In Vivo Bioluminescence Imaging of Cobalt Accumulation in a Mouse Model

Article abstract of DOI:10.1021/acs.analchem.8b00391

As a trace element nutrient, cobalt is critical for both prokaryotes and eukaryotes. In the current study, a turn-on Cobalt Bioluminescent Probe 1 (CBP-1) for the detection of cobalt has been successfully developed based on oxidative C-O bond cleavage. This probe exhibited high selectivity and sensitivity toward cobalt over other analytes. By using CBP-1, the successful in vivo imaging of cobalt accumulation was carried out in a mouse model. Such an ability to determine cobalt in living animals provides a powerful technology for studying the system distribution, toxic potency, and biological effect of Co²⁺.

Full text of DOI:10.1021/acs.analchem.8b00391

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In vivo bioluminescence imaging of cobalt accumulation in a mouse model
Bowen Ke, Lin Ma, Ting Kang, Wei He, Xueyan Gou, Deying Gong, Lupei Du, and Minyong Li
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00391 • Publication Date (Web): 28 Mar 2018
Downloaded from http://pubs.acs.org on March 28, 2018
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In vivo bioluminescence imaging of cobalt accumulation in a mouse
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Bowen Ke,b, ‡ Lin Ma,a, ‡ Ting Kang,b Wei He,b Xueyan Gou,b Deying Gong,b Lupei Du a,* and Minyong Li a,*
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong
University, Jinan, Shandong 250012, China. Tel./Fax: +86- 531-8838-2076; E-mail: mli@sdu.edu.cn
b Department of Anaesthesiology & Critical Care Medicine, Translational Neuroscience Center, West China Brain
Research Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
ABSTRACT: As a trace element nutrient, cobalt is critical for both prokaryotes and eukaryotes. In the current study, a
turn-on Cobalt Bioluminescent Probe 1 (CBP-1) for the detection of cobalt has been successfully developed based on
oxidative C-O bond cleavage. This probe exhibited high selectivity and sensitivity towards cobalt over other analytes. By
using CBP-1, the successful in vivo imaging of cobalt accumulation was carried out in a mouse model. Such an ability to
determine cobalt in living animals provides a powerful technology for studying the system distribution, toxic potency,
and biological effect of Co2+.
As an essential trace element, cobalt is well known for
its critical roles in biological processes not only in
prokaryotes, but also in eukaryotes.1,2 Basically, cobalt is
essential to the metabolism of all animals as a key
constituent and the active center of cobalamins, several
chemical forms of vitamin B12, which performs various
physiological functions in the normal functioning of
nervous system, the formation of red blood cells, DNA
synthesis, and amino acid metabolism as well.3-5 It has
been demonstrated that the lack of cobalt results in
cardiovascular, anemia, osteomyelitis and glaucoma and
other diseases. The benefit or detriment of cobalt
depends on its level or consumption. This excessive
redox-active metal ions can catalyze the production of
reactive oxygen species (ROS) and compete with other
essential metal ions to inhibit the biological function of
enzymes, which result in the high toxicity of cobalt ions
toward living cells.6 As such, long-term environmental
exposure to cobalt or acute intake of cobalt may lead to a
variety of diseases, including contact dermatitis,
pneumonia, allergic asthma, and lung cancer.7 Briefly, this
element is closely associated with human health.
adsorptive voltammetric and inductively coupled plasma
atomic emission spectrometry (ICP - AES).8-12 However,
the bulk traditional analysis methods typically require
sophisticated sample preparation procedures, highly-
trained individuals, and costly instruments. More
importantly, none of them are compatible with living
organisms. Among this collections, the optical probes,
due to good sensitivity and bioavailability, construct a
great figure as a valuable chemical tool to effectively
monitor and track analytes of interest in the biological
specimens/living organisms and therefore, are extremely
beneficial for the broader scientific community.13 To date,
some fluorescent and colorimetric probes have been
reported for the detection of cobalt,14-21 and none of them
has been applied to in vivo imaging. Therefore, there is an
urgent demand on developing novel strategies for
monitoring cobalt flux, in particular, in the whole animal.
Since cobalt contributes to physiological and
pathological processes, the underlying mechanism of the
correlation between its biological occurrence/trafficking
and healthy& disease status is still unclear. Therefore, the
detection of cobalt in the environmental system and
living organs has drawn intensive attention. In this
content, a number of techniques are known to be widely
applied in cobalt analysis, such as HPLC, flame atomic
absorption spectroscopy (FAAS), spectrophotography,
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Scheme 1. Design and synthesis of Cobalt Bioluminescent
Probe 1 (CBP-1), a selective Co2+-mediated release of the D-
luciferin substrate.
