A highly selective, cell-permeable fluorescent nanoprobe for ratiometric detection and imaging of peroxynitrite in living cells

Article abstract of DOI:10.1002/chem.201100148

Peroxynitrite (ONOO^-) is a highly reactive species implicated in the pathology of numerous diseases and there is currently great interest in developing fluorescent probes that can selectively detect ONOO^- in living cells. Herein, a p

Full text of DOI:10.1002/chem.201100148

DOI: 10.1002/chem.201100148
A Highly Selective, Cell-Permeable Fluorescent Nanoprobe for Ratiometric
Detection and Imaging of Peroxynitrite in Living Cells
Jiangwei Tian, Huachao Chen, Linhai Zhuo, Yanxia Xie, Na Li, and Bo Tang*[a]
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Chem. Eur. J. 2011, 17, 6626 – 6634

FULL PAPER
Abstract: Peroxynitrite (ONOOꢀ) is a
highly reactive species implicated in
the pathology of numerous diseases
and there is currently great interest in
developing fluorescent probes that can
selectively detect ONOOꢀ in living
avoids the influences from enzymatic
tive properties, such as good water sol-
ubility, photostability, biocompatibility,
and near-infrared excitation and emis-
sion. Fluorescence imaging experi-
ments by confocal microscopy show
that this nanoprobe is capable of visu-
alizing ONOOꢀ produced in living cells
and it exhibits very low toxicity and
good membrane permeability. We an-
ticipate that this technique will be a
potential tool for the precise pathologi-
cal understanding and diagnosis of
ONOOꢀ-related human diseases.
C
reaction and high-concentration OH
and ClOꢀ. The improved ONOOꢀ se-
lectivity of the nanoprobe is achieved
by a delicate complementarity of prop-
erties between the nanomatrix and the
embedded molecular probe (BzSe-Cy).
This nanoprobe also has other attrac-
cells. Herein,
a polymeric micelle-
based and cell-penetrating peptide-
coated fluorescent nanoprobe that in-
corporates ONOOꢀ indicator dye and
reference dye for the ratiometric detec-
tion and imaging of ONOOꢀ has been
developed. The nanoprobe effectively
Keywords: bioimaging · fluorescent
probes · nanoparticles · peptides ·
peroxynitrite
ꢀ
C
Introduction
lar, interferences from OH and ClO are severe obstacles
ꢀ
ꢀ
C
in detecting ONOO because of the similarity of OH, ClO ,
and ONOOꢀ with regard to their strong oxidation capaci-
ty.[6] To solve this problem, a few fluorescent probes, such as
ketone oxidation-based HKGreen[7] and lanthanide-com-
Peroxynitrite (ONOOꢀ), formed in vivo from a diffusion-
C
controlled reaction between nitric oxide ( NO) and superox-
ide (O2 ),[1] is a highly reactive species implicated in the
Cꢀ
pathogenesis of numerous diseases, including inflammatory
processes, ischemic reperfusion injury, multiple sclerosis, and
neurodegenerative disorders.[2] This peroxide easily crosses
biological membranes, and despite a relatively short half-life
(ꢁ1 s) under physiological conditions, it can interact with
various biological molecules, such as DNA, lipids, proteins,
thiols, and metalloenzymes,[3] which may result in serious
damage to living cells. To fully understand the biological ef-
fects of ONOOꢀ, a major focus of research is to exploit
highly selective and sensitive methods to monitor the con-
centration changes of ONOOꢀ in biosystems. Among these
methods, spectrofluorimetry has become a powerful tool
due to its high sensitivity and simplicity in data collection in
imaging techniques.[4]
In recent years, many research efforts have focused on the
development of fluorescence techniques for detecting
ONOOꢀ. A variety of fluorescent probes, such as 3’-(p-ami-
nophenyl)fluorescein (APF), dihydrorhodamine 123 (DHR-
123) and dihydrodichlorofluorescein (DCFH) have been
widely used to monitor ONOOꢀ in various biosystems.[5]
However, their fluorescence signals suffer from interferenc-
es by other reactive oxygen species (ROS), reactive nitrogen
species (RNS) or unwanted enzymatic reactions. In particu-
plex-based [Eu3+/Tb3+(DTTA)][8]
ACHTUNGRETNNUNG , (DTTA=[4’-(2,4-dime-
thoxyphenyl)-2,2’:6’,2’’-terpyridine-6,6’’-diyl]bis(methyleneni-
trilo)tetrakis(acetic acid)) have been developed for the se-
lective detection of ONOOꢀ. All of them are based on the
specific reaction between
a small-molecule probe and
ONOOꢀ. However, utilizing a synergistic combination of
nanomatrix and embedded ONOOꢀ-specific molecular
probe to achieve higher selectivity for detecting ONOOꢀ
has not been reported to date.
