Bioluminescence Imaging of Carbon Monoxide in Living Cells and Nude Mice Based on Pd0-Mediated Tsuji-Trost Reaction

Article abstract of DOI:10.1021/acs.analchem.8b01102

Carbon monoxide (CO) is highly toxic and lethal to humans and animals because of its strong affinity for hemoglobin, while this silent killer is constantly generated in the body as a cell-signaling molecule of the gasotransmitter family in various pathological and physiological conditions. Up to now, designing fluorescent probes for real-time imaging of CO in living species is a continuous challenge due to background interference, light scattering, and photoactivation/photobleaching. Herein, a novel type of bioluminescence probe (allyl-luciferin) was synthesized and exploited to realize CO imaging with high signal-to-noise ratios. Based on Pd⁰-mediated Tsuji-Trost reaction, allyl-luciferin specifically reacted with CO to yield D-luciferin and thus generate a turn-on bioluminescence response, exhibiting high selectivity against bioactive small molecules such as reactive nitrogen, oxygen, and sulfur species. Furthermore, the new probe can be easily employed to detect exogenous CO in Huh7 cells and MDA-MB-231 cells, and CO production was enhanced greatly in these living cells after pretreatment with [Ru(CO)₃Cl-(glycinate)] (CORM-3). Through the use of PdCl₂-containing liposomes to improve poor membrane permeability of PdCl₂, endogenous CO stimulated by heme was also seen clearly. In addition, the probe was successfully used to monitor exogenous and endogenous CO in nude mice. Overall, our data proved that the allyl-luciferin is a promising tool for exogenous and endogenous CO detection and imaging within living species. This is the first demonstration of bioluminescence imaging obtained by a probe for CO. We anticipate that the good imaging properties of allyl-luciferin presented in this study will provide a potentially powerful approach for illuminating CO functions in the future.

Full text of DOI:10.1021/acs.analchem.8b01102

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Bioluminescence Imaging of Carbon Monoxide in Living Cells
0
and Nude Mice Based on Pd-Mediated Tsuji-Trost Reaction
Xiaodong Tian, Xinda Liu, Anni Wang, Choiwan Lau, and Jianzhong Lu
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01102 • Publication Date (Web): 11 Apr 2018
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Analytical Chemistry
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Bioluminescence Imaging of Carbon Monoxide in Living Cells and
Nude Mice Based on Pd0-Mediated Tsuji-Trost Reaction
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Xiaodong Tian, Xinda Liu, Anni Wang, Choiwan Lau, and Jianzhong Lu*
School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
Email: jzlu@shmu.edu.cn; Tel: 0086-021-51980058
ABSTRACT: Carbon monoxide (CO) is highly toxic and lethal to humans and animals because of its strong affinity for
hemoglobin, while this “silent killer” is constantly generated in the body as a cell-signaling molecule of gasotransmitter
family in various pathological and physiological conditions. Up to now, designing fluorescent probes for real-time imag-
ing of CO in living species is a continuous challenge due to background interference, light scattering and photoactiva-
tion/photobleaching. Herein, a novel type of bioluminescence probe (allyl-luciferin) was synthesized and exploited to
realize CO imaging with high signal-to-noise ratios. Based on Pd0-mediated Tsuji-Trost reaction, allyl-luciferin specifically
reacted with CO to yield D-luciferin and thus generate a turn-on bioluminescence response, exhibiting high selectivity
against bioactive small molecules such as reactive nitrogen, oxygen and sulfur species. Furthermore, the new probe can be
easily employed to detect exogenous CO in Huh7 cells and MDA-MB-231 cells, which exhibited CO production was en-
hanced greatly in these living cells after pre-treated with [Ru(CO)3Cl-(glycinate)] (CORM-3). Through the use of PdCl2-
containing liposomes to improve poor membrane permeability of PdCl2, endogenous CO stimulated by heme was also
seen clearly. In addition, the probe was successfully used to monitor exogenous and endogenous CO in nude mice. Over-
all, our data proved that the allyl-luciferin is a promising tool for exogenous and endogenous CO detection and imaging
within living species. This is the first demonstration of bioluminescence imaging obtained by a probe for CO. We antici-
pate that the good imaging properties of allyl-luciferin presented in this study will provide a potentially powerful ap-
proach for illuminating CO functions in the future.
