In Vivo Two-Photon Fluorescence Imaging of the Activity of the Inflammatory Biomarker LTA4H in a Mouse Pneumonia Model

Article abstract of DOI:10.1021/acs.analchem.7b04885

Severe atmospheric haze caused by industrial pollution has severely affected human health and led to the increasing incidence of cardiopulmonary diseases, including pneumonia. Conventional methods for diagnosis of pneumonia are complicated and tedious, and current clinical imaging techniques might cause organ injuries to some extent. Therefore, an accurate, fast, and intact imaging method must be developed to diagnose pneumonia in the early stages. In this study, we propose a new two-photon fluorescence probe, named as ASPC, for detection of the activity of the inflammatory biomarker LTA₄H through specific recognition and cleavage of amides containing the unnatural amino acid l-AspBzl. The activity of LTA₄H in the lung tissues of mice was rapidly and accurately monitored for the first time and could be an indicator for diagnosis of pneumonia. The severity of pneumonia in mice caused by haze particulate was determined through imaging the activity of LTA₄H as biomarker and confirmed using a commercial ELISA kit of interleukin-1β. This work provides a promising method for clinical detection of pneumonia and for screening specific depressors of LTA₄H as potential drug candidates.

Full text of DOI:10.1021/acs.analchem.7b04885

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In vivo Two-photon Fluorescence Imaging of the Activity of the
Inflammatory Biomarker LTA4H in a Mouse Pneumonia Model
Hui Wang, Ke Xue, Ping Li, Yuyun Yang, Zixu He, Wei Zhang, Wen Zhang, and Bo Tang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04885 • Publication Date (Web): 23 Apr 2018
Downloaded from http://pubs.acs.org on April 23, 2018
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Analytical Chemistry
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In vivo Two-photon Fluorescence Imaging of the Activity of the In-
flammatory Biomarker LTA4H in a Mouse Pneumonia Model
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Hui Wang, † Ke Xue, †Ping Li, * Yuyun Yang, Zixu He, Wei Zhang, Wen Zhang, and Bo Tang *
College of Chemistry, Chemical Engineering and Materials Science, Institute of Biomedical Sciences, Collaborative Innovaꢀ
tion Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and
Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shanꢀ
dong Normal University, Jinan 250014, PR China
*Corresponding author email: tangb@sdnu.edu.cn; lip@sdnu.edu.cn Phone: 86ꢀ531ꢀ86180010
ABSTRACT: Severe atmospheric haze caused by industrial pollution has severely affected human health and led to the increasing
incidence of cardiopulmonary diseases, including pneumonia. Conventional methods for diagnosis of pneumonia are complicated
and tedious, and current clinical imaging techniques might cause organ injuries to some extent. Therefore, an accurate, fast, and
intact imaging method must be developed to diagnose pneumonia in the early stages. In this study, we propose a new twoꢀphoton
fluorescence probe, named as ASPC, for detection of the activity of the inflammatory biomarker LTA4H through specific recogniꢀ
tion and cleavage of amides containing the unnatural amino acid LꢀAspBzl. The activity of LTA4H in the lung tissues of mice was
rapidly and accurately monitored for the first time and could be an indicator for diagnosis of pneumonia. The severity of pneumonia
in mice caused by haze particulate was determined through imaging the activity of LTA4H as biomarker and confirmed using a
commercial ELISA kit of interleukin (IL)ꢀ1β. This work provides a promising method for clinical detection of pneumonia and for
screening specific depressors of LTA4H as potential drug candidates.
crucial roles in the diagnosis but inflict considerable damages,
such as cellular canceration, chromosomal aberration, and
INTRODUCTION
Regional haze pollution and increasing pollution levels have
emerged in China and seriously threaten public health1,2. Studꢀ
ies of the 1952 London smog demonstrate that exposure to
high concentrations of particulate matter can significantly
increase the hospitalization and mortality rates associated with
cardiopulmonary diseases3,4. Global epidemiological surveys
show the direct relationship between the level of atmospheric
particulates, including PM2.5, and the rate of pulmonary dysꢀ
function or respiratory symptom diseases, such as pneumonia5-
8. In general, pneumonia is a disease of the alveoli and respiraꢀ
tory bronchioles caused by pathogens, which induce local and
systemic inflammatory responses in the patient9,10. Severe
complications of pneumonia include meningitis, systemic sepꢀ
sis, lung abscesses, brain damage, and hearing loss often ocꢀ
cur11-14. The mortality rate of patients with pneumonia remains
high for the past decades. Thus, accurate diagnosis is essential
for timely treatment of this disease. Clinical diagnosing pneuꢀ
monia requires multiple techniques and clinical indices, inꢀ
cluding chest Xꢀray and CT imaging, culture of blood and
expectorated sputum samples, and evaluation of several sugꢀ
gestive clinical features15,16. Chest Xꢀray and CT imaging play
germ cell abnormality, which restrict their application17-19
.
Therefore, effective and rapid imaging approaches must be
developed to accurately diagnose pneumonia.
