A mitochondria-targeted fluorescent probe for ratiometric detection of endogenous sulfur dioxide derivatives in cancer cells

Article abstract of DOI:10.1039/c5cc09092j

A new mitochondria-targeted fluorescent probe HCy-D, constructed by dansyl and hemicyanine fluorophores, for SO₂ derivatives (HSO₃^-/SO₃^2-) was presented. This probe was designed based on a new FRET platform. HCy-D showed a ratiometric, sensitive and rapid response to HSO₃^-/SO₃^2-. Importantly, HCy-D was successfully used for fluorescence imaging of endogenous bisulfite in HepG2 cells, which may benefit cancer diagnosis by discriminating liver cancer cells from normal liver cells.

Full text of DOI:10.1039/c5cc09092j

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A mitochondria-targeted fluorescent probe for ratiometric
detection of endogenous sulfur dioxide derivatives in cancer cells
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
a
a
a
a
Dong-Peng Li , Zhao-Yang Wang , Xiang-Jian Cao , Jie Cui , Xin Wang , Hao-Zhong Cui , Jun-Ying
b*
a*
Miao , Bao-Xiang Zhao
DOI: 10.1039/x0xx00000x
www.rsc.org/
5
h,5m,5o,5s
A
new mitochondria-targeted fluorescent probe HCy-D, wavelengths involved.
Thus, still a big room for
constructed by dansyl and hemicyanine fluorophore, for SO
2
improvement is there.
Förster resonance energy transfer (FRET) has been widely
-
2-
derivatives (HSO
3
/SO
3
) was presented. This probe was designed
based on a new FRET platform. HCy-D showed a ratiometric, used to construct ratiometric fluorescent probes, in which
-
2-
3 3
sensitive and rapid response toward HSO /SO . Importantly, nonradiative energy transfers from an excited dye donor to a
6
HCy-D was successfully used to fluorescence imaging of dye acceptor in the ground state. Generally, the FRET process
endogenous bisulfite in HepG2 cells, which may benefit cancer on/off is controlled by modulation of the acceptor molar
diagnosis by discriminating liver cancer cells from normal liver absorption coefficient (Scheme 1a) or tuning the donor-
6
a,6c
cells.
acceptor distance (Scheme 1b).
Here a new strategy to
construct ratiometric fluorescent probes based on FRET was
introduced. The FRET process may occur in the dyad to
prohibit the donor fluorescence and to enhance the acceptor
fluorescence. While upon reaction with analyte, the FRET
process will be cancelled because of the changed absorption
band of the acceptor to restore the donor fluorescence
2
Sulfur dioxide (SO ) is a main air pollutant which exists as
2
-
-
sulfite (SO
However, SO
mitochondria of cells during decomposition of sulfur-
3
) and bisulfite (HSO
3
) anions in aqueous solution.
2
can be produced enzymatically in cytosols and
1
2
containing amino acids. Like nitric oxide (NO), SO is a newly
recognized gaseous signal transmitter involved in many
(Scheme 1c). Thus, a new ratiometric fluorescent probe was
physiological processes (such as regulation of vascular smooth
2
muscle tone and lowering blood pressure). SO
designed (Scheme 1d). A dansyl fluorophore was selected as
7
2
may serve as
the donor for its good spectroscopic properties.
A
antioxidant and in recent years, a few favourable redox-
responsive fluorescent probes have been developed and
applied to explore the generation, transport, physiological
function, and pathogenic mechanisms of reactive oxygen
hemicyanine fluorophore was chosen as the acceptor because
it may not only serve as the quencher of donor fluorescence,
-
2-
5f,5h,5j-m
but also serve as HSO
3
/SO
3
receptor.
Importantly,
fluorescence emission band of the donor and UV-vis
absorption band of the acceptor overlap fully.
3
species and antioxidants. Nonetheless, the biological roles of
2 2
SO still remain largely unknown. The detection of SO in cells
is a bottleneck problem.