INTRODUCTION
have revolved around optical imaging with a particular
focus on fluorescence imaging. As we known, fluores-
cence is a common useful modality for optical imaging
that is broadly used for detection and monitoring of vari-
ous biological processes. Recently, several fluorescent
probes have indeed been reported for the specific detec-
tion of CO in living cells 2-3, 29, 34-37, but their depth pene-
tration of detection still need further improvement be-
cause of the inevitable interference of background fluo-
Carbon monoxide (CO) outcompetes oxygen when
binding to the iron center of hemeproteins, leading to a
reduction in blood oxygen level and acute poisoning. As
we known, CO is highly toxic and lethal to humans and
animals which are usually inhaled from common sources1-
2. However, emerging studies showed that this “silent kill-
er” is constantly generated in the body through the inter-
action of heme and heme oxygenase3-6, similar to other
two gasotransimitter molecules NO7-11 and H2S12-19. As a
second gas messenger, the biological function of CO has
been validated in modulating responses to both chemical
and physiological processes such as anti-inflammatory
response20-21, vascular smooth muscle22, neurotransmis-
sion23-24, vasorelaxation22 and antiproliferative activities25-
26. However, its abnormal metabolism has a significant
correlation with some severe diseases such as hyperten-
sion3, Alzheimer`s disease27, heart failure28, etc.
rescence,
light
scattering
and
photoactiva-
tion/photobleaching18.
Unlike fluorescence based assays, chemilumines-
cence (CL) and bioluminescence (BL) luminophores are
considered as one of the most sensitive families of probes
for detection and imaging applications, as no excitation
light source is required and background signal is not af-
fected by phenomena like autofluorescence38-42. Thus, it is
imperative to develop CL/BL probes with high signal-to-
noise ratios so as to eliminate the auto-interference.
Based on these considerations, we first proposed to de-
sign and synthesize a novel type of BL probe that is capa-
ble of tracing CO in aqueous buffer and in biological sys-
tems. Recently we have developed a turn-on reaction-
based BL probe for imaging H2S18 by taking advantage of
its specific reduction ability. In contrast, CO is not a par-
ticularly electrophilic or nucleophilic reagent, and thus
Currently, a major barrier to further illuminate po-
tential functions of CO is lack of ways for selectively
tracking this transient small molecule within biological
systems. Available techniques are limited for detecting
CO, especially under living specimens, including electro-
chemical29-30, colorimetric31-33 and gas chromatography34,
etc. Significant effort has gone into the development of
molecular imaging technologies and most approaches
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we have to find a new approach to solve this problem. was added and the temperature of the mixture was heated
Herein we sought to employ one Pd0-mediated Tsuji-
Trost reaction for the development of a BL turn-on probe
for CO detection. As depicted in Scheme 1, a novel BL
probe allyl-luciferin was designed where one allyl ether
substituent would cage the hydroxyl group of D-luciferin
and thus hide its interaction with firefly luciferase and
consequently quench BL emission. Meanwhile, the allyl
ether is anticipated to be the reaction site that can be
easily removed via Pd0-mediated Tsuji-Trost reaction.
