Hydrocyanines: A class of fluorescent sensors that can image reactive oxygen species in cell culture, tissue, and in vivo

Article abstract of DOI:10.1002/anie.200804851

Accurate and tunable: The title compounds can detect reactive oxygen species (ROS) in cell culture, tissue explants, and for the first time in vivo. The hydrocyanines are synthesized by reduction of the cyanine dyes with NaBH₄. They can accurat

Full text of DOI:10.1002/anie.200804851

Angewandte
Chemie
DOI: 10.1002/anie.200804851
Fluorescence Imaging
Hydrocyanines: A Class of Fluorescent Sensors That Can Image
Reactive Oxygen Species in Cell Culture, Tissue, and In Vivo**
Kousik Kundu, Sarah F. Knight, Nick Willett, Sungmun Lee, W. Robert Taylor, and
Niren Murthy*
The development of fluorescent probes for superoxide and
the hydroxyl radical is a central problem in the field of
chemical biology.[1] Superoxide and the hydroxyl radical play
a significant role in a variety of inflammatory diseases, and
probes that can detect these reactive oxygen species (ROS)
have tremendous potential as medical diagnostics and
research tools.[2–6] Fluorescent sensors for superoxide and
the hydroxyl radical, such as dihydroethidium (DHE), have
been developed; however, they have had limited applicability
because of their spontaneous autoxidation, rapid photo-
bleaching, low emission wavelengths, and multiple reaction
products with ROS.[7] New chemical probes for superoxide
and the hydroxyl radical are therefore greatly needed.
Herein, we present a new family of fluorescent ROS sensors,
termed the hydrocyanines, which can be synthesized in one
step from the commercially available cyanine dyes and can
detect superoxide and the hydroxyl radical in living cells,
tissue samples, and for the first time in vivo. We anticipate
widespread interest in the hydrocyanines given their physical/
chemical characteristics and ease of synthesis.
The synthesis of and mechanism by which hydrocyanines
Figure 1. a) Synthesis of hydrocyanines by a one-step reduction with
NaBH4. Reaction with superoxide or the hydroxyl radical oxidizes the
hydrocyanines to produce the fluorescent cyanine dyes. b) Hydro-IR-
676 has negligible fluorescence emission; however, oxidation with
superoxide causes a 100-fold increase in fluorescence (lex =675 nm,
lem =693 nm).
image ROS are shown in Figure 1a. The hydrocyanines are
synthesized by reducing the iminium cations of the cyanine
dyes with NaBH4. Hydrocyanines detect ROS through
fluorescent imaging; they are weakly fluorescent because of
their disrupted p conjugation; however, oxidation with either
superoxide or the hydroxyl radical dramatically increases
their fluorescence by regenerating their extended p conjuga-
tion. Figure 1b demonstrates the proof of principle of this
methodology. Hydro-IR-676 has minimal fluorescence; how-
ever, oxidation with superoxide causes a 100-fold increase in
its fluorescence intensity. The cyanine dyes comprise a family
of approximately 40 dyes,[8–9] and the reduction methodology
presented in Figure 1a was used to synthesize several new
fluorescent ROS sensors, which have the physical and
chemical properties needed to detect intracellular and
extracellular ROS, both in vitro and in vivo. For example,
five new ROS sensors, termed hydro-Cy3, hydro-Cy5, hydro-
Cy7, hydro-IR-783, and hydro-ICG, were synthesized by
reduction with NaBH4 (Table 1). These molecules have
negligible fluorescence, as a result of reduction of their
iminium cations; however, after reaction with either super-
oxide or the hydroxyl radical, they fluoresce at 560, 660, 760,
800, and 830 nm, respectively (see Table 1 and the Supporting
Information for details). Hydro-Cy3, hydro-Cy5, hydro-IR-
676, and hydro-Cy7 have the physical properties needed to
detect intracellular ROS, in that they are initially membrane-
permeable molecules; however, oxidation with intracellular
ROS converts them into charged and membrane-imperme-
able molecules, which should accumulate within cells that are
overproducing ROS. In contrast, hydro-ICG and hydro-IR-
[*] K. Kundu, S. Lee, Prof. W. R. Taylor, Prof. N. Murthy
The Wallace H. Coulter Department of Biomedical Engineering and
Parker H. Petit Institute of Bioengineering and Bioscience
Georgia Institute of Technology, Atlanta, GA 30332 (USA)
Fax: (+1)404-894-4243
E-mail: niren.murthy@bme.gatech.edu
S. F. Knight, N. Willett, Prof. W. R. Taylor
Cardiology Division, Department of Medicine
Emory University School of Medicine, Atlanta, GA 30322 (USA)
Prof. W. R. Taylor
Cardiology Division
Atlanta VA Medical Center, Decatur, GA 30032 (USA)
[**] This work was supported by the Georgia Tech/Emory Center for the
Engineering of Living Tissues (funded by NSF-EEC-9731643)
(N.M.), NSF-BES-0546962 Career Award (N.M.), NIH UO1
HL80711-01 (N.M.), NIH R21 EB006418 (N.M.), and a J&J/GT
Health Care Innovation Seed Grant Proposal (N.M.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200804851.
