A bioluminescent probe for imaging endogenous hydrogen polysulfides in live cells and a murine model of bacterial infection

Article abstract of DOI:10.1039/c9cc01699f

In this work, we report the first bioluminescent probe BP-PS for detecting H₂S_n with high specificity and sensitivity. Owing to the bioluminescence imaging without requiring an excitation light source, tissue autofluorescence is eliminated and BP-PS shows a high signal-to-noise ratio. Moreover, BP-PS was successfully utilized to visualize endogenous H₂S_n in live cells and a murine model of bacterial infection.

Full text of DOI:10.1039/c9cc01699f

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DOI: 10.1039/C9CC01699F
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A Bioluminescent Probe for Imaging Endogenous Hydrogen
Polysulfides in Live Cells and Murine Model of Bacterial Infection†
Received 00th January 20xx,
Accepted 00th January 20xx
Jun-Bin Li, Qianqian Wang, Hong-Wen Liu, Lin Yuan, and Xiao-Bing Zhang*
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this work, we report the first bioluminescent probe BP-PS for depth, phototoxicity or low photostability of photobleaching,
detecting H with high specificity and sensitivity. Owing to the limiting the in vivo applications.
bioluminescence imaging without requiring an excitation light Bioluminescence imaging is a powerful technique for visualizing
2 n
S
source, tissue autofluorescence is eliminated and BP-PS shows a biological events (such as the change of reactive species levels, the
high signal-to-noise ratio. Moreover, BP-PS was successfully activity of an enzyme, the expression of a gene, or the tumor growth
utilized to visualize endogenous H
model of bacterial infection.
2
S
n
in live cells and murine and metastasis) occurring in live cells or in vivo.21-23 For firefly
luciferase-luciferin system, the firefly luciferase (fLuc) catalyzes
substrate D-luciferin into oxyluciferin to emit a visible light in the
Hydrogen polysulfides (H
2
S
n
, n > 1) are known as reactive sulfur
2+ 24
2
presence of ATP, O , and Mg . Compared with fluorescence
species (RSS), playing prominent roles in biological systems.1
imaging, bioluminescence imaging does not require external
Reactions caused by cystathionine β-synthase (CBS) or
excitation which features its minimal phototoxicity, low
cystarhionine-γ-lyase (CSE) could generate endogenous H
redox form of H S, H work with H S collectively to maintain the
balance of sulfur redox. Though H S has been widely studied as a
gasotransmitter, the functions of H are still poorly understood. In
some physiological activities, it was thought to be mediated by H S,
while it may be actually mediated by H . For example, H S was
originally thought to react with cysteine residues to yield persulfides.
Actually, this reaction is mainly mediated by H
.4 Abnormally
high concentrations of H are found in inflammation, in which
CSE is overexpressed.5 Thus, H
are closely associated with
2
S
n
.2, 3 As
autofluorescence and deep tissue penetration in vivo imaging.25 To
2
2
S
n
2
date, this tool has been developed for bioluminescence imaging of
various analytes. The analytic objects have included metal ions,26
small molecules,27 reactive oxygen/nitrogen species,28-30 and
enzyme,31 etc. With advantages of high signal-to-noise contrast and
excellent biocompatibility, bioluminescence imaging holds great
2
2
S
n
2
2
S
n
2
promise to realize highly sensitive detection of H
2
S
n
in vivo.
2 n
S
However, bioluminescent H
best knowledge.
2 n
S probes are still unavailable, to our
2 n
S
2
S
n
Herein, we presented the first bioluminescent probe BP-PS for
detecting H in vitro and in vivo. We constructed BP-PS by
-responded moiety onto a fLuc substrate.13 BP-PS
installing a H
can be converted to D-luciferin through reaction with H
generating bioluminescence (Scheme 1a). BP-PS showed high
specificity and sensitivity to H . With excellent biocompatibility,
we successfully applied BP-PS to visualize the change of H in
live cells. More importantly, BP-PS was used to visualize
endogenous H in bacterial infection of live mice.
diverse physiological and pathophysiological conditions. And, in
order to better understand its role in biological processes, it is
significant to develop accurate and sensitive tools for sensing and
2 n
S
2 n
S
2 n
S ,
bioimaging of H
2
S
n
in vivo.