Hence, we envisaged that this allyl ether-based probe
would have the same mechanism for particular detection
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to 70 ℃ for overnight. As the completion of the reaction
under TLC monitoring, the reaction mixture was diluted
with ethyl acetate after cool down and washed with satu-
rated salt water for three times. The organic phase was
dried over anhydrous magnesium sulfate, the solvent was
then removed under reduced pressure and the crude solid
product was purified via semi-prepared silica gel plate
(petroleum ether: ethyl acetate = 3:1, v/v) to afford allyl-
CBT as a white solid ( 150.6 mg, yield 82 %).1H NMR (600
MHz, CDCl3) δ = 8.07 (d, J=9.1, 1H), 7.36 (d, J=2.5, 1H), 7.26
(dd, J=9.1, 4.6, 1H), 6.09 (ddt, J=17.2, 10.5, 5.3, 1H), 5.47
(ddd, J=17.3, 3.0, 1.5, 1H), 5.37 (dq, J=10.5, 1.3,1H), 4.65 (dt,
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of CO 29, 35, 43-44
.
13
J=5.3, 1.5, 2H). C NMR (151 MHz, CDCl3) δ = 159.5, 147.0,
137.5, 133.5, 132.3, 126.0, 119.0, 118.7, 113.4, 104.2, 69.7.
C11H8N2OS [M + H]+ calcd. 217.0, found 217.0.
Synthesis of Allyl-luciferin: Allyl-luciferin was synthe-
sized according to a modified literature procedure45. D-
Cysteine hydrochloride monohydrate (184.4 mg, 1.05
mmol) was charged in PBS buffer (10 mM pH 7.4). 150.6
mg of allyl-CBT (0.70 mmol) was placed in dichloro-
methane and methanol (dichloromethane / methanol
=1/5.5, V/V), and then, the above-prepared D-cysteine
hydrochloride monohydrate solution was slowly added
into the allyl-CBT solution. After the reaction mixture was
refluxed at 50℃ for overnight, the solvent was then re-
moved under reduced pressure and the crude solid prod-
uct was purified using reverse-phase preparative-HPLC
(Dikma C18 column 250*10.0mm, 5 μm, using a isocratic
run of MeCN (38 %) and 0.1 % aqueous trifluoroacetic
acid (TFA) at 4.5 mL/min) to provide the desired com-
1
pound ( 69.4 mg, yield 31 %). H NMR (600 MHz, MeOD)
Scheme 1. The proposed sensing mechanism for biolumines-
cence detection of CO by a new probe allyl-luciferin via Pd0-
mediated Tsuji-Trost reaction.
δ = 7.98 (d, J=9.0, 1H), 7.57 (d, J=2.4, 1H), 7.22 (dd, J=9.0,
2.5, 1H), 6.12 (ddd, J=22.4, 10.5, 5.2, 1H), 5.47 (dd, J=17.3, 1.6,
1H), 5.41 (t, J=9.0, 1H), 5.31 (dd, J=10.6, 1.4, 1H), 4.67 (d,
13
J=5.2, 2H), 3.78 (m, 2H). C NMR (151 MHz, MeOD) δ =
EXPERIMENTAL SECTION
Materials and Instruments:
173.5, 167.8, δ = 160.0, 159.7, 149.1, 139.2, 134.5, 126.0, 118.8,
118.2, 106.2, 79.7, 70.6, 36.0. C14H12N2O3S2 [M + H]+ calcd.
321.0, found 321.0.
Millipore water was used throughout the analytical
experiments. The bioluminescence spectra were deter-
mined with an IVIS Kinetic (Caliper Life Sciences, USA)
equipped with a cooled charge coupled device (CCD)
camera for bioluminescent imaging of the cells and nude
mice. TLC plates were employed for monitoring a reac-
tion progress with UV light (254 and 365 nm). All NMR
data was taken on a 600 MHz spectrometer (Bruker Co.,
Ltd., Germany). Mass spectra were recorded on an Agilent
1100 LC/MS system. Luciferase was obtained from
Promega. Heme and ATP were bought from Sigma-
Aldrich. CORM-3 was purchased from Target Molecule
Corp.
