Organic Nanoprobe Cocktails for Multilocal and Multicolor Fluorescence Imaging of Reactive Oxygen Species

Article abstract of DOI:10.1002/adfm.201700493

Hypochlorite (ClO^?) as a highly reactive oxygen species not only acts as a powerful “guarder” in innate host defense but also regulates inflammation-related pathological conditions. Despite the availability of fluorescence probes for detection

Full text of DOI:10.1002/adfm.201700493

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Fluorescence Imaging
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Organic Nanoprobe Cocktails for Multilocal and Multicolor
Fluorescence Imaging of Reactive Oxygen Species
Chao Yin, Houjuan Zhu, Chen Xie, Lei Zhang, Peng Chen, Quli Fan,* Wei Huang,
and Kanyi Pu*
Among them, hypochlorite (ClO−) is pro-
duced by immune cells through the reac-
Hypochlorite (ClO−) as a highly reactive oxygen species not only acts as a
powerful “guarder” in innate host defense but also regulates inflammation-
related pathological conditions. Despite the availability of fluorescence probes
for detection of ClO− in cells, most of them can only detect ClO− in single
cellular organelle, limiting the capability to fully elucidate the synergistic effect
of different organelles on the generation of ClO−. This study proposes a nano-
probe cocktail approach for multicolor and multiorganelle imaging of ClO− in
cells. Two semiconducting oligomers with different π-conjugation length are
synthesized, both of which contain phenothiazine to specifically react with
ClO− but show different fluorescent color responses. These sensing compo-
nents are self-assembled into the nanoprobes with the ability to target cellular
lysosome and mitochondria, respectively. The mixture of these nanoprobes
forms a nano-cocktail that allows for simultaneous imaging of elevated level of
ClO− in lysosome and mitochondria according to fluorescence color variations
under selective excitation of each nanoprobe. Thus, this study provides a gen-
eral concept to design probe cocktails for multilocal and multicolor imaging.
tion between hydrogen peroxide (H2O2)
and chloride ions catalyzed by myelop-
eroxidase (MPO),[2] and can serve as a
powerful “guarder” in innate host defense
to kill invading microbes.[3] In addition,
ClO− is often upregulated in many inflam-
matory pathological conditions such as
neurondegeneration,[4] cancer,[5] cardio-
vascular diseases,[6] atherosclerosis,[7] and
rheumatoid arthritis.[8] Thereby, imaging
of ClO− using activatable fluorophores is
critical to understanding the pathological
functions of ClO− and developing inno-
vative therapeutic approach to treat these
diseases.[9]
Lysosome and mitochondria are two
major organelles in almost all eukaryotic
cells related to the production of ClO−.
Lysosome has degradation machinery with
abundant enzymes, which contains MPO
to catalyze the generation of ClO−;[10] whereas, mitochondria
are the cellular “plants of power” that host respiration chain
and are the major sources of ROS including ClO−.[11] Although
fluorescent molecular probes were developed to detect ClO− in
mitochondria or lysosome, most of them only can detect it in
one organelle[10b,12] or in the two organelles separately.[13] How-
ever, to understand the synergistic effect of lysosome and mito-
chondria on the production of ClO−, development of probes that
can simultaneously image ClO− in both organelles is essential,
which remains elusive.
In this contribution, we report a cocktail probe design
approach based on organic semiconducting nanoparticles
(OSNs) for in vitro multilocal and multicolor imaging of ClO−.
OSNs composed of semiconducting polymers or oligomers
have recently evolved into a new generation of photonic mate-
rials for molecular imaging. The structural versatility of OSNs
has led to different biological applications including tumor
imaging,[14] neuroinflammation imaging,[15] lymph node map-
ping,[16] ultrafast hemodynamic imaging,[17] and neuron acti-
vation.[18] Although OSNs have been developed for imaging
of ROS, selective fluorescence imaging of ClO− has yet to be
demonstrated.[19]
1. Introduction
Reactive oxygen species (ROS) are chemical mediators that
play crucial roles in signal transductions of living organisms.[1]
C. Yin, Dr. L. Zhang, Prof. Q. Fan, Prof. W. Huang
Key Laboratory for Organic Electronics and Information
Displays & Institute of Advanced Materials (IAM)
Jiangsu National Synergetic Innovation Center for Advanced Materials
(SICAM)
Nanjing University of Posts & Telecommunications
Nanjing 210023, China
E-mail: iamqlfan@njupt.edu.cn
C. Yin, Dr. H. Zhu, Dr. C. Xie, Prof. P. Chen, Prof. K. Pu
School of Chemical and Biomedical Engineering
Nanyang Technological University
70 Nanyang Drive, Singapore 637457, Singapore
E-mail: kypu@ntu.edu.sg
Prof. W. Huang
Key Laboratory of Flexible Electronics (KLOFE) & Institute
of Advanced Materials (IAM)
Jiangsu National Synergetic Innovation Center for Advanced Materials
(SICAM)
Nanjing Tech University (NanjingTech)
Nanjing 211816, China
To make the probe cocktails, two semiconducting oli-
gomers (4 and 5, Scheme 1) and two amphiphilic block copoly-
mers (9 and 10, Scheme 1) are synthesized and used as the
building blocks. Both oligomers contain phenothiazine as the
The ORCID identification number(s) for the author(s) of this article
can be found under http://dx.doi.org/10.1002/adfm.201700493.
DOI: 10.1002/adfm.201700493
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Scheme 1. Design and synthesis of the phenothiazine-based semiconducting oligomers (4 and 5) and the amphiphilic block copolymers (9 and 10) that
target the lysosome and mitochondria, respectively. Reaction conditions: (i) 1-bromohexane, NaH, anhydrous N,N-dimethylformamide (DMF), room
temperature. (ii) NBS/anhydrous DMF, room temperature. (iii) tributyl(vinyl)tin, Pd(PPh3)2Cl2/2,6-di-tert-butylphenol, anhydrous toluene, 100 °C. (iv)
Pd(OAc)2/P(o-tolyl)3, anhydrous DMF, triethylamine, 100 °C. (v) Pd(OAc)2/P(o-tolyl)3, anhydrous DMF, triethylamine, 100 °C. (vi) Sodium azide, anhy-
drous DMF, room temperature. (vii) Sodium azide, anhydrous DMF, room temperature. (viii) NaH, anhydrous tetrahydrofuran (THF), 3-bromoprop-
1-yne, room temperature. (ix) CuSO4•5H2O, sodium ascorbate, anhydrous DMF, room temperature. (x) CuSO4•5H2O, sodium ascorbate, anhydrous
DMF, room temperature.