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Table 1: Hydrocyanines synthesized in high yields with tunable physical
properties.
Hydrocyanine
Yield
[%][a]
Intensity
increase
[fold][b]
lex/lem
[nm][c]
97
95
118
97
535/560
635/660
Figure 2. Hydro-Cy3 has high specificity for superoxide and radical
oxidants over other reactive oxygen and nitrogen species. (See the
Supporting Information for details). RFU=relative fluorescence unit,
TBHP=tert-butyl hydroperoxide, TBO·=tert-butyl peroxyl radical,
NOC-5=3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene,
SIN-1=3-(4-morpholinyl)sydnone imine hydrochloride, GSH=
g-l-glutamyl-l-cysteinyl-glycine.
93
100
675/693
arise from a reaction mechanism involving two consecutive
one-electron oxidations, similar to DHE oxidation. There-
fore, we anticipate that the hydrocyanines will provide
information about the production of superoxide and other
radical oxidants in cell culture and in vivo.
We investigated the ability of the hydrocyanines to
quantify the hydroxyl radical[11] in vitro, and its sensitivity
was compared against that of DHE. Figure 3 demonstrates
95
93
104
83
735/760
765/800
93
86
750/830
[a] Yields of isolated products. [b] Fold increase was calculated by
dividing the fluorescent intensity of oxidized hydrocyanine by the
hydrocyanine fluorescence intensity. Concentrations of hydrocyanines
and oxidized hydrocyanines were 7.5 mm in 1 mL methanol. Hydro-
cyanines were oxidized with 20 mm KO2 in 20 mL of DMSO. [c] Excitation
and emission wavelengths for the corresponding oxidized products.
Figure 3. Sensitivity of hydro-Cy3, hydro-Cy7, and DHE towards the
hydroxyl radical. Both hydro-Cy3 and hydro-Cy7 have nanomolar
sensitivity towards the hydroxyl radical and are significantly more
sensitive than DHE.
783 are charged and membrane-impermeable molecules and
have the physical properties needed for measuring extrac-
ellular ROS production. Finally, several of the hydrocyanines,
such as hydro-Cy7 and hydro-ICG, have near-infrared emis-
sion wavelengths and are therefore suitable for detecting
ROS in vivo. Thus, NaBH4 reduction of the cyanine dyes
represents a powerful synthetic methodology for generating
fluorescent ROS sensors.
Cells produce several different types of reactive oxygen
and nitrogen species. We therefore investigated the specificity
of the hydrocyanines towards different reactive oxygen and
nitrogen species.[10] Figure 2 demonstrates that hydro-Cy3 has
high selectivity towards radical oxidants and superoxide and
generates negligible fluorescence in the presence of Fe2+,
singlet oxygen, hydrogen peroxide, nitric oxide, peroxyni-
trates, and various other oxidizing agents (hydro-Cy7 also has
a similar specificity profile; see the Supporting Information
for details). The selectivity of the hydrocyanines to ROS may
that both hydro-Cy3 and hydro-Cy7 can detect nanomolar
levels of radical oxidants. They both had linear relationships
(r2 = 0.99) between fluorescence intensity and hydrogen
peroxide concentration (1–30 nm range) in the Fentonꢀs
reagent. This correlation was significantly better than that
of DHE, which had an r2 value of only 0.36 in this concen-
tration range. The hydrocyanines also react rapidly with ROS;
for an equimolar concentration of Fentonꢀs reagent and
hydro-Cy3 (50 mm), the oxidation of hydro-Cy3 occurred with
a half-life of 3 min (see the Supporting Information).