Twoꢀphoton (TP) fluorescence imaging technology is a nonꢀ
invasive method that uses highly sensitive and selective fluoꢀ
rescence probes20,21. In particular, the excitation at the nearꢀ
infrared wavelength (690ꢀ950 nm) leads to increased tissue
penetration depth, high temporal resolution, and reduced phoꢀ
totoxicity to tissues22. This imaging method can be used in
vivo for study on cell metabolism and disease processes at the
molecular level23. Therefore, TP fluorescence imaging using
ideal fluorescent probes with high selectivity and sensitivity
can be used to detect pneumonia biomarkers and effectively
diagnose the disease in tissues of living systems24. LTA4H (EC
3.3.2.6) is a key protein that mediates the degradation of pepꢀ
tides in host defense mechanism25,26. The expression level of
LTA4H significantly increases during inflammation and is
related to the development and progression of inflammaꢀ
tion27,28. Therefore, LTA4H could be an effective biomarker for
detecting pneumonia and estimating the effectiveness of the
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treatment and as a potential molecular target for pneumonia
therapy19,29. However, existing detection kits for LTA4H activiꢀ
ty are only applied to the purified LTA4H in vitro and cannot
be used to obtain real physiological data in situ30. Moreover,
the activity loss of LTA4H caused by sample preparation may
result in severe measurement error. Thus, a fluorescence probe
with sensitive and selective response toward LTA4H must be
developed and used to accurately diagnose pneumonia by TP
fluorescence imaging of the enzyme activity fluctuations in
realꢀtime.
5ꢀdiphenyltetrazolium bromide (MTT) were purchased from
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Sigma. Ubenimex was from Energy Chemical. Silica gel (200ꢀ
300mesh) used for flash column chromatography was purꢀ
chased from Qingdao Haiyang Chemical Co,Ltd. A549 human
lung adenocarcinoma cell line and human hepatic stellate cells
(LXꢀ2) were purchased from Cell Bank of the Chinese Acadꢀ
emy of Sciences (Shanghai, China). Recombinant human Leuꢀ
kotriene A4 Hydrolase (LTA4H) was purchased from Abcam.
Aminopeptidases N (APN), leucine aminopeptidases (LAP)
were purchased from CloudꢀClone Corp and methionine amiꢀ
nopeptidases (METAP) was purchased from Novoprotein.
Mouse interieukinꢀ1β ELISA kit was purchased from Sinoꢀ
bestbio (ShangHai, China).
1HNMR and 13CNMR spectra were determined by using
Bruker NMR spectrometers at 400 MHz and 100 MHz. The
high resolution mass spectra (HRMS) were measured with
Bruker maxis ultraꢀhigh resolutionꢀTOF MS system. The fluoꢀ
rescence spectra measurements were performed using FLSꢀ
920 Edinburgh fluorescence spectrometer. Absorption spectra
were measured with a TUꢀ1900 UVꢀvis spectrophotometer
(Beijing Purkinje General Instrument Co., Ltd.). Absorbance
was measured with a microplate reader (RT 6000, Ra yto,
USA). Fluorescence imaging in cells and in vivo were perꢀ
formed with Zeiss LSM 880 NLO Confocal Laser Scanning
Microscope. PM2.5 was collected from Jinan atmosphere by
using an integrated intelligent air total suspended particulate
sampler with a PM2.5 cutter (Laoying 2030, Laoshan Appliꢀ
cable Technology Institute, Qingdao, China).
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In this study, we propose a novel twoꢀphoton fluorescent
probe (ASPC) for detection of LTA4H activity in vivo. The
structure of ASPC contained an amide bond linking the unꢀ
natural amino acid LꢀAspBzl as reaction site and 7ꢀaminoꢀ4ꢀ
methylcoumarin (AMC) as chromophore31. LTA4H could speꢀ
cifically hydrolyze the amide bond between the carboxyl
group of LꢀAspBzl and the amino group in AMC. Moreover,
AMC was selected as the fluorescence reporting group beꢀ
cause of its desirable photophysical properties, such as high
fluorescent quantum yield, good photostability, and large
Stokes shifts32. In ASPC, the reduction in the electronꢀcloud
density of the nitrogen atom by the carbonyl group weakens
the electron donor–acceptor interactions in the conjugated
system of AMC, leading to decreased fluorescence quantum
yield of AMC. After LTA4H recognizes the substrate Lꢀ
AspBzl in the active center and cleavages the amide bond, the
fluorescence intensity of the probe recovers (Scheme 1).
ASPC was used to measure the activity of LTA4H for the first
time and obtain the TP fluorescence images of LTA4H in livꢀ
ing cells and tissues. Based on the detected activity of LTA4H
in the lung tissues, mice exposed to PM2.5 were also diagꢀ
nosed with pneumonia.
Synthesis of ASPC. ASPC was synthesized according to
the procedure in Scheme S1, and its structure characterization
was described in the Supporting Information.
Specificity of ASPC toward LTA4H. Fluorescence reꢀ
sponses of ASPC (0.1 ꢂM) reacted with different kinds of
species for 90 min at 37 °C in a final volume of 200 ꢂL of
TrisꢀHCl (100 mM, pH 7.4) and NaCl (100 mM). Such as
Zn2+ (50 mM), Ca2+ (50 mM), Mg2+ (50 mM), alanine (Ala,
1.0 mM), arginine (Arg, 1.0 mM), cysteine (Cys, 1.0 mM),
glycine (Gly, 1.0 mM), lysine (Lys, 1.0 mM), glucose (50
mM), Vitamin C (Vc, 1.0 mM) and LTA4H (4 ng mLꢀ1).
Cleavage of ASPC (0.1 ꢂM) by other aminopeptidases such as
APN, LAP and METAP was also investigated33ꢀ35. The enꢀ
zymes were tested in the following conditions: APN (5.0 ꢂg
mLꢀ1) in TrisꢀHCl buffer (100 mM, pH 7.5), LAP (5.0 ꢂg mLꢀ1)
in PBS buffer (50 mM, pH 7.4) and MAP (5.0 ꢂg mLꢀ1) in
Hepes buffer (50 mM, pH 7.4) and NaCl (100 mM). The reacꢀ
tions were performed in a final volume of 200 ꢂL and incubatꢀ
ed for 90 min at 37 °C. At the end of the reaction, fluorescence
was measured on a FLSꢀ920 Edinburgh fluorescence specꢀ
trometer (λex = 320 nm, λem = 400 nm). Each experiment was
repeated at least three times.
