Activity-based near-infrared fluorogenic probe for enabling in vitro and in vivo profiling of neutrophil elastase

Article abstract of DOI:10.1021/acs.analchem.8b04455

Neutrophil elastase (NE), a typical hematopoietic serine protease, has significant roles in inflammatory and immune responses, and thus is highly associated with various diseases such as acute lung injury (ALI) and lung cancer. Rapid and accurate measurement of NE activity in biological systems is particularly important for understanding the role of NE in inflammatory diseases, as well as clinical diagnosis. However, the specific detection and noninvasive imaging of NE in vivo remains a challenge. To address this issue, a small-molecule substrate based near-infrared fluorogenic probe (NEP) for NE was constructed via incorporating pentafluoroethyl as the recognition group with a hemicyanine dye-based fluorophore. This initially quenched probe possesses more than 25-fold red fluorescence enhancement upon the catalysis of human NE, and the detection limit is about 29.6 ng/mL. In addition, the high specificity and the long emission wavelength (?"_emmax = 700 nm) of NEP allowed the direct monitoring of NE-trafficking, exogenous NE uptake, and endogenous NE upregulation at the cellular level. Moreover, the successful spatiotemporal imaging of NE in ALI model mice also made it a promising new tool in clinical diagnosis for ALI and other lung diseases.

Full text of DOI:10.1021/acs.analchem.8b04455

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Article
Activity-based Near-Infrared Fluorogenic Probe Enables
In Vitro and In Vivo Profiling of Neutrophil Elastase
Shi-Yu Liu, Hao Xiong, Rong-Rong Li, Wen-Chao Yang, and Guang-Fu Yang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04455 • Publication Date (Web): 10 Jan 2019
Downloaded from http://pubs.acs.org on January 10, 2019
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Activity-based Near-Infrared Fluorogenic Probe Enables In
Vitro and In Vivo Profiling of Neutrophil Elastase
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Shi-Yu Liu,†,§ Hao Xiong,†,§ Rong-Rong Li, Wen-Chao Yang, and Guang-Fu
†
*,†
Yang*,†‡
†
Key Laboratory of Pesticide & Chemical Biology of Ministry of Education,
International Joint Research Center for Intelligent Biosensor Technology and Health,
and Chemical Biology Center, College of Chemistry, Central China Normal
University, Wuhan 430079, P.R. China
‡
Collaborative Innovation Center of Chemical Science and Engineering, Tianjin
30071, P.R. China.
—————————
§
These two authors made equal contributions.
*
Wen-Chao Yang, e-mail: tomyang@mail.ccnu.edu.cn; Tel: 86-27-67867706; Fax:
86-27-67867141; Guang-Fu Yang, e-mail: gfyang@mail.ccnu.edu.cn; Tel: 86-27-67867800;
Fax: 86-27-67867141.
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Abstract
Neutrophil elastase (NE), a typical hematopoietic serine protease, has significant roles
in inflammatory and immune responses, and thus highly associated with various
diseases such as acute lung injury (ALI) and lung cancer. Rapid and accurate
measurement of NE activity in biological systems is particularly important for
understanding the role of NE in inflammatory diseases, as well as the clinic diagnosis.
However, the specific detection and non-invasive imaging of NE in vivo remains a
challenge. To address this issue, a small-molecule substrate-based near-infrared
fluorogenic probe (NEP) for NE was constructed via incorporating pentafluoroethyl
as the recognition group with a hemicyanine dye-based fluorophore. This initially
quenched probe possesses over than 25-fold red fluorescence enhancement upon the
catalysis of human NE and the detection limit is about 29.6 ng/mL. In addition, the
high specificity and the long emission wavelength (λemmax = 700 nm) of NEP
allowed the direct monitoring of NE-trafficking, exogenous NE up taking and
endogenous NE up-regulation at the cellular level. Moreover, the successfully
spatiotemporal imaging of NE in ALI model mice also made it a promising new tool
in clinical diagnosis for ALI and other lung diseases.
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Keywords
Neutrophil elastase, fluorogenic probe, intracellular trafficking, acute lung injury.
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INTRODUCTION
Neutrophils are viewed as critical immune cells in inflammatory response for
preventing invasion by micro-organisms and clearance of extracellular pathogens.1
When neutrophils are recruited to site of inflammation, neutrophil elastase (NE) can
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promote inflammatory responses in many human diseases. Human NE (EC 3.4.21.37)
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consisting of 267 amino acid residues , is of high proteolytic activity which could
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resulted in protease-dependent tissue destruction in the inflammatory sites and also is
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implicated in regulation of cytokine network . Therefore, the activation of human NE
and its distribution is highly crucial for the regular and abnormal inflammatory
responses.
