvant (IFA). To evaluate
the therapeutic efficacies of glutathione (GSH) and MTX, one
group of the CIA mice were administrated with saline, another
two groups of CIA mice were administrated with GSH (200
mg/kg body weight) and MTX (5.0 mg/kg body weight), Scheme 1. Structures and the proposed mechanism toward ONOO . (A) The
respectively. All mice were intravenously administrated every 3 ONOO .
−
synthesis of Ratio-A and Ratio-B; (B) The proposed mechanism of Ratio-A toward
−
days, and all treatments were repeated six times for each group.
Fluorescence imaging was performed at 3 days after the last 3.2. Spectroscopic properties of Ratio-A and Ratio-B
injection.
The spectroscopic properties of Ratio-A (10 μM) and Ratio-B
10 μM) were evaluated in physiological condition (20 mM PBS,
(
pH 7.4, 0.1% DMSO). The UV-vis absorption responses of Ratio-
A and Ratio-B were investigated. As shown in Figure 1A, the free
probe Ratio-A possessed a protruding absorption peak at 600
3
. Results and discussions
3
.1. Design and Synthesis of ratiometric fluorescent probes
−
nm and a weak peak at 414 nm. After the addition of ONOO
Rhodamine dyes has excellent optical characteristics, such as
3
6
with the concentration of 0-10 μM, the absorption peak at 600
nm was reduced and the the absorption peak at 414 nm was
enhanced. Meanwhile, the solution colour was changed from
blue to yellow (Figure S21). We then tested the fluorescence
good photo-stability and high fluorescence quantum yields.
However, the Stokes shift of Rhodamine derivatives is generally
less than 50 nm, which results in background interference in
fluorescence imaging. We selected the structural improved
Rhodamine fluophore, QF for skeleton and obtained Ratio-A
and Ratio-B (Scheme 1) after condensing with cinnamaldehydes
with QF. The Stokes shifts of Ratio-A and Ratio-B were more
than 100 nm. Furthermore, the probes can avoid interference
factors by showing optical changes at two emission bands.
The detection mechanism towards ONOO is anticipated that
butadienyl bridge is oxidized into a carbonyl fluorophore, which
is confirmed by high resolution mass spectrum.
−
response of Ratio-A to different concentrations of ONOO , and
the corresponding fluorescence emission peak at 564 nm was
enhanced gradually as well as the fluorescence emission at 700
nm was decreased (Figure 1B and 1C). The fluorescence changes
7,38 can be ascribed to the disruption of ICT process induced by a
3
−
−
specific ONOO -triggered butadienyl bridge cleavage. There
2
was a linear (R = 0.993) relationship between logarithm of the
−
3
9,40
fluorescence ratio (F564 nm/F700 nm) and ONOO concentration (0-
The mass
1
0 µM) (Figure 1D). The detection limit was calculated as 28.06
signal (m/z) at 390.1701 indicated the carbonyl fluorophore P1
nM based on 3σ/k. Similarly, the absorption peaks of Ratio-B
were 680 nm and 414 nm (Figure S2A). The solution colour was
changed from glaucous to yellow (Figure S21). Upon addition of
(calculated value: 390.1700) was formed, and the mass signal
(m/z) at 151.0406 represented the carboxylic phenyl compound
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−
ONOO , corresponding emission peak at 808 nm was gradually distinct changes in the ratio values of Ratio-B. The pH effects
View Article Online
DOI: 10.1039/D0TB01970D
diminished, and the emission peak at 564 nm increased on the Ratio-A and its sensing reaction in different pH buffer
gradually (Figure. S2B, 2C). The linear (R = 0.967) relationship were also investigated. The results showed that Ratio-A was
between logarithm of the ratio (F564 nm /F808 nm) and ONOO (0- stable in the range of pH 4.5‒8.5, and the recognition of ONOO
0 µM) was shown in Figure S2D. The detection limit was
2
−
−
1
was not affected in physiological condition (Figure S3).
calculated as 652.03 nM. The absolute quantum yields of Ratio- Furthermore, the fluorescence ratio values of Ratio-A were
A (Φ = 4.67 %) at 700 nm were much higher than Ratio-B (Φ = investigated in varied polarity of solvents (Figure. S4). The
0
(
.068 %). The fluorescence lifetimes of Ratio-A were 4.3226 ns results showed that the ratio values were essentially constant,
dominant, 85.32 %) and 100 ns (minor, 14.68 %) (Figure S19), which indicated that Ratio-A was insensitive to different
whereas the lifetimes of Ratio-B were 0.6965 ns (dominant, polarity.
