An Optical/Photoacoustic Dual-Modality Probe: Ratiometric in/ex Vivo Imaging for Stimulated H2S Upregulation in Mice

Article abstract of DOI:10.1021/jacs.9b09181

Tracking signaling H₂S in live mice demands responsive imaging with fine tissue imaging depth and low interferences from tissue scattering/autofluorescence and probe concentration. With complementary advantages of fluorescence and photoacoustic (PA) imaging, optical/PA dual-modality imaging was suggested for in/ex vivo H₂S imaging. Therefore, a meso-benzoyloxyltricarboheptamethine cyanine, HS-CyBz, was prepared as the first ratiometric optical/PA dual-modality probe for H₂S, profiting from a keto-enol transition sensing mechanism. Tail intravenous injection of this probe leads to probe accumulation in the liver of mice, and the endogenous H₂S upregulation triggered by S-adenosyl-l-methionine has been verified by ratiometric optical/PA imaging, suggesting the promising potential of this ratiometric dual-modality imaging.

Full text of DOI:10.1021/jacs.9b09181

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Communication
An Optical/Photoacoustic Dual Modality Probe: Ratiometric
in/ex Vivo Imaging for Stimulated H2S Upregulation in Mice
Zhongyan Chen, Xueling Mu, Zhong Han, Shiping Yang,
Changli Zhang, Zijian Guo, Yang Bai, and Weijiang He
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b09181 • Publication Date (Web): 28 Oct 2019
Downloaded from pubs.acs.org on October 28, 2019
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An Optical/Photoacoustic Dual Modality Probe: Ratiometric in/ex
Vivo Imaging for Stimulated H2S Upregulation in Mice
Zhongyan Chen,† Xueling Mu,# Zhong Han,† Shiping Yang,*,# Changli Zhang,† Zijian Guo,*,†,‡ Yang
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Bai,† and Weijiang He*,†
† State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, School of Chemistry and
Chemical Engineering, Nanjing University, Nanjing 210023, P. R. China
‡ Chemistry and Biomedicine Innovation Center, Nanjing University，Nanjing 210023, P. R. China
# Key Laboratory of Resource Chemistry, Shanghai Normal University, Shanghai, 200234, P. R. China
Supporting Information Placeholder
ratiometric imaging in overcoming these interferences,8 NIR
ratiometric optical/PA dual modality probe is required for
in/ex vivo SSM imaging.
ABSTRACT: Tracking signaling H2S in live mice demands
responsive imaging with fine tissue imaging depth and low
interferences from tissue scattering/autofluorescence and
probe concentration. With complementary advantages of
fluorescence and photoacoustic (PA) imaging, optical/PA dual
modality imaging was suggested for in/ex vivo H2S imaging.
Therefore, a meso-benzoyloxyltricarboheptamethine cyanine,
HS-CyBz, was prepared as the first ratiometric optical/PA dual
modality probe for H2S, profiting from a keto-enol transition
sensing mechanism. Tail intravenous injection of this probe
leads to probe accumulation in the liver of mice, and the
endogenous H2S upregulation triggered by S-adenosyl-L-
methionine has been verified by ratiometric optical/PA
imaging, suggesting the promising potential of this ratiometric
dual modality imaging.
As the third gaseous signaling molecule, H2S is involved in
vasodilation, angiogenesis, anti-oxidation, anti-inflammation,
and central nervous regulation.9 Many fluorescence probes
have been developed for intracellular H2S imaging,10 in vivo
optical or PA imaging for H2S is still not satisfying since most
of them are based on turn-on probes.11 In this study, the first
optical/PA dual modality probe showing ratiometric response
to H2S, HS-CyBz, was developed (Scheme 1), and in/ex vivo
ratiometric optical and PA imaging for endogenous H2S
upregulation stimulated by the cystathionase activator S-
adenosyl-L-methionine (SAM) was realized in mice with this
probe.