Extensive studies showed that NE was elevated in various inflammatory diseases
especially lung diseases, such as acute lung injury (ALI) and chronic obstructive
pulmonary disease (COPD).5-7 In those diseases, NE was recruited and gathered in
lung tissue, and thus caused the tissue damage and endothelial cell injury8-9 This
systemic inflammatory response syndrome induced organ dysfunction further leads to
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increased permeability, vasodilation, and activation of the coagulation cascade. Thus,
human NE is validated as a biomarker of disease progression, and a therapeutic target
for drug discovery. Selective inhibition of NE activity was found to be an effective
solution for treatment of the above diseases. Sivelestat, the only commercial human
NE inhibitor, was reported to significantly prolonged survival in animal model
study6-7 and clinical investigation . However, another clinical trial revealed that ALI
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patients treated with sivelestat did not associated with decreased mortality and a new
generation of NE selective inhibitor, AZD9668, failed in the phase Ⅱ clinical trial of
COPD.12 The failure of the above therapeutics may be partially attributed to the
insufficient understanding of the NE-related pathological mechanism. Besides, NE is
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also known as an important hallmark of lung cancer , which has a critical role in
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accelerate of lung tumor growth and promotion of tumor invasion and metastasis
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and even connected with insulin resistance . Thus, developing a tool for
spatiotemporal monitoring of NE in living cells and in vivo is of great importance,
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which would facilitate the thorough and comprehensive understanding of the function
of NE in lung diseases.
Small molecule-based fluorescent substrate for protease has aroused great
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interests over the last two decades. With the high spatial and temporal resolution , it
is capable of tracing enzymes trafficking of its subcellular location in real-time
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manner17-18, studying the related biological function of enzymes19-20 and monitoring
of regulation of protein level in cell level animal disease model in vivo21-22. Previously,
we discovered the first non-peptide-based fluorogenic substrate for elastase. However,
the green fluorophore of that probe and its limited specificity impede its application
for NE detection and imaging in living organisms. Hence, we herein report the
identification of a small molecule-based near-infrared fluorogenic probe (named NEP)
for human NE and the application in cellular an in vivo imaging (Scheme 1). On the
basis of the comprehensive characterization, NEP displays excellent features for the
specific human NE detection in physiologically relevant environment with ~25-fold
higher fluorescence enhancement and ~29.6 ng/mL of the detection limit. More
importantly, this probe enabled the spatially resolved monitoring of cellular
NE-trafficking, exogenous NE uptake and endogenous NE up-regulation. In addition,
it could image the elevated NE level in ALI model mice compared to health control.
These findings indicate that NEP could be a valuable tool to be applied in a definitive
diagnosis of ALI and molecule-level pathophysiological investigations of lung
diseases.
Scheme 1. Sensing mechanism for human NE with designed probe.
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EXPERIMENTAL SECTION
Organic synthesis.
Reagents. The reactants used in this article were all commercially available and
purchased from regular manufacturers. The solvents used in silica gel column
chromatography (CC) were purchased from Chinasun Specialty Products Co., Ltd.
and Wuhan Shenshi Chemical & Instrument Co., Ltd. Meanwhile, the silica gel
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(200-300 mesh) used in chromatography was brought from Qingdao Haiyang
Chemical Co., Ltd.
Instrumentation. The cuvette (Hellma, Germany) on the microplate reader
(SpectraMax M5, Molecular Devices) was used to measure the UV-Vis absorption
spectrum and fluorescence spectrum, while the black 96-well microplate (Corning,
America) one the same microplate reader was applied in the inhibitory studies. The
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structure of key intermediate was characterized through GC-MS (ESI) and H NMR
(Varian Mercury 400 MHz Plus), while the target compound was identified through
HRMS (MALDI; AB SCIEX TOF/TOF TM 5800 System), H/ C NMR (Varian
Mercury 400 or 600 MHz Plus), respectively. The fluorescence images were acquired
by inverted fluorescence microscope (Olympus IX71, Japan) and the in vivo imaging
study was carried out with IVIS Lumina XRMS III (PerkinElmer, USA).
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The preparation of HCY-NH
HCY-NH
intermediate HCY-NH
-nitrophenol (347 mg, 2.5 mmol) as starting materials and purified by column
2
(Scheme 2). The intermediates I-1, I-2, Cy7Cl and
were synthesized according to the published procedures.22-23 The key
2
2
(277 mg) was prepared with Cy7Cl (639 mg, 1.0 mmol) and
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chromatography (DCM/MeOH = 40/1 to 5/1, V/V) with the yield of 52.8%. H NMR
(600 MHz, DMSO-d ) δ 8.45 (d, J = 14.4 Hz, 1H), 7.68 (s, 2H), 7.50 – 7.38 (m, 2H),
6
7.36 – 7.28 (m, 1H), 6.87 (s, 1H), 6.73 (d, J = 8.6 Hz, 1H), 6.66 (s, 1H), 6.27 (d, J =
14.4 Hz, 1H), 4.27 (s, 2H), 2.69 (d, J = 18.8 Hz, 4H), 1.89 – 1.77 (m, 2H), 1.71 (s,
+
6H), 1.32 (t, J = 7.2 Hz, 3H). GC-MS (ESI) calcd for [C27
H
29
N
2
O] , 397.22; found,
397.54.
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Scheme 2. The synthesis of the intermediates I-1, I-2, Cy7Cl, I-3 and HCY-NH .