6
.15%) and 4.4136 ns (minor, 93.85%) on account of the various
Characteristics of high stability and fast recognition are necessary
to the desirable probes. The reaction kinetics of Ratio-A on ONOO
−
installed groups (Figure S20).
was studied. As shown in Figure 1E, the fluorescence ratio values of
Ratio-A reached maximum in 10 s and stayed constant for 15 min
−
after the addition of ONOO . The reaction kinetics of Ratio-B on
−
ONOO was shown in Figure S2E, Ratio-B also exhibits fast
recognition and stability.
−
3
2
.4. Exogenous and endogenous ONOO imaging in RAW
64.7 cells
After investigating the chemical performance of Ratio-A, we
next evaluated the capability of Ratio-A in RAW264.7 cells for
−
detection of exogenous and endogenous ONOO . Cytotoxicity
was assessed by Cells Counting Kit-8 (CCK-8) assay. As illustrated
in Figure S17, Ratio-A showed low cytotoxicity even in the
concentration of 100 μM, thus the probe was suitable for living
cell imaging. RAW264.7 cells were selected as the cell model,
ROS and RNS donors were employed to evaluate the selectivity
of Ratio-A in RAW264.7 cells. Fluorescence imaging was
displayed in Figure 2A, and the pseudo-color ratiometric images
were presented to illustrate the ratios of green channel versus
red channel. The fluorescence signal in the control group
showed weak fluorescence in green channel and strong
fluorescence in red channel, which were consistent with the
fluorescence spectra of free probe Ratio-A (Figure 2B, 2C). As
shown in Figure 2A, the blue channel indicated that the probes
did not enter into the nucleus of the cells. Obvious
enhancement of the intracellular fluorescence ratios were
observed after treating with SIN-1, rather than treated with
Figure 1. Spectral properties of Ratio-A (10 μM) in PBS solution (pH=7.4, 20 mM, with
−
0
.1% DMSO). Data were recorded 10 min after the addition of ONOO . (A) UV absorption
−
spectra of Ratio-A in the presence of ONOO (0−10 μM). (B) Emission spectra of Ratio-A
−
at 564 nm with concentrations of ONOO (0−10μM). (λex = 480 nm). (C) Emission spectra NOC-18 and MSB. The increased fluorescence ratio in SIN-1-
of Ratio-A at 700 nm (λex = 600 nm). (D) The linear relationship between log(F564nm/F700nm
)
treated cells were attenuated after addition of
−
−
and ONOO . Insert: ratio values with different concentrations of ONOO . (E) Time
−
ONOO scavenger FeTMPyP, which was suggested that Ratio-A
−
dependent fluorescence ratios (F564 nm/F700 nm) of Ratio-A toward ONOO (0 μM, 10 μM)
exhibited significant stability to other biological species and
during 0-900 s, probe was added at 120 s. (F) Ratiometric fluorescence responses
−
564nm/F700nm) of Ratio-A toward ONOO− (10.0 μM) and other ROS/RNS/RSS (20
showed high selectivity toward ONOO in living cells. The
(F
equivalents), metal ions (20 equivalents). The error bars represent ± S.D. (n=3).
ratiometric fluorescence variations in flow cytometry analysis
further verified the selective ONOO detecting capacity of
Ratio-A (Figure 2A and 2B).
−
3
.3. Selectivity studies of Ratio-A and Ratio-B
The intracellular retention of Ratio-A was evaluated by
means of fluorescence ratio values (Fgreen/Fred) (Figure S18A),
and the ratio values of Ratio-A in RAW264.7 cells stayed
constant over two hours (Figure S18C). The fluorescence ratio
values were further confirmed by flow cytometry analysis
(Figure S18B). The results suggested that Ratio-A and the
reaction products can retain in cells due to the negatively
To further evaluate the selectivity of Ratio-A and Ratio-B
−
toward ONOO , the fluorescence spectra were recorded in the
absence and presence of various biologically relevant species.