A major problem with DHE is its autoxidation in aqueous
solutions, which creates high levels of background fluores-
cence and interferes with ROS measurements. The hydro-
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cyanines should have higher stability against autoxidation
than DHE, because of their lower kinetic and thermodynamic
oxidation driving force, resulting from lower resonance
stabilization of oxidation intermediates and products. We
therefore measured the stability of the hydrocyanines to
autoxidation. Figure 4 demonstrates that the hydrocyanines
Figure 5. Detection of ROS in live cells and tissue explants by hydro-
cyanines: a–c) confocal fluorescent images of live RASM cells:
a) RASM cells incubated with 100 mm hydro-Cy3; b) RASM cells treated
with Ang II (100 nm) for 4 h and incubated with 100 mm hydro-Cy3;
c) RASM cells incubated with Ang II (100 nm) for 4 h followed by
5 mm TEMPOL, prior to the addition of hydro-Cy3 (100 mm);
d–f) confocal fluorescent images of rat aortic tissue. Fluorescent
image of the aorta section of d) the untreated mouse incubated with
hydro-Cy3 for 15 min, e) the mouse treated with LPS incubated with
hydro-Cy3 for 15 min, and f) the mouse treated with LPS followed by
incubation with 5 mm TEMPOL and hydro-Cy3 for 15 min. Slides (d–f)
were stained with 4’,6-diamidino À2-phenylindole (DAPI) to identify
cell nuclei (blue dots).
Figure 4. Stability profile of hydro-Cy3, hydro-Cy7, and DHE towards
autoxidation. The stability was measured in PBS buffer (pH 7.4) at
378C (see the Supporting Information for details).
have higher stability to autoxidation than DHE. For example,
hydro-Cy7 and hydro-Cy3 both had half-lives of approxi-
mately 3 days in aqueous pH 7.4 buffer at 378C and were two
orders of magnitude more stable than DHE, which had a half-
life of only 30 min (Figure 4). The hydrocyanines therefore
have the stability, sensitivity, and reactivity needed to detect
ROS in biological samples.
ditions and therefore provides details about ROS production
that cell culture cannot. C57Bl/6 mice were treated with either
16 mgkgÀ1 lipopolysaccharide endotoxin (LPS) or phosphate-
buffer saline (PBS) for 16 h and then euthanized. The aortas
of these mice were then isolated and incubated with either
hydro-Cy3 or hydro-Cy3 and TEMPOL for 15 min. Sections
of the aorta were made, fixed with 10% formalin, and then
mounted en face. Figure 5d–f demonstrates that hydro-Cy3
can image ROS production in live tissue explants. Mice
incubated with LPS and hydro-Cy3 displayed significantly
higher fluorescence intensity than mice treated with just PBS
and hydro-Cy3 (Figure 5e vs. d). For example, the total
integrated fluorescence intensity of the LPS-treated slide
(Figure 5e) was 183.2 versus 46.9 in the PBS-treated mice
(Figure 5d); importantly, incubation with TEMPOL reduced
the fluorescence of the LPS-treated slides to control levels of
only 34.9 (Figure 5 f), demonstrating that the hydro-Cy3
fluorescence was due to ROS production.
A key benefit of the hydrocyanines is their high emission
wavelengths, which makes them suitable for imaging ROS
production in vivo. We investigated the ability of hydro-Cy7
to image ROS production in vivo generated by activated
macrophages and neutrophils in an LPS model of acute
inflammation. Nine C57Bl/6 mice were divided into three
groups. One group was given an intraperitoneal (i.p.)