analytical methods, such as UV-vis
in
Conventional
2 n
H S
2 n
S
6
spectroscopy, are not sensitive and suitable for detection of H
2
S
n
2 n
S
live cells and in vivo. To date, the use of fluorescent probe as a
detection and imaging tool has numerous advantages, including
simplicity, fast analysis, and real-time observation.7-10 Although
2 n
S
BP-PS was successfully synthesized following the procedure
showing in Scheme 1b and the structures of the compounds were
verified by MS and NMR. The spectroscopic bioluminescence
various fluorescent H
image
in live cells or tissues,11-20 they suffer from
autofluorescence interference, limited exciting light penetration
2 n
S probes have been reported and applied to
2 n
H S
2 n
emission and absorption characteristics of the probe BP-PS for H S
were carried out in PBS. As we expected, BP-PS itself only
exhibited very weak bioluminescence signal. However a significant
Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of enhancement of bioluminescence intensity was observed after
Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical
Engineering, Hunan University, Changsha 410082, China. E-mail:
xbzhang@hnu.edu.cn
adding 100 μM Na
absorption wavelength of BP-PS red-shifted to 345 nm for blending
2 4
S to BP-PS solution (Fig. 1a). And the
with Na
2
S
4
in the UV-vis spectrum as shown in Fig. 1b. The
†
Electronic Supplementary Information (ESI) available: Synthesis, experimental
details and supplementary figures. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
a
View Article Online
DOI: 10.1039/C9CC01699F
O
S
N
N
S
COOH
H2Sn
HO
S
N
N
S
COOH
a
O
S
O
BP-PS
D-luciferin
0.2
0.4
0.6
0.8
1.0
2+
8
2
ATP, O2, Mg2+
Luciferase ATP, O , Mg
2
Radiance (× 10 p/sec/cm /sr)
Luciferase
Bioluminescence
Bioluminescence
b
COOH
S
b
COOH
SH
Cl
a
b
+
O
HO
S
O
CN
N
1
(yield: 78%)
O
S
COOH
O
S
N
N
S
c
CN
O
S
O
N
O
S
O
2
(yield: 60%)
BP-PS (yield: 50%)
Scheme 1 (a) Strategy for the design of BP-PS for detecting H
procedure of BP-PS. Reagent and reaction conditions: a) NaHCO , H O, 0 °C, 15 min, RT,
h; b) EDCI, DMAP, CH Cl , RT, 6 h; c) D-cysteine hydrochloride, K CO , CH Cl /MeOH
1:1, v/v), RT, 15 min.
2 n
S . (b) Synthesis
3
2
1
2
2
2
3
2
2
(
Fig. 2 (a) Bioluminescence images of 20 μM BP-PS to different Na
.1, 0.2, 0.5, 1, 2, 5, 10, 50, 100, 200 μM). (b) Quantification of bioluminescence signal
spectra were presumably attributed to the release of D-luciferin intensity with different Na2S4 concentrations. Inset of (b): The fitted calibration line
-triggered reaction. We further confirmed our between the total flux and Na concentration.
conjecture by HPLC analysis (Fig. S1, ESI†). The data showed that
2 4
S concentrations (0,
increased bioluminescence intensity and red-shift in absorption
0
caused by Na
2
S
4
2 4
S
BP-PS (HPLC retention time, T
R
=13.2 min) was converted to D-
As we know, selectively recognizing its target analyte is an
1
luciferin (T
R
= 4.0 min). And H NMR spectrometry analysis of the essential feature for a probe. To further evaluate whether BP-PS had
purified compound in mixed solution of BP-PS and Na
corresponded to D-luciferin (Fig. S2, ESI†). These data verified the relevant analytes, we incubated 20 μM BP-PS with H S and other
2
S
4
a much higher selectivity for H2Sn relative to other biologically
2
n
response mechanism of BP-PS to H
To study the sensitivity of BP-PS, we treated BP-PS with we observed high turn-on bioluminescence intensity in the well
different concentrations of Na (0-200 μM) in black 96-well plate, containing of H S and no obvious increase in the bioluminescence
then added fLuc solution into every set of solution. As shown in Fig. intensity in other wells. It indicated that our probe BP-PS had an
2
S
n
.
potentially interfering species in 96-well plate. As shown in Fig. 3,
2 4
S
2
n
2
(
, with addition of 100 μM Na
total flux) was 7.3-fold higher than that without Na
although high concentration of Na may slightly affect the activity
of fLuc (Fig. S3, ESI†). Notably, obvious bioluminescence intensity
was still observed with Na concentration as low as 0.1 μM,
indicating the high sensitivity of BP-PS for bioluminescence
detection of Na . The bioluminescence intensity showed a good
linear relationship with the Na concentration in the range of 0.1 to
μM (Fig. 2b). The detection limit was calculated to be 30 nM
based on 3σ rule). The concentration of endogenous H has been
reported to be in the nanomolar levels.32 Therefore, BP-PS is
promising to visualize endogenous H in living systems.