The results of Ratio-A were illustrated in Figure 1F. Ratio-A
exhibited 94-fold ratiometric fluorescence enhancement
−
towards ONOO compared with the blank group, while other
species caused negligible ratiometric signal changes even in the
presence of 20 equivalents. The results of Ratio-B were shown
in Figure S2F, the •OH radical caused a 12-fold ratiometric
4
1
charged carboxylates.
Ratio-A was used to explore the imaging of endogenous ONOO
−
1
in RAW264.7 cells after stimulation. As shown in Figure 3A and
2 2 2
enhancement. •NO radicals, TBHP, O and H O can induce
4
| J. Name., 2012, 00, 1-3
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C, the ratio of green channel versus red channel was enhanced
View Article Online
DOI: 10.1039/D0TB01970D
distinctly after stimulation with LPS/IFN-γ/PMA.
Figure 2. Validation the selectivity of Ratio-A (10 μM) with ROS/RNS donors by confocal imaging and flow cytometry analysis in RAW264.7 cells. (A). Cells were loaded
−
•−. FeTMPyP (50 μM) was used to
with Ratio-A and then treated with SIN-1 (50 μM), NOC-18 (500 μM), and MSB (100 μM) respectively to produce ONOO , •NO, O
2
−
decomposite ONOO . (B). Flow cytometry analysis of the cells in corresponding group of (A). (C) The fluorescence ratio values (Fgreen/Fred) of (A). Scale bar represents 20
μm. The experiments were repeated three times and the data were shown as mean (± S.D.).
−
Figure 3. Confocal imaging of endogenous ONOO with Ratio-A (10 μM) in stimulated RAW264.7 cells. (A) Cells were treated with LPS (1μg/ml), and IFN-γ (100 ng/mL), followed by
PMA (10 nM), and then stained with Ratio-A before imaging. AG (5 mM) and apocynin (100 μM) were added at the onset of immune stimulation to reduce the level of •NO or O
respectively. (B). Flow cytometry analysis of the cells in corresponding group of (A). (C) The fluorescence ratio values (Fgreen/Fred) of (A). Scale bar represents 20 μm. The
experiments were repeated three times, the error bars represent ± S.D. (n=3)
2
•−,
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2
4
After the cells were incubated with NOX inhibitor, apocynin was thought to be of pathogenic significance in RA. the
DOI: 10.1039/D0TB01970D
(100 μM) and iNOS inhibitor aminoguanidine (AG, 5 mM), the possibility of Ratio-A for assessing the treatment of RA model in
green fluorescence of Ratio-A was effectively suppressed. mice. As shown in Figure 5A and 5B, CIA mice treated with saline
These results demonstrated that the fluorescence changes of showed a significant decrease in red fluorescence channel and
−
Ratio-A in murine RAW264.7 cells can ascribed to the ONOO an obvious enhancement in green fluorescence channel after
generation. The fluorescence ratios detected by flow cytometry injected with Ratio-A, while the fluorescence ratio signal
analysis were consistent with the cell imaging using confocal (Fgreen/Fred) at the ankle joints were reduced after treating with
microscope (Figure 3B)
.
Collectively, it was suggested that GSH and MTX. The above results indicated that Ratio-A was
−
−
Ratio-A was suitable for ONOO selectively imaging in living capable of monitoring ONOO in CIA mice, and GSH as well as
−
45,46
cells.
MTX can restrain the levels of ONOO .
Subsequently, the
−
3
.5. ONOO -related inflammatory response in mice
mice were sacrificed, and the knee joints were collected.