ClO− sensing unit but show different multicolor responses
toward ClO−; whereas, the copolymers
and 10 have
1. Bromination of 1 was carried out carefully using N-bromosuc-
cinimide (NBS) to yield a single bromine substituted compound
2. Then 10-hexyl-3-vinyl-10H-phenothiazine (3) was synthesized
in 48.1% yield by heating the mixture of 2 and tributyl(vinyl)tin in
9
morpholine and triphenylphosphine groups to target lysosome
and mitochondria, respectively. Nanoprecipitation between the
semiconducting oligomer and the amphiph-
ilic copolymer leads to the organelle-targeted
nanoprobes: lysosome-targeted nanoprobe
(LNP) composed of 4 and 9, and mitochon-
dria-targeted nanoprobe (MNP) composed
of 5 and 10. By selective light excitation, the
cocktail of LNP and MNP allows for multi-
local and multicolor fluorescence imaging of
ClO− in living cells (Figure 1).
2. Results and Discussion
2.1. Synthesis and Characterization
The designs of fluorescence molecular probes
for ClO− sensing are generally based on oxida-
tion and cleavage.[20] For instance, p-methoxy-
phenol and spirothioether have been used as
the reaction site to trigger the probe activation
by ClO−.[21] However, fluorescence turn-on
signals rather than fluorescence ratiometric
signals are generated. To realize multicolor
sensing, the phenothiazine-based semicon-
ducting oligomers (4 and 5) were synthesized
as shown in Scheme 1. Phenothiazine was
first alkylated with 1-bromohexane in presence
of sodium hydride (NaH) to obtain compound
Figure 1. The illustration of organelle-differentiated multilocal and multicolor fluorescence
imaging of endogenous ClO− in macrophage cells using the organic nanoprobe cocktails com-
posed of lysosome-targeted nanoprobe (LNP) and mitochondria-targeted nanoprobe (MNP).
LNP targets lysosome and changes its fluorescence color from green to blue upon activation by
ClO−; while MNP targets mitochondria and changes its fluorescence color from red to orange
upon activation by ClO−.
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Figure 2. Characterization of the lysosome-targeted nanoprobe (LNP) and mitochondria-targeted nanoprobe (MNP). a) Representative transmission
electron microscopy (TEM) of LNP. b) Dynamic light scattering (DLS) of the LNP. c) Average hydrodynamic diameters of the LNP stored in PBS for
different time periods. d) Representative TEM of MNP. e) DLS result of the MNP. f) Average hydrodynamic diameters of the MNP stored in PBS for
different time periods. The error bars represent standard deviations (SD) of three separate measurements.
toluene using Pd(PPh3)2Cl2/2,6-di-tert-butylphenol as catalyst at
100 °C for 24 h. Heck reaction was then conducted between com-
pound 3 and 1,4-dibromobenzene, yielding the semiconducting
oligomer 4 (1,4-bis((E)-2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)-
benzene). The oligomer 5 (4,7-bis((E)-2-(10-hexyl-10H-phenothi-
azin-3-yl)vinyl)benzo[c][1,2,5]thiadiazole) was synthesized via a
similar procedure using compound 3 and 4,7-dibromobenzo[c]
[1,2,5]thiadiazole. To synthesize the organelle-targeted poly-
mers, poly(ethylene glycol)-block-poly(propylene glycol)-block-
poly(ethylene glycol) (PEG-b-PPG-b -PEG) was first reacted with
3-bromoprop-1-yne to introduce the terminal alkynyl, yielding 8.
Then, click reaction was used to conjugate 8 with 6 or 7 to yield
the lysosome-targeted or mitochondria-targeted polymers (9 or
10). The correct chemical structures of all the intermediates and
the final products were confirmed by 1H NMR and mass spectra
(Supporting Information).
Co-nanoprecipitation method was used to synthesize the
organelle-targeted nanoprobes. The lysosome- and mitochon-
dria-targeted amphiphilic triblock copolymers (9 and 10) were
respectively coprecipitated with the semiconducting oligomers
4 and 5 to obtain LNP and MNP. Transmission electron micros-
copy (TEM) indicated the spherical morphology for both
LNP and MNP (Figure 2a,d). Dynamic light scattering (DLS)
2.2. Optical Responses toward ROS
The optical responses of the nanoprobes toward ROS were
investigated. LNP had the maximum absorption peak at
405 nm, which underwent an immediate spectral evolution
with addition of ClO−. The absorption at 405 nm gradually
decreased, concomitant with the emergence of a new blue-
shifted band centered at 352 nm (Figure 3a). Accordingly,
the solution color changed from yellow green to colorless
(Figure 3g). Similarly, MNP exhibited the maximum absorption
peak at 503 nm, which gradually blue-shifted to 463 nm with
addition of ClO− (Figure 3d), and the solution color changed
from red to orange (Figure 3h). LNP and MNP emitted green
and red fluorescence with the quantum yields of 0.52 and
0.14, respectively. With the addition of ClO−, the emission of
LNP gradually decreased at 535 nm, and shifted to 480 nm
with a shoulder peak at 450 nm with the quantum yield of
0.16 (Figure 3b); whereas, the emission of MNP gradually
decreased at 690 nm, and shifted to 610 nm with the quantum
yield of 0.23 (Figure 3e). Such spectral changes led to the fluo-
rescence multicolor responses of these nanoprobes toward
ClO−: green to blue for LNP (Figure 3g) and red to orange for
MNP (Figure 3h). Furthermore, the fluorescence spectra evo-
lution allowed the signal quantification using the ratiometric
fluorescence signals. A good linear correlation between the log-
showed the average hydrodynamic sizes of 22.6
0.52 and
24.5 0.86 nm for LNP and MNP, respectively (Figure 2b,e). The
average hydrodynamic diameters of LNP and MNP remained
almost unchanged even after storage in phosphate buffered
saline (PBS, pH = 7.4) for 30 d (Figure 2c,f), indicating the high
stability of the nanprobes probably due to good water-solubility
provided by PEG segment from the amphiphilic copolymers as
well as strong hydrophobic interactions between the PPG seg-
ment and the semiconducting oligomer.
arithmic value of ratiometric fluorescence signals (ln(I480/I535
)
for LNP and ln(I610/I690) for MNP) and the concentration of
ClO− was observed with the limit of detection of 0.15 × 10−6 and
0.19 × 10−6 m for LNP and MNP, respectively (Figure 3c,f).