2
S
4
, the bioluminescence intensity excellent selectivity for H S and possibility of detecting H S in real
2
n
2 n
2 4
S addition, biological samples.
2 4
S
a
2 4
S
0.2
0.4
0.6
0.8
1.0
2 4
S
8
2
Radiance (× 10 p/sec/cm /sr)
2 4
S
2
(
b
2 n
S
2 n
S
a
b
Fig. 3 (a) Bioluminescence images of 20 μM BP-PS with different species (2-14:
2
−
2
00 μM): (1) BP-PS only, (2) NaClO, (3) H
2
O
2
, (4) H
, (10) ascorbic acid, (11) Cys, (12) Hcy, (13) GSSG, (14) S
15) GSH (5 mM), (16) Na (100 μM) and (17) Na (100 μM). (b)
2 3
S, (5) HNO, (6) NO, (7) SO ,
Fig. 1 (a) Bioluminescence spectra of 20 μM BP-PS in PBS with 10 mM MgCl
mM ATP and fLuc before (black) and after (red) Na (100 μM) treatment. (b)
Absorbance of 20 μM BP-PS in PBS before (black) and after (red) Na (100 μM)
2
, 2
-
•−
2 3
O
2-,
(8) ONOO , (9) O
2
2 4
S
(
S
2 2
2 4
S
2 4
S
Quantification of bioluminescence signal intensity of BP-PS with different species.
2 4
treatment for 30 min. Inset of (a): A photo of BP-PS (left) and BP-PS after Na S
treatment (right) after addition of fLuc solution.
2
| J. Name., 2012, 00, 1-3
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bioluminescence signal exhibits no differences (VFiewigA.rtiScl5e aO,nlinbe,
DOI: 10.1039/C9CC01699F
ESI†). All these demonstrated that BP-PS had the ability to
a
1
15 30 45 60 90 120 150 180 / min
PAG +
BP-PS
monitor H
We further applied BP-PS for bioluminescence imaging H
in vivo. As an inflammatory mediator, LPS can induce the CSE
overexpression and H could be generated enzymatically by
2 n
S levels in live cells.
2 n
S
BP-PS
2 n
S
Na S +
2
4
BP-PS
CSE. After administration of LPS for 12h, fLuc and BP-PS
were locally injected (Fig. 5a). Then we measured the
bioluminescence signal and observed an obvious enhancement
in LPS-stimulated inflammation tissues in contrast with the
normal tissues (Fig. 5b). Previous studies suggested that
bacterial infection can cause severe inflammation.36
Furthermore, a murine model of bacterial infection was
established by subcutaneous injection of E. coli into right legs
of mice (Fig. 5d). And after 12 h, we found that the tissues
infected with E. coli displayed an obviously enhanced
bioluminescence signal in contrast with the normal tissues (Fig.
2.0
4.0
6.0
8.0
5
2
Radiance (× 10 p/sec/cm /sr)
b
5e). Moreover, when the inflammation tissues were first treated
with NEM (a RSS scavenger), the bioluminescence signal was
significantly weakened (Fig. S6, ESI†). Taken together, these
results proved that probe BP-PS could be used to visualize
2 n
endogenous H S in live inflamed and bacterial infected mice.
a
b
c 2.0
12 h
6
.0
LPS BP-PS+fLuc
Fig. 4 (a) Bioluminescence images of MDA-MB-231-luc cells preincubated with PAG
100 μM) for 30 min and then with 20 μM BP-PS (first row), incubated with 20 μM BP-
PS (second row), preincubated with Na (100 μM) and then with 20 μM BP-PS (third
row). Bioluminescence images were captured at 1, 15, 30, 45, 60, 90, 120, 150, and 180
min. (b) bioluminescence signal intensity quantification of the bioluminescence images.