Scheme 1. Schematic of design and mechanism of fluorescent
probes for LTA4H activity.
EXPERIMENTAL SECTION
Materials and Methods. All reagents were purchased
commercially and used without further purification. All chemꢀ
icals and solvents used were of analytical grades. Ultrapure
water (18.2Mꢁ cmꢀ1) was used throughout the analytical exꢀ
periments. BocꢀLꢀaspartic acid 4ꢀbenzyl ester (BocꢀLꢀAsp
(OBzl)ꢀOH) was purchased from Aladdin and 7ꢀAminoꢀ4ꢀ
methylcoumarin (AMC) was purchased from Ark Pharm. Inc,
Lipopolysaccharide (LPS) and 3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,
Kinetic parameters of ASPC toward LTA4H. Determinaꢀ
tion of the Michaelis constant (Km) was performed in initial
rate conditions. For this purpose, recombinant human LTA4H
(4 ng mLꢀ1) was added to increasing concentrations of ASPC
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Analytical Chemistry
reader. Cell viability was expressed as a percentage of MTT
in a final volume of 200 ꢂL of TrisꢀHCl (100 mM, pH 7.4) and
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NaCl (100 mM) and incubated at 37 °C for 40 min. The subꢀ
strate concentrations for Km determination ranged from 0.05 to
50 ꢂM. The AMC concentration and corresponding Relative
Fluorescence Unit was calculated by total hydrolysis of indeꢀ
pendent substrates at the same concentration. The average
value was used for further calculations. Each experiment was
repeated at least three times and results are presented as mean
± SD. The Km values were calculated using GraphPad Prism 6
reduction with the untreated cells as 100%.
siRNA Transfection. LTA4H protein expression in A549
was knocked out using LTA4Hꢀspecific siRNA (sense, 5′ꢀ
CAA AGG ACC UUA CAA UAG ATTꢀ3′, antisense, 5′ꢀUCU
AUU GUA AGG UCC UUU GTTꢀ3′; GenePharma, Shanghai,
China). To verify transfection efficiency, we used FAMꢀ
siRNA (sense, 5′ꢀUUC UCC GAA CGU GUC ACG UTTꢀ3′;
antisense, 5′ꢀACG UGA CAC GUU CGG AGA ATTꢀ3′) as
negative control. One day before transfection, A549 cells
(4×105 total) were seeded in 6ꢀwell dishes by medium without
antibiotics. For each transfection sample, prepare siRꢀ
NA:Lipofectamine 2000 complexes as follows: Dilute the
siRNA (3 ꢂL) and Lipofectamine 2000 (5 ꢂL) in 200 ꢂL Optiꢀ
MEM I, respectively. And then mix them gently before add
the siRNA: Lipofectamine 2000 complexes to each well. Fiꢀ
nally, incubate the cells at 37 °C for 48 h until they are ready
to assay for gene knockdown.
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software, and the Kcat was determined using the equation Kcat
=
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Vmax/[E]. Fluorescence was measured on a FLSꢀ920 Edinꢀ
burgh fluorescence spectrometer (λex = 320 nm, λem = 400 nm).
Inhibitory potency measurements. To determine the in
vitro inhibitory potencies or inhibitory constant (Ki) values of
ubenimex toward LTA4H, recombinant enzyme (4 ng mLꢀ1)
was preincubated for 30 min at 37°C in TrisꢀHCl (100 mM,
pH 7.4) and NaCl (100 mM), with increasing concentrations
of inhibitor (from 10ꢀ9 to 10ꢀ3 M final concentration). The fluoꢀ
rescent substrates ASPC (10 ꢂM) were added in a final volꢀ
ume of 200 ꢂL and incubated at 37°C for 40 min. The fluoresꢀ
cent values were measured on a FLSꢀ920 Edinburgh fluoresꢀ
cence spectrometer (λex = 320 nm, λem = 400 nm). Samples
with 0% hydrolysis were obtained by adding the substrate to
the buffer, and samples with 100% relative activity were preꢀ
pared without the inhibitor. The percentage of cleavage was
evaluated and compared with 100% relative activity, and the
IC50 values were determined accordingly. The IC50 values
were calculated using GraphPad Prism 6 software. The Ki valꢀ
ues of the inhibitors (mean of at least three independent assays
in duplicate) were calculated using the equation Ki = IC50/(1 +
[S]/Km), assuming their mode of binding to be competitive.
Quantitative RealꢀTime PCR. RTꢀPCR was performed
with the Linegene 9620 detection system using Power SYBR
Green PCR master mix (Applied Biosystems). Total RNA was
extracted using UNlQꢀ10 Column total RNA Purification Kit
(Sangon Biotech). All data were normalised to the level of the
βꢀactin (sense,5′ꢀTCCCTGGAGAAGAGCTACGꢀ3′; antisense,
5′ꢀGTAGTTTCGTGGATGCCACAꢀ3′) in the sample. Three
independent technical replicates were performed for each reacꢀ
tion.
Western Blot. A549 cells grown in 6ꢀwell dishes were
washed twice with cold PBS and lysed in 200 ꢂL of RIPA
lysis buffer containing protease inhibitors (1 mM PMSF). Lyꢀ
sate samples were incubated for 25 min at 4 °C. Samples were
homogenized using cell scrapers and centrifuged at 12,000
rpm for 7 min at 4 °C. The supernatant was recovered, and
protein concentration was determined by the Bradford assay.