To investigate the selectivity of the nanoprobes, the ratios of
the fluorescence ratiometric signals in the presence of ROS to
that in the absence of ROS were calculated and summarized
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Figure 3. Optical responses of the lysosome- and mitochondria-targeted nanoprobe (LNP and MNP) toward ROS. a) UV–vis absorption spectra of LNP
(10 µg mL−1) upon addition of ClO− from 0 to 20 × 10−6 m at the interval of 2 × 10−6 m. b) Gradual evolution of fluorescence spectra of LNP solution
(10 µg mL−1) upon treatment of ClO− from 0 to 20 × 10−6 m at the interval of 2 × 10−6 m (λex = 405 nm). c) The logarithmic value of ratiometric fluores-
cence signals of LNP (ln(I480/I535)) as a function of ClO− concentration. The red line represents linear fitting from [ClO−] = 0 to 18 × 10−6 m. d) UV–vis
absorption spectra of MNP (10 µg mL−1) upon addition of ClO− from 0 to 20 × 10−6 m at the interval of 2 × 10−6 m. e) Fluorescence spectra of MNP
solution (10 µg mL−1) upon addition of ClO− from 0 to 20 × 10−6 m at the interval of 2 × 10−6 m with the excitation at 450 nm. f) The logarithmic value
of ratiometric fluorescence signals of MNP (ln(I610/I690)) as a function of ClO− concentration. The red line represents linear fitting from [ClO−] = 0 to
20 × 10−6 m. g) Photographs of the LNP solution before and after addition of ClO− under ambient light and a black lamp (λ = 365 nm). h) Photographs
of the MNP solution before and after addition of ClO− under ambient light and a black lamp (λ = 365 nm). i) The ratios of ratiometric fluorescence
intensity of LNP (I480/I535) and MNP (I610/I690) in the presence of ROS to that in the absence of ROS. The error bars represent standard deviations
(SD) of three separate measurements.
in Figure 3i. Upon the activation of ClO−, I480/I535 and I610/I690
were respectively increased by 14.26- and 5.43-fold, while no
obvious changes were detected for other ROS including H2O2,
processes were not susceptible to pH as shown by the pH-
independent response of both LNP and MNP toward ClO−
(Figure S12, Supporting Information), showing the ability of
the nanoprobes to detect ClO− regardless of pH in different
organelles.
−
ONOO−, O2 , 1O2, and •OH. These data demonstrated the high
selectivity of LNP and MNP to ClO−.
The sensing mechanism of the nanoprobes toward ClO− was
studied by testing the reaction product of 4 or 5 after reaction
with ClO− using liquid chromatograph mass spectrometer
(LC-MS). The results showed an increased molecular weight
of ≈32 in both compounds (4 and 5) after reaction with ClO−
(Figure S11, Supporting Information), which was equal to the
molecular weight of oxygen. Thus, it revealed that ClO− medi-
ated oxidation occurred, turning the divalent sulphur into
sulfoxide in 4 or 5 (Figure 4). Such ClO− mediated oxidation
To enable the multilocal and multicolor imaging of ClO−,
the optical responses of the LNP/MNP cocktail toward ClO−
were then investigated. The weight ratio of LNP to MNP was
optimized to be at 7:1, wherein the absorbance of MNP was
low and hidden in the absorption peak of LNP (Figure S13,
Supporting Information). Selective excitation experiments
were conducted to test the ability of differentiating the signals
from different probes. Upon excitation at 405 nm for LNP, the
single emission peak at 535 nm was observed for the probe
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activation by ClO−, I472/I535 (λex = 405 nm)
and I610/I690 (λex = 570 nm) were respec-
tively increased by 22.8- and 16.3-fold,
while no obvious changes were detected for
−
other ROS including H2O2, ONOO−, O2 ,
1O2, and •OH. This proved the high selec-
tivity of the cocktail toward ClO−. The exci-
tation-dependent fluorescence ratiometric
responses indicated that it is feasible to use
the nanoprobe cocktail to image ClO− in dif-
ferent cellular organelles according to their
fluorescence multicolor changes.
Figure 4. The proposed reaction mechanism of oligomers a) 4 and b) 5 with ClO−. The resulted
sulfoxide compounds have different fluorescent colors from their corresponding original forms.
cocktail, which gradually changed to the dual peaks at 472 and
590 nm with nearly identical intensities after activation by
ClO− (20 × 10−6 m) (Figure 5b). The spectral evolution of the
cocktail toward ClO− allowed us to quantify the signals using
the ratiometric fluorescence intensity at 472 nm to that at
535 nm (I472/I535). A good linear correlation between the loga-
rithmic value of ratiometric fluorescence signals (ln(I472/I535))
and the concentration of ClO− was detected with the limit
of detection of 0.17 × 10−6 m (Figure 5c). Upon excitation at
570 nm for MNP, the emission peak at 690 nm was observed
and gradually blue-shifted to 610 nm after activation by ClO−
(20 × 10−6 m) (Figure 5d). Similarly, a good linear correlation
between the logarithmic value of ratiometric fluorescence sig-
nals (ln(I610/I690)) and the concentration of ClO− was observed
with the limit of detection of 0.19 × 10−6 m (Figure 5e). Upon
2.3. Validation of Targeting Capability
To examine the subcellular targeting ability, LNP or MNP
was incubated with murine macrophage cells (RAW 264.7)
and costaining was conducted with LysoTracker Deep Red or
MitoTracker Green. It should be noted that only the signals
from red channel can be detected from LysoTracker- or MNP-
treated cells, while only green fluorescence can be observed
from MitoTracker- or LNP-treated cells (Figure S14, Supporting
Information). These results confirmed no signal crosstalk
between the nanoprobes (LNP and MNP) and their corre-
sponding trackers (LysoTracker and MitoTracker), indicating
the feasibility of using these trackers for colocalization studies.
