Hypoxia-sensitive fluorescent probes for in vivo real-time fluorescence imaging of acute ischemia

Article abstract of DOI:10.1021/ja105937q

Based on the findings that the azo functional group has excellent properties as the hypoxia-sensor moiety, we developed hypoxia-sensitive near-infrared fluorescent probes in which a large fluorescence increase is triggered by the cleavage of an azo bond.

Full text of DOI:10.1021/ja105937q

Published on Web 10/27/2010
Hypoxia-Sensitive Fluorescent Probes for in Vivo Real-Time Fluorescence
Imaging of Acute Ischemia
Kazuki Kiyose,†,‡ Kenjiro Hanaoka,†,‡ Daihi Oushiki,†,‡ Tomomi Nakamura,§ Mayumi Kajimura,§
Makoto Suematsu,§ Hiroaki Nishimatsu,| Takehiro Yamane,†,‡ Takuya Terai,†,‡ Yasunobu Hirata,‡,|
and Tetsuo Nagano*,†,‡
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan, CREST, JST Agency, Kawaguchi, Saitama, Japan, Keio UniVersity, School of Medicine,
Shinanomachi, Shinjuku-ku, Tokyo, Japan, and The UniVersity of Tokyo Hospital, Hongo, Bunkyo-ku, Tokyo, Japan
Received July 5, 2010; E-mail: tlong@mol.f.u-tokyo.ac.jp
ࠗ
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This paper contains enhanced objects available on the Internet at http://pubs.acs.org/jacs.
effectively quench the fluorescence, electron-withdrawing groups
such as nitroaromatics have to be placed in the vicinity. At the
same time, to effectively serve as a substrate for reductases,
however, the same groups have to be rather distant from the
fluorophore moiety to prevent interference with the enzymatic
activity.
Abstract: Based on the findings that the azo functional group
has excellent properties as the hypoxia-sensor moiety, we
developed hypoxia-sensitive near-infrared fluorescent probes in
which a large fluorescence increase is triggered by the cleavage
of an azo bond. The probes were used for fluorescence imaging
of hypoxic cells and real-time monitoring of ischemia in the liver
and kidney of live mice.
To fulfill seemingly two opposing requirements, we employed
Fo¨rster resonance energy transfer (FRET). FRET occurs efficiently
when the distance between donor and acceptor is within 10 nm.
With this distance between the fluorophore moiety and hypoxia-
sensitive moiety, the fluorophore moiety should have little effect
on the enzymatic reactivity. The FRET efficiency can be altered
either by changing the distance to the FRET donor or by changing
the overlap integral between the emission spectrum of the FRET
donor and the absorption spectrum of the FRET acceptor.13,14 In
order to change the distance or overlap integral, the hypoxia-
sensitive moiety should act as a linker between the FRET donor
and acceptor or as the acceptor itself. However, considering the
structure of nitroaromatic or quinone groups, hypoxic reduction of
nitro groups does not appear to induce a drastic change in the
distance or absorption, so it is likely to be difficult to develop
hypoxia-sensitive fluorescent probes based on nitro aromatic groups.
Therefore, we focused on an azo structure as a hypoxia-sensitive
alternative to nitroaromatic or quinone moieties.
Hypoxia, which is caused by an inadequate oxygen supply, is a
feature of various diseases, including cancer, cardiopathy, ischemia,
and vascular diseases.1 Therefore, hypoxia-specific molecular probes
would be useful as diagnostic agents. Hypoxia-selective probes
developed for positron emission tomography (PET),2 fluorescence
imaging, immunostaining,3 and phosphorescence imaging4,5 exploit
the properties of two key intracellular responses caused by hypoxia.
One is stabilization of hypoxia inducible factor-1 (HIF-1), and the
other is the imbalance of cellular redox states leading to an increase
in the reducing equivalent.
One of the earliest cellular responses is stabilization of HIF-1, a
transcription factor of genes encoding a series of proteins involved
in glycolytic metabolism and neovasculization. Since the stabiliza-
tion is easily reversed by oxygenation through activation of a prolyl
hydroxylase-domain protein (PHD) and ubiquitin-proteasome sys-
tem, in ViVo imaging of hypoxia has been achieved by using a fusion
protein of the HIF-1R-binding domain called HRE with appropriate
reporter proteins.6,7 However, it is very difficult to apply this method
in living animals due to the need for genetic modification.
A decrease in oxygen causes an increase in reductive stress.
Indeed many small molecule-based probes to detect hypoxia take
advantage of this phenomenon, and most probes employ nitroaro-
matic or quinone derivatives as hypoxia-sensitive moieties.8-11 But
the fluorescent probes previously developed are inadequate for in
ViVo imaging of ischemia, because of the low sensitivity response
to hypoxia and/or use of short wavelengths for photoemission that
potentially renders living cells and tissues vulnerable. We speculated
that the poor reactivity in hypoxic reduction might be due to the
probes being poor substrates for reductases. Nitroaromatic groups
or quinone groups have very low LUMO energy, so that fluores-
cence quenching occurs Via electron transfer, which is known as a
photoinduced electron transfer (PeT) mechanism.12 Generally, to
Scheme 1
Generally, azobenzene derivatives are reduced stepwise by
various reductases to aniline derivatives (Scheme 1).15 Through a
series of reactions, the azo bond is cleaved and the distinctive
absorption of the azo moiety is lost. In other words, a drastic change
in distance and overlap integral becomes possible by using azo
compounds. In the case of nitroaromatics, the formation of
nitroanion radical compounds in the first step of reduction is
reversible. This step is also strongly influenced by oxygen, and
when the reaction system is abundant in oxygen, back oxidation
readily occurs; thus, reduction is completed.16 Azobenzene deriva-
tives look like “oxidized amines”, similar to nitroaromatics, which
leads us to hypothesize that the reduction of azobenzene would
also be strongly influenced by the oxygen concentration.
† The University of Tokyo.
‡ CREST, JST Agency.
We first evaluated the reduction potential of representative
nitroaromatic compounds, such as nitroimidazole and nitrofuran,
§ Keio University.
| The University of Tokyo Hospital.
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15846 J. AM. CHEM. SOC. 2010, 132, 15846–15848
10.1021/ja105937q  2010 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. (a) Design strategy of QCys. (b) Chemical structure of QCys.
