Reversible off-on fluorescence probe for hypoxia and imaging of hypoxia-normoxia cycles in live cells

Article abstract of DOI:10.1021/ja310049d

We report a fully reversible off-on fluorescence probe for hypoxia. The design employs QSY-21 as a F?rster resonance energy transfer (FRET) acceptor and cyanine dye Cy5 as a FRET donor, based on our finding that QSY-21 undergoes one-electron bioreduction to the radical under hypoxia, with an absorbance decrease at 660 nm. At that point, FRET can no longer occur, and the dye becomes strongly fluorescent. Upon recovery of normoxia, the radical is immediately reoxidized to QSY-21, with loss of fluorescence due to restoration of FRET. We show that this probe, RHyCy5, can monitor repeated hypoxia-normoxia cycles in live cells.

Full text of DOI:10.1021/ja310049d

Communication
pubs.acs.org/JACS
Reversible Off−On Fluorescence Probe for Hypoxia and Imaging of
Hypoxia−Normoxia Cycles in Live Cells
Shodai Takahashi,† Wen Piao,‡ Yuriko Matsumura,§ Toru Komatsu,‡ Tasuku Ueno,‡ Takuya Terai,‡
Toshiaki Kamachi,§ Masahiro Kohno,§ Tetsuo Nagano,‡ and Kenjiro Hanaoka*,‡
‡
†Faculty of Pharmaceutical Sciences and Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan
§Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi,
Kanagawa 226-8501, Japan
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S
* Web-Enhanced Feature * Supporting Information
be involved. However, no method was available to visualize the
dynamics of cycling hypoxia in real time. Therefore, we set out
to develop a reversible fluorescence probe for hypoxia.
Existing redox-sensitive reversible fluorescence probes, such
as nitric oxide,19 ROS,20,21 and peroxynitrite22 probes, detect
oxidative stress but not reductive stress, so a key requirement
for a reversible hypoxia probe is the choice of a suitable
detector moiety.
ABSTRACT: We report a fully reversible off−on
fluorescence probe for hypoxia. The design employs
QSY-21 as a Forster resonance energy transfer (FRET)
̈
acceptor and cyanine dye Cy5 as a FRET donor, based on
our finding that QSY-21 undergoes one-electron bio-
reduction to the radical under hypoxia, with an absorbance
decrease at 660 nm. At that point, FRET can no longer
occur, and the dye becomes strongly fluorescent. Upon
recovery of normoxia, the radical is immediately reoxidized
to QSY-21, with loss of fluorescence due to restoration of
FRET. We show that this probe, RHyCy5, can monitor
repeated hypoxia−normoxia cycles in live cells.
We decided to employ the Forster resonance energy transfer
̈
(FRET) mechanism for our probe design. Namely, we required
a dye that shows a reversible spectral change between its
reduced and oxidized forms for use as a FRET acceptor (Figure
1). We examined several candidates (Supporting Information
ypoxia is a feature of various diseases, including tumors,1
H
cardiovascular diseases,2 and stroke.3 So far, various
approaches have been used to selectively detect hypoxic regions
by utilizing immunostaining,4,5 PET imaging,6,7 phosphor-
escence imaging,8,9 magnetic resonance imaging,10,11 and
fluorescence imaging.12−15 Among them, fluorescence imaging
offers various advantages, including high sensitivity and ease of
use.
Hypoxia induces two distinctive biological responses, i.e.,
stabilization of hypoxia-inducible factor-1 (HIF-1)16 and
accelerated bioreductive reaction.17 The latter has been the
preferred target for the design of hypoxia-sensitive imaging
probes because bioreductive reactions proceed efficiently under
hypoxia, while they do not proceed under normoxia. Several
hypoxia-sensitive fluorescence probes, whose fluorescence
intensity increases upon bioreduction under hypoxia, have
been developed, employing a nitro group,12,13 quinone group,14
or azo group15 as the hypoxia-sensing moiety. However, the
structural change of the functional group to the “fluorescence
on” state is irreversible in these probes. In other words, once
these probes become strongly fluorescent in a hypoxic region,
they remain strongly fluorescent even after the region returns
to normoxia.
Figure 1. Design strategy for a reversible fluorescence probe for
hypoxia.
Figure S1). 2-Aminoanthraquinone and malachite green were
selected as candidates because it is reported that quinones and
malachite green can be enzymatically reduced,23,24 and QSY-21
was selected because of the similarity of its chemical structure
to that of malachite green. A solution of each compound was
incubated with rat liver microsomes, which contain various
reductases, at 37 °C in air (∼20% pO2; normoxia) or in an
argon atmosphere (∼0.1% pO2; hypoxia), and the changes of
the absorption spectra were measured. QSY-21 showed a
spectral change which suggested that it might be suitable as a
hypoxia-sensing moiety for a reversible fluorescence probe
(Figure 2).