Polymeric micelles, self-assembled nanoparticles from am-
phiphilic block copolymers, have many advantages in con-
structing multifunctional fluorescent nanoprobes. Micelle
structures can incorporate different hydrophobic dyes in a
single nanoparticle without complicated synthetic and sepa-
rating procedures, so they offer a fascinating strategy for
constructing ratiometric probes. The nanoparticles can be
excited at the near-infrared (NIR) range by encapsulating
NIR dyes, which leads to minimized photodamage and cell
autofluorescence,[9] and also have stability in aqueous media
and surface functionalization to act as biological probes.[10]
Moreover, such nanoparticles exhibit higher brightness and
better photostability than small-molecule dyes, owing to
large numbers of chromophores per particle as well as the
protective matrix.[11] As ONOOꢀ is a highly permeable
ROS,[12] another appealing feature of using a micelle struc-
ture is that the nanomatrix can enhance the selectivity of
the embedded molecular probe toward ONOOꢀ over other
biologically relevant substances. Such induced selectivity can
be achieved by a delicate complementarity of properties be-
tween the nanomatrix and the embedded molecular
probe.[13] Based on the above, we attempted to develop a
polymeric micelle-based nanoprobe that is highly specific
for ONOOꢀ.
[a] J. Tian, H. Chen, L. Zhuo, Y. Xie, N. Li, Prof. B. Tang
College of Chemistry, Chemical Engineering and Materials Science
Key Laboratory of Molecular and Nano Probes
Engineering Research Center of Pesticide and Medicine Intermediate
Clean Production, Ministry of Education
Shandong Provincial Key Laboratory of Clean Production
of Fine Chemicals
Shandong Normal University, Jinan 250014 (P. R. China)
Fax : (+86)531-8618-0017
E-mail: tangb@sdnu.edu.cn
To further improve the cell-uptake efficiency of the
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201100148.
nanoprobe for intracellular ONOOꢀ imaging, a strong impe-
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B. Tang et al.
tus has been placed on the development of a nanoprobe
with good cell permeability. Most methods that have been
investigated to achieve the efficient intracellular delivery of
nanoparticles (e.g., electroporation[14]) are limited to in vitro
applications and often cause unwanted cellular effects, such
as high cytotoxicity.[15] In recent years, use of cell-penetrat-
ing peptides (CPPs) conjugated to nanoparticles as an alter-
native strategy to facilitate their intracellular uptake has at-
tracted broad interest and attention. CPPs which can enter
cells in a nontoxic and highly efficient manner have been
used successfully for the intracellular delivery of nanoparti-
cles into the cell without disturbing the stability of the cell
membrane and with low cytotoxic effects.[16] Thus, the use of
these CPPs offers several advantages over conventional
techniques because it is efficient for a range of cell types.
sulting in a dramatic fluorescence decrease, whereas the
fluorescence of IRhB does not change, which leads to the
selectively ratiometric detection of ONOOꢀ. Herein, the
spectral characteristics of the nanoprobe were investigated
under a simulated physiological environment, and the selec-
tivity of free BzSe-Cy and the nanoprobe toward ONOOꢀ
were evaluated. Finally, the nanoprobe was applied to imag-
ing of ONOOꢀ in living macrophages (RAW 264.7), normal
human liver cells (HL-7702), and human hepatoma cells
(HepG2).
Results and Discussion
Design and synthesis of BzSe-Cy: We designed a hydropho-
bic NIR molecular probe (BzSe-Cy) that is sensitive to
ONOOꢀ (see Scheme 3 and Experimental Section). The rel-
ative fluorescence quantum yield of BzSe-Cy was 0.106,
Herein,
a
hybrid polypeptide–polymer–organic dye
nanoprobe for ONOOꢀ has been designed and synthesized.