2
The synthesis of NEP (Scheme 3). In, HCY-NH (155 mg, 0.3 mmol) was added to
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the solution of anhydrous dichloromethane, and then triethylamine (606 mg, 0.6 mmol)
was added dropwise into the round-bottomed flask by a syringe at room temperature
until the reactants were sufficiently dissolved. After a while, the solution of
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,2,3,3,3-pentafluoropropanoic anhydride (CAS ID: 356-42-3; 186 mg, 0.6 mmol)
dissolved in the same dichloromethane was added slowly with a dropping funnel in an
ice bath. The reaction mixture was stirred for 3 h at room temperature until
HCY-NH
sequentially by H
after the reaction completed. Then the organic layer was dried over anhydrous
Na SO and concentrated with the rotary evaporator. The combined residue was
2
almost ran out (TLC monitoring). The reaction solution was washed
2
O (2 ×) and saturated sodium chloride solution (1×) immediately
2
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eventually separated and purified by the silica gel chromatography (DCM/MeOH =
60/1 to 20/1, V/V) to generate a purple solid NEP (72 mg) as final product with the
1
yield of 36.4%. H NMR (400 MHz, DMSO-d
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) δ 11.81 (s, 1H), 8.60 (d, J = 15.1 Hz,
1
H), 7.91 (d, J = 2.0 Hz, 2H), 7.84 (d, J = 7.4 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.68
dd, J = 8.5, 2.0 Hz, 1H), 7.62 – 7.54 (m, 2H), 7.50 (t, J = 7.4 Hz, 1H), 7.43 (s, 1H),
.68 (d, J = 15.1 Hz, 1H), 4.50 (q, J = 7.0 Hz, 2H), 3.10 (q, J = 7.4 Hz, 3H), 2.72 (dt,
J = 25.4, 6.3 Hz, 4H), 1.87 – 1.81 (m, 2H), 1.77 (s, 6H), 1.40 (t, J = 7.2 Hz, 3H), 1.18
t, J = 7.2 Hz, 6H). 13C NMR (150 MHz, DMSO-d
) δ 177.80, 159.19, 157.92,
55.68, 152.36, 145.37, 142.45, 140.97, 139.05, 131.05, 129.59, 129.06, 128.16,
(
6
(
6
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27.63, 123.00, 119.19, 117.83, 114.28, 113.56, 107.48, 105.70, 50.76, 45.55, 40.64,
+
8.67, 27.15, 23.56, 19.82, 12.91, 8.53. HRMS (MALDI) calcd for [C30
H
28
F
5
N
2
O
2
] ,
43.2065; found, 543.1636.
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Scheme 3. The synthesis of the target probe NEP.
Specific detection of NE in aqueous solution. The sensing capability of NEP (10
μM) toward human NE was tested in 10 mM phosphate buffer at different pH and
temperature. Under the optimized condition, both the time- and
concentration-dependent fluorescence response of NEP in the presence of human NE
were then measured by fluorescence increasement of the reaction system at the
maximum emission wavelength of 700 nm (λexmax = 670 nm). The specificity study
of NEP toward NE and other commonly used enzymes (0.01 mg/mL) was also
performed by evaluating the final increased fluorescence intensity (F-F
detection of NE activity in mouse serum and broncho-alveolar lavage (BAL), each
00 μL of mouse serum or BAL were pre-incubated with or without 10 μM sivelestat
0
). For the
1
for 10 min, followed by the addition of 10 μM NEP for 30 min, the enhanced
fluorescence intensity at 700 nm was measured by microplate reader.
Cell culture and cellular imaging. Human non-small cell lung carcinoma cells were
grown in 1640 (Gibco, USA) medium supplemented with 10% (V/V) fetal bovine
serum (Gibco, USA). Rat basophilic leukemia cell RBL-2H3 cells were grown in
MEM (Gibco, USA) medium supplemented with 15% (V/V) heat-activated fetal
bovine serum (Gibco, USA). Both cell lines were maintained at 37 °C and 5% CO ,
2
5
and were seeded at a density around 3×10 cells on a glass-bottom cell culture dishes
(NEST, 15 mm) in the day before the experiment. Next day, after washed with PBS
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for three times, the cells pre-incubated with 10 μM inhibitors (DHPI or sivelestat) or
DMSO for 2 h were treated with NEP accordingly. For the NE trafficking
experiments, cells were pre-incubated with or without 2 μM Brefeldin A (BFA) for 16
h before cell imaging. Similarly, cells were pre-incubated with lipopolysaccharide
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(
LPS, 20 μg/mL), interleukin 6 (IL-6, 100 ng/mL) or tumor necrosis factor alpha
TNF-α, 100 ng/mL) for 24 h, respectively, and then imaged for the inflammation
(
study. To understand the cellular up-take of human NE, cells were pre-incubated with
0 μM dynasore before incubating with or without 80 nM human NE for 1 h prior to
4
addition of probe NEP. All the above pre-treated cells were incubated with probe
NEP (1 μM) for 30 min and Hoechst 33342 (10 μM) for 5 min at 37 °C, and the
fluorescence images were acquired with an inverted fluorescence microscope
(Olympus IX71, Japan). The fluorescence image data were analyzed by quantifying
the fluorescence intensity of NEP in each cell using Image J software (National
Institutes of Health, America). Each experiment was performed in three independent
biological replicates.