Then, lysate samples added to SDSꢀPAGE loading buffer and
heated for 5 min at 95 °C. After that, the analyzed by Western
blotting were accomplished by Yuci Biotechnology Company.
Cells culture. A549 human lung adenocarcinoma cells and
human hepatic cells (HLꢀ7702) were respectively cultured in
Roswell Park Memorial Institute (RPMIꢀ1640) and Dulbecco's
Modified Eagle Medium (DMEM) supplemented with 10%
fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin
at 37 °C in a 5%CO2/95% air incubator MCOꢀ15AC (SANYO,
Tokyo, Japan). Two days before imaging, the cells were deꢀ
tached and replanted on glassꢀbottomed dishes or 6ꢀwell dishꢀ
es.
OP and TP fluorescence imaging of HLꢀ7702 cells and
A549 cells. For the detection of endogenously produced
LTA4H, the cells were divided into three groups and treated
with different conditions. (a) Blank cells: The cells were only
incubated with ASPC (10 µM) in TrisꢀHCl (100 mM) at room
temperature for 40 min. (b) LPSꢀtreated cells: The cells were
incubated with 10 µg mLꢀ1 LPS in RPMI 1640 at 37 °C for 2 h.
Then the cells were treated with ASPC (10 µM) in TrisꢀHCl
(100 mM) at room remperature for 40 min. (c)
LPS+ubenimexꢀtreated cells: The cells were incubated with 10
µg mLꢀ1 LPS in RPMI 1640 at 37 °C for 2 h. After 2 h, the
cells were treated with ubenimex (1 µM) in TrisꢀHCl at room
temperature for 40 min, and then cells were treated with
ASPC (10 µM) for another 40 min. The cells were washed
three times with TrisꢀHCl prior to the imaging. Oneꢀphoton
and twoꢀphoton fluorescent images were acquired on a LSM
880 NLO Confocal Laser Scanning Microscope with a water
Cytotoxicity assays. The cytotoxicity of ASPC towards
A549 cell lines was measured by 3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀ
yl)ꢀ2, 5ꢀdiphenyltetrazolium bromide (MTT) assay. A549 cells
were seeded in a 96ꢀwell plate at a concentration of 1×105
cells wellꢀ1 in 100 ꢂL of RPMIꢀ1640 medium with 10% fetal
bovine serum, 1% penicillin, and 1% streptomycin and mainꢀ
tained at 37 °C in a 5%CO2 incubator for 12 h. Then, cells
were exposed to different concentrations of ASPC (0.01, 0.1,
1, 5, 10, 20 µM) for 24 h. The cells were washed with 37 °C
PBS and MTT solution (5 mg mLꢀ1, 20 ꢂL) was added to each
well and continuously incubated for 4 h at 37 °C. After 4 h,
MTT solution was removed and DMSO (150 ꢂL) was added
to each well to dissolve the dark blue formazan crystals. Abꢀ
sorbance was measured at 490 nm in a Triturus microplate
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objective lens (×20). The excitation wavelength was 405 nm route for 12 and 24 h; and the two other groups of blank mice
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and 690 nm, respectively.
were injected with the same amount of PBS.
TP fluorescence imaging and flow cytometry imaging of
A549 cells after siRNA transfection. The cells were divided
into five groups and treated with different conditions. (a)
Blank cells (Blank group): A549 cells were pretreated with
TrisꢀHCl (100 mM) for 2 h, and then incubated with ASPC
(10 ꢂM) for 40 min. (b) LPS cells (LPS group): A549 cells
were pretreated with LPS (10 µg mLꢀ1) for 2 h, and then incuꢀ
bated with ASPC (10 ꢂM) for 40 min. (c) LPS+ubenimex
cells: A549 cells were pretreated with LPS (10 µg mLꢀ1) for 2
h, then treated with ubenimex (1 µM) for 40 min, and then
cells incubated with ASPC (10 ꢂM) for 40 min. (d) siRNA
cells (siRNA group): A549 cells pretreated with LTA4Hꢀ
specific siRNA were pretreated with TrisꢀHCl (100 mM) for 2
h, and then incubated with ASPC (10 ꢂM) for 40 min. (e)
LPS+siRNA cells (siRNA LPS group): A549 cells treated with
LTA4H siRNA were pretreated with LPS (10 µg mLꢀ1) for 2 h,
and then incubated with ASPC (10 ꢂM) for 40 min. Twoꢀ
photon fluorescent images were acquired on a LSM 880 NLO
Confocal Laser Scanning Microscope with a water objective
lens (× 20). The excitation wavelength was 690 nm.
In fluorescence imaging experiments, mice were injected
with ASPC (200 µM, 50 µL) in right lung after LPS adminꢀ
istration (0 h, 12 h, 24 h). The mice were performed to expose
the lung by a thoracotomy. Twoꢀphoton fluorescent images
were acquired on a LSM 880 NLO Confocal Laser Scanning
Microscope with an objective lens (×10). The excitation waveꢀ
length was 690 nm.
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Measurement of lung homogenate Interleukin (IL)ꢀ1β
levels. After the mice model was constructed successfully, the
mice were killed by cervical dislocation. The lungs from mice
were excised and washed 3 times repeatedly with 100 mL
saline. Then, the lungs were abraded and lung homogenate
was obtained. Each sample was added 1 mL saline and centriꢀ
fuged at 4 °C, 3000 rpm and 10 min. The supernatant was
collected. After that, we could measure the concentration of
ILꢀ1β by using standard ELISA kit. The ILꢀ1β ELISA kit inꢀ
cludes a set of calibration standards. The calibration standards
are assayed at the same time as the samples and allow the opꢀ
erator to produce a standard curve of Optical Density versus
ILꢀ1β concentration. The concentration of ILꢀ1β in the sample
is then determined by comparing the O.D. (450 nm) of the
sample to the standard curve.