Ratiometric fluorescent probes can detect trace amounts of
analyte accurately by built-in correction with two emission
4
bands and eliminating the environmental impacts. Till now, a
-
few well-behaved ratiometric fluorescent probes for HSO
2
3
-
5
have been developed. Even so, these probes showed
/SO
3
5
a-
some drawbacks such as unsatisfactory detection limits,
d,5g,5n
5a-d,5f,5i,5p,5q,5s
long response time,
g,5j,5p
inferior selectivity over
5b-e,5k,5r
ultraviolet light excitation or two excitation
5
H
2
S,
Scheme 1 (a) and (b) Two known approaches of FRET dyad. (c) Our new strategy of the
FRET dyad toward bisulfite. (d) Modulation of the FRET in probe HCy-D. Green : donor;
Red: acceptor.
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Probe HCy-D was synthesized by two steps with good yield calculated to be 0.1
Journal Name
ꢀ
Scheme S1, ESI†). The structure was fully characterized by IR, superior to some reported probes
M (S/N = 3, Fig. S3, ESI†), which was
5
f,5g,5p,5r
(
and sensitive enough
5v,5w,8
1
13
-
H NMR, C NMR and HRMS (see ESI). The response to HSO
2
3
to quantitatively detect endogenously produced bisulfite
3
SO was investigated in PBS (10 mM, pH = 7.4, containing 30% Hemicyanine fluorophore was reported to be able to
-
/
-
2-
-
5f,9
DMF). NaHSO
3
was used as source of HSO
coexisted in neutral aqueous solution with a molar light on the reaction mechanism, two compounds, Donor and
ratio of about 3:1. The red-color solution of HCy-D became Acceptor (Scheme S1, ESI†), were synthesized and their
3
/SO
3
since HSO
3
capture nucleophilic agents via 1,2- or 1,4- addition. To shed
2
-
and SO
3
-
2-
colorless upon addition of 10 equiv. of NaHSO
light. Correspondingly, the red fluorescence changed into of the Acceptor was quenched in the presence of 20 equiv. of
green at 365 nm irradiation (Fig. 1a). Thus HCy-D showed great NaHSO , whereas that of the donor was unaffected (Fig. S4,
potential in colormetric and ratiometric fluorescence detection ESI†). What’s more, upon addition of NaHSO to the buffered
. Subsequently, time-dependent fluorescence solution of probe HCy-D, the absorption band peaked at 508
3
under visible reactivity toward HSO
3
/SO
3
was examined. The fluorescence
3
3
-
2-
of HSO
3 3
/SO
-
2-
response to HSO /SO was examined (Fig. 1b, and Fig. S1, nm decreased gradually (Fig. S5, ESI†) and the color of the
3
3
ESI†), which showed that the reaction could be completed in 2 probe solution changed from red to colorless (Fig. 1a),
min, and I530/I582 (the intensity ratio of two emission bands at indicating the interruption of the conjugated structure in HCy-
5
30 nm and 582 nm) peaked and stayed unchanged in 1 h. D. Thus the hemicyanine fluorophore of HCy-D might be
-
Such a rapid response is among the fastest ones (Table S1, responsible for the reaction between probe HCy-D and HSO
2
ESI†), which is appealing for real-time detection. The influence /SO3
of solution pH was also investigated (Fig. 1c). Probe HCy-D
itself is stable in the range of pH 4 to 8.3. After addition of 6 dominant peaks at m/z 579.2951 and 661.2666 were attributed to
3
-
.
Furthermore, the addition was confirmed by HRMS in which two
+
equiv. of NaHSO , I530/I582 increased greatly and peaked from probe HCy-D and [HCy-D + H SO ] , respectively (Fig. S6, ESI†). This
3
2
3
1
pH 6 to 8.3, implying that HCy-D could be used in a wide pH strongly suggested the formation of a 1:1 addition product. Then H
range including physiological conditions.