Live imaging in ALI model mice. Wild type C57 mice and nude mice were
purchased from Huafukang Bioscience Co., Ltd. (Beijing, China). Mice were
intravenous injected with or without 5 mg/kg LPS and further kept for 48 h. After
barbered and intravenous injected with 200 μL NEP (50 μM) for 30 min, all the mice
were imaged with IVIS Lumina XRMS III in vivo imaging system (PerkinElmer,
USA).
RESULTS AND DISCUSSIONS
Probe design and characterization
In our previously work, we reported the discovery of a pentafluoroethyl conjugated
7
-amino-4-trifluomethylcoumarin as the first non-peptide fluorescent substrate for
human NE.24 Unfortunately, the specificity and the green fluorescence of the
fluorophore restricted its further application in cells and animal model. To obtain a
specific NE probe with improved properties for real-time monitoring and visualizing
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human NE in living cells and animals, we designed a new near-infrared probe with a
hemicyanine based fluorophore which already showed its excellent performance in
biological samples and living system.22, 25 Thus, we further synthesized this probe and
the detail is described in the experimental section, the chemical structure is also fully
verified (Figures S1-S5). As expected, the intact NEP is well quenched, and generated
strong red fluorescence upon the addition of human NE.
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We firstly studied the effects of pH and the temperature for NEP characterization
at aqueous solution. It was found that the optimum pH and temperature are around pH
=
7.0 and 37 ℃, respectively, which are very close to physical environment (Figures
S6 and S7). Under the optimized conditions, we examined the time-dependent
fluorescence spectra of the NEP when incubating with appropriate amount of human
NE (Figure 1a). Similarly, we also tested the human NE dose-dependent enhancement
of the fluorescence intensity of NEP at the maximum emission wavelength of 700 nm
(Figure 1b). Interestingly, the hydrolysis of NEP catalyzed by human NE was very
fast, and the fluorescence intensity (λemmax = 700 nm) reached the maximum value
within 20 min and the enhancement is about 25-fold comparing to the basal level. In
addition, the fluorescence response of NEP vs the different concentrations of human
NE (0-1.0 μg/mL) was in a good linearity from which the limit of detection (LOD)
was determined to be 29.42 ng/mL (Figure 1c). Moreover, the specificity of NEP was
also investigated against various analytes. Considering the previous report that a
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rhodamine probe containing trifluoroacetamide can be hydrolyzed by PLE , we
screened some common used esterases or amidases in aqueous buffer solution to
figure out the specificity of NEP. As anticipated, NEP shows very high specificity for
human NE, since the responses caused by other amidases (chymotrypsin, trypsin,
carboxypeptidase A, carboxypeptidase B) were negligible (Figure 1d). Although PLE
did show weak response toward NEP, the two major human sources of liver esterase
(BChE and carboxylesterase) displayed almost no interference against NE detection
with NEP. Meanwhile, no significant interference by amino acids and metal anions
was observed for sensing human NE with NEP (Figure S8). To explain why the use of
the hemicyanine based fluorophore is more sensitive and selective for NE compared
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to the aminocoumarin based fluorophore , molecule docking was carried out to get
their stimulated binding modes in the active site of HNE, respectively (Figure S9). It
revealed that NEP shows a closer distance between its amide bond and the key
catalytic residue Ser195, suggesting that NEP could be more prone to be attacked by
Ser195. This data taking together indicates that NEP can be a useful tool for NE
profiling in rather complicated biological environments.
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Figure 1. Kinetic characterization of NEP as a fluorogenic substrate for human NE.
(
a) Time-dependent fluorescent spectrum of NEP after the addition of 10 μg/mL
human NE. (b) Fluorescence intensity (λemmax = 700 nm) of NEP when incubating
with various concentration of (0, 2, 5, 10 μg) human NE. (c) Linearity curve of
fluorescence intensity vs the concentration of human NE ranging from 0 to 1 μg/mL.
(
d) Fluorescence responses of NEP (10 µM) against different hydrolases (10 μg/mL).
AChE refers to acetylcholinesterase, BChE refers to butyrylcholinesterase and PLE
represents to porcine liver esterase.
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Visualization of NE trafficking in living cells.
As is well known, NE is specifically expressed in immune cells that are usually
suspended in growth medium, which is unfavorable for live imaging. We hence chose
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a chemical-treated adherent growth leukemia cell line RBL-2H3 as the model for
living cell image. Prior to the cell imaging studies, we tested the cytotoxicity of NEP
by MTT assay and observed no apparently toxicity against RBL-2H3 cells at various
concentrations for 24 h (Figure S10). As shown in Figure 2a, the incubation with NEP
and the routine nuclear staining with Hoechst 33342 for 30 min led to the robust red
and blue fluorescent response in cells, respectively. It is worth to mention that some
strong red fluorescent puncta were observed, and we initially thought it might be
localized in cell membrane because the localization of active form of endogenous NE
is in cell membrane.27 However, we co-stained the cells with NEP and the
commercial cell membraned dye D4292, and found that the signal was not localized
in cell membrane (Figure S10). Considering the cation containing fluorophore of NEP,
we speculated that the free fluorophore might gather into the mitochondria. Thus, we
further stained the cells with NEP and commercial dye mito-tracker green together,
and found that the they were highly colocalized (Figure S11), indicating that signal of
NEP is eventually gathered in mitochondria. Two common NE inhibitors, DHPI and
the commercial drug sivelestat, were pre-incubated with cells prior to the addition of
NEP. Consequently, the red fluorescence intensity significantly declined around 50%
comparing to the DMSO-treated control group (Figure 2b). More importantly, the
fluorescence signal in cell membrane was almost eliminated after the pre-incubation
with sivelestat, which further indicated that the fluorescent signal was attributed to the
NE activity.