As shown in Figure 6, the green fluorescence of LNP and the
red fluorescence of MNP overlapped well with the signals from
Figure 5. Optical responses of the LNP/MNP cocktail toward ClO−. a) Illustration of the fluorescence evolution of the cocktail nanoprobe upon ClO−
treatment. b) Fluorescence spectra evolution of the cocktail nanoprobe solution (10 µg mL−1) upon treatment of ClO− from 0 to 20 × 10−6 m at the
interval of 2 × 10−6 m (λex = 405 nm). The fluorescence intensities were normalized at 535 nm. c) The logarithmic value of ratiometric fluorescence
signals (ln(I472/I535)) as a function of ClO− concentration. The red line represents linear fitting from [ClO−] = 0 to 18 × 10−6 m. d) Normalized fluores-
cence spectra of the cocktail nanoprobe solution (10 µg mL−1) upon addition of ClO− from 0 to 20 × 10−6 m at the interval of 2 × 10−6 m (λex = 570 nm).
e) The logarithmic value of ratiometric fluorescence signals (ln(I610/I690)) as a function of ClO− concentration. The red line represents linear fitting from
[ClO−] = 0 to 18 × 10−6 m. f) The ratios of ratiometric fluorescence intensity of the cocktail (I472/I535 and I610/I690) in the presence of ROS to that in the
absence of ROS. The error bars represent SDs of three separate measurements.
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Figure 6. Intracellular localization of LNP and MNP in macrophage cells. a) Images of macrophage cells incubated with LNP (20 µg mL−1) for 24 h, and
subsequently LysoTracker (1.0 × 10−6 m) for another 15 min. Red, LysoTracker fluorescence; green, LNP fluorescence; yellow, merged signal. b) Intensity
profile of linear regions of interest across macrophage cells (white arrow in (a)). Red and green lines represent the intensity of the LysoTracker and
LNP, respectively. c) Correlation plot of LysoTracker and LNP intensities. d) Images of macrophage cells treated with MNP (20 µg mL−1) for 24 h,
and subsequently MitoTracker (1.0 × 10−6 m) for another 15 min. Green, MitoTracker fluorescence; Red, MNP fluorescence; yellow, merged signal.
e) Intensity profile of linear regions of interest across macrophage cells (white arrow in (d)). Red and green lines represent the intensity of the MNP
and MitoTracker, respectively. f) Correlation plot of MitoTracker and MNP intensities.
LysoTracker and MitoTracker, respectively. The changes in the
intensity profiles of linear regions of interest between LNP and
LysoTracker (Figure 6b) or MNP and MitoTracker (Figure 6e)
were also synchronous and well-overlapped. Furthermore, the
intensity of the correlation plot (Figure 6c,f) reflected high
overlap coefficients for LysoTracker and LNP (0.90), and
MitoTracker and MNP intensities (0.84). These data demon-
strated the excellent intracellular targeting capabilities of LNP
and MNP toward lysosome and mitochondria, respectively. In
combination with the specific responses toward ClO− and good
cytocompatibility (Figure S15, Supporting Information), the
nanoprobes should have the potential for ClO− imaging at the
subcellular level.
channels under the same condition (Figure 7a). This proved
that the nanoprobes remained unactivated state both in lyso-
some and mitochondria in the resting cells. In contrast, when
the cells were stimulated with LPS/IFN-γ prior to incuba-
tion with the nanoprobe cocktail, strong fluorescence signals
(Figure 7b) were detected for the blue and orange channels.
Note that the relatively strong fluorescence in the green
channel was caused by the activation of MNP, as the activated
MNP could be excited at 405 nm and emit the fluorescence
in the range from 500 to 800 nm. Nevertheless, the images
in Figure 7b indicated that the nano-cocktails were activated
by ClO− in both lysosome and mitochondria of stimulated
macrophage cells. This was also confirmed by reduced signals
in both blue and orange channels for the cells cotreated with
LPS/IFN-γ and NAC (a ROS scavenger). Furthermore, overlap-
ping the blue and orange channels allowed for simultaneous
visualization of the ClO− distribution in both lysosome and
mitochondria (Figure 7b).
The ratiometric fluorescence signals of IBlue/IGreen (Figure 7d)
and IOrange/IRed (Figure 7e) of cells treated with LPS/IFN-γ were
calculated to be 0.32 and 1.5, respectively. According to the
calibration curves in Figure 5c and Figure 5e, the average ClO−
concentrations in lysosomes and mitochondria in activated mac-
rophage cells were estimated to be ≈9.0 × 10−6 and 8.0 × 10−6 m,
respectively. Such estimated concentrations were consistent with
the values reported in literatures, showing that ClO− can be gen-
erated at 20 × 10−6– 400 × 10−6 m h−1 by activated immune cells
under inflammatory conditions.[2] These data confirmed the
capability of the nanoprobe cocktail for multicolor fluorescence
imaging of ClO− in mitochondria and lysosome in a simulta-
neous manner. In addition, another nanoprobe cocktail could
be made by pairing 4 with 10 and 5 with 9, which exhibited
different color variation toward ClO− (Figure S18, Supporting
Information).
2.4. Multilocal and Multicolor Imaging of ClO−
The nanoprobe cocktail was tested for multilocal and multi-
color imaging of endogenous ClO− in murine macrophage
cell line (RAW 264.7). To mimic the inflammatory conditions,
the cells were pretreated with bacterial cell wall lipopolysac-
charide (LPS) and a dimerized soluble cytokine, interferon-γ
(IFN-γ) which stimulated resting macrophages to produce
ROS including ClO−. The negative control cells were pre-
treated with
a free-radical scavenger, N-acetyl-L-cysteine
(NAC), in addition to LPS/IFN-γ to scavenge macrophage-
generated ROS. The multilocal and multicolor fluorescence
imaging were conducted by selective excitation of LNP at
405 and MNP at 570 nm to detect the signals in lysosome and
mitochondria, respectively. After incubating the resting mac-
rophage cells with the nanoprobe cocktail, strong fluorescence
signals were detected for both the green (520–550 nm) and
red (690–750 nm) channels, while almost no fluorescence was
observed for the blue (420–460 nm) and orange (580–610 nm)
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Figure 7. Multilocal and multicolor imaging of ClO− in murine macrophage cells (RAW 264.7). a) RAW 264.7 cells were incubated with the nanoprobe
cocktail (30 µg mL−1) for 24 h without any pretreatment. b) Cells were pretreated with LPS (100 ng mL−1)/IFN-γ (20 ng mL−1) for 24 h and then incubated
with the nanoprobe cocktail (30 µg mL−1) for another 24 h. c) Cells were pretreated with LPS (100 ng mL−1)/IFN-γ (20 ng mL−1) and NAC (1 × 10−3 m) for
24 h, and then incubated with the nanoprobe cocktail (30 µg mL−1) for another 24 h. All the imaging signals were collected through four channels: blue
(420–460 nm, λex = 405 nm), green (520–550 nm, λex = 405 nm), orange (580–610 nm, λex = 570 nm), and red (690–750 nm, λex = 570 nm). d) Quanti-
fication of the ratiometric fluorescence signals of the blue channel to that of the green channel (IBlue/IGreen) and their logarithmic values (ln(IBlue/IGreen))
of the macrophage cells upon different treatments: control, +LPS/IFN-γ, and +LPS/IFN-γ+NAC. e) Quantification of the ratiometric fluorescence signals
of the orange channel to that of the red channel (IOrange/IRed) and their logarithmic values (ln(IOrange/IRed)) of the macrophage cells upon different treat-
ments: control, +LPS/IFN-γ, and +LPS/IFN-γ+NAC. The error bars represent standard deviations (SDs) of three separate measurements.