The Kinetics of Allyl-luciferin in Bulk PBS: Because of
its poor solubility in water, allyl-luciferin was prepared in
DMSO solution. Then the probe was diluted with PBS
buffer (10 mM pH 7.4). The reaction of allyl-luciferin and
PdCl2 (100 μM each) with CORM-3 (100 μM) was started
in PBS buffer. As a blank control experiment, PBS buffer
was employed instead of CORM-3 solution. The above
reactions (100 μL) were carried out at room temperature
in the dark for 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 and 55
min, respectively, followed by 1 μL (100 mM) of ATP and 2
μL (100 μg/mL) of luciferase for each tube. BL measure-
ment was carried out with a BPCL chemiluminescence
analyzer (Beijing, China). BL signal for each tube was re-
ported in photons per second.
Synthesis of Allyl-CBT: 2-Cyano-6-hydrocybenzo-
thiazole (150 mg, 0.85 mmol) was placed in DMF and
K2CO3 (352.4 mg, 2.55 mol), as a base, was added into the
reaction solution. After the mixture was stirred at room
temperature for 15 min, allyl bromide (369 μL, 4.26 mmol)
Selectivity Measurement: The mixture solutions (100 μL)
of allyl-luciferin and PdCl2 (100 μM each) were reacted
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Analytical Chemistry
seeded onto black 96-well plates (5
×
105 cells per well) and
with the same concentration of analytes (100 μM each),
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including NaHCO3, NaHS, Na2SO4, Na2CO3, NaNO2,
Na2S2O3, Na2SO3, cysteine (Cys), glutathione (GSH), NaF,
NaNO3, NaAc, NaHSO4, KI, NaClO, tert-butyl hydroper-
oxide (t-BuOOH), NO, H2O2 and CORM-3 in PBS buffer.
The reactions were carried out at room temperature
without light for 15 min, followed by 1 μL of ATP (100 mM)
and 2 μL of luciferase (100 μg/mL) for each tube. BL signal
for each tube was reported in photons per second.
allowed to grow for overnight at 37 ℃ in a 5 % CO2 humid
incubator. Following that, the heme (200, 300 and 400
μM) was added into the wells, as a vehicle control, equal
volume of PBS buffer was added into relative wells instead
of heme. After 1 h incubation, the DMEM was removed
from the chambers containing cells and was washed with
PBS buffer. The cells in the bottom of wells were finally
covered in PBS buffer, simultaneously. Then, allyl-
luciferin and the PdCl2-containing liposomes (100 μM
each) were added into the corresponding wells and incu-
bated for about 2 h prior to imaging.
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Sensitivity Measurement: The mixture solutions (100 μL)
of allyl-luciferin and PdCl2 (100 μM each), were respec-
tively incubated with series concentrations of CORM-3 in
PBS buffer. As a blank control, a same volume of PBS was
added to the mixture solution of allyl-luciferin and PdCl2
(100 μM) instead of CORM-3. The blank control and sam-
ple reactions were carried out at room temperature with-
out light for 15 min, followed by 1 μL of ATP (100 mM) and
2 μL of luciferase (100 μg/mL) for each tube. BL signal for
each tube was reported in photons per second.
Bioluminescence Imaging of Exogenous CO in Mice:
On the first day, we choose two nude mice, 200 μL of ally-
luciferin (5 mM with 40% DMSO) and 50 μL of PdCl2-
containing liposomes were intraperitoneally and intra-
tumorally injected into each nude mouse as control
groups. BL imaging was captured for 60 s using the IVIS
Imager. On the second day, the same mice received 200
μL of ally-luciferin (5 mM) and then an intraperitoneal
injection of 50 μL CORM-3 (10 and 15 mM), followed by
an intratumoral injection of 50 μL PdCl2-containing lipo-
somes (100 μM) for each mouse. BL imaging was also
measured for 60 s by the IVIS Imager.
