Dynamic-Reversible Photoacoustic Probe for Continuous Ratiometric Sensing and Imaging of Redox Status in Vivo

Article abstract of DOI:10.1021/jacs.9b10353

Here, we report a reversible photoacoustic (PA) probe, BDP-DOH, to image the localized redox state in vivo via monitoring the dynamic changes of the redox couple, superoxide anion (O₂^a￠-) and glutathione (GSH). The probe features a s

Full text of DOI:10.1021/jacs.9b10353

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Communication
Dynamic-Reversible Photoacoustic Probe for Continuous
Ratiometric Sensing and Imaging of Redox Status in Vivo
Judun Zheng, Qin Zeng, Ruijing Zhang, Da Xing, and Tao Zhang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b10353 • Publication Date (Web): 26 Nov 2019
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Dynamic-Reversible
Ratiometric Sensing and Imaging of Redox Status in Vivo
Judun Zheng,†‡ Qin Zeng,†‡ Ruijing Zhang,†‡ Da Xing,*†‡ and Tao Zhang *†‡
Photoacoustic
Probe
for
Continuous
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†MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, Guangdong Provincial Key Laboratory of
Laser Life Science, South China Normal University, Guangzhou 510631, P. R. China
‡College of Biophotonics, South China Normal University, Guangzhou 510631, P. R. China
KEYWORDS. redox status, superoxide anion, glutathione, photoacoustic imaging, boron-dipyrromethene
Supporting Information
microenvironmental species, such as hypoxia,31 pH,32-33 metal
ABSTRACT: Here, we report a reversible photoacoustic (PA)
probe BDP-DOH to image the localized redox state in vivo via
ions (eg. Cu2+, Hg2+, Ca2+),34-37 and some oxidants (eg. NO,
•OH, peroxynitrite ONOO-, H2O2)38-43 and reductants (eg.
monitoring the dynamic changes of the redox couple, superoxide
anion (O2•−) and glutathione (GSH). The probe features a
significant shift in absorption between 680 and 750 nm during
hydrogen sulfide H2S, GSH)44-45. Recently, Tang et al
achieved a favorable step in sensing the redox couple of
H2O2/GSH by containing two molecule photoacoustic probes
within a nanomicelle.46 Even considering the high demand for
the continuous monitoring of redox dynamics, however, there
is still no report of a reversible photoacoustic probe towards
this challenge.
•−
selective oxidization with O2 and the reductive recovery of
GSH, thus showing a reversible responsiveness in photoacoustic
intensity ratio (PA750/PA680) to successfully visualize the redox
status of the tissue in situ. This redox probe with specific and
sensitive photoacoustic response provides a potential technical
tool towards understanding pathological formation processes.
Herein, we first report a single-molecular reversible PA
probe, BDP-DOH, based on
a
near-infrared boron
dipyrromethene (BODIPY) dye to visualize the localized
redox status in vivo via dynamically and continuously
monitoring the redox cycle of O2 and GSH (Scheme 1).
Homeostasis of cellular redox status plays critical role in
various physiological and pathological processes.1-2 Among
these, reactive oxygen species (ROS) are recognized as the
major oxidants that are involved in vital cellular signaling
pathways, ranging from pathways involved in immunity to
metabolic processes.3-4 Given the mechanism of cellular self-
protection, on the other hand, various biologic antioxidants or
reductants, and in which glutathione (GSH) is the most
abundant reductant containing nonprotein thiol,5-6 effectively
scavenge free ROS to prevent oxidative stress.7 Since
superoxide anion (O2•−) is the precursor of other ROS,8-9 such
as hydrogen peroxide (H2O2) and hydroxyl radical (•OH),
dynamically and sensitively evaluating the localized redox
cycle of O2•− and GSH is of great importance.