Recently, it was reported that cycling hypoxia, i.e., repeated
cycles of hypoxia−reoxygenation, induces increased HIF-1
activity of tumor cells, and this phenomenon is associated with
a decreased success rate of radiotherapy or chemotherapy.18
The mechanism of HIF-1 stabilization is unclear, although
production of reactive oxygen species (ROS) is considered to
Received: October 11, 2012
© XXXX American Chemical Society
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absorbance in the visible region (Figure S3). However, the
reduced form of QSY-21 showed an absorbance peak at 470 nm
in the visible region, as shown in Figure 2a. It is reported that
rhodamine dyes, whose absorbance lies in the range of 491−
564 nm, can be reversibly photoreduced by thiols to form stable
radicals in the absence of O2.26 These radicals have absorbance
at shorter wavelength than that of rhodamine dyes themselves,
but still in the visible region, i.e., 386−432 nm. In addition,
triphenylmethyl radical, the first organic radical, discovered by
Gomberg,27 is reported to be stable because the free electron is
delocalized and the radical center is shielded by three phenyl
groups, and this radical also has absorption at around 330 nm.28
So, we thought that a radical might have been formed from
QSY-21 by the bioreduction.
Electron spin resonance (ESR) measurement showed that an
ESR signal (g = 2.0032, indicating carbon center radical29)
appeared after bioreduction of QSY-21 under hypoxia but was
not observed under normoxia (Figures 3 and S4). The same
Figure 2. In vitro assay of QSY-21 and RHyCy5. (a) Chemical
structure of QSY-21 and changes of the absorption spectrum of 5 μM
QSY-21 under normoxia, hypoxia, and hypoxia-to-normoxia transition.
Spectra were measured every 5 min for 30 min at 37 °C in 0.1 M
potassium phosphate buffer (pH 7.4) containing rat liver microsomes
(0.25 mg protein/mL), 50 μM NADPH as a coenzyme, and 0.5%
DMF as a cosolvent. Red lines show the initial and subsequent spectra
(some are overlapping), and green lines show the final spectra at 30
min. (b) Chemical structure of RHyCy5. (c) Time-dependent changes
of fluorescence intensity of 1 μM RHyCy5 under normoxic and
hypoxic conditions. Data were measured at 37 °C in 0.1 M potassium
phosphate buffer (pH 7.4) containing rat liver microsomes (0.25 mg
protein/mL), 50 μM NADPH as a coenzyme, and 0.1% DMF as a
cosolvent. NADPH addition: NADPH was added at the point
indicated by the arrowhead. The transition of hypoxia to normoxia
was achieved by exposing the hypoxic solution to air. Ex/Em = 650/
670 nm.
Figure 3. Investigation of the mechanism of the fluorescence increase
of RHyCy5 by bioreduction under hypoxia. (a) ESR spectra of QSY-
21 under normoxia or hypoxia were measured in 0.1 M potassium
phosphate buffer (pH 7.4) containing 0.5% DMF as a cosolvent. The
solution contained rat liver microsomes (0.25 mg protein/mL) and 50
μM NADPH as a coenzyme. Mn2+ was used as an internal standard.
(b) Proposed structure of the radical form of QSY-21 generated upon
bioreduction. (c) Proposed mechanism of reversible detection of
hypoxia by RHyCy5.
We found that QSY-21,25 a dark quencher of fluorescence in
the red to near-infrared (NIR) region, was enzymatically
reduced under hypoxia. Concomitantly, the absorbance at 660
nm decreased and the absorbance at 470 nm increased (Figure
2a). No spectral change was observed under normoxia.
Moreover, the absorbance at 660 nm recovered and the
absorbance at 470 nm decreased within 1 min after exposure of
the hypoxic solution to air (Video 1). Based on these
characteristics of QSY-21, we designed and synthesized a
novel reversible fluorescence probe for hypoxia, RHyCy5
(Reversible Hypoxia Cy5 Probe) (Figure 2b). RHyCy5 showed
extremely weak fluorescence under normoxia (Figure S2, Table
S1) that increased 7- to 8-fold (Φfl = 0.002−0.022) under
hypoxia and then rapidly decreased to the initial level after
exposure to air and subsequently remained unchanged (Figure
2c, Table S2). Thus, RHyCy5 works as a reversible fluorescence
probe for hypoxia.
ESR signal was also detected upon addition of dithionite, a
chemical reducing agent (Figure S5a). Further, addition of
dithionite to QSY-21 in potassium phosphate buffer (pH 7.4)
resulted in the same change in the absorption spectrum as that
seen in Figure 2a (Figure S5b). Upon addition of dithionite to
RHyCy5 in potassium phosphate buffer (pH 7.4), the
fluorescence intensity increased 7- to 8-fold (Φfl = 0.002−
0.021), as was the case in the bioreduction shown in Figure 2c
(Figure S5c, Table S2).