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To meet the stringent criteria of in vivo imaging of
cobalt in the whole animal, we sought to utilize
bioluminescence imaging (BLI) as a noninvasive strategy
to establish detection platform. Different from
fluorescence, bioluminescence generates photon signal
catalyzed by an enzyme in the absence of external light as
a natural phenomenon widely spread among marine and
terrestrial species.22,23 As a result, it obviates the intrinsic
limitations of fluorescence and affords high sensitivity
and low background which render it suitable for imaging
in deep tissue and long-term studies of living animal. In
the past decade, diverse bioluminescent probes have been
developed based on luciferin/luciferase system.24-28 Using
caging strategy, appropriate protective groups are
employed to cage the reactivity of the substrate toward
the enzyme and to block the luminescence production.
Analytes can selectively uncage bioluminescent probes
and release luciferin substrates. Subsequent oxidation of
luciferin can transfer the chemical energy of ATP to a
light signal for analyte detection. It should be noted that
recently, along with this line, several bioluminescent
probes were developed for detecting accumulation of
metal cations in vivo, including Cu2+ and Fe2+.29,30 Herein,
we described the development of the first bioluminescent
cobalt probe, as well as its initial applications in in vivo
imaging of cobalt accumulation in a mouse model.
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Scheme 2. Proposed mechanism of CBP-1 for the detection
of Co2+.
By utilizing the N3O ligand with a C-O benzyl ether
bond, we successfully prepared CBP-1 based on D-
luciferin via three straight-forward steps (Scheme 1).
More synthetic details can be found in the Supporting
Information (Scheme S1).
Initial experiments evaluated the efficiency of CBP-1 to
detect Co2+ in aqueous buffer system (Tris buffer at pH 7.4,
with 10 mM MgCl2). Consistent with our expectation,
CBP-1 was no longer a light-emitting substrate for firefly
luciferase so that produced a weak signal in the absence
o
of analyte. After pretreated with 30 µM CoCl2 at 37 C for
60 min followed by addition of luciferase, CBP-1 (15 µM)
displayed a 40-fold bioluminescence emission (Figure S1).
We subsequently evaluated the capability of this probe to
detect Co2+ in a concentration-dependent manner. Such a
probe (15 µM) was incubated with various concentrations
Our design strategy in establishing such a reaction-
based bioluminescent probe is to introduce a cobalt
reporter into 6'-hydroxyl group of luciferin. The
modification of luciferin at this position normally affords
the blockade of identifiability of substrate toward
luciferase. The tetradentate ligand N3O was chosen as an
appropriate trigger for cobalt since it has been
demonstrated to be feasible for turn-on detection of
Co2+.31,32 We envisioned that, taking advantage of the
cobalt-mediated C-O bond cleavage, the bioluminescent
probe can release the luciferin substrate in the presence
of Co2+, thus generating a bioluminescent readout upon
reacting with luciferase (Scheme 2). To validate this
proof of concept, we designed and synthesized a Cobalt
o
of Co2+ in a Tris-HCl buffer at 37 C for 60 min in 96-well
plate. After adding luciferase into the plate, clear
bioluminescent signals were observed and recorded.
Bioluminescence signals generated from such a probe
consistently increased with gradual concentrations of
CoCl2 before Co2+ concentration reached 30 µM. And then
the bioluminescence intensity began to decline. All these
results indicated that CBP-1 possesses the considerable
ability for detecting Co2+ at diverse concentrations in the
aqueous environment.
Having determined that CBP-1 has the excellent ability
to react with Co2+, we turn our attention to examine the
selectivity of the probe toward Co2+ over other cations. A
solution of CBP-1 (15 µM) was treated with a variety of
relevant metal ions (30 µM), including Na+, K+, Mg2+, Ca2+,
Fe2+, Fe3+, Cu+, Cu2+, Co2+, Mn2+, Zn2+, Hg2+, Cr3+, Ag+, Pd2+,
Cd2+ and Vitamin B12 (Vit. B12), at 37 oC for 60 min,
respectively (Figure 1). In the presence of ATP and
luciferase, there was little to no bioluminescence response
to other cations except Cu+ and Co2+. Furthermore, the
intensity of bioluminescence signal generated from the
reaction of the probe with Co2+ is 40-fold higher than with
other ions. The concern was also addressed as to whether
copper levels would interfere with the probe in a
biologically-relevant setting. CBP-1 with Co2+ treatment
Bioluminescent Probe (CBP-1),
a
new turn-on
chemosensor for selective detection of Co2+.