QCy5 (R1 ) R2 ) R3 ) R4 ) H), QCy5.25 (R1 ) R2 ) CHdCH, R3 ) R4
) H), QCy5.5 (R1 ) R2 ) R3 ) R4 ) CHdCH). (c) Time-dependent change
of the fluorescence intensity of 1 µM QCy5 in the presence of rat liver
microsomes (50 µL/3 mL) under hypoxic (red line) or normoxic (blue line)
conditions. Measurements were performed in 0.1 M potassium phosphate
buffer (pH 7.4) containing 0.1% DMSO as a cosolvent and 50 µM NADPH
as an electron donor. Excitation and emission wavelengths were 650 and
670 nm.
Figure 2. Oxygen-dependent fluorescence increase of QCys inside living
MCF-7 cells, and oxygen dependency of QCy5 fluorescence. (a) Confocal
laser scanning microscopy (left) or white light image (right) of 1 µM QCy5,
QCy5.25, QCy5.5 loaded MCF-7 cells incubated under hypoxic (less than
0.1% O2) or normoxic (20% O2) conditions for 6 h. The QCys emission
was obtained using excitation at 650 nm (QCy5) or 670 nm (QCy5.25 and
QCy5.5). Scale bar, 50 µm. (b) Oxygen dependency of QCy5 fluorescence.
MCF-7 cells loaded with 1 µM QCy5 were incubated at various oxygen
levels. Scale bar, 50 µm.
and azobenzene derivatives, such as methyl red and disperse red,
by means of cyclic voltammetry (Table S1). The reduction potentials
of azobenzene derivatives are nearly equal to those of nitroaromatic
compounds; in other words, azobenzene derivatives are reduced
as efficiently as nitroaromatic compounds. Thus azobenzene deriva-
tives appear to be good candidates for hypoxia-sensitive moieties.
In order to develop fluorescent probes for in ViVo imaging, it is
desirable that the fluorophore has near-infrared (NIR) absorption
and emission since tissue penetration is best in that wavelength
range. However, most of the azobenzene derivatives have absorp-
tions and emissions in the visible region. We, therefore, focused
on the Black Hole Quencher (BHQ) series. Among them, BHQ-3
has a very wide absorption from the visible to NIR and can quench
NIR emissions. To examine the oxygen sensitivity of BHQ-3, we
conducted an enzymatic assay of BHQ-3 using rat liver microsomes,
which contain diverse metabolic enzymes. Under hypoxia, but not
under normoxia, the absorption of BHQ-3 disappeared within 10
min (Scheme 1 and Figure S1). Hence we concluded that BHQ-3
is an excellent candidate for a hypoxia-sensitive moiety. We then
designed and synthesized hypoxia-sensitive fluorescent probes
combining an NIR fluorophore and BHQ-3. Under normoxic
conditions, BHQ-3 is not reduced and the probes are nonfluorescent
owing to the FRET mechanism. Under hypoxic conditions, BHQ-3
is readily reduced and loses its absorption, so FRET efficiency is
decreased and the probes become fluorescent (Figure 1a). A great
advantage of this design is that various probes with a wide range
of absorption and emission wavelengths can be produced by simply
changing the combination of fluorophores and quenchers.
We chose dicarbocyanines as candidate NIR fluorophores. First,
we synthesized three dicarbocyanines (Schemes S1 and S2) and
confirmed the stability of them in the presence of reductases under
hypoxic conditions. The absorption spectra of all the dyes showed
essentially no change under aerobic or hypoxic conditions, indicating
that the dyes are stable (Figure S2). Therefore, we adopted this design
strategy and synthesized three hypoxia-sensitive probes (QCy5,
QCy5.25, QCy5.5) which have different absorption characteristics
(Scheme S3, Figures 1b and S3, Table S2). All the probes had
extremely weak fluorescence, and the emission could hardly be
detected. After enzymatic reactions under hypoxic conditions, a 50-
to 100-fold fluorescence increase was observed, and the fluorescence
intensity reached a plateau within 10 min (Figures 1c and S4). On the
other hand, under normoxic conditions, no fluorescence increase
occurred. Furthermore, fluorescence intensities of QCys were insensi-
tive to the pH change between 4.0 and 11.0. These results indicated
that QCys could detect hypoxia selectively and rapidly, being superior
to known fluorescent probes, in these respects.
Next, we applied the probes to living cells and investigated
whether they could detect intracellular hypoxia. The three probes
were loaded into MCF-7 breast cancer cells and incubated under
normoxic conditions in a CO2 incubator (20% O2, 5% CO2) or
hypoxic conditions generated with an AnaeroPack (Mitsubishi Gas
Corp.) (>0.1%O2, 5% CO2) for 6 h. Each probe showed strong
fluorescence only in cells incubated under hypoxic conditions. These
results indicated that QCys could detect hypoxia inside living cells
(Figure 2a). We next investigated whether QCy5 could distinguish
oxygen concentrations inside cells; i.e., MCF-7 cells loaded with
1 µM QCy5 were incubated under various oxygen concentrations
(20%, 10%, 3%, 1%, 0.1%) for 6 h, and fluorescence images were
obtained. In this experiment, strong fluorescence was detected from
the cells incubated at 1% oxygen or less. Furthermore, the
fluorescence intensity of the cells under 0.1% oxygen was twice
that of the cells under 1% oxygen. These results indicated that QCy5
fluorescence was oxygen concentration dependent (Figures 2b and
S5). We also examined other cell lines and obtained similar data.
Hence, QCy5 could visualize the hypoxic status of a wide variety
of cell lines. Interestingly, the oxygen sensitivity varied from cell
line to cell line, presumably reflecting the origin of each cell line
(Figure S5).
We next examined whether QCy5 can detect hypoxia in the intact
animal in ViVo using an ischemia model of the mouse liver. At 30
min after an intravenous injection of QCy5 (0.5 mg/kg), the portal
vein was ligated to induce ischemia. Immediately after the ligation,
the fluorescence intensity rapidly increased, suggesting that the
uptake of QCy5 by hepatocytes occurred. Results indicate that QCy5
was readily reduced in the ischemic liver, but not in the normal
liver. Thus, we achieved real-time imaging of liver ischemia using
QCy5 (Figures S6 and S7, Supporting Video 1).
Next, we examined ischemic imaging at the whole-body level.
A solution of QCy5 (0.5 mg/kg) was administered to mice by
intravenous injection, and the portal vein and renal vein were
ligated. Just after ligation, the fluorescence intensity of each organ
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J. AM. CHEM. SOC. VOL. 132, NO. 45, 2010 15847