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Figure 2. Live image with NEP in RBL-2H3 cells. (a) Fluorescence images of
RBL-2H3 cells pre-treated with or without NE inhibitors for 3 h before incubation
with 1 μM NEP for 30 min. (b) Analysis of relative FI shown in left panel. (**P <
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.01, data analyses were performed with an independent samples t-test with equal
variances, means ± s.d., n = 3 independent experiments. λex = 590-650 nm, λem
60-730 nm.)
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To investigate the probability of demonstrating subcellular trafficking of active
NE by NEP, brefeldin A (BFA), an inhibitor that can arrest protein transport to Golgi
and redistribution to endoplasmic reticulum (ER)27 was applied in this study. As
depicted in Figure 3a, the fluorescence is mainly distributed outer cellular when the
cells were treated with NEP (Figure 3a). However, upon the stimulation with BFA for
16 h, the fluorescent signal appeared to be constitutively internalized, which
suggested that the transport of matured NE to cell membrane was blocked by BFA
(Figure 3b). Moreover, after removing of BFA and further incubated the cells for 30
min, the internalization of NEP signal was relieved (Figure 3c), indicating the
transportation of intracellular NE was recovered. Intriguingly, in the control
experiment with the fluorophore HCY-NH (Figure S12), the fluorescent signal was
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Figure 3. Monitoring NE trafficking in RBL-2H3 cells with NEP. (a-b) RBL-2H3
cells pre-treated with 0.5% DMSO or 2 μM BFA for 16 h before incubation with 1
μM NEP for 30 min. (c) RBL-2H3 cells pre-treated with 2 μM BFA for 16 h and then
removal of BFA for 30 min before incubation with 1 μM NEP for 30 min. (λex
90-650 nm, λem = 660-730 nm.)
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association of NE with lung cancer claimed that exogenous NE could be uptaken into
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cancer cells and the growth was promoted accordingly. To further in situ image the
process of NE uptaking, NEP was applied to visualize A549 cells stimulated with
exogenous NE. As shown in Figure 4a, pre-incubated with 80 nM human NE resulted
in robust and punctate fluorescence in single cell imaging, while the background
fluorescence in control group treated with only DMSO was very negligible. To obtain
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plausible evidence, we also employed dynasore, a dynamin inhibitor to block the cell
permeability by inhibiting clathrin-mediated endocytosis28 before the subsequent
treatment with external human NE and probe NEP. Intriguingly, the fluorescence
intensity of the A549 cells pre-incubated with dynasore indeed become weaker than
that of cells only treated with human NE and probe NEP. Besides, we also applied
NEP to study the uptake of NE in MDA-MB-231 cells with same method described in
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the literature, and obtained the similar result (Figure S13). The above observations
lead us to conclude that, NEP is a reliable and specific tool to visualize NE trafficking
and up-taking processes.
Figure 4. Monitoring of NE-uptake in A549 cells by NEP. (a) Fluorescent image
with A549 cells pre-incubated with or without 80 nM human NE for 1h before
incubated with 1 μM NEP for 30 min. (b) Fluorescent image taken from A549
pre-incubated with or without 40 μM dynasore for 1h before incubated with 80 nM
human NE for 1h. (c) Analysis of relative FI shown in panel b. (**P < 0.01, data
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analyses were performed with an independent samples t-test with equal variances,
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To evaluate the performance of NEP in the context of lung diseases related
mechanism study, we imaged and quantitatively analyzed the regulation of
endogenous NE activity in cells in the presence of stimulating factors. As mentioned
before, NE plays important role in immune system and elevated in inflammatory
response. LPS is a typical macromolecule that can induce inflammation in cells and
animals,30 and two pro-inflammation factors IL-6 and TNF-α,31-32 are common
reagents to induce inflammatory response. As displayed in Figure 5, pronounced
increases of the red fluorescence in cells separately treated with LPS, IL-6 and TNF-α.
After quantifying the fluorescence intensities for each group of cells, all the
pre-treated cells showed over 50% elevation when compared with DMSO-treated
control. This data suggests that the up-regulation of NE triggered by stimulation
factors can be directly visualized with NEP at the cellular level.
Figure 5. Up-regulation of NE in RBL-2H3 cells induced by LPS and
pro-inflammation factors. (a) Fluorescence images of RBL-2H3 cells pre-treated with
DMSO (0.5%), LPS (20 μg/mL), IL-6 (100ng/mL) and TNF-α (100ng/mL) before
incubation with 1μM NEP for 30 min. (b) Analysis of relative fluorescence intensity
shown in left panel. (**P < 0.01, data analyses were performed with an independent
samples t-test with equal variances, means ± s.d., n = 3 independent experiments. λex
=
590-650 nm, λem = 660-730 nm.)