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exhibited 8-fold higher bioluminescent signal intensity
than that with Cu+ (1.42 e10 vs 1.86 e9), even at low
concentration (30 µM). This evidence suggested the high
selectivity of CBP-1 toward Co2+ under the same
condition, which confirms this probe as a promising
chemical toolkit for the detection of cobalt in the aqueous
environment.
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Figure 2. The bioluminescence intensities of ES-2-Luc cells
incubated with CBP-1 with various concentrations of a CoCl2
solution and the linearity between bioluminescence intensity
and Co2+ concentrations. All assays were performed in
triplicate and represented as the mean ± SEM.
After confirming that CBP-1 has distinct advantages in
aqueous solution and in cell culture, we continued to
examine whether the performance of this probe was
translated to the whole animal and whether its favorable
properties could be kept in in vivo study. Evaluation of
CBP-1 for in vivo imaging was performed with the
transgenic FVB-luc+ mice, ubiquitously expressing firefly
luciferase. D-luciferin control study was performed for in
vivo assays as it is not clear what effects cobalt has on
overall enzyme activity and viability. In control studies,
we mimic the conditions for the experimental studies to
test whether cobalt at different levels would affect the
bioluminescence system in FVB-luc+ mice. In vivo
imaging results showed that even high levels (200 µL, 10
mM) of Co2+ did not interfere the luminescent signals
from animals in this experiment (Figure S3), which
indicated that Co2+ did not disturb the expression of
luciferase and the level of cofactors (ATP, O2 and Mg2+)
under the experimental condition. To account for the
ability of the probe to detect Co2+, normal saline (200 µL)
and a sublethal dose of CoCl2 (200 µL, 10 mM) was
injected into FVB-luc+ mice from a tail vein. At 60 min
after intravenous (i.v.) injection of Co2+, 200 µL CBP-1 (1
mM) was injected into the intraperitoneal (i.p.) cavity in
transgenic mice. The bioluminescence signals produced
from transgenic FVB-luc+ animal models were monitored
in real-time using CCD camera per 3 min. During the
extension of the tracking time, a robust increase in
bioluminescence signals generated from pretreating FVB-
luc+ mice was observed in a time-dependent manner, and
the highest bioluminescence intensity was detected
(Figures 3A and 3B). Moreover, a 4-fold enhancement in
bioluminescence intensity was clearly measured
compared with the vehicle-treated control animal, which
strongly indicated the practicability of CBP-1 for BLI in
vivo. Under the experimental conditions, mice still
showed a significant bioluminescence signal within 24h,
Figure 1. The relative bioluminescence responses of 15 µM
CBP-1 toward 30 µM different cations (Na+, K+, Mg2+, Ca2+,
Fe2+, Fe3+, Cu+, Cu2+, Co2+, Mn2+, Zn2+, Hg2+, Cr3+, Ag+, Pd2+,
Cd2+ and Vit. B12). All assays were performed in triplicate and
represented as the mean ± SEM.
Since these impressive data in hand, we next sought to
apply CBP-1 to the detection of Co2+ in living cells by
taking advantage of its high selectivity and fast response
ability. ES-2-Luc cells (luciferase transfected cell line)
were employed in the cell-based bioluminescence
imaging. We first evaluated the cytotoxicity of the probe
and Co2+ on ES-2-Luc cell by using 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (Figure S2). CBP-1 exhibited low toxicity
(IC50 > 1000 µM) to cell lines in the experiment, which
suggested this probe possesses high biocompatibility. The
effect of Co (II) on living cells has also been investigated.
Loss of cell viability was observed when ES-2-Luc cells
were exposed to increasing Co (II) concentrations for 24 h.
IC50 values of cobalt was 731 µM in the MTT assay. To
examine whether CBP-1 was interacting with dynamic
Co2+ in intracellular space, ES-2-Luc cells were pretreated
with different concentrations of CoCl2 solution ranging
from 0 to 750 μM at 37 oC for 30 min. After the removal of
the medium, cells were washed with phosphate buffered
saline and then incubated with 50 µM probe for an
additional 5 min. As shown in (Figure 2), a gradual
increasing bioluminescence signal generated from living
cells was recorded upon addition of CBP-1. Moreover,
there is a good linearity (R2 = 0.997) between the
bioluminescence intensities and Co2+ levels. These data
demonstrated that CBP-1 is capable of detecting
fluctuation in Co2+ levels in living cells.
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indicating that there is still a fairly high cobalt ion level.