The nanoarchitecture is composed of two key components:
1) a polymeric micelle that has
a hydrophobic core made of
poly(d,l-lactic acid) (PLA) and
a hydrophilic shell consisting of
polyethylene glycol (PEG) con-
jugated to CPPs; and 2) benzyl-
selenide-tricarbocyanine (BzSe-
Cy) as ONOOꢀ indicator dye
(Scheme 1) and isopropylrhod-
ACHTUNGTRENNUNGamine B (IRhB) as reference
Scheme 3. Synthesis of BzSe-Cy; see the Experimental Section for details.
which was obtained by comparison with IR-786 in methanol
(F=0.159), and the absorption coefficient was
28778mꢀ1 cmꢀ1 (see Supporting Information). The design
strategy for BzSe-Cy is inspired by the specific reaction in
which ONOOꢀ oxidizes divalent selenium to quadrivalent
selenium.[17] As indicated in Scheme 1, treatment of BzSe-
Cy with ONOOꢀ to generate the oxidized BzSe-Cy results
in a significant fluorescence decrease. BzSe-Cy and its oxi-
dized product have been verified by ESI-MS (Figure S1,
Supporting Information) and 77Se NMR spectroscopy (Fig-
ure S2).
Scheme 1. Reaction of BzSe-Cy with ONOOꢀ.
dye, which are simultaneously encapsulated in the hydro-
phobic core of the micelle for ratiometric ONOOꢀ measure-
ment. The details of this strategy are shown in Scheme 2.
Because of its high diffusibility, ONOOꢀ can rapidly diffuse
into the core of the nanomatrix that blocks the entry of in-
terferential bioanalytes and react with BzSe-Cy, thereby re-
Synthesis and characterization of the nanoprobe: BzSe-Cy
and IRhB were loaded into polymeric micelles by nanopre-
cipitation and self-assembly[18] at 2.9 and 0.05 wt%, respec-
tively. In this study, amphiphilic block copolymers consisting
of maleimide-terminated poly(ethylene glycol)-block-
poly(d,l-lactide) (MAL-PEG-PLA) and methoxy-terminat-
ed poly(ethylene glycol)-block-poly(d,l-lactide) (MPEG-
PLA) were used for micelle formation. Different amounts
of MPEG-PLA were introduced to control the density of
maleimide, which ultimately determined the density of CPPs
on the micelle surface (0 and 10% of all PEG chains). CPPs
were attached to the micelle surface through a covalent
thiol–maleimide linkage.[19]
Transmission electron microscopy (TEM) and dynamic
light scattering (DLS) were used for characterization of the
Scheme 2. Schematic illustration of the fabrication of a hybrid polypep-
tide–polymer–organic dye nanoprobe.
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Fluorescent Nanoprobe for Detection of Peroxynitrite in Living Cells
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prepared nanohybrid. The TEM results indicate that the re-
sulting particles in aqueous solution are well dispersed with-
out aggregation, with mean hydrodynamic radius 54.7 nm as
determined by DLS (Figure 1). The absorption spectrum
*
Figure 2. Absorption spectrum of the free dyes ( ) in 0.1m PBS solution
*
(CH3CN/H2O, 2:8, pH 7.4) and the nanoprobes ( ) in 0.1m PBS solution
(pH 7.4).
therefore investigated the photostability of free BzSe-Cy
and the nanoprobe. Figure 4 shows changes in the relative
fluorescence intensity (F/F0) of free BzSe-Cy and the nano-
AHCTUNGTREpGNNUN robe as a function of radiation time. The F/F0 of free BzSe-
Cy decreases by 50% within 60 min due to photon-induced
Figure 1. Dynamic light scattering curve of the nanoprobe. DLS samples
were measured at a concentration of 0.25 mgmLꢀ1 in 0.1m phosphate-
buffered saline (PBS, pH 7.4) at room temperature. Inset: Representative
TEM image of the nanoprobe negatively stained with 2.0% phospho-
tungstic acid.
chemical damage, whereas the F/F0 of the nanoprobe does
shows that the nanoprobe has
two maximum absorption wave-
lengths, 555 nm for IRhB and
788 nm for BzSe-Cy (Figure 2).
When the two dyes are finally
encapsulated in the core of
polymeric micelles, a slight red-
shift of the absorption spectrum
of IRhB and BzSe-Cy occurs,
which is caused by the confine-
ment of dye molecules in the
structures.[20]
micelle The
nanoprobe can be excited at
two different wavelengths to
give different emission wave-
Figure 3. Confocal fluorescence images of the nanoprobe. Laser lines at a) 532 nm with emissions from 535 to
600 nm and b) 635 nm with emissions from 700 to 800 nm were used sequentially; c) overlay of panels (a) and
(b). Scale bar: 25 mm.
lengths (lem =575, 810 nm). Confocal laser scanning micros-
copy (CLSM) studies were carried out to demonstrate that
both IRhB and BzSe-Cy are in the same nanoparticle
(Figure 3). When nanoparticles are excited at 532 nm, only
IRhB can be excited and its associated green fluorescence is
observed. Similarly, when the nanoparticles are excited at
635 nm, only BzSe-Cy can be excited and its associated red
fluorescence is observed. When these two images are over-
laid, they overlap completely, which demonstrates that both
fluorophores are encapsulated in the same nanoparticle.