time-of-flight mass spectrometry (MALDI-TOF-MASS) was performed
on a Bruker autoflex under the reflector mode for data acquisition.
3. Conclusion
TEM images were obtained on a JEM 1230 transmission electron
microscope with an accelerating voltage of 200 kV. DLS was performed
In conclusion, we have proposed a probe cocktail approach
toward multicolor and multiorganelle imaging of ClO− in living
on the Malvern ZetaSizer Nano S. UV–vis spectra were recorded on a
cells. The nanoprobe cocktail was made of two organic nano-
Shimadzu UV-2450 spectrophotometer. Fluorescence measurements
particles (LNP and MNP) self-assembled from different pairs
were carried out on a Fluorolog 3-TCSPC spectrofluorometer (Horiba
of semiconducting oligomers and targeted amphiphilic poly-
mers. Phenothiazine unit within the semiconducting oligomers
endowed the nanoprobe specific and sensitive multicolor fluo-
rescent responses toward ClO−; whereas, morpholine and tri-
phenylphosphine groups of the amphiphilic polymers led to
the capability of the nanoprobe cocktails to target lysosome and
mitochondria, respectively. In combination with good water-
solubility, small sizes (less than 25 nm), and low cytotoxicity,
the nanoprobe cocktail was able to efficiently internalize the
cells, precisely target lysosome and mitochondria, and color-
fully delineate the ClO− levels in these organelles with different
fluorescence responses. Our study not only provides a new
generation of organic nanoparticles that show fluorescence
ratiometric responses toward ClO− but also highlights a feasible
methodology to image one biomarker in multiple subcellular
sites by simply formulating a cocktail of nanoprobes.
Jobin Yvon). Confocal laser scanning microscope was conducted on an
LSM 710 Meta Confocal Scope.
Chemicals: All chemicals were obtained from Sigma-Aldrich unless
otherwise stated. LysoTracker Deep Red and MitoTracker Green were
purchased from Molecular Probes Company.
10-Hexyl-10H-phenothiazine (1): A 250 mL round-bottom flask with
magnetic stirring bar was charged with a mixture of sodium hydride
(60% in mineral oil, 1.35 g, 33.75 mmol) and 1-bromohexane (4.95 g,
30.18 mmol) under an argon atmosphere. Then 5 g of 10H-phenothiazine
(dissolved in 25 mL anhydrous DMF) was slowly added into the round-
bottom flask. The mixture was stirred at room temperature overnight,
which was poured into water and extracted with diethyl ether. The
separated organic layer was dried over Na2SO4 and the solvent removed
using rotary evaporation. The crude product was purified by column
chromatography (silica gel, petroleum ether) to afford 1 as a colorless
1
oil (6.40 g, 90%). H NMR (300 MHz, Acetone-d6, ppm) δ: 7.22–7.12
(m, 4H), 7.04–6.90 (m, 4H), 3.93 (t, 2H), 1.84–1.72 (m, 2H), 1.52–1.39
(m, 2H), 1.35–1.22 (m, 4H), 0.86 (t, 3H). ESI-MS m/z: 284.16 [M+H]+.
3-Bromo-10-hexyl-10H-phenothiazine
(2):
N-Bromosuccinimide
(2.85 g, 16.00 mmol) was slowly added to a solution of 1 (4.53 g,
16.00 mmol) in anhydrous DMF (25 mL). The mixture was stirred
at room temperature overnight, which was poured into water and
extracted with diethyl ether. The organic layer was washed with brine,
and then dried over Na2SO4. After the solvent was removed under
reduced pressure, the pure product was obtained as a light yellow oil
(4.36 g, 75.04%) by column chromatography (silica gel, petroleum ether:
4. Experimental Section
Methods: NMR spectra were recorded on a Bruker Ultra Shield Plus
300 MHz NMR. LC-MS was performed on Thermo LCQ Fleet LC-MS
with ESI mode (America). Matrix-assisted laser desorption/ionization
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dichloromethane = 20:1). 1H NMR (300 MHz, Acetone-d6, ppm) δ:
7.34–7.10 (m, 4H), 7.00–6.83 (m, 3H), 3.86 (t, 2H), 1.80–1.66 (m, 2H),
1.49–1.33 (m, 2H), 1.30–1.19 (m, 4H), 0.83 (t, 3H). ESI-MS m/z: 364.15
[M+H]+.
dialysis membrane. After freeze-drying, 8 (0.65 g, 65%) was obtained as
white floccules. 1H NMR (300 MHz, CDCl3, ppm) δ: 4.20 (d, 4H), 3.91–
3.58 (m, 783H), 3.56–3.45 (m, 106H), 3.44–3.24 (m, 61H), 2.44 (t, 2H),
1.21–1.04 (m, 167H).