2
NMR titration was performed in DMSO-d6 (containing 20% D O)
(
Scheme S2c, ESI†). The proton shifts of H1 and H2 disappeared
which indicated that 1,4-addition instead of 1,2-addition was
involved in the reaction. What’s more, two sets of proton signals
5
h,5m,5j,5k,5t
were presented in the spectrum. According to literatures,
we attributed the two sets of signals to compounds adduct a and
adduct b, respectively. We attempted to isolate the two products
but failed due to the starting material (HCy-D) would be retrieved
upon purification, even though the products are stable in reaction
solution. To clarify the reaction mechanism involved, a clean
spectrum (Scheme S2d, ESI†) was got in DMSO-d6 (containing 80%
2
D O) which could be assigned to adduct a and used to compare
with the spectrum that presented two sets of proton signals. Based
on above findings, we deduced that the ratio of adduct a and
adduct b may be influenced by the content of water. The proposed
5
h
mechanism was presented in Scheme S2a.
Notably, the
Fig. 1 (a) Color change of HCy-D solution (10 ꢀM, in 10 mM PBS containing 30% DMF) in
-
2-
the absence or presence of 10 equiv. NaHSO
spectra of HCy-D (5 ꢀM) in the presence of 6 equiv. of NaHSO
fluorescence spectra of HCy-D (5 ꢀM) in the absence or presence of 6 equiv. of NaHSO
d) Fluorescence titration spectra of HCy-D (5 ꢀM) upon addition of NaHSO (0-35 ꢀM).
λex = 410 nm, slit: 10 nm/12 nm.
3
. (b) Time-dependent fluorescence fluorescence change of the probe when it responded to HSO /SO
3 3
3
. (c) pH-Dependent
will not be distinguished by adduct a or adduct b because the
conjugation system in hemicyanine fluorophore was undoubtedly
blocked. The fluorescence of adduct a or b is dominated by the
dansyl fluorophore, whose structure was unaffected by the
reaction.
3
.
(
3
Upon addition of NaHSO3 (0-7 equiv.) to the buffered
solution of probe HCy-D, a fluorescence titration curve was
The fluorescence emission band of the Donor had a high overlap
obtained (Fig. 1d). The characteristic emission band of the level with the absorption band of the Acceptor (Fig. S7, ESI†). The
probe (centered at 582 nm) decreased gradually with a new energy transfer efficiency from the Donor to the Acceptor was
emission band (centered at 530 nm) emerged and increased. A calculated to be 95%. So for HCy-D, the FRET will proceed from the
23-fold enhancement of the fluorescence ratio I530/I582 was dansyl fluorophore to the hemicyanine fluorophore once the energy
observed when about 6 equiv. of NaHSO was added (Fig. S2, donor is excited, which could weaken or even quench the
3
ESI†). This proved the high reaction efficiency between HCy-D
fluorescence of the dansyl fluorophore. However, addition of
-
and HSO /SO , compared with more than 10 equiv. of
2-
3
3
NaHSO will interrupt the π-conjugation system in the hemicyanine
3
5
g-i,5l,5n,5q
NaHSO or Na SO needed for some reported probes.
3
2
3
fluorophore to cancel the FRET process due to the low overlap level
I530/I582 was good linearly proportional to the concentrations of
NaHSO over a range of 0-15 M, and the detection limit was
(
Fig. S7a, ESI†) and restore the fluorescence emission of the dansyl
ꢀ
3
structure. What’s more, both probe HCy-D and the Donor can be
2
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excited at 410 nm but the Acceptor cannot, which further confirms
For a further biological study, we investigated whether the
the FRET process in probe HCy-D (Fig. S7b, ESI†) Theoretical probe could be used for the detection of endogenously
calculation methods have been powerful tools for many chemical produced bisulfite inside cells. Thiosulfate sulphurtransferase
10
processes. Recently, theoretical calculations have been applied to (TST) is a widespread enzyme in nature which commonly
5
i,5l
discuss the sensing mechanism of ICT-based fluorescent probes.
localizes in mitochondrial membrane and mitochondrial
1
matrix. Biological bisulfite can be produced endogenously
2
The sensing mechanism of probe HCy-D toward bisulfite could also
be rationalized by theoretical calculations. The results are
1
from thiosulfate via TST. As TST is abundant in mammalian
3
1
3
5
i,5l
livers, HepG2 cells (human liver cancer cells) and L-02 cells
human normal liver cells) were selected to evaluate the
consistent with reported probes (Scheme S3 and Table S2).