•−
Upon encountering the O2•−, the ortho-phenolic hydroxyl
groups on BODIPY can be readily and selectively oxidized, as
a result of which the absorbance at 680 and 750 nm were
decreased and increased, respectively, leading to a large
PA750/PA680 ratio. The BDP-DOH in oxidized state can be
reduced back by GSH, leading to the recovery of the
photoacoustic ratio. Using the ratiometric PA750/PA680 as a
detection signal allows for reversibly monitoring and imaging
of O2•− and GSH in vitro and in vivo, realizing the mapping of
the localized redox status.
To date, fluorescent probes have made significant progress
in monitoring redox changes.10-16 Specifically, several
reversibly fluorescent sensors based on organic molecules
have been reported to dynamically monitor the redox
•−
couples,17-24 including the O2 and GSH,25-26 which greatly
broadens the redox detection toolkit. However, a major
challenge associated with these fluorescent probes lies in the
poor resolution beyond an imaging depth of 1 mm owing to
the limited tissue penetration, seriously impeding the
exploration of the localized redox state. Alternatively,
photoacoustic (PA) molecular imaging is an emerging
noninvasive technique in bioimaging and sensing that
possesses strong optical absorption contrast and high
ultrasonic resolution of deep tissue.27-30 Given this, some PA
sensors have recently enabled the detection and imaging of
Scheme 1. Schematic of the reversible photoacoustic sensing of
O2•− and GSH.
To obtain a reversible photoacoustic probe for tracking the
dynamic changes of redox status in situ, BDP-OH and BDP-
DOH were designed and synthesized for sensing the redox
cycle of O2•−/GSH (Scheme 2). The absorption spectra of
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BDP-OH and BDP-DOH were first recorded, showing a large
extinction coefficient centered at 640 nm (3.7  104 M−1
cm−1) and 680 nm (4.2  104 M−1 cm−1), respectively (Figure
S1). In view of the tissue penetration of excitation light, BDP-
DOH was rationally chosen as the latent reversible probe.
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Photoacoustic tomography is an advanced imaging modality
based on the detection of ultrasonic waves generated by the
nonradiative relaxation of a light-absorbing contrast under
pulsed laser irradiation, where the photoacoustic amplitude has
been described proportional to the absorption coefficient and
photothermal conversion efficiency (Figure 1a).47-48 In the
presence of O2•−, BDP-DOH showed a significant redshift
from 680 nm to 750 nm in absorption resulting from the
increased conjugation along with the oxidation of
pyrocatechol moieties (Figure 1b). The absorption of as-
oxidized BDP-DOH then demonstrated a blueshift recovery
through reduction with GSH. The reaction between BDP-
Figure 1. Representation of the photoacoustic effect and
absorbance behaviors of BDP-DOH. a). Schematic illustration of
photoacoustic effect generation. b). Absorbance changes of BDP-
DOH in DMSO solution (black) upon treated with O2
(potassium superoxide KO2, red) and subsequent addition of GSH
(blue).
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•−
The reversibility of the probe was further tested to ensure
the dynamic detection of the redox cycle. As shown in Figure
2e, the O2•−-induced increase of PA750/PA680 could be
recovered to nearly the original value after addition of GSH.
Significantly, this reversible redox cycle can be repeated more
than three times with different concentration of O2•− and GSH
in turn. The results together with its satisfactory stability in
recycling validated BDP-DOH as a reliable redox indicator
•−
1
DOH and O2 was also validated by H spectrometry (Figure
S2), where variations of chemical shift peaks between 9.0-10.0
ppm were in accordance with the previous oxidation
mechanism.23
•−
(Figure S6). Next, the selectivity of BDP-DOH towards O2
was firstly studied via recording the photoacoustic responses
and absorption changes after treatment of other suspected
1
analytes, including H2O2, O2, hypochlorite (ClO-), ONOO-,
Fe3+ and Cu2+ (Figure 2f and S7). The results revealed that
•−
only addition of O2 caused a prominent change in
photoacoustic signals, whereas the variations with the other
analytes were negligible. On the flip side, we found that the
sensing oxidation was liable to be reversed by GSH (Figure
S8). Detailed investigations on the selectivity of the probe
over the suspected major interfering analytes H2O2 and
cysteine, revealed that the sensing reaction should be
dominated by either thermodynamics or kinetics (Figure S9
and S10). Taken together, BDP-DOH could act as a reliable
photoacoustic indicator for ratiometrically monitoring the
redox dynamics.