Scheme 1. H2S sensing mechanism of HS-CyBz and
chemical structure of HS-CyBz-1.
Many small molecule species including metal cations,
reactive oxygen species, and gasotransmitters are essentially
involved in signaling pathways and pathogenesis.1,2 Beyond
intracellular fluorescence imaging of signaling small molecules
(SSMs),3 in/ex vivo SSMs tracking in animal model especially in
mice is appealed in pathological and clinical study. To evaluate
the signaling behavior and pathology, in/ex vivo SSMs imaging
is required to track SSM fluctuation besides offering SSM
distribution pattern. Therefore imaging technique showing
deep tissue imaging depth and SSM response is demanded. PET
and SPECT are incapable to provide SSMs fluctuation
information due to their irresponsive probe. Although MRI
responsive probes have been reported,4 optical imaging is
more attractive for its quick response and high sensitivity.5
However, the limited tissue penetration depth of fluorescence
(<1 mm) makes optical imaging visualize only superficial SSMs.
This disadvantages can be overcome by photoacoustic (PA)
imaging with imaging depth of several centimeters in tissues,6
although the sensitivity and resolution of PA imaging are lower.
Therefore, fluorescence/PA dual modality imaging, which
possesses the advantages of both modalities, is expected to
improve SSMs imaging efficacy in mice.7 As fluorescence and
enol form
I
H2S
OH
+
I
O
O
N
N
Cl
N
N
O
SH
IBS-
Acid
Cl
HS-CyBz, enol ester
ketone form
O
O
O
N
N
N
N
Cl
HS-CyBz-1
Cy-Ketone
As benzoic esters of a meso-hydroxyltricarboheptamethine
cyanine, both probe HS-CyBz and its analogue without ortho-
iodo substituent, HS-CyBz-1 were prepared in this study. Their
cyanine moiety was adopted to provide NIR absorption and
emission.12 It is expected that nucleophilic substitution of the
ester by HS- would release enolic heptamethine cyanine, which
undergoes keto-enol tautormerization to form Cy-ketone
(Scheme 1). This tautomerization would shift emission and
absorption distinctly, showing ratiometric fluorescent and
photoacoustic response to HS-. Both compounds were
prepared via reacting the corresponding benzoic chloride with
meso-hydroxyltricarboheptamethine cyanine.
PA
signals
can
be
interfered
by
tissue
scattering/autofluorescence and probe concentration, single
channel optical and PA imaging is not reliable for tracking
stimulated SSM fluctuation. Considering the advantage of
Emission spectrum of HS-CyBz exhibits a main emission
band centered at 805 nm and a minor broad band centered at
630 nm (ex, 595 nm). Na2S addition resulted in the drop of
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former band and drastic enhancement of the latter one (Figure
pH 5.0 to pH 8.5 (Figure S13). The colorimetric sensing
behavior suggested that this probe might enable ratiometric
photoacoustic sensing for HS-. PA imaging of HS-CyBz solutions
revealed that the PA signal upon excitation at 825 nm (isobestic
point) was weak and stable upon HS- titration, while the signal
upon excitation at 775 nm decreased distinctly in the process
(Figure 2c), and the ratio PA825/PA775 increased almost linearly
from ~0.2 to ~0.7 with [HS-]total increasing from 0 to 1 mM
(Figure S12c). Moreover, other tested biochemical species did
not induce obvious change of PA825/PA775, and the presence of
biothiols such as GSH, Hcy and Cys did not interfere with the
HS--induced PA825/PA775 ratio enhancement (Figures 2b and
S12b), showing the ratiometric photoacoustic sensing ability
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1a). The large hypsochromic emission shift of ~175 nm and
well separated dual emission bands indicated the excellent
ratiometric response behavior of HS-CyBz toward H2S. A linear
enhancement of emission ratio (F630/F805) was observed upon
Na2S titration from 0 to 1 mM (Figure S7), and the H2S detection
limit of this probe was determined to be 0.5 μM. This showed
the potential of HS-CyBz for endogenous H2S detection (10-
600 M in human brain, 10-100 M in human blood10a,13). HS-
CyBz-1 possesses a similar emission spectrum. Temporal
tracking of ratio F630/F805 after Na2S addition revealed that HS-
CyBz reacted with HS- rapidly and attained equilibrium in 10
min, while HS-CyBz-1 reacted with HS- much more slowly and
reaction equilibrium could not be attained even after 0.5 h.