The
(
FRET process was also verified by theoretical calculations with
Gaussian09 (Fig. S8). Compared to most reported ratiometric
probes designed on modulation of the ICT process alone (Table S1,
potential of probe HCy-D for endogenous bisulfite detection.
-
2-
ESI†), our probe for HSO
Scheme S4, ESI†) process.
Selectivity of HCy-D toward HSO
relevant anions and small molecules was investigated (Fig. S9 and
3
/SO
3
was based on a special FRET-ICT
(
-
2-
3
3
/SO
over various biological
-
2-
S10, ESI†). Only HSO
3
and SO
3
led to a big enhancement of
I
530/I582, while others hardly brought about any change to I530/I582
.
-
2-
The excellent selectivity for HSO
3
/SO
3
is desirable, considering the
fact that hemicyanine fluorophores can also be attacked by H S or
2
9
,11
thiols.
3
What’s more, the response of HCy-D to NaHSO was
almost unaffected among potential competitive species (Fig. S11,
ESI†), even in the presence of H S or biological thiols.
2
Now that probe HCy-D had shown a good optical response
-
2-
to HSO /SO in vitro, cell imaging with HCy-D was first
3
3
investigated in HeLa cells. HCy-D was photostable in living cells
(Fig. S12, ESI†) and with negligible cytotoxicity at a
Fig. 3 (a) The first row (vertically): HepG2 cells were incubated with HCy-D (1 ꢀM) for 1
concentration of 1-10
ꢀM (Fig. S13, ESI†). As cationic cyanine h; The second row: HepG2 cells were incubated with HCy-D (1 ꢀM) for 1 h, and then
5
k,5l,9
dyes may accumulate in mitochondria,
a colocalization with 500 ꢀM GSH and 250 ꢀM Na
2
S
2
O
3
for 0.5 h; The third row: HepG2 cells were
incubated with HCy-D (1 ꢀM) for 1 h, then with 10 mM TNBS for 0.5 h, followed by 500
M GSH and 250 ꢀM Na for another 0.5 h; The fourth row: HepG2 cells were
assay with Mito Tracker Deep Red (a mitochondrial dye) and
HCy-D was conducted (Fig. 2). The fluorescence of HCy-D
overlaid very well with that of Mito Tracker Deep Red and an
ꢀ
2 2 3
S O
incubated with HCy-D (1 ꢀM) for 1 h, then with 500 ꢀM GSH for another 0.5 h. (b) From
left to right: the relative ratio of green/red fluorescence intensity of row 1, 2, 3 and 4 in
overlap coefficient was calculated to be 0.94 (Fig. 2d), thereby (a). The ratio images were all obtained as Fgreen/Fred. Images were acquired from 405-
implying a preferential distribution of HCy-D in mitochondria.
555 nm for green fluorescence, and from 560-700 nm for red fluorescence. λex = 405
nm.
HepG2 cells were incubated with probe HCy-D (1
h, washed with PBS buffer, and further incubated with 500
GSH and 250 M Na for 0.5 h before the images were
taken. A clear fluorescence change was observed in HepG2
ꢀM) for 1
ꢀ
M
ꢀ
2 2 3
S O
Fig. 2 HeLa cells were incubated with HCy-D (1 ꢀM) for 1 h, followed by Mito Tracker cells (Fig. 3). By contrast, if HCy-D loaded HepG2 cells were just
Deep Red (1 ꢀM) for 0.5 h. (a) The fluorescence image of HCy-D. λex = 405 nm, λem =
incubated with GSH, no significant fluorescence change was
5
60-640 nm. The red fluorescence was colored as green for discrimination. (b) The
observed. Moreover, HCy-D loaded HepG2 cells were pre-
treated with 10 mM TNBS (2,4,6-trinitrobenzenesulphonate,
known as a TST inhibitor) and then were incubated with 500
fluorescence image of Mito Tracker Deep Red. λex = 639 nm, λem = 640-700 nm. (c)
Merged images of (a) and (b). (d) Colocalization coefficient (Pearson's coefficient) of
HCy-D and Mito Tracker Deep Red was 0.94.