Mice. All female KM mice and female BALB/c mice were
purchased from Shandong University Laboratory Animal Cenꢀ
ter. All the animal experiments were carried out in accordance
with the relevant laws and guidelines issued by the Ethical
Committee of Shandong University.
PM2.5 model of mice. PM2.5 was collected from Jinan atꢀ
mosphere by using an integrated intelligent air total suspended
particulate sampler with a PM2.5 cutter. PM2.5 was collected
on glass fiber filters placed inside the sampler beforehand, and
the flow rate was set to 120 L minꢀ1 for 24 h. The filters were
collected and washed with distilled water by using an ultrasonꢀ
ic cleaning machine. The residue of the filters was removed,
and the particles were freeze dried in vacuum. The obtained
PM2.5 was stored at 4 °C in the dark. The suspensions were
prepared before instillation.
LPSꢀinduced abdominal inflammation model of Mice.
All female KM mice (16ꢀ20 g) were divided into three groups
and treated with different conditions. (A) Blank mice: the mice
were preꢀtreated with PBS for 4 h and then injected ASPC for
30 min. (B) LPSꢀinduced mice: the mice were preꢀtreated with
LPS (2 mg mlꢀ1, 400 µL) for 4 h and injected ASPC for 30
min. (C) LPS+ubenimexꢀtreated mice: the mice were preꢀ
treated with LPS for 4 h and then injected ubenimex for 30
min, then the mice were injected ASPC for 30 min. Then,
mice were anesthetized with 4% chloral hydrate (3 mL kgꢀ1)
by intraperitoneal injection and the hair and skin on the ventral
surface of the abdomen was removed. Twoꢀphoton fluorescent
images were acquired on a LSM 880 NLO Confocal Laser
Scanning Microscope with a water objective lens (×20). The
excitation wavelength was 690 nm (6 mW).
Six to eightꢀweekꢀold female BALB/c mice were randomꢀ
ly divided into three groups, which were injected with 20 ꢂL
of normal saline (control group), 20 ꢂL of lowꢀdose
PM2.5 suspensions (7.5 mg mLꢀ1 in normal saline) and 20 ꢂL
of highꢀdose PM2.5 suspensions (15 mg mLꢀ1 in normal saline),
respectively, through tracheal instillation for 3 days. After 24
h, we incubated mice with the ASPC and performed twoꢀ
photon fluorescence imaging.
In vivo lung inflammation model of Mice. Six to eightꢀ
weekꢀold female BALB/c mice were anesthetized with 4%
chloral hydrate (3 mL kgꢀ1) by intraperitoneal injection. While
anesthetized, the mice were located on a 45° plate and the
mouse's tongue was pulled by tweezers. The mice were treated
with LPS (167 µg mLꢀ1) through intranasal administration.
After that, hold the mouse's nose immediately. 20s later, the
mice were released of the tongue and nose and made them to
wake up in the cage naturally. The mice were randomly dividꢀ
ed into four groups: two groups of LPSꢀtreated mice were
injected with 20 ꢂL of LPS (167 ꢂg ꢂLꢀ1) through intranasal
Statistical Analyses. The results are presented as the mean
±SEM. The statistical significance was evaluated using twoꢀ
tailed Student’s tꢀtest, *P < 0.05, **P < 0.01, ***P < 0.001.
RESULTS AND DISCUSSION
Chemical synthesis of ASPC. Fluorescent probe ASPC
could be readily synthesized in a yield of 25.6% under mild
conditions (Scheme S1). The structure of ASPC was well
characterized by 1H NMR, 13C NMR and HRMS (Figure S7ꢀ
S9).
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Photophysical properties and selectivity of ASPC. The
ASPC exhibits good selectivity for LTA4H over other biologiꢀ
cally relevant species. Thus, the ASPC could be used to detect
the activity of LTA4H in complex biological systems.
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spectral properties of the ASPC were investigated under physꢀ
iological conditions (100 mM Tris buffer, pH 7.4) (Figure
S1aꢀS1d). The ASPC displayed a distinct absorption maxiꢀ
mum band at 320 nm and emitted a faint fluorescence at
around 400 nm under excitation at 320 nm (Figure 1a). Upon
the addition of LTA4H, the fluorescence intensity at 400 nm
increased, and the fluorescence quantum yield increased from
0.04 to 0.61. The probe exhibited a large stoke shift of about
80 nm, thereby alleviating any efficiency loss attributed to
selfꢀabsorption and reduced excitation interference. The fluoꢀ
rescence emission spectra of ASPC were also examined 120
min after adding LTA4H. Figure 1b shows that the ASPC
displays an eightfold fluorescence enhancement at 400 nm
after 120 min in the presence of LTA4H. The TP properties of
the ASPC were also investigated under same physiological
conditions. The ASPC is excited at 690 nm and emits at 400
nm. Upon treatment with LTA4H, the TP fluorescence intensiꢀ
ty increased dramatically at 400 nm and the twoꢀphoton cross
section of the ASPC is 25 GM36. All these data reveal that the
ASPC can detect changes in LTA4H activity.
To prove that the ASPC can be applied in complex biologiꢀ
cal systems, we investigated the cytotoxicity of the probe toꢀ
ward cells through standard MTT assay. No obvious cytotoxiꢀ
city was observed at concentrations up to 20 ꢂM (Figure S1e).
Additionally, the fluorescence photostability of the ASPC in
living cells was investigated (Figure S4). All these data
demonstrate that the ASPC exhibit low cytotoxicity and good
photostability. Hence, the ASPC can act as an excellent fluoꢀ
rescence probe in vivo.