Moreover, the HS--induced ratio enhancement factor of HS-
CyBz is much higher than that of HS-CyBz-1 (Figure S8c).
Therefore HS-CyBz is more suitable for H2S sensing due to its
high sensitivity and rapid response, and the electron
withdrawing iodo group favoring ester cleavage via HS-
nucleophilic attacking might be the origin.
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for H2S.
0.5
(a)
1: probe only; 2-3: Na2S, S2n- (300 μM);
4-22: K+, Ca2+, Na+, Mg2+, Ac-, SCN-, NO3- , NO2- ,
H2PO4- , SO24-, SO23-, S2O32-, CO32-, ClO-, H2O2,
(b)
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1: probe only; 2-3: Na2S, S2n-(300 μM);
0.4
0.3
0.2
0.1
0.0
4-22: K+, Ca2+, Na+, Mg2+, Ac-, SCN-, NO3- ,
NO2- , H2PO4- , SO24-, SO23-, S2O32-, CO32-, ClO-,
H2O2, EtNH2,EtOH, Cys, GSH (1 mM);
23: Hcy (200 μM); 24: CE (0.5 U/mL)
EtNH2, EtOH, Cys, GSH (1 mM);
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23: Hcy (200 μM); 24: CE (0.5 U/mL)
4
2
Absorption spectrum of HS-CyBz shows a sharp absorption
band at 775 nm ( ~ 1.8105 M-1cm-1) and a shoulder band (708
nm). Probe reaction with Na2S at pH 7.4 led to the distinct
decrease of the sharp band and a minor increase at 850 nm,
resulting in an isobestic point at 825 nm (Figures 1b, S8b and
S9). This colorimetric sensing behavior suggested that this
0
probe might be a ratiometric PA probe for HS-.
2.0
4
3
2
1
0
Figure 2. Emission ratio F630/F805 (a) and PA ratio PA825/PA775
(b) of HS-CyBz (10 μΜ) in HEPES buffer in the presence of
different analytes. (c) PA image of probe (10 M) solutions
upon Na2S titration (dual channel mode: ex 775 and 825 nm).
(a)
(b)
20 min
1.5
1.0
0.5
0.0
0 min
Na2S
Na2S
0 min
0 min
Na2S
20 min
20 min
Na2S
20 min
0 min
650
700
750
800
850
500
600
700
800
900
Wavelength (nm)
Wavelength (nm)
Figure 1. Emission (a, ex 595 nm) and absorption (b) spectra
of HS-CyBz (10 μΜ) in HEPES buffer (pH 7.4) determined after
the addition of Na2S (1 mM).
ESI mass determination of HS-CyBz solution mixed with
Na2S displayed a signal of m/z 493.25 in positive mode
spectrum and a signal of m/z 262.80 in negative mode
spectrum. The two signals were assigned as [Cy-ketone+H]+
and [IBS-Acid-H]-, respectively (Figure S10). Cy-Ketone has
been separated from the reaction mixture in a medium yield.
All these indicated that HS- nucleophilic substitution of benzoic
ester
would
release
enolic
meso-
hydroxyltricarboheptamethine cyanine and trigger keto-enol
transition, which is responsible for the HS--induced
spectroscopic response. The quantum yield of HS-CyBz and Cy-
Ketone were determined as 0.02 and 0.17. The emission ratio
F630/F805 for HS-CyBz increased from 0.3 to 54.9 (183-fold)
after 100 equiv Na2S being added.