ꢀ
2 2 3
M GSH and 250 ꢀM Na S O for 0.5 h, still no significant
-
2-
fluorescence change was observed (Fig. 3). These results
demonstrated that the probe was capable of detecting
endogenous bisulfite in HepG2 cells, which was comparable to
Then HCy-D was applied to image HSO
3
/SO
3
in living Hela
M) for 1
cells. After Hela cells were incubated with HCy-D (1
ꢀ
h, strong fluorescence in the red channel and weak
fluorescence in the green channel were observed (Fig. S14,
ESI†). Further incubated with different concentrations of
1
3
probes reported.
L-02 cells were incubated with HCy-D (1
concentrations of NaHSO for 0.5 h to induce significant
ꢀ
M) and different
3
3
NaHSO for 0.5 h, the cells displayed a fluorescence decrease
fluorescence change. However, no significant fluorescence
change was observed in L-02 cells incubated with probe HCy-D
in the red channel and a fluorescence increase in the green
channel. The results demonstrated a ratiometric and dose-
-
2-
2 2 3
(1 ꢀM) and GSH/ Na S O (Fig. S15). A comparison of above
results demonstrated that HCy-D could potentially be applied
dependent response of probe HCy-D toward HSO
living cells (Fig. S14b, ESI†).
3
/SO
3
in
to differentiate between liver cancer cells and normal liver
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cells on the basis of their different endogenous bisulfite levels.
This may be ascribed to the different TST content or activity,
Yu, Analyst, 2013, 138, 3018; (h) M. J. Peng, X. F. Yang, B. Yin,
Y. Guo, F. Suzenet, D. En, J. Li, C. W. Li, and Y. W. Duan,
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2
-
different membrane permeability of GSH or S
2
O
3
for these
two kinds of cells or other reasons. Whatever, the new way
may be of prognostic significance at cellular level. Present
cancer diagnostic methods such as magnetic resonance
imaging (MRI), ultrasound, positron emission tomography
2
015, 56, 1; (m) Q. Zhang, Y. Zhang, S. Ding, H. Zhang and G.
(PET) imaging, X-ray imaging exhibit drawbacks including
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limited spatial resolution, high instrument cost and radiological
1
4
hazards. Besides, these methods are often not effective until
1
5
the middle and last stages of cancer. However, this new,
easy-operating method put forward here for the recognition of
liver cancer cells was specific, for relatively abundant TST was
reported only in liver tissues. Our method was more
straightforward because most presented methods for cancer
diagnosis are based on the specific recognition of intracellular
2
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6
or extracellular biomarkers (e.g. over-expressed enzymes).
We anticipate that our method could be applied to liver cancer
related researches in the near future.
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In summary, a ratiometric fluorescent probe based on a new
FRET platform was developed for rapid, sensitive and selective
-
2-
6
(a) L. Yuan, W. Lin, K. Zheng and S. Zhu, Accounts Chem. Res.,
2
3 3
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system should pave a new way for designing ratiometric
fluorescent probes. The probe was mitochondria-targeted and
was successfully applied to detect endogenous bisulfite in
ratiometric and dose-dependent manner, which may be
7
8
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This study was supported by the Natural Science Foundation
of Shandong Province (ZR2014BM004), Major Subject of
Shandong Province (2015ZDJS04003) and the National Natural
Science Foundation of China (91313303).
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