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In vivo imaging of NE in ALI model mice.
Human NE is a major cause for acute lung injury (ALI) due to its elevated
proteolytic activity that destroy the normal lung tissue.6-7 However, the detailed
response of lungs to ALI remains unclear. Fluorescent probes are attractive to tracking
the desired enzyme level in vivo, but no small-molecular fluorogenic probe are
available for NE imaging in live animals. Thus, NEP was applied in the constructed
ALI model mice generated by LPS injection. In general, healthy mice and ALI model
mice were intravenous injected with 200 μL NEP (50 μM) for 30 min before gas
anesthesia and fluorescence image. To our delight, very low fluorescent signal was
detected in control group mice (left in Figure 6a), whereas remarkable fluorescent
signal is acquired in ALI model mice group (right in Figure 6a). However, the signal
were observed in the abdomen of the mice instead of the chest. To understand this
phenomenon, we further investigated the distribution of NEP signal in ALI nude mice
model. As shown in Figure S14, the fluorescent signal in ALI nude mouse was
emerged only after 5 min, quickly enhanced within 10 min, suggesting that the NEP
hydrolysis catalyzed by NE is quite fast. Based on our understanding, there may be
two reasons behind this observation: a) the NEP doesn't possess preference toward
lung, and could accumulate in multiple organs. Thus the signal is occurred in both
lung and other organs; b) the free fluorophore released after the NE catalysis can be
quickly metabolized by liver, kidney or intestine. Anyhow, the significantly enhanced
fluorescent signal in both ALI model C57 mice and nude mice provides solid
evidence that NE was highly elevated in in vivo ALI model. We further collected the
two most widely used biological samples, bronchoalveolar lavage fluid (BAL) and
serum6, 30 from both healthy control and ALI model mice, and utilized NEP to
examine the relative contents of NE (Figures. 6b and 6c). No surprise that the NE
activity in the biological samples of ALI group is much higher than those of control
group, which are in good consistent with the result of the live mouse imaging. To
avoid false results, the commercial NE inhibitor, sivelestat, was set to be a positive
control group for NE detection in BAL and mice serum. As anticipated, no obvious
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fluorescent signal could be detected when the samples were pre-incubated with 10 μM
of sivelestat for 10 min. Taking together, our results suggest that NEP may serve as a
valuable tool for clinical diagnosis of ALI and other NE related disease.
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Figure 6. In vivo imaging of NEP in ALI mice model. (a) Live image of control mice
(left) and ALI mice (right) intravenously injected with 200 μL 50 μM NEP. (b)
Measure of NE activity in BAL from control and ALI mice with NEP with or without
pre-incubated with 10 μM of sivelestat for 10 min. (c) Measure of NE activity in
serum from control and ALI mice with 10 μM NEP with or without pre-incubation
with 10 μM of sivelestat for 10 min. (**P < 0.01, data analyses were performed with
an independent samples t-test with equal variances, means ± s.d., n = 3 independent
experiments. λex = 660 nm, λem = 670-730 nm.)
CONCLUSION
In summary, we reported a near-infrared fluorescent probe (NEP) based on
hemicyanine dye for the highly specific detection of human NE in vitro and in vivo.
With this new off-on fluorescent reporter, we have the convenient and fast tool other
than complicated and time-consuming immunofluorescence assay, to timely and
spatially analyze several important cellular events, such as NE trafficking, NE up
taking and LPS induced NE up-regulation. Moreover, we further applied NEP in ALI
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model mice and demonstrated the capability for quantitative analysis of NE in living
mice and biological samples. As far as we know, NEP is the first small molecular
near-infrared fluorescent probe to detect exogenous and endogenous NE in cells and
living organisms. Therefore, it is envisioned that NEP may serve as a potential tool
for diagnosis and pathological mechanism study of NE-related disease.
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ACKNOWLEDGMENT
This work was funded by the National Key Research and Development Program of
China (No. 2017YFA0505200) and National Natural Science Foundation of China
(No. 21332004, and 21672079); Hubei Province Natural Science Foundation (No.
2018CFA072); the self-determined research funds of Central China Normal
University (CCNU18ZDPY01 and CCNU18TS007) from the colleges’ basic research
and operation of MOE”.
SUPPORTING INFORMATION
Organic synthesize, characterization figures and cell imaging experiments are
provided in the supporting information. This material is available free of charge via
the Internet.
REFERENCES
1
.
Mantovani, A.; Cassatella, M. A.; Costantini, C.; Jaillon, S., Neutrophils in the
activation and regulation of innate and adaptive immunity. Nat Rev Immunol
2
011, 11 (8), 519-31.
2
3
.
.
Pham, C. T., Neutrophil serine proteases: specific regulators of inflammation.
Nat Rev Immunol 2006, 6 (7), 541-50.
M. Gautier., A. J. P. A., S. Janos, H. William, B. Erika Neutrophil Elastase as a
Target in Lung Cancer. Anti-Cancer Agents in Medicinal Chemistry 2012, 12,
565-579.