Aqueous dispersions of the nanoprobe are clear and stable
(Figure S3, Supporting Information), with negligible dye
leakage (less than 1% at pH 7.4 in 80 h).
Figure 4. Relative fluorescence, F/F0!Ft=t/Ft=0, of free BzSe-Cy (5 mm) in
0.1m PBS solution (CH3CN/H2O, 2:8, pH 7.4) and the nanoprobe
(0.25 mgmLꢀ1) in 0.1m PBS, pH 7.4, after different radiation times with a
770 nm laser. The fluorescence emission intensity was measured at
800 nm.
Photostability investigations: A highly desirable property of
fluorescent probes is high resistance to photobleaching. We
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B. Tang et al.
not change over the same time period owing to the protec-
tion of the nanomatrix.[21] Similar results can be obtained for
F/F0 changes from free IRhB and the nanoprobe (Figure S4,
Supporting Information). Therefore, the nanomatrix can ef-
fectively stabilize the fluorophore against oxidative degrada-
tion and makes a remarkably stable fluorescent nanoprobe.
therefore has the sensitivity to detect ONOOꢀ in biosys-
tems.
Kinetic assay: The fluorescence quenching kinetics of the
ONOOꢀ–nanoprobe system was determined by real-time re-
cording of the fluorescence intensity changes at lex/lem 788/
810 nm after addition of ONOOꢀ. As shown in Figure 6,
upon the addition of ONOOꢀ at room temperature, a
marked decrease in the intensity was obtained instantly up
to 8 s and leveled off thereafter, which indicated the rapid
response toward ONOOꢀ. Therefore, the nanoprobe has
good reactivity for the detection of ONOOꢀ.
Ratiometric detection of ONOOꢀ: Spectroscopic properties
of the nanoprobe toward ONOOꢀ were evaluated under si-
mulated physiological conditions (0.1m PBS, pH 7.4). Fig-
ure 5a shows the fluorescence emission spectra at 800–
900 nm taken from the nanoprobe with ONOOꢀ. As expect-
ed, the fluorescence intensity is reduced successively with in-
creasing ONOOꢀ concentration. However, the fluorescence
intensity at 575 nm is not influenced (Figure 5b). The fluo-
rescence emission intensity ratio (F810/F575) varies from 2.49
in the absence of ONOOꢀ to 0.16 after ONOOꢀ treatment.
Figure 5b shows the linear correlation (R=0.9982) between
the F810/F575 ratio and the relative amounts of added
ONOOꢀ, within the concentration range of 0–5 mm. The de-
tection limit was calculated to be 50 nm based on triplicate
measurements of the relative standard. The nanoprobe
Figure 6. Fluorescence quenching kinetics curves of the nanoprobe
(0.25 mgmLꢀ1) at 0 (top) and 5 mm (bottom) ONOOꢀ. Spectra were ac-
quired at room temperature in 0.1m PBS, pH 7.4 (lex =788 nm, lem
=
810 nm).
Test for probe selectivity: The selectivity of the nanoprobe
towards ONOOꢀ was examined by monitoring the changes
in F810/F575 ratio upon exposure to various interfering agents,
1
C
such as H2O2, O2, and OH. As shown in Figure 7, a 96%
decrease in F810/F575 ratio can be observed when the nano-
Figure 5. a) Fluorescence response of the nanoprobe (0.25 mgmLꢀ1) to-
wards ONOOꢀ with 788 nm excitation. Spectra shown are for ONOOꢀ
concentrations of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mm.
Spectra were acquired at room temperature in 0.1m PBS, pH 7.4.
b) Changes in fluorescence intensity at 575 nm with 555 nm excitation
(squares, right Y scale) of the nanoprobe and the fluorescence intensity
ratio F810/F575 (circles, left Y scale) as a function of ONOOꢀ.
Figure 7. F810/F575 ratio of the nanoprobe (0.25 mgmLꢀ1) in 0.1m PBS so-
lution (pH 7.4) with various biological analytes. Data were acquired at
room temperature and the final concentrations of various analytes were
ꢀC
C
5 mm for ONOOꢀ, 500 mm for H2O2, 1O2, O2
,
NO, NO2ꢀ, NO3ꢀ, and
ꢀ
C
C
ROO , 450 mm for OH, 250 mm for ClO , and 10 mm for HRP.