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We subsequently investigated the response of the ASPC
toward different concentrations of LTA4H. The probe was
incubated with various concentrations of LTA4H in Tris buffer
at 37 °C for 1 h. Upon the addition of LTA4H, the fluoresꢀ
cence intensity at 400 nm was considerably enhanced (Figure
1c). A good linear relationship was obtained between the fluoꢀ
rescence intensity of the ASPC and the concentrations of
LTA4H (0ꢀ6.0 ng mLꢀ1), with a detection limit of 0.2183 ng
mLꢀ1 (R2 > 0.99). Hence, the ASPC exhibited satisfactory reꢀ
sponse to the enzyme and can detect the activity of LTA4H
with excellent sensitivity under physiological conditions.
Figure 1. Photophysical properties and selectivity of ASPC. (a)
Oneꢀphoton excitation and emission spectra for ASPC (0.1 µM)
before (●,○) and after (▲,△) addition of LTA4H (4 ng mLꢀ1).
Inset: TP emission spectra for ASPC before (○) and after (△)
addition of LTA4H. (b) Fluorescence emission spectra scan of
ASPC reacted with LTA4H. (c) Linear relationship between fluoꢀ
rescence intensity of ASPC and LTA4H concentrations (0ꢀ6.0 ng
mLꢀ1). (d) Fluorescence responses of ASPC reacted with different
kinds of species for 90 min. (100 mM Tris buffer, 100 mM NaCl,
pH = 7.4, 0.30 % DMSO, v/v, 37 °C). Error bars represent standꢀ
ard deviation (n = 3).
In addition, the kinetic parameters of ASPC toward LTA4H
were also investigated. Under the optimized condition, the
calculated values of Michaelis constant Km and Kcat were 21.18
± 3.23 ꢂM and 0.58 ± 0.04 Sꢀ1 (Figure S2b), respectively,
indicating that ASPC binds to LTA4H with excellent affinity.
In order to prove that ASPC could be used to assess inhibitor
potency, the Ki value of an aminopeptidase inhibitorꢀubenimex,
was determined using ASPC. Results showed that ubenimex
inhibited the activity of LTA4H with Ki 58.5 ± 1.21 nM (Figꢀ
ure S2a), which is approximate with the previous report37.
Therefore, we believe that ASPC is promising to be a convenꢀ
ient tool for accurately screening specific depressors of
LTA4H.
Endogenous LTA4H imaging in live cells with ASPC. To
assess the applicability of the ASPC as a twoꢀphoton fluoresꢀ
cent probe for imaging intracellular LTA4H activity in biologꢀ
ical systems, we tested the probe in living cells. The twoꢀ
photon confocal fluorescence image of LTA4H was applied to
human hepatic cells (HLꢀ7702). A cellular model of inflamꢀ
mation was successfully established by LPS stimulation,
where inflammatory cytokines, such as LTA4H, were overꢀ
expressed38,39. After treating two groups of HLꢀ7702 cells with
TrisꢀHCl (100 mM) (as control group) or LPS (10 µg mLꢀ1)
for 2 h, the cells were incubated with the ASPC (10 ꢂM) at
37 °C for 40 min and washed with TrisꢀHCl to remove residuꢀ
al probes. The weak OP and TP fluorescence images were
observed in Blank cells (Figure 2). By contrast, the fluoresꢀ
cence intensity of the ASPC increased obviously upon the
addition of LPS. This finding indicates the higher activity of
LTA4H in LPSꢀtreated cells than that in Blank cells. To conꢀ
To further evaluate the selectivity of the ASPC to LTA4H,
we examined various biologically relevant molecules, such as
aminopeptidases (APN, METAP, METAP), amino acids (glyꢀ
cine, alanine, cysteine, lysine, arginine), metal ions (Zn2+,
Mg2+, Ca2+), and glucose. The fluorescence intensity of ASPC
did not evidently increase in the presence of the other relevant
substances (Figure 1d). After coꢀculture with LTA4H under
physiological conditions, the fluorescence intensity of the
ASPC obviously increased. These results indicate that the
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was extracted from Blank cells, LPS cells, siRNA cells and siRꢀ
NA+LPS cells respectively.
firm that the increase in the fluorescence intensity could be
due to the high activity of LTA4H, we added the inhibitor of
LTA4H into LPSꢀtreated cells and observed changes in fluoꢀ
rescence. We selected ubenimex as the inhibitor of LTA4H
because it can competitively occupy the activity center of
LTA4H and inhibit the catalytic activity40,41. LPS+ubenimexꢀ
treated cells showed lower fluorescence intensity than LPSꢀ
treated cells (Figure 2g and 2h). Hence, the ASPC exhibits
high sensitivity and selectivity to LTA4H and can be applied
for fluorescence imaging of enzyme activity in live cells.
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To demonstrate the selective fluorescence response of the
ASPC to intracellular LTA4H, we performed lta4h gene silencꢀ
ing in A549 cells by using small interfering RNA (siRNA)42.
The transfection results of FAMꢀlabeled siRNA indicated an
average transfection efficiency of approximately 92.6% (Figꢀ
ure S5). The efficiency of gene silencing was verified by
Western blot (Figure 3g) and realꢀtime PCR analyses (Figure
S1f)43,44. All these results suggest that the expression of
LTA4H was successfully suppressed in siRNAꢀtreated cells.
Compared with blank A549 cells without any stimulation,
cells pretreated with LTA4H siRNA showed reduced fluoresꢀ
cence enhancement fold from 1 to 0.5 (Figure 3a and 3d).