Scheme 2. Synthetic scheme of BDP-DOH and BDP-OH.
•−
Having shown that BDP-DOH can photoacoustically
respond to O2•−/GSH in vitro, we next intended to assess the
capability of our probe in sensing the cellular endogenous
O2•−/GSH. After being with BDP-DOH, the cell pellets were
loaded into a homemade round sample slot for photoacoustic
imaging. As shown in Figure S11, the ratio of PA750/PA680
showed an significant changes in the EMT6 cells after treated
with phorbol myristate acetate (PMA) and -lipoic acid
To concretely examine the response of BDP-DOH to O2
,
the absorption changes towards various concentration of the
analytes were measured (Figure 2a). We found a linear
relationship between the ratio of Abs750/Abs680 and O2•− from 2
to 10 μM (Figure S3). Afterwards, different contents of GSH
were introduced, and
a high responsiveness between
Abs750/Abs680 and GSH was recorded (Figure 2b and Figure
S4).
(LPA), which was respectively validated as the stimulator for
We then explored its photoacoustic sensing behaviors in
details. As shown in Figure 2c, the BDP-DOH exhibited a
•−49-50
the generation of cellular O2
and GSH51-52 (Figure S12).
We also found that BDP-DOH exhibited reversible on-off-on
high photoacoustic signal at 680 nm (PA680) and a low signal
•−
type of fluorescence response after treatment with O2 and
•−
at 750 nm (PA750). Upon the addition of O2
, the
GSH in turn. The dynamic cellular redox changes controlled
by PMA and LPA were successfully monitored by using our
probe via confocal laser scanning microscopy (Figure S13).
Those assays strongly indicated that the photoacoustic ratio
changes could be attributed to the dynamic alterations of
photoacoustic ratio (PA750/PA680) showed a 9.84-fold increase,
•−
exhibiting a good linearity between the PA750/PA680 and O2
concentration in the range of 0–4 × 10−6 M with a high
correlation coefficient of 0.98, and the detection limit was
measured to be 0.03 × 10−6 M. Following with the treatment of
GSH, the value of PA750/PA680 decreased (Figure 2d), which
was consistent with the responses in absorption. Point
scanning exhibited the clear variations in the amplitudes of
PA680 and PA750 during the detection (Figure S5). These results
confirmed the excellent ratiometrically photoacoustic sensing
of O2•− and GSH with BDP-DOH.
•−
intracellular O2 and GSH levels, inferring the feasibility of
our reversible probe in visualizing the redox state of living
systems.
Our final focus was to validate the potential of using BDP-
DOH to photoacoustically map the in vivo localized redox
status. Considering the specificity of microenvironmental
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redox in tumors, as a proof of concept, we herein built a
found to maintain a relatively high level over 4~6 h, which
was selected as the optimal detection window. Then,
photoacoustic imaging of three groups of mice pretreated via
intratumorally injecting with physiological saline, PMA and
PMA+LPA, respectively, was conducted after intravenously
loaded BDP-DOH/LP. Ultrasound images were also provided to
show the skin and tumor boundaries in the overlaid ratiometric
PA images. As shown in Figure 3c and 3d, the photoacoustic
images of those PMA-treated mice in the channel of 750 nm and
680 nm were significantly brightened and weakened, respectively,
resulting in a 6.3-fold enhancement of the PA750/PA680 (Figure 3e
and Figure S20). By contrast, the value of PA750/PA680 in the
tumor treated with LPA became lower, indicating the increase of
the endogenous GSH. The dynamic changes of O2•−/GSH were
then further evaluated via ratiometric photoacoustic imaging in
real-time. As shown in Figure 3f and 3g, the PA750/PA680 ratio of
the tumor loaded with BDP-DOH/LP reached maximum at 4 h
postinjection but gradually decreased owning to the cellular self-
protection over time. These results indicated that BDP-DOH
could be used as a reliable indicator for detecting the dynamic
changes of localized redox status with high resolution.