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Fluorescent Nanoprobe for Detection of Peroxynitrite in Living Cells
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A
either SNAP (Figure 8c) or xanthine/xanthine oxidase (Fig-
ure 8d). The mean fluorescence intensity ratio from the
living cells was also quantified with Image-Pro software
(Figure 8e), and demonstrated that the SIN-1-treated cells
had an approximately 45% decrease in fluorescence intensi-
ty ratio, which is much more than that of other groups (5
and 8%, respectively). Thus, we conclude that this nanop-
robe can be used for the selective imaging of ONOOꢀ pro-
duced in cultured cells. Moreover, bright-field measure-
ments and Hoechst-33258 staining confirmed that the cells
are viable throughout the imaging studies (Figure 8b and
Figure S7, Supporting Information). Taken together, these
results suggest that the nanoprobe has the potential for
studying biological processes involving ONOOꢀ within
living cells.
ꢀC
ces, including H2O2, 1O2, O2
(500 mm), H2O2/horseradish peroxidase (HRP; 500/10 mm),
C
change of F810/F575 ratio (change <5%). We also investigat-
ed the fluorescence responses of free BzSe-Cy to various an-
alytes. As shown in Figure S6a and S6b in the Supporting In-
formation, BzSe-Cy is specific for ONOOꢀ when other ana-
C
C
NO, NO2 , NO3 , ROO
ꢀ
ꢀ
,
ꢀ
OH (450 mm), and ClO (250 mm), could not induce the
C
lytes are in a certain concentration range (50 mm for OH
and ClOꢀ, 500 mm for others). The selectivity to ONOOꢀ is
due to the specific reaction in which ONOOꢀ oxidizes diva-
lent selenium to quadrivalent selenium, and to the relatively
high oxidation potential of free BzSe-Cy (0.622 V, see Sup-
porting Information), which meant that BzSe-Cy was not
oxidized easily by ROS.[22] However, as shown in Figure S6b
in the Supporting Information, it suffers interferences from
enzymatic reaction (H2O2/HRP) and highly oxidizing ana-
Fluorescence imaging in HL-7702 and HepG2 cells: Consid-
ering that the majority of cells in the human body are non-
phagocytic, confocal fluorescence imaging experiments were
carried out in other biological models to further verify the
feasibility of the nanoprobe. Extensive studies demonstrate
that tumor occurrence is related to ROS.[25] Therefore, we
selected HL-7702 and HepG2 cells to investigate whether
this nanoprobe can detect ONOOꢀ in nonphagocytic cells
(Figure 9). Images reveal that there are fluorescence distinc-
tions between normal cells and tumor cells. These results in-
dicate that the probe can be used for detecting ONOOꢀ in a
variety of cells, which foreshadows broad application pros-
pects of the nanoprobe in biological systems.
C
lytes with high concentrations (450 mm for OH and 250 mm
for ClOꢀ). In contrast, when BzSe-Cy was encapsulated in
the nanoparticle, it showed excellent selectivity even in the
high-concentration ROS solution (Figure 7). The enhanced
specificity of the nanoprobe is attributed to the nanomatrix
that blocks the interferences from other biological analytes.
Significantly, ONOOꢀ has high diffusibility (the permeabili-
ty coefficient is 8.0ꢁ10ꢀ4 cmsꢀ1 which is ꢁ400 times greater
ꢀC
[23]
than that of O2
)
and could rapidly diffuse into the hy-
drophobic core of the nanomatrix and react with BzSe-Cy.
By contrast, other ROS such as O2 and ClO with relative-
ly low diffusibility find it hard to permeate the nanomatrix.