injection of LPS (1 mg in 400 mL saline), a second group
was given saline (400 mL), and a third control group was
untreated. After 6 h, the mice were anaesthetized, their
abdominal fur was removed, and the LPS- and saline-treated
mice were injected i.p. with hydro-Cy7 (5 nmol in 50 mL
methanol). The mice were imaged as triplets, one from each
group, by using a Kodak FX in vivo imager with a 700 nm
We measured the ability of hydro-Cy3 to detect ROS
production in rat aortic smooth muscle (RASM) cells, during
angiotensin (Ang) II mediated cell signaling.[12] Ang II medi-
ated ROS production in aortic smooth muscle cells is
implicated in the development of atherosclerosis and hyper-
tension.[12] There is therefore great interest in measuring ROS
production from RASM cells stimulated with Ang II. Fig-
ure 5a–c demonstrates that hydro-Cy3 can detect ROS
production in RASM cells during Ang II mediated cell
signaling. For example, RASM cells incubated with Ang II
and hydro-Cy3 displayed intense intracellular fluorescence
(Figure 5b), whereas RASMs incubated with just hydro-Cy3
displayed significantly lower fluorescence (Figure 5a). Impor-
tantly, application of the superoxide dismutase mimetic
TEMPOL resulted in a dramatic decrease in fluorescence
from RASM cells treated with hydro-Cy3 and Ang II,
demonstrating that the cellular fluorescence was due to
intracellular ROS production (Figure 5c). Importantly,
hydro-Cy3 caused no cellular toxicity under these experi-
mental conditions (100 mm) and also at higher concentrations
(1 mm), as determined by the trypan blue cell viability assay
(see the Supporting Information for details).
We also investigated the ability of hydro-Cy3 to image
ROS production from live, explanted mouse aortas, after
lipopolysaccharide (LPS) stimulation. The detection of ROS
in live tissue provides information about ROS production in a
physiologic environment that closely resembles in vivo con-
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excitation laser and a 790 nm emission filter. Figure 6a shows
In summary, the hydrocyanines are a new family of ROS
sensors, synthesized by NaBH4 reduction of commercially
available cyanine dyes, which have excellent stability to
autoxidation, tunable emission wavelengths, and nanomolar
sensitivity to ROS. Furthermore, the hydrocyanines can
image superoxide and the hydroxyl radical in cell culture,
tissue explants, and for the first time in vivo. On the basis of
these results, we anticipate that the hydrocyanines will have
numerous applications as research tools and medical diag-
nostics.
a representative fluorescent image of mice treated with LPS
and hydro-Cy7 versus those treated with just saline and
hydro-Cy7, and demonstrates that the LPS-treated mice had
greater fluorescence intensity than saline-treated mice. The
mean fluorescent intensity from the abdominal area from the
three sets of mice was also quantified by using Image-Pro
software (Figure 6b), and demonstrates that the LPS-treated
mice had approximately twofold higher fluorescence intensity
than the saline group. Taken together, these results demon-
strate that hydro-Cy7 can image ROS production in vivo.
Experimental Section
Representative procedure for the synthesis of hydrocyanines: Cy3
(100 mg, 0.2 mmol) was dissolved in methanol (5 mL), placed in a
four-drum vial, and covered with aluminum foil. NaBH4 (3 mg,
0.08 mmol in 0.5 mL methanol) was added dropwise to the purple Cy3
solution, which was stirred for 10 min to generate a colorless solution.
The reaction mixture was then stirred for an additional 10 min, and
the solvent was removed under reduced pressure. The resulting solid
was dissolved in 10 mL dichloromethane and 5 mL water and
vigorously shaken. The organic layer was extracted with additional
dichloromethane (5 mL ꢁ 2), dried over anhydrous sodium sulfate,
and the solvent was removed under reduced pressure. The reduced
dye thus obtained was used without further purification. See the
Supporting Information for detailed characterization of the products.
Detection of the hydroxyl radical (Figure 3): The hydroxyl radical
was generated in situ by treating hydrogen peroxide with Fe2+ for the
following experiments. To
a solution (1 mL) of hydro-Cy3 in
methanol (78 mm), various quantities of a hydrogen peroxide stock
solution were added to generate hydrogen peroxide concentrations in
the range 2–30 nm. Aqueous Fe2+ (1 mm) was then added to the hydro-
Cy3/H2O2 solution to generate a 200 nm concentration. The resulting
solution was kept at ambient temperature for 5 min, and the
fluorescence intensity was measured (lex/lem = 535/560 nm) against a
reagent blank at the same time. The sensitivity of DHE (lex/lem = 515/
560 nm) and hydro-Cy7 (lex/lem = 735/760 nm) towards the hydroxyl
radical was measured under similar experimental conditions.
Received: October 4, 2008
Published online: December 8, 2008
Keywords: cyanines · fluorescence · hydrocyanines ·
.
reactive oxygen species · superoxide
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