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tumor-bearing mouse model to demonstrate the capability of
BDP-DOH in monitoring localized redox dynamics. Owing to
the limited aqueous solubility and short tumor retention of the
molecular probe (Figure S14), the nanoagent BDP-DOH/LP
was constructed by self-assembly of BDP-DOH with an
amphiphilic
phosphoethanolamine-N-[carboxyl poly(ethylene glycol)-
2000] (DSPE-PEG2000-COOH), to enhance the
lipid
(LP),
1,2-distearoyl-sn-glycero-3-
biocompatibility of BDP-DOH and deliver it into the tumor
more efficiently via the enhanced permeability and retention
(EPR) effect (Figure 3a).53-54 The obtained BDP-DOH/LP was
assessed by transmission electron microscopy and dynamic
light scattering, exhibiting good dispersal with an average
diameter of  50 nm (Figure S15, S16 and S17). BDP-
DOH/LP showed characteristic absorption of BDP-DOH (680
nm), and the capacity of BDP-DOH in this nanomicelle was
determined as 72.1% (w/w) (Figure S18). The photoacoustic
responsive behavior of BDP-DOH/LP towards O2•− was found
to agree well with that of BDP-DOH (Figure 3b). These
characterizations demonstrated that the fabricated BDP-
DOH/LP should be suitable for localized redox monitoring.
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Figure 3. Photoaoustic imaging of the redox cycle of O2•−/GSH in
vivo. a). Formation of BDP-DOH/LP. b). Photoacoustic signals
of BDP-DOH/LP at 680 (green) and 750 (red) nm before and
after treated with O2•−. c). Photograph of the tumor-bearing mouse
and the red dashed frame is the photoacoustic (PA) scanning area.
d). Photoacoustic and ultrasound (gray) images of the tumor in
different treatment groups with intravenous injection of BDP-
DOH/LP (power density: 0.5 w/cm2). e). Corresponding
quantified photoacoustic intensity at 680 (green) and 750 (red) nm
from d). f). Real-time photoacoustic and ultrasound (gray) images
of the redox status in mice pretreated with PMA. g). Quantitative
statistics for the recorded data of f).
Figure 2. BDP-DOH sensing O2•−/GSH. Absorbance changes of
BDP-DOH upon titration with a). O2 (0 to 20 M) and
•−
following titration with b). GSH (0 to 50 M). The PA750/PA680
intensity of BDP-DOH against with different concentrations of c).
•−
O2 and subsequently adding with d). GSH. e). The redox cycles
of 10 M BDP-DOH by the addition of O2•− (20, 5 and 1 M) and
following GSH (50, 10 and 5 M) in PBS buffer solution (pH =
7.4). f). The PA750/PA680 intensity towards O2•− and other analytes.
Insert: photoimages of BDP-DOH solution in the presence of the
analytes.
Thus application of BDP-DOH/LP for photoacoustic
imaging of O2•−/GSH in vivo was then examined. First, after
intravenous injection of BDP-DOH/LP, photoacoustic images
at the time points of 1, 2, 4, 6, 12 and 24 h postinjection were
captured under the pulsed laser at 690 nm (Figure S19). The
intensity of the undisturbed PA690 signal in tumor region was
In summary, we have reported a reversible photoacoustic
molecular probe BDP-DOH to achieve map the localized
redox homeostasis via dynamically monitoring the redox cycle
of the superoxide anion (O2•−) and glutathione (GSH). The
detection principle was based on the reversible absorption shift
between 680 nm and 750 nm of the probe du6ring selectively
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sensing O2•−/GSH, which leads to the changes of the
photoacoustic signal ratio (PA750/PA680). As proof of concept,
BDP-DOH was successfully applied to photoacousically
visualize O2•−/GSH in tumor microenvironment with the
delivery of a nanomicelle. The photoacoustic probe with a
specific and reversible redox response provides a potential
candidate for understanding various redox-related pathological
events.