ꢀC
ꢀ
C
OH has the shortest lifetime (nanoseconds or shorter)
Test for cell permeability of the nanoprobe: To enhance cell
permeability of the nanoprobe for intracellular imaging,
CPPs were attached to the micelle surface to facilitate the
internalization of the nanoprobe. Flow cytometry and
CLSM were used to evaluate the effect of CPPs on nanopar-
ticle uptake. Figure 10 shows the flow cytometry histogram
after 0 and 10% CPP micelles were incubated with macro-
phages for 60 min. An obvious increase in cell uptake is ob-
served with 10% CPP micelles over 0% CPP micelles, thus
demonstrating that the CPP-encoded nanoprobe is more
cell-permeable for intracellular imaging. To further study
the cell permeability of the nanoprobe (10% CPP micelles),
the interactions of the cells with nanoprobe at 4 and 378C
was investigated. Nanoprobe internalization was studied at
48C to mitigate energy-dependent endocytic pathways. As
shown in Figure S9 in the Supporting Information, the intra-
cellular fluorescence is still apparent at 48C, which confirms
that the nanoprobe has good cell permeability and is suit-
among all the ROS, and does not have enough time to pene-
trate into the nanomatrix. The nanomatrix resistance to
HRP interference is due to the large size of the HRP, which
is too large to penetrate into the nanomatrix. The same size
exclusion may also eliminate potential confounding interac-
tions from other enzymes or macromolecules. It is quite evi-
dent that the nanomatrix is beneficial to the enhanced spe-
cificity of the nanoprobe for ONOOꢀ.
Fluorescence imaging of intracellular ONOOꢀ in RAW
264.7 macrophages: We next sought to apply such a nano-
ACHTUNGTRENNUNG
probe to ratiometric fluorescence imaging of ONOOꢀ in
living cells. We investigated the potential of the nanoprobe
to image ONOOꢀ, generated by activated macrophages, in a
3-morpholinosydnonimine (SIN-1) model.[24] The ratiometric
fluorescence images were monitored in RAW 264.7 macro-
phages treated with different oxidants, namely the ONOOꢀ
C
donor SIN-1, the NO donor S-nitroso-N-acetyl-dl-penicill-
G
ACHTUNGTRENaNGNU ble for fluorescence imaging in biological samples.
ꢀC
amine (SNAP), and the O2 donor xanthine/xanthine ox-
idase. The macrophages were incubated with nanoprobes
(0.03 mgmLꢀ1) for 60 min and then washed three times with
PBS buffer. The ratio images were constructed from 700 to
800 nm (red) and from 535 to 600 nm (green) fluorescence
collection windows. As expected, treatment of nanoprobe-
loaded cells with 10 mm SIN-1 triggers a dramatic decrease
in the red-to-green emission ratio (Figure 8b), but not with
MTT assay: To evaluate the cytotoxicity of the nanoprobe,
we performed MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide) assay in macrophages with nano-
AHCTUNGTREGpNNUN robe concentrations of 0.005, 0.01, 0.015, 0.02, 0.03, 0.05,
0.10, and 0.25 mgmLꢀ1. The results show that the nanoprobe
exhibits almost no cytotoxicity or side effects for the incuba-
tion (Figure S10, Supporting Information).
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B. Tang et al.
Figure 8. a)–d) Confocal, ratiometric fluorescence images of RAW 264.7 macrophage cells. Top: Images displayed in pseudocolor represent the ratio of
emission intensities collected in optical windows of 700–800 (red) and 535–600 nm (green). Bottom: Bright-field images. The macrophages were incubat-
ed with 0.03 mgmLꢀ1 nanoprobe for 60 min at 378C and then subjected to different treatments. a) Control; b)10 mm SIN-1; c) 10 mm SNAP; d) 100 mm
xanthine and 0.1 IU xanthine oxidase. Scale bar: 25 mm. e) Bar graph representing the integrated intensity from 700 to 800 nm over the integrated fluo-
rescence intensity from 535 to 600 nm. Values are the mean ratio generated from the intensity from five randomly selected fields. Error bars represent
standard error measurement (s.e.m.).
Experimental Section
Conclusion
Materials: Unless stated otherwise, solvents were dried by distillation.