PEG-b-PPG-b-PEG-Morpholine (9): Compound 8 (105 mg, 8.26 µmol),
compound 6 (5.6 mg, 32.94 µmol), CuSO4•5H2O (8 mg, 0.032 mmol)
and sodium ascorbate (6.54 mg, 0.032 mmol) were placed in a 50 mL
round-bottom flask under an argon atmosphere. Then anhydrous DMF
(10 mL) was injected into the mixture. The reaction was allowed to
proceed at room temperature for 2 d. After the solvent was removed,
the crude product was suspended in water and purified by dialysis using
3500 Da molecular weight cut-off dialysis membrane. After freeze-drying,
9 (63 mg, 60%) was obtained as grey floccules. 1H NMR (300 MHz,
CDCl3, ppm) δ: 7.69 (s, 2H), 3.98–3.57 (m, 709H), 3.56–3.44 (m, 100H),
3.43–3.28 (m, 57H), 2.88–2.65 (br, 8H), 2.41–2.22 (br, 4H), 2.09–1.87
(br, 4H), 1.16-1.05 (m, 166H).
PEG-b-PPG-b-PEG-Triphenylphosphonium (10): Compound 8 (100 mg,
7.87 µmol), compound 7 (6.93 mg, 15.75 µmol), CuSO4•5H2O (4 mg,
0.016 mmol) and sodium ascorbate (3.17 mg, 0.016 mmol) were placed
in a 50 mL round-bottom flask under an argon atmosphere. Then
anhydrous DMF (10 mL) was injected into the mixture. The reaction
was allowed to proceed at room temperature for 2 d. After the solvent
was removed, the crude product was suspended in water and purified
by dialysis using 3500 Da molecular weight cut-off dialysis membrane.
After freeze-drying, 10 (74.4 mg, 69.62%) was obtained as grey floccules.
1H NMR (300 MHz, CDCl3, ppm) δ: 8.08 (s, 2H), 7.92–7.57 (m, 25H),
3.90–3.58 (m, 783H), 3.56–3.46 (m, 127H), 3.44–3.32 (m, 73H), 1.17–
1.07 (m, 202H).
Preparation of Lysosome- or Mitochondria-Targeted Nanoprobe: To
synthesize the lysosome-targeted nanoprobe, compound 9 (20 mg) and
4 (0.25 mg) were dissolved in 1.0 mL of THF, and then swiftly dropped
into water (10 mL) under sonication. THF was then removed by nitrogen
blowing on the solution surface under stirring at room temperature.
After filtering through a 0.22 µm filter, a bright yellow-green aqueous
solution was obtained. The mitochondria-targeted nanoprobe was
prepared as a bright red aqueous solution through the similar procedure
using compound 10 (20 mg) and 5 (0.25 mg) as the precursors.
Cell Culture: Murine RAW 264.7 cell lines were grown as monolayers
in 75 cm2 flasks containing Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 1% penicillin
streptomycin glutamine (GIBCO) at 37 °C in a humidified incubator
of 5% CO2. Cells were carefully harvested and split when they reached
80% confluence to maintain exponential growth.
Cytotoxicity Test: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assays were performed to assess the metabolic
activity of macrophage cells. The cells were seeded in 96-well plates
(Costar, Chicago, IL) at an intensity of 2 × 104 cells mL−1. After 24 h of
incubation, the medium was replaced by the nanoprobe solution at the
concentration of 5, 10, 15, 20, 25, and 30 µg mL−1, and the cells were
then incubated for 24 h. After the designated time intervals, the wells
were washed twice with PBS buffer, and 100 µL MTT solution (0.5 mg
mL−1) was added to each well. After incubation for 3 h at 37 °C, the
MTT medium solution was carefully removed and the formazan crystals
were solubilized with 200 µL of dimethylsulfoxide for 15 min. The
absorbance value was recorded at 490 nm using a microplate reader.
The absorbance of the untreated cells was used as a control with its
absorbance as the reference value for calculating 100% cellular viability.
Cellular Imaging: For determining the subcellular location of the
nanoprobe, live RAW 264.7 macrophage cells cultured in 35 mm glass
bottom culture dishes were incubated with 20 µg mL−1 nanoprobe
(LNP or MNP) for 24 h. Then LysoTracker (1.0 × 10−6 m) or MitoTracker
(1.0 × 10−6 m) was added for another 15 min. Then the cells were washed
by PBS prior to imaging. To investigate multicolor cellular imaging of
different organelles, the experiment can be divided into three groups.
For one group, the macrophage cells were cultured with the LNP/MNP
nanoprobe cocktail (30 µg mL−1) for 24 h prior to imaging. In the second
group, macrophage cells were pretreated with LPS (100 ng mL−1)/
IFN-γ (20 ng mL−1) for 24 h, then treated with LNP/MNP nanoprobe
10-Hexyl-3-vinyl-10H-phenothiazine (3):
2 (4.00 g, 11.02 mmol),
tributyl(vinyl)tin (4.21 g, 13.22 mmol), Pd(PPh3)2Cl2 (0.685 g,
0.97 mmol) and 2, 6-di-tert-butylphenol (95 mg, 0.46 mmol) were placed
in a 50 mL round-bottom flask and the reaction vessel was degassed
and purged with N2. Then 20 mL of anhydrous toluene was injected into
the mixture, which was subjected to three freeze-pump-thaw cycles to
remove O2. The mixture was vigorously stirred at 100 °C for 24 h. After
the organic solvent was distilled out, the crude product was purified by
column chromatography (silica gel, petroleum ether: dichloromethane =
1:1) to give yellow-green oil (1.64 g, 48.1%). 1H NMR (300 MHz, CDCl3,
ppm) δ: 7.22–7.09 (m, 4H), 6.94–6.75 (m, 3H), 6.65–6.52 (m, 1H),
5.61 (d, 1H), 5.13 (d, 1H), 3.83 (t, 2H), 1.86–1.73 (m, 2H), 1.48–1.38
(m, 2H), 1.34–1.25 (m, 4H), 0.88 (t, 3H). ESI-MS m/z: 310.24 [M+H]+.