C O M M U N I C A T I O N S
Promoted Research 22000006 to T.N. and 20689001 and 21659204
to K.H.), and by the industrial Technology Research Grant Program
in 2009 from the New Energy and Industrial Technology Develop-
ment Organization (NEDO) of Japan (to T.T.). K.H. was also
supported by Sankyo Foundation of Life Science. M.K. and M.S.
are supported by JST, ERATO, Suematsu Gas Biology Project in
Tokyo 160-8582.
Supporting Information Available: Full experimental procedures,
characterization data for all compounds, spectral properties of cyanine
dyes and QCys, and the pH profiles of QCys. This material is available
free of charge via the Internet at http://pubs.acs.org.
Figure 3. (a) Fluorescence images of living mouse after injection of QCy5
followed by vessel ligation as described in the text. Fluorescence images
were obtained for 30 min, with excitation at 620 nm and emission at 680
nm. (b) Fluorescence images of living mouse after injection of QCy5 without
vessel ligation.
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rapidly increased, and after 30 min, the fluorescence intensity was
saturated. The fluorescence intensity of these organs in nontreated
mice did not change (Figures 3 and S8, Supporting Video 2). In
this experiment, we could detect a fluorescence increase within 1
min, which means that real-time monitoring of the ischemic organs
was achieved with QCy5. Because acute ischemia of the liver or
kidney is often problematic in transplant surgery,17 these probes
have the potential to become powerful tools for clinical use. To
our knowledge, QCys are the first fluorescent probes suitable for
real-time imaging of acute ischemia in living animals.
In summary, we identified azobenzene derivatives as a novel
type of hypoxia-sensitive moiety and developed a series of NIR
fluorescent probes, QCys, for detecting hypoxia. These probes could
distinguish hypoxia in Vitro and were also suitable for fluorescence
imaging of ischemic organs in live mice. The in ViVo fluorescence
response was rapid, occurring within 1 min after vessel ligation.
These probes should be suitable for many biological applications,
such as real-time monitoring of cerebral infarction.
Acknowledgment. This work was supported in part by a Grant-
in-Aid for JSPS Fellows, by the Ministry of Education, Culture,
Sports, Science and Technology of Japan (Grant Nos. Specially
JA105937Q
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