4. Korkmaz, B.; Moreau, T.; Gauthier, F., Neutrophil elastase, proteinase 3 and
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Analytical Chemistry
1
2
3
4
5
6
7
8
9
cathepsin G: Physicochemical properties, activity and physiopathological
functions. Biochimie 2008, 90 (2), 227-242.
5
6
.
.
Owen, C. A., Roles for proteinases in the pathogenesis of chronic obstructive
pulmonary disease. Int J Chron Obstruct Pulmon Dis 2008, 3, 253-268.
Tao, W.; Miao, Q. B.; Zhu, Y. B.; Shu, Y. S., Inhaled neutrophil elastase inhibitor
reduces oleic acid-induced acute lung injury in rats. Pulm Pharmacol Ther 2012,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
5 (1), 99-103.
7
8
.
.
Ishii, T.; Doi, K.; Okamoto, K.; Imamura, M.; Dohi, M.; Yamamoto, K.; Fujita,
T.; Noiri, E., Neutrophil elastase contributes to acute lung injury induced by
bilateral nephrectomy. Am J Pathol 2010, 177 (4), 1665-73.
Taggart, C. C.; Greene, C. M.; Carroll, T. P.; O'Neill, S. J.; McElvaney, N. G.,
Elastolytic proteases: inflammation resolution and dysregulation in chronic
infective lung disease. Am J Respir Crit Care Med 2005, 171 (10), 1070-6.
9. Fujii, M.; Miyagi, Y.; Bessho, R.; Nitta, T.; Ochi, M.; Shimizu, K., Effect of a
neutrophil elastase inhibitor on acute lung injury after cardiopulmonary bypass.
Interact Cardiovasc Thorac Surg 2010, 10 (6), 859-62.
10. Miyoshi, S.; Hamada, H.; Ito, R.; Katayama, H.; Irifune, K.; Suwaki, T.;
Nakanishi, N.; Kanematsu, T.; Dote, K.; Aibiki, M.; Okura, T.; Higaki, J.,
Usefulness of a selective neutrophil elastase inhibitor, sivelestat, in acute lung
injury patients with sepsis. Drug Des Devel Ther 2013, 7, 305-16.
1
1. Iwata, K.; Doi, A.; Ohji, G.; Oka, H.; Oba, Y.; Takimoto, K.; Igarashi, W.;
Gremillion, D. H.; Shimada, T., Effect of Neutrophil Elastase Inhibitor
Sivelestat Sodium) in the Treatment of Acute Lung Injury (ALI) and Acute
(
Respiratory Distress Syndrome (ARDS): A Systematic Review and
Meta-Analysis. Internal Medicine 2010, 49 (22), 2423-2432.
1
1
2. Kuna, P.; Jenkins, M.; O'Brien, C. D.; Fahy, W. A., AZD9668, a neutrophil
elastase inhibitor, plus ongoing budesonide/formoterol in patients with COPD.
Respir Med 2012, 106 (4), 531-9.
3. Wilson, D. O.; Weissfeld, J. L.; Fuhrman, C. R.; Fisher, S. N.; Balogh, P.;
Landreneau, R. J.; Luketich, J. D.; Siegfried, J. M., The Pittsburgh Lung
ACS Paragon Plus Environment

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Page 20 of 23
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
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1
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5
5
5
5
5
5
5
5
5
5
6
Screening Study (PLuSS): outcomes within 3 years of a first computed
tomography scan. Am J Respir Crit Care Med 2008, 178 (9), 956-61.
4. Houghton, A. M.; Rzymkiewicz, D. M.; Ji, H.; Gregory, A. D.; Egea, E. E.; Metz,
H. E.; Stolz, D. B.; Land, S. R.; Marconcini, L. A.; Kliment, C. R.; Jenkins, K.
M.; Beaulieu, K. A.; Mouded, M.; Frank, S. J.; Wong, K. K.; Shapiro, S. D.,
Neutrophil elastase-mediated degradation of IRS-1 accelerates lung tumor
growth. Nat Med 2010, 16 (2), 219-23.
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
5. Talukdar, S.; Oh, D. Y.; Bandyopadhyay, G.; Li, D.; Xu, J.; McNelis, J.; Lu, M.;
Li, P.; Yan, Q.; Zhu, Y.; Ofrecio, J.; Lin, M.; Brenner, M. B.; Olefsky, J. M.,
Neutrophils mediate insulin resistance in mice fed a high-fat diet through
secreted elastase. Nat Med 2012, 18 (9), 1407-12.
6. Liu, H. W.; Li, K.; Hu, X. X.; Zhu, L.; Rong, Q.; Liu, Y.; Zhang, X. B.;
Hasserodt, J.; Qu, F. L.; Tan, W., In Situ Localization of Enzyme Activity in Live
Cells by a Molecular Probe Releasing a Precipitating Fluorochrome. Angew
Chem Int Ed Engl 2017, 56 (39), 11788-11792.
17. Asanuma, D.; Sakabe, M.; Kamiya, M.; Yamamoto, K.; Hiratake, J.; Ogawa, M.;
Kosaka, N.; Choyke, P. L.; Nagano, T.; Kobayashi, H.; Urano, Y., Sensitive
beta-galactosidase-targeting fluorescence probe for visualizing small peritoneal
metastatic tumours in vivo. Nat Commun 2015, 6, 6463.