Investigating sensing behavior of HS-CyBz for biochemical
species of interest revealed that only Na2S led to a distinct
enhancement of emission ratio F630/F805 (~32-fold, 30 equiv
HS-), while other species such as cations (K+, Na+, Ca2+, Mg2+, 1
Figure 3. In vivo ratiometric optical (a-e, Ch 1: 62020 nm, Ch
2: 79020 nm, ex, 560 nm) and PA (f-j, Ch 3: 775 nm, Ch 4: 825
nm) imaging for H2S in mice injected subcutaneously with HS-
CyBz (P, 100 μL, 20 μM) in region of interests (A, B on the hind
limbs; C, D on the back). (a, c) Images of probe-injected mouse;
(b, d) images of mouse in (a, c) 30 min post saline (ROI A) and
Na2S (ROI B) injection (100 L, 1 mM); (e) normalized F620/F790
of ROIs A and B determined in (a-d). Scale bar: 1 cm. (f, h) PA
images of probe-injected mouse; (g, i) PA images of mouse in (f,
h) 30 mins post injection with saline (ROI C) and Na2S (ROI D);
(j) PA825/PA775 ratios for ROIs C and D. Scale bar: 3 mm.
mM), anions (Sn2-, 300 M; Ac-, HCO3 , NO3 , NO2 , SO42-, SO3
,
-
-
-
2-
H2PO4 -, SCN- , S2O32-, 1 mM), reactive oxygen species (H2O2, ClO-,
1 mM), biothiols (GSH, Cys, 1 mM; Hcy 200 µM), and
carboxylesterase (0.5 U/mL) triggered minor change in
F630/F805. The presence of biothiols GSH, Cys and Hcy showed
no obvious interference with the HS--induced ratio
enhancement (Figure S12a). Moreover, the emission ratio
F630/F805 of HS-CyBz showed no distinct pH-dependence from
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Without commercial available apparatus uniting optical and
12 h post SAM injection. (a) Fluorescence image of mice
injected with SAM and saline. Bandpath 79020 nm, ex 720
nm; (b) ex vivo fluorescence images of isolated livers from
mouse in (a) acquired at 620 (left) and 790 (middle) nm, and
ratiometric image (right), ex 560 nm; Scale bar, 1 cm. (c-d) PA
images of livers from the SAM- and saline-injected mice
recorded by channel 775 nm (c) and channel 825 nm (d), and
the related PA775/PA825 ratios (e). Scale bar, 6 mm.
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PA imaging, ratiometric optical and PA imaging ability of HS-
CyBz for H2S were investigated in mice, respectively. In vivo
ratiometric optical imaging was realized initially in a 6-week-
old mouse with the hind limbs being injected with probe
subcutaneously (ROIs A and B). The image from channel 1
(62020 nm) showed that the weak fluorescence of free probe
in ROIs
A and B was almost concealed by tissue
autofluorescence. The subsequent Na2S injection in ROI B led
to the fluorescence enhancement (~1.60-fold, Figures 3b and
S14a), which was still interfered by tissue autofluorescence.
The control (ROI A) injected with saline showed a less
enhancement factor of ~1.21-fold. Images from Channel 2
(79020 nm) showed no obvious autofluorescence
interference, and Na2S injection led to fluorescence decrease
with a factor of ~0.63-fold, and that for control (ROI A) was
In/ex vivo imaging for endogenous H2S upregulation in mice
was explored, and SAM was utilized to upregulate H2S
expression.14 The mouse was injected intravenously with HS-
CyBz 12 h post SAM intraperitoneal injection. Optical imaging
revealed that the fluorescence in the epigastrium of SAM-
injected mouse was much lower than that in mouse with saline
injection, implying SAM-injection enhanced H2S level distinctly
(Figure 4a). The ex vivo optical imaging of isolated viscera
disclosed the liver accumulation of this probe (Figure S15), and
ex vivo ratiometric image revealed that F620/F790 ratio in the
liver of SAM-injected mouse was distinctly higher than that in
mouse with no SAM injection (Figure 4b), confirming SAM-
injection inducing endogenous H2S enhancement in the liver.