We have developed a hybrid polypeptide–polymer–organic
dye nanoprobe that is highly sensitive and selective for the
ratiometric detection of ONOOꢀ. The molecular probe
BzSe-Cy is specific for ONOOꢀ, and the selectivity can be
further improved by encapsulating BzSe-Cy in the nanopar-
ticle for using as a nanoprobe. Additional experiments dem-
onstrate that the new probe has the unique advantages of
good water solubility, photostability, biocompatibility, and
NIR excitation and emission. Furthermore, ratiometric fluo-
rescence imaging establishes the good cell permeability and
capability of visualizing intracellular ONOOꢀ of this nano-
All reagents were of commercial quality and used without further purifi-
cation. Isopropylrhodamine B (IRhB) and 2-[4-chloro-7-(1-ethyl-3,3-di-
methylindolin-2-ylidene)]-3,5-(propane-1,3-diyl)-1,3,5-(heptatrien-1-yl)-1-
ethyl-3,3-dimethyl-3H-indolium (Cy.7.Cl) were synthesized in our labora-
tory. Methoxy-polyethylene glycol (MPEG, Mn =2000 Da), stannous(II)
octoate (Sn
ACHTUNGRTEN(NNUG Oct)2), 3-morpholinosydnonimine (SIN-1), 3-(4,5-dimethyl-
AHCTUNGTERGtNNUN hiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2’-azobis(2-ami-
dinopropane) dihydrochloride (AAPH), xanthine (X; 1 mm) in 10 mm
NaOH solution, xanthine oxidase (XO; 5 Umlꢀ1), S-nitroso-N-acetyl-dl-
penicillamine (SNAP), sodium nitroferricyanideACTHNUTRGNE(NUG III) dihydrate (SNP),
and horseradish peroxidase (HRP) were purchased from Sigma Chemical
Company. d,l-Lactide (99% pure) and dibenzyl diselenide (95+ %)
were purchased from Alfa Aesar Chemical Company. Maleimide–poly-
ethylene glycol (maleimide-PEG-OH, Mn =2900 Da) was purchased from
Shanghai Yare Biotech, Inc. TAT CPPs (Gly-Arg-Lys-Lys-Arg-Arg-Gln-
Arg-Arg-Arg-Pro-Pro-Gln-Cys) were purchased from Chinese Peptide
ACHTUNGTRENNUNGprobe. We anticipate that this nanoACHUTNGTRENNpUNG robe will be a potential
tool for the precise pathological understanding and diagno-
sis of ONOOꢀ-related human diseases.
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Fluorescent Nanoprobe for Detection of Peroxynitrite in Living Cells
FULL PAPER
Figure 9. Confocal fluorescence images of living HL-7702 (a–d) and HepG2 (e–h) cells. HL-7702 and HepG2 cells were separately incubated in
0.03 mgmLꢀ1 nanoprobe for 60 min and then were rinsed three times with PBS. The green channel (a,e) represents the fluorescence collected at 535–
600 nm, and the red channel (b,f) represents the fluorescence collected at 700–800 nm. Images c) and g) represent merged images of red and green chan-
nels. Images d) and h) represent bright-field images of cells in panels a)–c) and e)–g), respectively. Scale bar: 25 mm.
was measured in a Triturus microplate reader in the MTT assay. Flow cy-
tometry analysis was carried out on a CytomicsTM FC 500 flow cytome-
ter.
Synthesis of BzSe-Cy: According to the synthesis of a series of NIR cya-
nine dyes proposed by Peng,[26] with some modification, we synthesized
the new probe BzSe-Cy (Scheme 3). Solid NaBH4, (0.26 g, 6.9 mmol) was
added over a 15 min period to a suspension of dibenzyl diselenide
(1.021 g, 3 mmol) in absolute ethanol (60 mL) cooled to 08C under a
stream of nitrogen. After completion of the addition, the mixture was al-
lowed to warm to room temperature and stirred for 30 min to give a
clear solution. This reaction was quenched with 5% HCl (20 mL) and ex-
tracted with ether/pentane (1:1, 3ꢁ10 mL). The organic phase was dried
over anhydrous sodium sulfate and the solvent was evaporated. The ben-
zylselenol was obtained as a colorless foul-smelling oil. DMF (15 mL)
was added to benzylselenol, and then cyanine (0.196 g) was added to the
solution. The mixture was stirred at 438C under Ar for 48 h. Finally, the
Figure 10. Flow cytometry histogram of nanoprobe uptake in RAW 264.7
macrophages as a function of encoding CPPs. Error bars represent s.e.m.
mixture was purified by silica gel chromatography with elution by ethyl
acetate/methanol (4:1 v/v) to give BzSe-Cy as a green solid (55% yield).