The Toxicity Analysis of Allyl-luciferin, PdCl2 and
PdCl2-containing Liposomes: Cell viability was assessed
by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium
bromide in HEK 293T cell line. HEK 293T cells were
grown in Gibco DMEM supplemented with 10 % FBS, 1 %
streptomycin and 1 % penicillin. Cells were seeded onto
white 96-well plates (5000 cells per well) and incubated
to ~80 % density. Following that, various concentrations
of allyl-luciferin, PdCl2 and PdCl2-containing liposomes (0,
0.1, 1, 10, 25, 50, 100, 250 and 500 μM) were placed into
each well, respectively. After 12 h of treatment at 37 ℃, 10
μL of CCK8 solution was added into each well. After 2 h
incubation, absorbance of the solution was then measured
Bioluminescence Imaging of Endogenous CO in Mice:
On the first day, we choose another two nude mice, 200
μL of allyl-luciferin (5 mM) and 50 μL of PdCl2-containing
liposomes were intraperitoneally and intratumorally in-
jected into each nude mouse as control groups, respec-
tively. BL imaging was then measured for 60 s using the
IVIS Imager. On the second day, the same mice received
an intraperitoneal injection of 50 μL heme (20 and 40
mM). After 3 h, 200 μL of allyl-luciferin (5 mM) was in-
jected intraperitoneally into the mice, and then 50 μL
PdCl2-containing liposomes (100 μM) was intratumorally
injected for each mouse. BL imaging was also measured
for 60 s by the IVIS Imager.
at 450 nm with a microplate reader
.
Bioluminescence Imaging of CORM-3-triggered Ex-
ogenous CO in Cells: Huh7 cells and MDA-MB-231 cells
were grown in Gibco DMEM supplemented with 10 % FBS,
1 % streptomycin and 1 % penicillin. Cells were seeded
into 35 mm glass bottom dishes for 24 h incubation to
render the confluence up to around 80%. Equal volume of
RESULTS AND DISCUSSION
Synthesis:
cells were charged into black 96-well plates (5
105 cells
×
per well). Series concentrations of CORM-3 (0 - 1500 μM)
were incubated with Huh7 cells and MDA-MB-231 cells
for 10 min. Following that, allyl-luciferin and PdCl2 (100
μM each) were immediately added into the plates and
incubated for 40 min. Bioluminescence signal (photons
per second) was immediately measured for 30 s using a
Xenogeny IVIS spectrum imaging system (Camera Temp:
-80℃; Stage Temp: 30℃; Excitation Filter: Block; Emis-
sion Filter: Open).
Our initial study began with the introduction of function-
al reaction site: Allyl bromide was reacted with commer-
cially available starting material (6`-hydroxyl of 2-cyano-
6-hydroxybenzothiazole) through Williamson ether syn-
thesis as shown in Figure 1. Then a cyclization reaction
could spontaneously proceed between D-cysteine hydro-
chloride monohydrate and allyl-CBT in methanol and PBS
buffer (10 mM pH 7.4) to obtain allyl-luciferin. The details
of synthesis can be found in the Experimental Section and
the structures of allyl-CBT and allyl-luciferin were con-
firmed by ESI-MS and 1H NMR and 13C NMR spectroscopy
(See supporting information Figures S4-9).
Bioluminescence Imaging of Heme-triggered Endog-
enous CO in Cells: Huh7 cells and MDA-MB-231 cells
were grown in Gibco DMEM supplemented with 10 % FBS,
1 % streptomycin and 1 % penicillin. Then, the cells were
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CORM-3, 100 μM each;) in PBS buffer (10 mM, pH 7.4) at
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3
room temperature without light for 0, 5, 10, 15, 20, 25, 30, 35,
40, 45, 50 and 55 min, then 1 μL (100 mM) of ATP and 2 μL
(100 μg/mL) of luciferase were added respectively.
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Selectivity Measurement: The selectivity of allyl-
luciferin was tested by comparing BL intensities among
allyl-luciferin and bioactive small molecules such as reac-
tive nitrogen, oxygen, and sulfur species, including Na-
HCO3, NaHS, Na2SO4, Na2CO3, NaNO2, Na2S2O3, Na2SO3,
Cys, GSH, NaF, NaNO3, NaCl, NaAc, NaHSO4, KI, NaClO,
t-BuOOH, NO, H2O2 and CORM-3 in PBS buffer. To our
delight, as depicted in Figure 3, the turn-on BL response
for allyl-luciferin was found to have excellent selectivity
for capturing CO over other substances, due to that the
selective recognition of CO by allyl-luciferin is based on
CO-induced Pd0-mediated Tsuji-Trost reaction. However,
other testing species almost did not trigger BL responses,
which may be due to that these molecules can’t reduce
PdCl2 to Pd0.