Page 4 of 6
produced from these mice were measured within 60 min
following injection. The total photon flux integrated over
1 h for each mouse exhibited a significant dose-dependent
growth. To evaluate the accumulation of Co2+ in the living
animal, FVB-luc+ mice were i.g. administrated with a
sublethal dose of CoCl2 solution (200 µL, 100 mM). Cobalt
enrichment in the blood, kidney, and liver of Kun Ming
(KM) mice was independently confirmed by GFAAS
analysis (Figure S4). At the 1st, 24th, 48th and 72nd h after
the administration of Co2+, CBP-1 (200 µL, 1 mM) was
injected into the intraperitoneal cavity of these mice, and
subsequently signals generated from mice were measured.
A robust increasing bioluminescence signal was detected
in a real-time manner. The total photon flux integrated
over 1 h indicated that Co2+ can metabolize gradually in
living animal, which provided evidence that mice can
excrete most of Co2+ within 24 h (Figures 4C and 4D).
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Howerve, compared with the animals in the control group,
there is nearly no bioluminescence signals increased were
observede at, 48th h and 96th h, respectively (Figure 3C).
It suggested that exogenetic Co2+ could be metabolized or
excreted within 48
h and there is no excessive
accumulation of Co2+ in mice. Notably, CBP-1 produced
weak but measurable background in the vehicle-treated
control animal. Luciferase recognition of the caged
substrate, the endogenous degredation of the probe, or
the response to Cu+ and Co2+ from the diet may be a
reasonable cause.
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Figure 4. CBP-1 can monitor labile cobalt dynamics in
luciferin-expressing mice administrated i.g. with CoCl2. (A)
Representative imaging of FVB-luc+ mice injected i.p. with
CBP-1 (200 µL, 1 mM) after i.g. administration of various
concentrations (0, 10, 25, 50 and 100 mM) of 200 µL CoCl2
solutions; (B) Photon intensities from above these mice; (C)
Bioluminescence imaging and (D) total flux (photon/s)
output of FVB-luc+ mice injected i.p with CBP-1 after i.g.
administration of Co2+ (200 µL, 100 mM) at the 1st, 24th, 48th
and 72nd h, respectively. All assays were performed in
triplicate and represented as the mean ± SEM. **: P < 0.01, *:
P < 0.05.
Figure 3. (A) The bioluminescence imaging of FVB-luc+ mice
injected i.p. with 200 µL CBP-1 (1 mM) after i.v. injection of
CoCl2 solution (200 µL, 10 mM); (B) Bioluminescent
intensities measured at various time points (3, 6, 9, 12, 15, 18,
21, 24, 27, 30, 35, 40, 45, 50, 55 min), in the presence or
absence of Co2+; (C) Quantification of the total flux
(photon/s) from the whole-body area except tail in the
presence or of Co2+ (200 µL, 10 mM) at the 1st, 24th, 48th and
96th h, respectively. All assays were performed in triplicate
and represented as the mean ± SEM. **: P < 0.01.
In this study, a new bioluminescent probe was designed
and developed for the detection of cobalt. By using this
probe, we successfully carried out the first in vivo imaging
of cobalt. Our work represents the first attempt to
monitor fluctuation of cobalt level in real time in an
intact, living animal model. Such information may be
correlated with other analyses to provide a valuable
opportunity for studying system distribution, toxic
potency, and biological effect of Co2+, as well as to
understand the complicated molecular processes of cobalt
in the biological system.
Generally, Co2+ in nature is taken into the organism
orally, subsequently, we next turned our attention to
performing CBP-1 to monitor labile cobalt dynamics in
living animals with Co2+ intragastric (i.g.) administration.
FVB-luc+ mice were i.g. administrated with several
concentrations (0, 10, 25, 50 and 100 mM) of 200 µL CoCl2
solutions for 1 h, before i.p. injection of CBP-1 (200 µL, 1
mM). The bioluminescent imaging and signal (Figures
4A and 4B) produced from FVB-luc+ mice were recorded
in real-time using
a cooled CCD camera. In this
experiment, differences in bioluminescent signals
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AUTHOR INFORMATION
Corresponding Author
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* Minyong Li. Tel./fax: +86-531-8838-2076; E-mail:
mli@sdu.edu.cn
* Lupei Du. Tel./fax: +86-531-8838-2006; E-mail:
dulupei@sdu.edu.cn
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
‡ These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful for financial support from the National
Natural Science Foundation of China (Nos. 81773685 and
81673393), the 1.3.5 Project for Disciplines of Excellence, West
China Hospital, Sichuan University (Nos. ZY2016101 and
KY2016203), the Taishan Scholar Program at Shandong
Province, the Qilu/Tang Scholar Program at Shandong
University, the Key Research and Development Project of
Shandong Province (No. 2017CXGC1401) and the Major
Project of Science and Technology of Shandong Province (No.
2015ZDJS04001).
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
ACS Paragon Plus Environment