After stimulating with LPS for 2 h, the fluorescence enhanceꢀ
ment decreased from 5.8ꢀfold in LPSꢀtreated cells to 0.9ꢀfold
in LPS+siRNAꢀtreated cells (Figure 3b and 3e). Hence, the
ASPC can monitor the activity of endogenous LTA4H in livꢀ
ing cells with high sensitivity and excellent selectivity. Moreꢀ
over, TPM fluorescence imaging using the ASPC as probe is a
powerful method to effectively detect inflammation at the
cellular level.
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Figure 2. Fluorescence images of ASPC in human hepatic stellate
cells (HLꢀ7702). Oneꢀphoton (OP) and twoꢀphoton (TP) fluoresꢀ
cence images of HLꢀ7702 cells incubated with 10 ꢂM ASPC for
40 min. (a) Blank cells. (b) LPSꢀtreated cells. (c) LPS+ubenimexꢀ
treated cells. Oneꢀphoton images were acquired using 405 nm
To investigate the applicability of the ASPC to high
throughput screening, we used ImageStreamX platform in
analyzing the fluorescence intensity of the ASPC in different
cells. The capability of the ASPC to detect LTA4H in different
cells was investigated, and the mean fluorescence intensity in
thousands of cells was precisely calculated. LPSꢀpretreated
A549 cells exhibited the strongest fluorescence intensity (the
mean FI = 66706.96) compared with cells subjected to the
other pretreatments (Figure 4). Hence, the ASPC could be an
imaging tool for highꢀefficiency, highꢀthroughput monitoring
of LTA4H activity in vivo with high sensitivity.
excitation and emission windows 410ꢀ520 nm. TP imaging: λex
=
690 nm, λem = 380ꢀ520 nm. Scale bar = 200 ꢂm. (d) Relative OP
and TP fluorescence intensity of ASPCꢀlabeled cells from images.
Error bars represent standard deviation (n = 3). Statistical analysis
performed with a twoꢀtailed Student's tꢀtest, ***P < 0.001.
Figure 3. TP confocal images of LTA4H in A549 cells. (a) Blank
cells. (b) LPS cells. (c) LPS+ubenimex cells. (d) siRNA cells. (e)
LPS+siRNA cells. TP imaging: λex = 690 nm, λem = 380ꢀ520 nm.
Scale bar = 200 ꢂm. (f) Relative TP fluorescence intensity of
ASPCꢀlabeled cells from images. Error bars represent standard
deviation (n = 3). Statistical analysis performed with a twoꢀtailed
Student's tꢀtest, **P < 0.01, ***P < 0.001. (g) LTA4H levels were
analyzed with Western blot. From left to right, LTA4H protein
Figure 4. Determination of LTA4H in A549 cells by imaging flow
cytometry with four different treated conditions. (a) BF: bright
field; SSC: fluorescence of side scatter; FI: fluorescence of ASPC.
(b,c) Internalization score ( > 8000 cells).
In vivo fluorescence imaging of LTA4H in mice by ASPC.
The performance of the ASPC as fluorescent probe for animal
bioimaging in vivo was further investigated. A mouse model
of abdominal inflammation was constructed successfully by
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intraperitoneal injection of LPS into living mice45. After 4 h,
d) BALB/c mice were injected with 20 ꢂL LPS (167 µg µLꢀ1) for
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12 h and 24 h respectively. Labels from 50ꢀ250 ꢂm indicate scanꢀ
ning depths of the lung tissue. (e) Fluorescence intensity enhanced
ratios from images with different time. (f) The concentration of
ILꢀ1β was measured at different time in lung homogenate of mice.
Error bars represent standard deviation (n = 4). Statistical analysis
performed with a twoꢀtailed Student's tꢀtest, **P < 0.01, ***P <
0.001. TP imaging: λex = 690 nm, λem = 380ꢀ520 nm. Scale bar =
200 ꢂm.
the mice were injected with the ASPC prior to TP imaging.
Figure 5 shows that the fluorescence intensity in the abꢀ
dominal tissues of inflamed mice (LPSꢀinduced mice) is 4.2
times higher than that in healthy mice (Blank mice without
stimulus). After pretreatment with the inhibitor of LTA4Hꢀ
ubenimex, the fluorescence intensity in mice (LPS+ubenimex
ꢀtreated mice) is only 2.1 times higher than that in healthy
mice. This finding reveals that the LTA4H protein in LPSꢀ
induced mice was overꢀexpressed, leading to the strong fluoꢀ
rescence intensity of the ASPC. Meanwhile, adding ubenimex
to the ASPC could inhibit the catalytic activity of LTA4H and
prevent fluorescence enhancement. Hence, the ASPC shows
high selectivity for detecting the activity of LTA4H in mice.
9
Since LTA4H in mice and human share highly identical prot
einsequences (http://www.ncbi.nlm.nih.gov/guide/proteins/),
we used female mice as the live model. To verify the applicaꢀ
bility of the ASPC for accurate diagnosis of pneumonia, we
performed twoꢀphoton fluorescence imaging with the ASPC
for detecting the activity of LTA4H in LPSꢀinduced mice with
pneumonia29,46. Six to eightꢀweekꢀold female BALB/c mice
(16–18 g) were randomly divided into four groups: two groups
of LPSꢀtreated mice were injected with 20 ꢂL of LPS (167 µg
µLꢀ1) through intranasal route for 12 and 24 h; and the two
other groups of blank mice were injected with the same
amount of PBS. After injecting the ASPC for 30 min, twoꢀ
photon fluorescence imaging of the lung tissues of mice was
conducted. The fluorescence intensity in the lung tissues of
LPSꢀtreated mice is higher than that in blank mice (Figure 6).