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Figure 1. Synthesis of allyl-luciferin. (a) allyl bromide (5.0
equiv), K2CO3 (3.0 equiv), DMF, 70℃, overnight, 82 %. (b) H-
D-Cys-OH·HCl·H2O (1.5equiv), 50℃, overnight, 31 %.
The Kinetics of Allyl-luciferin in Bulk PBS: With allyl-
luciferin in hand, we tested its BL properties and CO reac-
tivity in PBS buffer. For in vitro analysis, a commercially
available and water soluble complex CORM-3 was utilized
as an easy-to-hand CO-releasing molecule2-3, 29, 34-37,43-44,46-47
(1 mole of CORM-3 can produce 1 mole of CO on the basis
of previous reports2, 29, 46). As expected, the modification
of allyl on the D-luciferin backbone rendering the mole-
cules incapable of interacting with luciferase to produce
light. A robust turn-on BL response was triggered, only
after the addition of 100 μM CORM-3 into a solution of
allyl-luciferin in the presence of firefly luciferase, ATP and
100 μM PdCl2 via CO-induced Pd0-mediated Tsuji-Trost
reaction. On the basis of the encouraging results, time-
dependent BL responses of allyl-luciferin to CO were
evaluated in PBS buffer by comparing the net BL intensity,
where the net BL intensity was defined as (BL intensity–
BL0 intensity), BL intensity represents the total flux of
samples and BL0 intensity represents the blank total flux.
As shown in Figure 2, kinetic experiments exhibited that
one 15-min reaction was optimum between allyl-luciferin
and CORM-3 in PBS solution.
4.00x106
3.00x106
2.00x106
1.00x106
0.00
1 2 3 4 5 6 7 8 9 101112131415161718192021
Figure 3. Bioluminescence changes of allyl-luciferin and
PdCl2 (100 μM each) with other bioactive small molecules
(100 μM each) in PBS (10 mM, pH 7.4): 1, PBS; 2, NaHCO3; 3,
NaHS; 4, Na2SO4; 5, Na2CO3; 6, NaNO2; 7, Na2S2O3; 8, Na2SO3;
9, Cys; 10, GSH; 11, NaF; 12, NaNO3; 13, NaCl; 14, NaAc; 15,
NaHSO4; 16, KI; 17,NaClO; 18, t-BuOOH; 19,NO; 20, H2O2; 21,
CORM-3. Other experimental parameters such as the reac-
tion temperature, reaction time, amounts of ATP and lucifer-
ase were the same as described in Figure 2.
1.00x107
8.00x106
6.00x106
4.00x106
Sensitivity Measurement: It is well known that the sen-
sitivity of a new probe is a key performance indicator. On
the basis of the 3σ method, a detection limit for CORM-3
was calculated to be about 58 nM and also good correla-
tions (R2=0.9828) were observed between the net BL in-
tensity and CORM-3 concentrations from 0.1 to 250 μM
(Figure 4). Overall, allyl-luciferin may be employed as an
effective tool for monitoring intracellular CO, which is
comparable or even better than most reported fluorescent
0
15
30
45
60
Time (min)
Figure 2. Bioluminescence time-dependence of allyl-
luciferin, PdCl2 with CORM-3 (allyl-luciferin, PdCl2 and
probes 2, 35, 37
.
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a)
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1.00
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y=0.8983x+4.6545
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10 25 50 100 250 500
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5
Probe (µM)
Lg[CORM-3]
b)
Figure 4. Calibration curve of the net BL intensity vs. the
concentration of CORM-3. Other experimental parameters
such as the concentrations of allyl-luciferin and PdCl2, the
reaction temperature, reaction time, amounts of ATP and
luciferase were the same as described in Figure 2.