As shown in single channel PA images of the isolated livers
(Figures 4c and 4d), it is difficult to judge whether SAM-
injection trigger H2S upregulation in liver. However, the PA
signal ratio PA825/PA775 was enhanced from 0.59 in the liver of
saline-injected mouse to 0.65 in the liver of SAM-injected
mouse (Figure 4e). This result indicated that SAM injection
caused H2S upregulation, confirming the advantage of dual
channel ratiometric PA imaging.
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~0.94-fold. Therefore, Na2S injection in ROI
B made
fluorescence ratio F620/F790 increase with a factor of ~2.54-
fold, indicating the enhanced HS- concentration. The control in
ROI A showed a minor ratio enhancement factor of ~1.28-fold
(Figure 3e). The Na2S-induced F620/F790 change in ROI B is
more distinct than the single channel signal change (F620). This
result indicated that in vivo ratiometric optical imaging is more
reliable and sensitive than single channel turn-on imaging, and
the interference from autofluorescence and deviated probe
concentration is effectively reduced.
The dual channel photoacoustic imaging was carried out on
the back of mouse with ROIs C and D injected subcutaneously
with the probe. The distinct photoacoustic signals of the probe
were observed in both channels (Figures 3f and 3h, ROIs C and
D) with Channel 4 showing the lower signal. The signal ratio
PA825/PA775 for both ROIs C and D with only probe injection is
similar (~0.51, Figures S14b and 3j). The subsequent Na2S
injection in ROI D made the signal in both channels decrease
(Figures 3g and 3i), which is different from the expected signal
decrease in Channel 3 and stable signal in Channel 4. The local
probe concentration decrease caused by diffusion and the
injection-induced dilution may be the origin. However, ratio
PA825/PA775 of ROI D was enhanced to 0.60 upon Na2S injection
(Figure 3j), while that for control in ROI C with saline injection
was 0.53. This confirmed that the HS- level in ROI D was
enhanced. All results revealed that the ratiometric PA imaging
is more reliable than single channel PA imaging in reducing
interference from the deviated local probe concentration.
In summary, the first ratiometric optical/PA dual modality
probe for H2S was prepared, profiting from the HS--induced
keto-enol
hydroxyltricarboheptamethine
absorption/emission, HS-CyBz
tautormerization
of
meso-
NIR
the
cyanine.
With
enables
optical/photoacoustic dual modality imaging for subcutaneous
H2S in mice, and the ratiometric imaging for both modes favors
the alleviation of interferences from tissue autofluorescence
and the deviated probe concentration. Tail intravenous
injection of probe resulted in accumulation in mice liver, and
the SAM-stimulated endogenous H2S enhancement in the liver
was confirmed by optical/PA dual modality imaging. This study
indicates that ratiometric optical/PA dual modality imaging
was an effective approach to tracking SSM upregulation in live
mice and tissues, and the perspective technique uniting optical
and PA imaging should promote SSM biology in the future.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website. Synthesis, characterization of probe
and experiment details for spectroscopic and imaging study.
AUTHOR INFORMATION
Corresponding Author
*shipingy@shnu.edu.cn
*zguo@nju.edu.cn
*heweij69@nju.edu.cn
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
Figure 4. Optical (a-b) and photoacoustic (c-e) imaging for
mice injected with SAM. HS-CyBz was injected intravenously
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Design principles of spectroscopic probes for biological applications.
Chem. Sci. 2016, 7, 6309-6315.
We thank the National Basic Research Program of China (No.
2015CB856300) and National Natural Science Foundation of
China (No. 21571099, 21977044 and 21731004) for financial
support.
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