Moreover, leuco QSY-21 was not oxidized to QSY-21 under
normoxia for at least 1 h (Figure S6). Based on these results, it
is considered that the radical form of QSY-21, i.e., the one-
electron reduction product of QSY-21 shown in Figure 3b, is
produced by bioreduction under hypoxia. Further, after
bioreduction of RHyCy5 under normoxia or hypoxia, the
solution was analyzed by HPLC (Figure S7). In the case of
hypoxia, the sample was subsequently exposed to air. Almost no
Next, we investigated the product formed upon bioreduction
of RHyCy5 under hypoxia. We initially considered that leuco
QSY-21, the two-electron reduction product of QSY-21, might
be produced by the bioreduction of QSY-21 and would have no
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Journal of the American Chemical Society
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peak at 650 nm was observed in the sample solutions incubated
under normoxia and hypoxia, except for that of RHyCy5. Thus,
we propose that the fluorescence off−on mechanism of this
reversible fluorescence probe is as shown in Figure 3c. The
immediate fluorescence decrease of RHyCy5 upon transition
from hypoxia to normoxia can be attributed to the rapid
oxidation of the radical form of RHyCy5 in the air. In general,
the fluorescence of fluorophores linked to radicals is known to
be quenched owing to electron transfer, but it has been
reported that the fluorescence of Cy5 conjugated to nitric
oxide, which is a radical, via a 2- or 5-methylene linker is not
quenched.30 The separation between Cy5 and QSY-21 in
RHyCy5 is larger than five methylenes, and this is probably
why the fluorescence of the radical form of RHyCy5 was not
quenched.
Finally, we applied RHyCy5 to living cells and investigated
whether this probe can reversibly detect repeated cycles of
hypoxia−normoxia in live cells. It has been shown that a pO2
gradient can be formed by putting a thin cover glass on top of
cells to prevent O2 diffusion from above.31 We used this
method to evaluate the suitability of RHyCy5 for real-time
imaging of hypoxia. Cycles of hypoxia−normoxia were
established by repeatedly placing a cover glass over the cells
and removing it. RHyCy5 was loaded into A549 cells, human
breast cancer cells, by the incubation of cells with RHyCy5 in
Dulbecco’s modified Eagle’s medium (DMEM) for 1 h. Almost
no fluorescence was observed under normoxia (Figure 4a).
change was observed when cells were incubated under
normoxia (air) for at least 3 h (Figure S8). Thus, this
fluorescence probe could visualize repeated hypoxia−normoxia
cycles in live cells. To our knowledge, this is the first reversible
fluorescence probe that is able to detect repeated hypoxia−
normoxia cycles by utilizing bioreductase activity under
hypoxia.
In summary, we found that QSY-21 is reversibly bioreduced
under hypoxia, and we identified the product of the
bioreduction as the radical form of QSY-21, which undergoes
rapid oxidation upon exposure to air. We utilized these findings
to design and synthesize a reversible fluorescence probe,
RHyCy5, based on the FRET mechanism by using QSY-21 as a
FRET acceptor and Cy5 as a FRET donor. This probe could
successfully detect repeated cycles of hypoxia−normoxia in live
cells. We anticipate that this probe will be a valuable tool for
exploring the response of organisms to hypoxia.
ASSOCIATED CONTENT
■
S
* Supporting Information
Full experimental procedures, characterization data for all
compounds, spectral properties of cyanine dyes and QSY-21
derivatives, compounds as candidates for a hypoxia-sensing
moiety other than QSY-21, ESR spectra, HPLC analyses, and
fluorescence imaging under normoxia. This material is available
free of charge via the Internet at http://pubs.acs.org.
W
* Web-Enhanced Feature
Video 1, showing exposure of a hypoxic solution of QSY-21 to
air, is available in the online versions.
AUTHOR INFORMATION
■
Corresponding Author
khanaoka@mol.f.u-tokyo.ac.jp
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by MEXT (Specially
Promoted Research Grant Nos. 22000006 to T.N., and Grants
24689003 and 24659042 to K.H.) and SENTAN, JST (to
K.H.). K.H. was also supported by Grants-in-Aid from Tokyo
Biochemical Research Foundation, Inoue Foundation for
Science, Takeda Science Foundation, and Astellas Foundation
for Research on Metabolic Disorders. T.T. was supported by
the Japan Society for the Promotion of Science (Grant-in-Aid
for Young Scientists (B) No. 21750135).
Figure 4. Fluorescence confocal microscopic images of A549 cells
loaded with 1 μM RHyCy5 and exposed to cycles of normoxia−
hypoxia. (a) Cells were incubated under normoxia for 1 h in DMEM
containing 1 μM RHyCy5, then washed with PBS (pH 7.4), and
placed in fresh DMEM. (b) The cells were incubated under a cover
glass (hypoxic condition) for 1 h after (a). (c) The cover glass was
removed and incubation was continued for 10 min under 5% CO2 in
air. (d) Cells were incubated under a cover glass for 1 h after (c). (e)
The cover glass was removed and incubation was continued for 10 min
under 5% CO2 in air. (f) The cells were incubated under a cover glass
for 1 h after (e). Scale bar: 50 μm.
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