Journal of the American Chemical Society
COMMUNICATION
Figure 2. (Left) Fluorescence spectra of 5 μM MMSiR before and after
reaction with various ROS in PBS at pH 7.4, containing 0.1% DMF. HOCl =
NaOCl (final 5 μM) was added and the mixture was stirred at 37 °C. •OH =
ferrous perchlorate (500 μM) and H2O2 (1 mM) were added at room
temperature. ONOOÀ = ONOOÀ (final 5 μM) was added and the mixture
was stirred at 37 °C. O2À• = KO2 (100 μM) was added and the mixture was
stirred at 37 °C for 30 min. H2O2 = H2O2 (100 μM) was added and the
mixture was stirred at 37 °C for 30 min. •NO = NOC7 (5 μM) was added
and the mixture was stirred at 37 °C for 30 min. BG = background. Excitation
wavelength was 620 nm. (Right) Absorbance and fluorescence spectra of
MMSiR before and after reaction with HOCl (0 to 5 μM). Inset shows a
linear response of fluorescence to the concentration of HOCl.
Figure 4. Comparison of white light (WL) and 750 nm fluorescence (FL)
images of unstimulated mouse (left) and the peritonitis model mouse
stimulated with zymosan and PMA (right). 50 μM wsMMSiR in 0.8 mL of
saline and 0.3 μg of PMA in 0.3 mL saline were successively administered by
intraperitoneal injection. Images were obtained just before (0 min) and 60
min after PMA injection. Representative data are shown (n = 3).
from Saccharomyces cerevisiae was added to MMSiR-loaded porcine
neutrophils, we observed that neutrophils engulfed zymosan particles,
followed by enhancement of the fluorescence signal inside the
phagosomes (Figure 3 and supporting movie). During the imaging
of phagocytosis, little fluorescence increase due to excitation laser-
induced autoxidation could be seen, and there was no observed
decrease of the fluorescence signal due to photobleaching after
completion of phagocytosis. Thus, MMSiR is extremely effective
for detecting HOCl generation during phagocytosis and should be
suitable for practical use in vitro.
We next assessed the ability of our probe to visualize HOCl in a
mouse peritonitis model. To obtain a sufficiently high concentration
for efficient in vivo imaging, we prepared the more hydrophilic
derivative wsMMSiR, bearing hydrophilic dicarboxylic acid struc-
ture. C57BL/6 mice were given an i.p. injection of zymosan to
induce neutrophils to invade the peritoneal cavity.24,25 After 4 h, the
mice were anesthetized, and the abdominal fur was removed. Then,
the mice were injected i.p. with wsMMSiR, and 5 min later, PMA
was i.p. injected. The fluorescence measured at the abdomen
increased markedly after PMA injection, and strong fluorescence
was observed at 60 min (Figure 4). In contrast, control, unstimu-
lated mice that were i.p. injected with the probe followed by saline
only (no zymosan or PMA) showed no significant fluorescence
enhancement. Thus, our far-red to NIR probe is applicable for not
only in vitro imaging but also in vivo imaging.
In summary, we have designed and synthesized a novel far-red to
NIR fluorescence probe, MMSiR, based on Si-rhodamine. MMSiR
can sensitively and selectively detect HOCl in real time, and MMSiR
and its oxidized product SMSiR have excellent properties for
biological applications, including pH-independence and tolerance
to autoxidation and photobleaching during excitation laser irradia-
tion. With MMSiR, we conducted real-time imaging of phagocytosis
by means of fluorescence microscopy, and, with the more hydro-
philic derivative wsMMSiR, we achieved noninvasive in vivo ima-
ging of HOCl generation in a mouse peritonitis model. Our probe is
expected to be a useful tool for investigation of the wide range of
biological functions of HOCl.
Figure 3. Fluoresence microscopic imaging of phagocytosis of opso-
nized zymosan by 1 μM MMSiR-loaded porcine neutrophil. Opsonized
zymosan particle is located near the neutrophil (0 s). The neutrophil
engulfs the zymosan (30 s). Phagosytosis is complete (90 s). HOCl
generated in the phagosome was detected with MMSiR (240 s).
W
b
A video of this imaging is available.
abdomen of mice after intraperitoneal administration was 1 order of
magnitude greater with the Si-rhodamine than with the visible light-
emitting O-rhodamine (Figure S1, SI), which clearly indicates the
superiority of MMSiR over HySOx for in vivo use.
Next, we examined the sensitivity and selectivity of MMSiR for
HOCl over other ROS (Figure 2). Although MMSiR was scarcely
fluorescent before detection of HOCl, when it reacted with HOCl, a
large and immediate increase of fluorescence intensity was observed,
owing to the formation of highly fluorescent SMSiR (Absmax/Emmax
=
652/670 nm, ε = 1.2 Â 105, Φfl = 0.31, in PBS), and the fluorescence
intensity change was linearly related to the concentration of HOCl. In
contrast, other ROS (•OH, ONOOÀ, O2À•, •NO, H2O2) produced
almost no fluorescence increase. Thus, MMSiR appears to be highly
sensitive and selective for the detection of HOCl. In addition, we
examined the fluorescence response of MMSiR in the presence of a
HOCl-generating enzymatic system (Figure S3, SI). The fluorescence
was increased, and the increase was suppressed by a MPO inhibitor.
Furthermore, we confirmed the photostability of SMSiR and the pH-
independence of its fluorescence (Figures S4 and S5, SI). Thus,
MMSiR can sensitively and selectively detect HOCl in real time, and
its oxidized product, SMSiR, has excellent properties as a fluorophore.
With these results in hand, we first applied MMSiR to image the
generation of HOCl during phagocytosis by porcine neutrophils,19,23
using a confocal microscope with the widely employed 633 nm
excitation laser and Cy5 filter set. When opsonized zymosan derived
’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis; experimental de-
b
tails; characterization of MMSiR, SMSiR, and wsMMSiR; and
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dx.doi.org/10.1021/ja111470n |J. Am. Chem. Soc. 2011, 133, 5680–5682
Koide, Yuichiro