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ASSOCIATED CONTENT
Supporting Information
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ratiometric electrochemical DNA sensor for monitoring intracellular redox
homeostasis. Chem. Commun. 2017, 53, 6215-6218.
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Zhu, L.; Lee, C. S.; Wong, W. T.; Law, G. L.; Wong, K. L., A smart "off-
on" gate for the in situ detection of hydrogen sulphide with Cu(ii)-assisted
europium emission. Chem. Sci. 2016, 7, 2151-2156.
1
The H NMR spectra, ESI-MS, DLS, TEM characterization and
Liang, Z.; Tsoi, T. H.; Chan, C. F.; Dai, L.; Wu, Y.; Du, G.;
UV−vis absorption of BDP-DOH or BDP-DOH/LP, the cell
viabilities and control experiments. This material is available free
of charge via the Internet at http://pubs.acs.org.
17.
Yu, F.; Li, P.; Wang, B.; Han, K., Reversible Near-Infrared
AUTHOR INFORMATION
Corresponding Author
Fluorescent Probe Introducing Tellurium to Mimetic Glutathione
Peroxidase for Monitoring the Redox Cycles between Peroxynitrite and
Glutathione in Vivo. J. Am. Chem. Soc. 2013, 135, 7674-7680.
Prof. Da Xing, E-mail: xingda@scnu.edu.cn
Prof. Tao Zhang, E-mail: zt@scnu.edu.cn
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Koide, Y.; Kawaguchi, M.; Urano, Y.; Hanaoka, K.; Komatsu,
T.; Abo, M.; Terai, T.; Nagano, T., A reversible near-infrared fluorescence
probe for reactive oxygen species based on Te-rhodamine. Chem.
Commun. 2012, 48, 3091-3.
Notes
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Zheng, J.; Wu, Y.; Xing, D.; Zhang, T., Synchronous detection
The authors declare no competing financial interests.
of glutathione/hydrogen peroxide for monitoring redox status in vivo with
a ratiometric upconverting nanoprobe. Nano Res. 2019, 12, 931-938.
20.
reversible fluorescent probe modulated by selenium for monitoring
peroxynitrite and imaging in living cells. J. Am. Chem. Soc. 2011, 133,
11030-3.
ACKNOWLEDGMENT
Yu, F.; Li, P.; Li, G.; Zhao, G.; Chu, T.; Han, K., A near-IR
This research was supported by the National Natural Science
Foundation of China (21771065 and 81630046), the Natural
Science Foundation of Guangdong Province, China
(2017A020215088), the Science and Technology Planning Project
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Kaur, A.; Kolanowski, J. L.; New, E. J., Reversible Fluorescent
Probes for Biological Redox States. Angew. Chem., Int. Ed. 2016, 55,
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Kong, F.; Tang, B., A near-infrared reversible fluorescent probe for real-
time imaging of redox status changes in vivo. Chem. Sci. 2013, 4, 1079.
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for Imaging Reversible Redox Cycles in Living Cells. J. Am. Chem. Soc.
2007, 129, 3458-3459.
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Liu, B.; Sun, J. Z.; Tang, B. Z., A two-channel responsive fluorescent
probe with AIE characteristics and its application for selective imaging of
superoxide anions in living cells. Chem. Commun. 2017, 53, 1653-1656.
of
Guangdong
Province,
China
(2015B020233016,
Xu, K.; Qiang, M.; Gao, W.; Su, R.; Li, N.; Gao, Y.; Xie, Y.;
2014B020215003), Pearl River Nova Program of Guangzhou,
Guangdong Province, China (201806010189). The Scientific and
Technological Planning Project of Guangzhou, Guangdong
Province, China (201805010002).
Miller, E. W.; Bian, S. X.; Chang, C. J., A Fluorescent Sensor
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Status in Vivo
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