1,4-Bis((E)-2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)benzene
(4): A Schlenk tube was charged with 3 (200 mg, 0.65 mmol),
1,4-dibromobenzene (76.66 mg, 0.33 mmol), Pd(OAc)2 (8 mg,
35.64 µmol), and P(o-tolyl)3 (36 mg, 117.6 µmol) before it was sealed
with a rubber septum. The Schlenk tube was degassed with three
vacuum-argon cycles to remove air. Then, DMF (3 mL) and triethylamine
(0.8 mL) were added to the Schlenk tube, and the mixture was frozen,
evacuated, and thawed three times to further remove air. The reaction
was then conducted at 100 °C for 2 d. After the organic solvent was
distilled out, the crude product was purified by column chromatography
(silica gel, petroleum ether: dichloromethane = 1:1) to afford yellow-
1
green oil (94.58 mg, 41.4%). H NMR (300 MHz, CDCl3, ppm) δ: 7.45
(s, 4H), 7.32–7.27 (m, 4H), 7.13 (d, 4H), 7.00–6.77 (m, 10H), 3.84 (t,
4H), 1.87–1.75 (m, 4H), 1.47–1.39 (m, 4H), 1.35–1.28 (m, 8H), 0.88 (t,
6H). ESI-MS m/z: 692.36 [M]+.
4,7-Bis((E)-2-(10-hexyl-10H-phenothiazin-3-yl)vinyl)benzo[c][1,2,5]
thiadiazole (5): Compound 5 was synthesized by a similar procedure
with compound 4 using 4, 7-dibromobenzo[c][1,2,5]thiadiazole instead
1
of 1, 4-dibromobenzene as the starting material. Yield: 38.2%. H NMR
(300 MHz, THF-d8, ppm) δ: 8.02 (d, 2H), 7.73 (s, 2H), 7.56 (d, 2H),
7.46–7.40 (m, 4H), 7.15–7.07 (m, 4H), 6.96 (t, 4H), 6.88d (t, 2H),
3.93 (t, 4H), 1.85–1.76 (m, 4H), 1.52–1.42 (m, 4H), 1.34–1.27 (m, 8H),
0.88 (t, 6H). MALDI-TOF, m/z: Calcd, 750.29; Found, 750.53 [M]+.
4-(3-Azidopropyl)morpholine (6): Sodium azide (0.78 g, 12 mmol)
was added to a solution of 4-(3-bromopropyl)morpholine (0.5 g,
2.42 mmol) in anhydrous DMF (20 mL) and the mixture was stirred
at room temperature for 24 h. After the solvent was removed by rotary
evaporation, the mixture was extracted with water and dichloromethane.
The pure product can be obtained without further purification after
removing the solvent of organic layer (0.39 g, 94.66%). 1H NMR
(300 MHz, CDCl3, ppm) δ: 3.70 (t, 4H), 3.34 (t, 2H), 2.51–2.33 (m, 6H),
1.80–1.71 (m, 2H). ESI-MS m/z: 171.03 [M+H]+.
(4-Azidobutyl)triphenylphosphonium Bromide (7): Sodium azide
(153 mg, 2.35 mmol) was added to a solution of (4-bromobutyl)
triphenylphosphonium bromide (0.73 g, 1.53 mmol) in anhydrous DMF
(15 mL) and the mixture was stirred at room temperature for 24 h.
After the solvent was removed by rotary evaporation, dichloromethane
was added to the mixture and the precipitate was removed. The
product was obtained as white crystals after distilling dichloromethane
(0.61 g, 89.97%). 1H NMR (300 MHz, CDCl3, ppm) δ: 7.93–7.59 (m,
15H), 3.89 (t, 2H), 3.42 (t, 2H), 2.10–1.89 (m, 2H), 1.80–1.58 (m, 2H).
ESI-MS m/z: 360.12 [M−Br−]+.
PEG-b-PPG-b-PEG-Alkyne (8): PEG-b-PPG-b-PEG (1 g, 0.079 mmol),
sodium hydride (60% in mineral oil, 38 mg, 0.95 mmol) and 10 mL
anhydrous THF were placed in a 50 mL round-bottom flask and the
mixture kept stirring for 1 h. Then 250 µL 3-bromoprop-1-yne (80% in
toluene) was added and the mixture was stirred at room temperature
for 24 h. After the precipitate was filtered away, the mixture was
concentrated and poured into diethyl ether. The precipitate was collected
and redissolved in water. Finally, the product was purified by dialysis
against distilled water for 2 d using a 5000 Da molecular weight cut-off
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cocktail (30 µg mL−1) for another 24 h prior to imaging. For the third
group, macrophage cells were stimulated with LPS (100 ng mL−1)/IFN-
γ (20 ng mL−1) and NAC (1 × 10−3 m) for 24 h, then treated with LNP/
MNP nanoprobe cocktail (30 µg mL−1) for another 24 h prior to imaging.
All the imaging signals were collected through four channels: blue
(420–460 nm, λex = 405 nm), green (520–550 nm, λex = 405 nm), orange
(580–610 nm, λex = 570 nm), and red (690–750 nm, λex = 570 nm).
[7] A. Daugherty, J. L. Dunn, D. L. Rateri, J. W. Heinecke, J. Clin. Invest.
1994, 94, 437.
[8] M. J. Steinbeck, L. J. Nesti, P. F. Sharkey, J. Parvizi, J. Orthop. Res.
2007, 25, 1128.
[9] a) T. Ueno, T. Nagano, Nat. Methods 2011, 8, 642; b) J. F. Lovell,
G. Zheng, J. Innovative Opt. Health Sci. 2008, 1, 45.
[10] a) W. M. Nauseef, Blood 1987, 70, 1143; b) X. J. Wu, Z. Li, L. Yang,
J. H. Han, S. F. Han, Chem. Sci. 2013, 4, 460.
[11] a) B. C. Dickinson, D. Srikun, C. J. Chang, Curr. Opin. Chem. Biol.
2010, 14, 50; b) M. P. Murphy, Biochem. J. 2009, 417, 1.