Histological analysis of knee joints was performed by
hematoxylin and eosin (H&E) staining. As shown in Figure 5C,
the articular cartilages were eroded and became roughly and
thinly in the RA mice model, the blue stained inflammatory cells
invaded into the hyperplastic synovium. It was suggested that
the degree of ankle tissue injury treated with MTX had been
alleviated. The above experiments results demonstrated that
Ratio-A can be utilized for evaluating the variations of
−
Studies demonstrated that excessively endogenous ONOO
8
production is a pivotal mediator in inflammation. Based on the
−
ONOO imaging capacity of Ratio-A, we further applied Ratio-A
−
for visualizing endogenous ONOO in acute inflammation mice
model. As displayed in Figure 4, the fluorescence intensity from
experimental group was remarkably increased in green channel,
while the fluorescence intensity in red channel was obviously
decreased compared with control group. As was shown in
Figure 4b, the ratio was increased by 30 times at 30 min
compared with control group. While the ratio of apocynin
treated group stayed unchanged (Figure 4c), which indicated
−
ONOO levels in CIA mice model. As an effective fluorescent
probe, Ratio-A can be used for RA diagnosis by monitoring
−
ONOO fluctuations.
−
that the generation of ONOO was blocked after treating with
apocynin. The results revealed that ONOO was transported and
−
accumulated in the inflamed sites. Thus, Ratio-A could be
−
employed to visualize the generation of endogenous ONOO in
acute inflammation mice model.
−
Figure 5. Visualization of ONOO -mediated inflammatory response in the RA model. (A).
Fluorescence images of mice in group a–d. a：control group; b: RA mice; c: GSH treated
RA mice; d: MTX treated RA mice. The mice were injected with Ratio-A (50 μM) before
imaging. (B). Fluorescence ratio values (Fgreen/Fred) in (A). (C). The corresponding H&E
staining of ankle joints tissues. Scale bar: 200 μm.
4
Conclusion
Figure 4. Imaging of endogenous ONOO− in acute inflammatory mice. Red channel:
In summary, two ratiometric fluorescent probes, Ratio-A and
6
70−750 nm, green channel: 520−600 nm. (A). (a). The control group were treated with
−
Ratio-B were synthesized for ONOO detection. Ratio-A
saline, and Ratio-A was injected into the inflamed site. (b). Mice were stimulated with
LPS for 4 h, and then the Ratio-A was injected. (c). Mice were treated with apocynin, and
then stimulated with LPS. Images were acquired at 30 min. (B). The ratiometric
fluorescence intensity (Fgreen/Fred) of (A). The experiments were repeated three times and mechanism of Ratio-A towards ONOO was confirmed by HR-
the data were shown as mean (± S.D.).
−
exhibited excellent selectivity towards ONOO compared to
Ratio-B under physiological conditions. The responding
−
1
13
MS and HPLC analysis, and HNMR, CNMR. Ratio-A exhibited
−
high sensitivity for ONOO with a 94-fold ratio enhancement
3
.6. Imaging of RA in Mice
and excellent selectivity over various biological species, the
Autoimmune arthritis models have become valuable research detection limit was as low as 28.06 nM. Moreover, the large
models for studying pathogenic mechanisms and developing stokes shift (> 100 nm) and low cytotoxicity endowed Ratio-A
new therapies.42 CIA model was widely used for studying the with the capacity of real time monitoring intracellular ONOO
−
4
3
process of rheumatoid arthritis (RA). There was an increasing fluctuations in immune stimulated RAW264.7 cells and acute
evidence to indicate that ROS play a vital role in the RA inflammatory model. Importantly, the ONOO− related
pathogenesis.2
3,44
The activated phagocytic cells, including inflammatory response in RA and the response of RA treatment
neutrophils and macrophages, can produce highly toxic ROS in were successfully demonstrated with the assist of Ratio-A. It is
the process of oxidative burst to kill the invading pathogens. envisioned that Ratio-A can contribute to future clinical
−
•−
ONOO was then formed by the fusion of •NO and O
2
, which researches on RA diagnosis, treatment response monitoring,
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25 S. Majumdar, M. E. Anderson, C. R. Xu, T. V. YaVkieowvAlertvicale,OLn.liCne.
Gu, T. R. Malefyt and T. J. Siahaan, J. .,
01, 3275–3291.
and the etiology studies of ONOO -mediated inflammatory
−
Pharm. Sci-U0TS 2012,
DOI: 10.1039/D B01970D
disorders, which can further illuminate the ONOO biology.
1
2
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
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Page 8 of 8
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DOI: 10.1039/D0TB01970D
−
Endogenous ONOO generation in rheumatoid arthritis mice was visualized and
confirmed assisted by ratiometric fluorescent probe Ratio-A