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The Toxicity Analysis of Allyl-luciferin, PdCl2 and
PdCl2-containing Liposomes: To employ allyl-luciferin
for the detection of CO in living cells, a key question is
whether the cells will survive during living cell imaging.
Therefore, the cytotoxicity of allyl-luciferin, PdCl2 and
PdCl2-containing liposomes were evaluated by the CCK8
assay. Figures 5a, 5b and 5c showed that cell viability re-
mained constant over the evaluated concentration range
of allyl-luciferin, PdCl2 and PdCl2-containing liposomes.
10 25 50 100 250 500
PdCl (µM)
2
c)
1.00
0.75
0.50
0.25
0.00
10 25 50 100 250 500
PdCl -liposomes (µM)
2
Figure 5. (a) CCK8 assay for the viability of HEK 293T cell
treated with various concentrations (0-500 μM) of allyl-
luciferin.. (b) CCK8 assay for the viability of HEK 293T cell
treated with various concentrations (0-500 μM) of PdCl2.. (c)
CCK8 assay for the viability of HEK 293T cell treated with
various concentrations (0-500 μM) of PdCl2-containing lipo-
somes.
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Bioluminescence Imaging of CORM-3-triggered Ex- Bioluminescence Imaging of Heme-triggered Endog-
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ogenous CO in Cells: We then sought to evaluate the
ability of allyl-luciferin to serve as BL agent for imaging
CO inside living cells. Huh7 cells and MDA-MB-231 cells
were selected to incubate with various concentrations of
CORM-3 respectively and then the cells were treated with
allyl-luciferin in the presence of 100 μM PdCl2 in the black
96-well plates. Data was acquired using a Xenogeny IVIS
Spectrum imaging system. A bright BL cell images was
found to be dependent on the concentration of CORM-3
(0-1500 μM) and 50 μM of CORM-3 was clearly seen in
both Huh7 cells (Figures 6a and 6b) and MDA-MB-231
cells (Figure S2). These results clearly demonstrated that
allyl-luciferin was suitable for the visualization of exoge-
nous CO in living cells by using BL imaging technique.
enous CO in Cells: To our knowledge, endogenous CO
can be generated via the interaction of heme and heme
oxygenases (HOS) 4-6, 48-49. Therefore, our major concern
was whether allyl-luciferin has capability to detect endog-
enously produced CO. Both Huh7 cells and MDA-MB-231
cells were seeded onto the black 96-well plates overnight.
Then 10 μL of heme (200, 300 and 400 μM) and PBS were
pre-incubated with these cells for 1 h, respectively, and
both Huh7 cells and MDA-MB-231 cells were added allyl-
luciferin and also PdCl2 for 2 h after washing with PBS
buffer. However, no apparent BL signal was found, and
thus we speculated that the main reasons may be possibly
due to the following three factors, (1) the endogenously
produced CO that stimulated with heme is supposed to
be quite low; (2) PdCl2 has poor membrane permeability;
(3) the cells were pre-washed to remove any interference
from complex matrix before imaging endogenous CO
with PBS buffer, leading to that those remaining attached
cells in the bottom of 96-well plates were much less than
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105 cells and could not emit enough BL signals. Since it
×
is unable to further increase the attached cells, and thus
we expected to increase the membrane permeability of
PdCl2 through the use of the PdCl2-containing liposomes
instead of free PdCl2. The liposomes have an aqueous
PdCl2 core surrounded by a hydrophobic lipid membrane.
The liposome bilayer can fuse with the cell membrane,
thus delivering PdCl2 to enter the cell according to "like
dissolves like". As expected, a clear and distinguishing BL
images were observed after using the PdCl2-containing
liposomes instead of free PdCl2 for imaging exogenous
CO (Figure S3), which proved that improving the mem-
brane permeability of PdCl2 may be a good way for BL
imaging of the endogenously produced CO in living cells.