1
8. Park, S. J.; Lee, H. W.; Kim, H.-R.; Kang, C.; Kim, H. M., A
carboxylesterase-selective ratiometric fluorescent two-photon probe and its
application to hepatocytes and liver tissues. Chemical Science 2016, 7 (6),
3
703-3709.
1
2
2
9. Kathayat, R. S.; Elvira, P. D.; Dickinson, B. C., A fluorescent probe for cysteine
depalmitoylation reveals dynamic APT signaling. Nat Chem Biol 2017, 13 (2),
1
50-152.
0. Hirayama, S.; Hori, Y.; Benedek, Z.; Suzuki, T.; Kikuchi, K., Fluorogenic probes
reveal a role of GLUT4 N-glycosylation in intracellular trafficking. Nat Chem
Biol 2016, 12 (10), 853-9.
1. Li, L.; Zhang, C. W.; Chen, G. Y.; Zhu, B.; Chai, C.; Xu, Q. H.; Tan, E. K.; Zhu,
ACS Paragon Plus Environment

Page 21 of 23
Analytical Chemistry
1
2
3
4
5
6
7
8
9
Q.; Lim, K. L.; Yao, S. Q., A sensitive two-photon probe to selectively detect
monoamine oxidase B activity in Parkinson's disease models. Nat Commun 2014,
5
, 3276.
2
2
2
2
2. He, X.; Li, L.; Fang, Y.; Shi, W.; Li, X.; Ma, H., In vivo imaging of leucine
aminopeptidase activity in drug-induced liver injury and liver cancer via a
near-infrared fluorescent probe. Chem Sci 2017, 8 (5), 3479-3483.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
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0
1
2
3
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5
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8
9
0
1
2
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4
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6
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8
9
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3. Xie, X.; Yang, X.; Wu, T.; Li, Y.; Li, M.; Tan, Q.; Wang, X.; Tang, B., Rational
Design of an alpha-Ketoamide-Based Near-Infrared Fluorescent Probe Specific
for Hydrogen Peroxide in Living Systems. Anal Chem 2016, 88 (16), 8019-25.
4. Sun, Q.; Li, J.; Liu, W. N.; Dong, Q. J.; Yang, W. C.; Yang, G. F.,
Non-peptide-based fluorogenic small-molecule probe for elastase. Anal Chem
2
013, 85 (23), 11304-11.
5. He, X.; Hu, Y.; Shi, W.; Li, X.; Ma, H., Design, synthesis and application of a
near-infrared fluorescent probe for in vivo imaging of aminopeptidase N. Chem
Commun (Camb) 2017, 53 (68), 9438-9441.
26. Abney, K. K.; Ramos-Hunter, S. J.; Romaine, I. M.; Goodwin, J. S.; Sulikowski,
G. A.; Weaver, C. D., Selective Activation of N,N'-Diacyl Rhodamine
Pro-fluorophores Paired with Releasing Enzyme, Porcine Liver Esterase (PLE).
Chemistry 2018, 24 (36), 8985-8988.
2
7. Tapper, H.; Källquist, L.; Johnsson, E.; Persson, A.-M.; Hansson, M.; Olsson, I.,
Neutrophil elastase sorting involves plasma membrane trafficking requiring the
C-terminal propeptide. Experimental Cell Research 2006, 312 (18), 3471-3484.
8. Macia, E.; Ehrlich, M.; Massol, R.; Boucrot, E.; Brunner, C.; Kirchhausen, T.,
Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 2006, 10 (6), 839-50.
9. Kerros, C.; Tripathi, S. C.; Zha, D.; Mehrens, J. M.; Sergeeva, A.; Philips, A. V.;
Qiao, N.; Peters, H. L.; Katayama, H.; Sukhumalchandra, P.; Ruisaard, K. E.;
Perakis, A. A.; St John, L. S.; Lu, S.; Mittendorf, E. A.; Clise-Dwyer, K.;
Herrmann, A. C.; Alatrash, G.; Toniatti, C.; Hanash, S. M.; Ma, Q.; Molldrem, J.
J., Neuropilin-1 mediates neutrophil elastase uptake and cross-presentation in
breast cancer cells. J Biol Chem 2017, 292 (24), 10295-10305.
2
2
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Analytical Chemistry
Page 22 of 23
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6
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3
0. Vlahos, R.; Wark, P. A.; Anderson, G. P.; Bozinovski, S., Glucocorticosteroids
differentially regulate MMP-9 and neutrophil elastase in COPD. PLoS One 2012,
7
(3), e33277.
1. Zimmermann, P.; Ziesenitz, V. C.; Curtis, N.; Ritz, N., The Immunomodulatory
Effects of Macrolides-A Systematic Review of the Underlying Mechanisms.
Front Immunol 2018, 9, 302.
0
1
2
3
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0
2. Barnes, P. J., The cytokine network in asthma and chronic obstructive pulmonary
disease. J Clin Invest 2008, 118 (11), 3546-56.
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