1H and 13C NMR spectra were recorded on a Bruker Avance 600 MHz
Company. Sartorius ultrapure water (18.2 MWcm) was used throughout
the analytical experiments. RAW 264.7 macrophage cells, human liver
(HL-7702) cells, and human hepatoma (HepG2) cells were purchased
spectrometer and 77Se NMR spectra were recorded on a Bruker 300 MHz
spectrometer. 1H NMR (600 MHz, [D6]acetone, 258C): d=8.80 (d, J=
12.6 Hz, 2H), 7.60 (d, J=7.2 Hz, 2H), 7.40–7.47 (m, 4H), 7.29 (t, J=
7.2 Hz, 2H), 7.25 (d, J=7.8 Hz, 2H), 7.16–7.23 (m, 3H), 6.37 (d, J=
14.4 Hz, 2H), 4.31 (q, J=6.6 Hz, 4H), 4.12 (s, 2H), 2.71 (t, J=6.6 Hz,
4H), 1.86 (m, 2H), 1.71 (s, 12H), 1.42 ppm (t, J=6.0 Hz, 6H); 13C NMR
(600 MHz, [D6]acetone, 258C): d=206.07, 172.95, 158.50, 150.14, 143.03,
142.15, 139.95, 134.98, 129.57, 129.56, 129.31, 127.92, 125.87, 123.34,
111.62, 101.75, 50.06, 39.92, 34.81, 27.91, 27.18, 21.87, 12.54 ppm;
77Se NMR (300 MHz, CD3CN, 258C): d=265 ppm; MS: m/z: calcd 647.9
[M]+, found 647.5; elemental analysis (%) calcd: C 63.6, H 6.1, N 3.6;
found C 63.7, H 6.2, N 3.7.
from the Committee on Type Culture Collection of the Chinese Acade-
my of Sciences.
Apparatus: Particle size was determined by the DLS method with a Dy-
naProTitan instrument (Wyatt Technology Corp., USA). Samples were
diluted and the hydrodynamic radius of a micelle was determined. TEM
was carried out on a JEM-100CX P electron microscope. Fluorometric
traces were obtained with a Cary Eclipse fluorescence spectrophotometer
(Varian, Inc., USA) equipped with a 1.0 cm quartz cell at the slits of 5/
10 nm. Absorption spectra were measured on a PharmaSpec UV-1700
UV–visible spectrophotometer (Shimadzu). The release tests of IRhB
and BzSe-Cy in vitro were performed with an Agilent 1100 Series HPLC
system. All pH measurements were performed with a pH-3c digital pH
meter (Shanghai Lei Ci Device Works, Shanghai, China) with a com-
bined glass–calomel electrode. 1H and 13C NMR spectra were obtained
on Bruker Avance 600 MHz and 77Se NMR Bruker 300 MHz spectrome-
ters. The fluorescence images of cells were taken by using an LTE confo-
cal laser scanning microscope (Germany Leica Co., Ltd.). Absorbance
Synthesis of MPEG-PLA and MAL-PEG-PLA: The MPEG-PLA and
MAL-PEG-PLA diblock copolymers were synthesized by ring-opening
polymerization as described previously.[27] In brief, predetermined
amounts of MPEG or maleimide-PEG and d,l-lactide were placed in a
dried round-bottomed bottle connected with a vacuum joint. In both
cases, an appropriate amount of stannous octoate was added as a solution
in dried toluene. The reactants were dried under reduced pressure at
Chem. Eur. J. 2011, 17, 6626 – 6634
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6633

B. Tang et al.
708C for 1 h, and then the reaction proceeded under vacuum at 1608C
for 4 h. The cooled product was dissolved in dichloromethane, and recov-
ered by precipitation into an excess of mixed solvent comprising ethyl
ether and petroleum ether. The precipitate was redissolved in acetone
and precipitated into excess water, with the purified copolymers dried in
a vacuum oven at 408C for 24 h and then stored in a desiccator under
vacuum. The two copolymers were analyzed by H NMR spectroscopy at
room temperature. The degree of polymerization of the PLA was calcu-
lated by comparing the integral intensity of the characteristic resonance
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1
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ꢀ
ꢀ
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3.64 ppm ( OCH2CH2 ) in the 1H NMR spectrum (Figure S11, Support-
ing Information). The amount of maleimide proton was calculated by
comparing the integral intensity of the characteristic resonance at
ꢀ
ꢀ
ꢀ
ꢀ
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The CPPs-encoding nanoprobe loaded with two dyes was prepared by
nanoprecipitation and self-assembly.[18] Briefly, MPEG-PLA (9 mgmLꢀ1),
MAL-PEG-PLA (1 mgmLꢀ1), BzSe-Cy (0.5 mm), and IRhB (0.01 mm)
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Acknowledgements
This work was supported by the National Basic Research Program of
China (973 Program, 2007CB936000), National Natural Science Funds
for Distinguished Young Scholars (No. 20725518), National Key Natural
Science Foundation of China (No. 21035003), Natural Science Founda-
tion of Shandong Province in China (No. ZR2010BZ001), and Science
and Technology Development Programs of Shandong Province of China
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Received: January 15, 2011
Published online: May 17, 2011
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