As we can see, the levels of endogenous CO of Huh7 cells
(Figures 7a and 7b) and MDA-MB-231 cells (Figures 7c
and 7d) were increased with the incubation of heme,
since the BL in the presence of heme were higher than
that of PBS control group, where the BL ratio was defined
as (BL intensity/BL0 intensity). Therefore, all data defi-
nitely indicated that allyl-luciferin is promising as an ef-
fective tool for monitoring endogenously produced CO.
b)
1.80x105
1.35x105
9.00x104
4.50x104
y=97.2546x+460.0435
2
R =0.9848
0.00
0
400
800
1200
1600
CORM-3 (µM)
Figure 6. Bioluminescence images of CORM-3 triggered CO
in luciferase-transfected Huh7 cells. (a) Various concentra-
tions of CORM-3 (0 - 1500 μM) were incubated with Huh7
cells for 10 min. Then, allyl-luciferin and PdCl2 (100 μM each)
were added and incubated for 40 min. Images was measured
using a Xenogeny IVIS spectrum imaging system. (b) Calibra-
tion curve of BL intensity vs. CORM-3 concentrations (0 -
1500 μM).
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Hence, a luciferase-transfected Huh7 tumor was xeno-
b
)
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grafted in the right forelimb of each nude mouse. Due to
individual differences in mice, the size and shape of tu-
mors are often different for different mouse, and thus the
same mouse was used as its own control. As shown in
Figure 8, compared with the blank control, BL signals
4
3
2
1
0
from treated CORM-3 groups were obviously enhanced
.
These results suggested that allyl-luciferin was applicable
for imaging exogenous CO activity in tumors in living
mice.
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0
200
300
400
Heme (
µ
M)
d
)
4
3
2
1
0
Figure 8. Bioluminescence imaging of nude mice that xeno-
grafted with luciferase-transfected Huh7 tumors. The images
obtained from two nude mice treated with different concen-
trations of CORM-3 (10 and 15 mM) and BL ratio were also
measured from each mouse that treated-CORM-3 group and
the relative control group.
0
200
300
400
Heme (µM)
Figure 7. Bioluminescence images of heme-triggered endog-
enous CO in luciferase-transfected Huh7 and MDA-MB-231
cells. The cells were treated with heme and PBS for about 1 h.
Then, allyl-luciferin and the PdCl2-containing liposomes (100
μM each) were added and incubated for 2 h. Images was
measured using a Xenogeny IVIS spectrum imaging system.
(a) BL images acquired from Huh7 cells treated with heme
(200, 300 and 400 μM) and PBS. (b) BL ratio was measured
from Huh7 cells treated with heme and PBS. (c) BL images
acquired from MDA-MB-231 cells treated with heme (200,
300 and 400 μM) and PBS. (d) BL ratio was measured from
MDA-MB-231 cells treated with heme and PBS.
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Bioluminescence Imaging of Endogenous CO in Mice:
After receiving an injection of allyl-luciferin and PdCl2-
containing liposomes, mice only displayed a weak BL in
the xenografted liver tumors. In contrast, BL signals (Fig-
ure 9) exhibited an obvious increase after incubation with
heme compared with the control group, demonstrating
that allyl-luciferin could even detect endogenous CO in
vivo. However, we should state that this in vivo CO imag-
ing is only partially successful, the maximum BL ratio
values of the probe were comparatively not high especial-
ly for endogenous CO in intact living mice, more work
still needs to be done to improve the probe for endogeous
CO imaging in mice.
Bioluminescence Imaging of Exogenous CO in Mice:
Based on the above studies, we explored the capability of
allyl-luciferin for exogenous CO visualization in vivo
.
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Supplementary experimental methods, probes characteriza-
tion are available free of charge via the Internet at
1
2
http://pubs.acs.org.”
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4
5
6
AUTHOR INFORMATION
Corresponding Author
*jzlu@shmu.edu.cn.
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Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the Natural Science Foundation
of China (No. 21675030).
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