In addition, the fluorescence intensity increased by 4.6ꢀ and
5.3ꢀfold under LPS stimulation for 12 and 24 h, respectively.
This result indicates that the LPSꢀtreated mice exhibit higher
LTA4H activity in the lung and might have pneumonia after
LPS stimulation. Interleukin (IL)ꢀ1β is one of the critical indiꢀ
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Figure 5. TP fluorescence imaging of LTA4H in the mice model
of abdominal inflammation. (a) Blank mice. (b) LPSꢀinduced
mice. (c) LPS+ubenimexꢀinduced mice. (d) Fluorescence intensity
enhanced ratios from images. TP imaging: λex = 690 nm, λem
=
380ꢀ520 nm. Scale bar = 200 ꢂm. Error bars represent standard
deviation (n = 4). Statistical analysis performed with a twoꢀtailed
Student's tꢀtest, **P < 0.01, ***P < 0.001.
cators of pneumonia47-49
.
To confirm whether LPSꢀtreated mice have pneumonia, we
assessed the amount of ILꢀ1β by using a commercial enzymeꢀ
linked immunosorbent assay (ELISA) kit. After stimulation
for 12 and 24 h, the concentration of ILꢀ1β is 2.9 and 3.6 times
higher in LPSꢀtreated mice than that in blank mice, respectiveꢀ
ly (Figure 6f). These data suggest that LPSꢀtreated mice suffer
from pneumonia, and their conditions continue to deteriorate
over time. During this process, the expression level of LTA4H
in the lung tissues continually increased, resulting in increased
fluorescence signal of the ASPC. Hence, LTA4H can be used
as an inflammatory biomarker for fluorescence imaging to
accurately diagnose pneumonia.
Figure 6. TP fluorescence imaging of LTA4H in the mice model
of lung inflammation with different time. Lung of mice were inꢀ
cubated with ASPC (10 ꢂM) for 30 min. (a, c) BALB/c mice
were injected with PBS (20 ꢂL) for 12 h and 24 h respectively. (b,
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probe is the first reported for specific detection of LTA4H
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activity in vivo. The ASPC possesses distinct advantages,
including low cytotoxicity and deep tissue penetration depth,
and can be used for rapid and real time TP fluorescence imagꢀ
ing of LTA4H in situ in cells and tissues. The probe was sucꢀ
cessfully applied for in situ visualization of LTA4H activity in
the lungs and for determining the gradual deterioration stage
of pneumonia caused by increasing haze particulates dosage in
the mouse model. With these outstanding advantages,
the ASPC can be an indicator of pneumonia in vivo and can
be used not only to diagnose pneumonia but also to estimate
the effectiveness of treatment. The novel ASPC probe and
imaging strategy could be a novel method for screening speꢀ
cific inhibitors of LTA4H as potential drug candidates in the
future.
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Figure 7. 3D volumeꢀrendered TP imaging of LTA4H in PM2.5
contaminated mice. (a) Blank group: mice were injected with
normal saline. (b) Mice were injected with lowꢀdose PM2.5 (7.5
mg mLꢀ1). (c) Mice were injected with highꢀdose PM2.5 (15 mg
mLꢀ1). (d) Fluorescence intensity enhanced ratios from images
with different PM2.5 concentration. (e) The concentration of ILꢀ
1β was measured by different PM2.5 concentration in lung hoꢀ
mogenate of mice. TP imaging: λex = 690 nm, λem = 380ꢀ520 nm.
Scale bar = 200 ꢂm. Error bars represent standard deviation (n =
4). Statistical analysis performed with a twoꢀtailed Student's
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Organic synthesis, characterization, experimental details and addiꢀ
tional figures (PDF)
tꢀtest, **
P < 0.01, ***P < 0.001.
Considering the successful establishment of a novel twoꢀ
AUTHOR INFORMATION
Corresponding Author
photon fluorescence imaging for accurately diagnosing pneuꢀ
monia, we investigated whether haze particulates can cause
pneumonia in mice7,8,50. A contaminated mouse model exꢀ
posed to PM2.5 was built successfully. Six to eightꢀweekꢀold
female BALB/c mice (16ꢀ18 g) were randomly divided into
three groups, which were injected with 20 ꢂL of normal saline
(control group), 20 ꢂL of lowꢀdose PM2.5 suspensions (7.5
mg mLꢀ1 in normal saline) and 20 ꢂL of highꢀdose PM2.5 susꢀ
pensions (15 mg mLꢀ1 in normal saline) through tracheal instilꢀ
lation for 3 days. After 24 h, we incubated mice with the
ASPC and performed twoꢀphoton fluorescence imaging. The
fluorescence intensity in the lungs of mice that received
PM2.5 suspensions is 1.6 (lowꢀdose) and 2.9 times (highꢀdose)
higher than that in the control group (Figure 7). This finding
indicates the increased LTA4H expression in the lungs of mice
and the gradual deterioration of pneumonia with increasing
particulate dosage. A commercial ELISA kit was adopted to
measure the amount of ILꢀ1β in the lung homogenates from
the mice. The amount of ILꢀ1β in the homogenates increased
with increasing dosage of PM2.5 (Figure 7e), consistent with
the result of the twoꢀphoton fluorescence imaging. Hence, the
contaminated mice that inhaled haze particulates (PM2.5) may
suffer from pneumonia.
tangb@sdnu.edu.cn
lip@sdnu.edu.cn
Author Contributions
†Hui Wang and Ke Xue contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by 973 Program (2013CB933800), Naꢀ
tional Natural Science Foundation of China (21405097,
21535004，21390411), the Project of Shandong Province Higher
Educational Science and Technology Program (J14LC04), and the
Primary Research Plan in Shandong Province (2016GSF201205).
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