CCD
Camera
4.0
(
1
.0
×107
2 4
S
2
.0
Radiance
(p/sec/cm /sr)
0
.0
2
Inflamed Normal
Inflamed Normal
d
e
f
It is known that the biocompatibility is a precondition for
12 h
E. coli BP-PS+fLuc
2.0
2 n
probe BP-PS to monitor H S in living systems. MTT assays
6
4
.0
.0
were conducted to assess its potential cytotoxicity. The data in
Fig. S4 (ESI†) showed that the cells viability had no marked
change even incubating the cells with high concentration of
BP-PS (200 μM) for 24 and 48 h. These results indicated that at
the tested concentrations, BP-PS possesses no significant
cytotoxicity. Then, we used BP-PS to monitor the changes of
CCD
Camera
107
×
1
.0
2
.0
Radiance
(p/sec/cm /sr)
0
.0
2
Infected Normal
Infected Normal
Fig. 5 (a) LPS was injected into right hind leg of each mouse. 12 h later, the mice were
2 n
H S in the cells. Hydrogen polysulfide-derivatives are known locally injected with fLuc and BP-PS. (b) Bioluminescence images of mice pre- (left) and
to be generated from L-cystine by CSE.2, 3 In addition, it is
post-injection (right) of fLuc and BP-PS. (c) Ratio of bioluminescence emission intensity
at normal and inflamed areas in b (right). (d) 100 μL of E. coli (5×106 CFU mL ) was
injected into right hind leg of each mouse. 12 h later, the mice were locally injected
with fLuc and BP-PS. (e) Bioluminescence images of mice pre- (left) and post-injection
-1
observed that CSE was expressed in MDA-MB-231 cells.33, 34
2 4
We treated the cells with PBS, Na S
or PAG (CSE inhibitor),35
respectively. Then we added the same doses of BP-PS into the (right) of fLuc and BP-PS. (f) Ratio of bioluminescence emission intensity at normal and
wells. The plates were imaged for 180 min. As shown in Fig. bacterial infected areas in e (right).
4
a, we found that the cells incubated with BP-PS exhibited
obvious light production. And the bioluminescence signal
increased with the incubation time increasing from 1 to 180 for detecting H S . Upon incorporating a H S sensitive moiety onto
In conclusion, we describe the first bioluminescent probe BP-PS
2
n
2 n
min. The results indicated that BP-PS was potential to detect of firefly luciferin, BP-PS is converted to free D-luciferin after reaction
endogenous H in live cells. For further confirmation, cells with H S , resulting in a bioluminescence response in the presence
were preincubated with Na
addition of Na , a further increase in the bioluminescence autofluorescence is eliminated and BP-PS was effectively applied to
intensity was measured compared to that of adding BP-PS only. the detection and imaging of H S at nanomolar concentration with
2 n
S
2
n
2 4
S and PAG, respectively. With of fLuc. Owing to the bioluminescence without excitation light,
2 4
S
2
n
It can be explained by the increased level of H
increased bioluminescence intensity. With addition of PAG, biocompatibility, BP-PS allowed for bioluminescence imaging of
reduction of generation leads to decreased endogenous H S in live cells and murine model of inflammation
2 n
S resulted in high signal-to-noise ratio. Additionally, with excellent
2 n
H S
2
n
bioluminescence intensity (Fig. 4b). Again, when cells were induced by LPS and bacterial infection. With these results, we
treated with the same doses of PAG and D-luciferin, believe that BP-PS will be a powerful and promising tool for finding
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the biological functions of H
2
S
n
in living systems.
22.
S. Iwano, M. Sugiyama, H. Hama, A. Watakabe, N.
View Article Online
Hasegawa, T. Kuchimaru, K. Z. TanaDkOaI,: M10..1T0a3k9a/Ch9aCshCi0, 1Y6.99F
Ishida and J. Hata, Science, 2018, 359, 935-939.
J. A. Prescher and C. H. Contag, Curr. Opin. Chem. Biol.,
This work was supported by the NSFC (Grants 21890744,
1521063, J1210040), and the science and technology project of
2
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Hunan Province (2016WK2002, 2016RS2009).
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S. M. Marques and J. C. Esteves da Silva, IUBMB life, 2009,
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Conflicts of interest
L. Mezzanotte, M. van‘t Root, H. Karatas, E. A. Goun and
C. W. Löwik, Trends Biotechnol., 2017, 35, 640-652.
A. T. Aron, M. C. Heffern, Z. R. Lonergan, M. N. Vander
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