[12] a) J. T. Hou, M. Y. Wu, K. Li, J. Yang, K. K. Yu, Y. M. Xie, X. Q. Yu,
Chem. Commun. 2014, 50, 8640; b) H. D. Xiao, K. Xin, H. F. Dou,
G. Yin, Y. W. Quan, R. Y. Wang, Chem. Commun. 2015, 51, 1442.
[13] L. Yuan, L. Wang, B. K. Agrawalla, S. J. Park, H. Zhu, B. Sivaraman,
J. J. Peng, Q. H. Xu, Y. T. Chang, J. Am. Chem. Soc. 2015, 137, 5930.
[14] a) L. H. Feng, C. L. Zhu, H. X. Yuan, L. B. Liu, F. T. Lv, S. Wang,
Chem. Soc. Rev. 2013, 42, 6620; b) C. G. Qian, J. C. Yu, Y. L. Chen,
Q. Y. Hu, X. Z. Xiao, W. J. Sun, C. Wang, P. J. Feng, Q. D. Shen,
Z. Gu, Adv. Mater. 2016, 28, 3313; c) C. F. Wu, D. T. Chiu,
Angew. Chem., Int. Ed. 2013, 52, 3086; d) C. Xie, X. Zhen, Q. Lei,
R. Ni, K. Pu, Adv. Funct. Mater. 2017, 27, 1605397; e) C. Xie,
P. K. Upputuri, X. Zhen, M. Pramanik, K. Pu, Biomaterials 2017, 119,
1; f) D. Cui, C. Xie, Y. Lyu, X. Zhen, K. Pu, J. Mater. Chem. B 2017,
DOI: 10.1039/C6TB03393H; g) Q. Miao, Y. Lyu, D. Ding, K. Pu,
Adv. Mater. 2016, 28, 3662; h) K. Pu, J. Mei, J. V. Jokerst, G. Hong,
A. L. Antaris, N. Chattopadhyay, A. J. Shuhendler, T. Kurosawa,
Y. Zhou, S. S. Gambhir, Z. Bao, J. Rao, Adv. Mater. 2015, 27,
5184; i) C. Yin, X. Zhen, H. Zhao, Y. Tang, Y. Ji, Y. Lyu, Q. Fan,
W. Huang, K. Pu, ACS Appl. Mater. Interfaces 2017, DOI: 10.1021/
acsami.7b02014; j) X. Zhen, X. Feng, C. Xie, Y. Zheng, K. Pu, Bioma-
terials 2017, 127, 97.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
K.P. thanks Nanyang Technological University (Start-Up grant:
NTU-SUG: M4081627.120) and Singapore Ministry of Education
(Academic Research Fund Tier 1: RG133/15 M4011559 and Academic
Research Fund Tier2 MOE2016-T2-1-098) for the financial support. W.H.
and Q.F. thank the National Basic Research Program of China (No.
2012CB933301), the National Natural Science Foundation of China (No.
21674048, 21574064, 61378081, 11404219, 61571239, and 61505076),
Synergetic Innovation Center for Organic Electronics and Information
Displays, and the Natural Science Foundation of Jiangsu Province
of China (No. BZ2010043, NY211003, BM2012010) for the financial
support.
Conflict of Interest
[15] X. Zhen, C. Zhang, C. Xie, Q. Miao, K. Lim, K. Pu, ACS Nano 2016,
10, 6400.
The authors declare no conflict of interest.
[16] K. Pu, A. J. Shuhendler, J. Jokerst, J. Mei, S. Gambhir, Z. Bao, J. Rao,
Nat. Nanotechnol. 2014, 9, 233.
[17] G. S. Hong, Y. P. Zou, A. L. Antaris, S. Diao, D. Wu, K. Cheng,
X. D. Zhang, C. X. Chen, B. Liu, Y. H. He, J. Z. Wu, J. Yuan, B. Zhang,
Z. M. Tao, C. Fukunaga, H. J. Dai, Nat. Commun. 2014, 5, 4206.
[18] a) D. Ghezzi, M. R. Antognazza, M. Dal Maschio, E. Lanzarini,
F. Benfenati, G. Lanzani, Nat. Commun. 2011, 2, 166; b) Y. Lyu,
C. Xie, S. Chechetka, E. Miyako, K. Pu, J. Am. Chem. Soc. 2016, 138,
9049.
Keywords
activatable probes, fluorescence imaging, nanoparticles, reactive oxygen
species
Received: January 26, 2017
Revised: March 5, 2017
Published online:
[19] a) J. Zhang, X. Zhen, P. Upputuri, M. Pramanik, P. Chen, K. Pu,
Adv. Mater. 2017, 29, 1604764; b) K. Pu, A. J. Shuhendler, J. Rao,
Angew. Chem., Int. Ed. 2013, 52, 10325; c) A. J. Shuhendler, K. Pu,
L. Cui, J. Uetrecht, J. Rao, Nat. Biotechnol. 2014, 32, 373; d) K. Pu,
N. Chattopadhyay, J. Rao, J. Controlled Release 2016, 240, 312;
e) Q. Miao, K. Pu, Bioconjugate Chem. 2016, 27, 2808; f) C. Yin,
X. Zhen, Q. Fan, W. Huang, K. Pu, ACS Nano 2017, DOI:10.1021/
acsnano.7b01092.
[20] a) S. I. Reja, V. Bhalla, A. Sharma, G. Kaur, M. Kumar, Chem.
Commun. 2014, 50, 11911; b) L. Long, D. Zhang, X. Li, J. Zhang,
C. Zhang, L. Zhou, Anal. Chim. Acta 2013, 775, 100; c) X. Chen,
X. Wang, S. Wang, W. Shi, K. Wang, H. Ma, Chem. Eur. J. 2008, 14,
4719.
[1] E. J. Pereira, C. M. Smolko, K. A. Janes, Front. Pharmacol. 2016, 7,
457.
[2] Y. W. Yap, M. Whiteman, N. S. Cheung, Cell. Signalling 2007, 19,
219.
[3] C. C. Winterbourn, M. B. Hampton, J. H. Livesey, A. J. Kettle, J. Biol.
Chem. 2006, 281, 39860.
[4] D. I. Pattison, M. J. Davies, Chem. Res. Toxicol. 2001, 14,
1453.
[5] B. Pan, H. Ren, X. F. Lv, Y. Y. Zhao, B. Q. Yu, Y. B. He, Y. J. Ma,
C. G. Niu, J. G. Kong, F. Z. Yu, W. B. Sun, Y. Y. Zhang, B. Willard,
L. Zheng, J. Transl. Med. 2012, 10, 65.
[6] C. Sand, S. L. M. Peters, M. Pfaffendorf, P. A. van Zwieten, Clin.
Exp. Pharmacol. Physiol. 2003, 30, 249.
[21] a) Y. Koide, Y. Urano, K. Hanaoka, T. Terai, T. Nagano, J. Am. Chem.
Soc. 2011, 133, 5680; b) W. J. Zhang, C. Guo, L. H. Liu, J. G. Qin,
C. L. Yang, Org. Biomol. Chem. 2011, 9, 5560.
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2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim