A water-soluble “turn-on” fluorescent probe for specifically imaging mitochondria viscosity in living cells

Article abstract of DOI:10.1016/j.saa.2018.05.121

Rational design of water-soluble probes for mitochondrial viscosity in practical biological applications remains a challenge. Herein, we described a novel hydro soluble benzothiazole salt derivative MitoSN, which exhibits specifically response and singular sensitivity to the mitochondria viscosity in living Hela cells. MitoSN displays an excellent fluorescence enhancement (ca. 35-fold) with the increase of the viscosity in the water-glycerol system. Moreover, confocal microscopy indicates that MitoSN is sensitive to the local viscosity and selectively stains mitochondria, the body of zebrafish as well. Importantly, MitoSN is capable to identify the viscosity difference of mitochondria in normal and nystatin treated Hela cells. The work provides a useful tool to monitor the changes of viscosity in the mitochondrial microenvironment.

Full text of DOI:10.1016/j.saa.2018.05.121

Accepted Manuscript
A water-soluble “turn-on” fluorescent probe for specifically
imaging mitochondria viscosity in living cells
Hui Wang, Bin Fang, Lufei Xiao, Di Li, Le Zhou, Lin Kong, Yan
Yu, Xiangzi Li, Yunjun Wu, Zhangjun Hu
PII:
S1386-1425(18)30521-3
doi:10.1016/j.saa.2018.05.121
SAA 16155
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 2 April 2018
Revised date:
Accepted
date:
27 May 2018
2
9 May 2018
Please cite this article as: Hui Wang, Bin Fang, Lufei Xiao, Di Li, Le Zhou, Lin Kong, Yan
Yu, Xiangzi Li, Yunjun Wu, Zhangjun Hu , A water-soluble “turn-on” fluorescent probe
for specifically imaging mitochondria viscosity in living cells. Saa (2017), doi:10.1016/
j.saa.2018.05.121
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ACCEPTED MANUSCRIPT
A water-soluble “turn-on” fluorescent probe for
specifically imaging mitochondria viscosity in living
cells
a, ,†
b,†
c,*
a
a
b
a
a
Hui Wang * , Bin Fang , Lufei Xiao , Di Li , Le Zhou , Lin Kong , Yan Yu , Xiangzi Li ,
Yunjun Wu , Zhangjun Hud
a
[
a] Department of Chemistry, Anhui provincial engineering research center for polysaccharide
drugs, Wannan Medical College, Wuhu 241002, People's Republic of China
[
b]Department of Chemistry, Key Laboratory of Functional Inorganic Material Chemistry of
Anhui Province, Anhui University, Hefei 230601, P. R. China
c] Department of Food and Environmental Engineering, Chuzhou Vocational and Technical
college, Chuzhou 239000, P. R. China;
d] Division of Molecular Surface Physics & Nanoscience, Department of Physics, Chemistry and
[
[
biology (IFM), Linköping University, 58183, Linköping, Sweden
E-mail address: wanghias@126.com; xlf3345@163.com
†
These authors contributed equally to this work and should be considered co-first authors.
Abstract
Rational design of water-soluble probes for mitochondrial viscosity in practical
biological applications remains a challenge. Herein, we described a novel hydro
soluble benzothiazole salt derivative MitoSN, which exhibits specifically response
and singular sensitivity to the mitochondria viscosity in living Hela cells. MitoSN
displays an excellent fluorescence enhancement (ca. 35-fold) with the increase of the
viscosity in the water-glycerol system. Moreover, confocal microscopy indicates that
MitoSN is sensitive to the local viscosity and selectively stains mitochondria, the
body of zebrafish as well. Importantly, MitoSN is capable to identify the viscosity
difference of mitochondria in normal and nystatin treated Hela cells. The work

ACCEPTED MANUSCRIPT
provides a useful tool to monitor the changes of viscosity in the mitochondrial
microenvironment.
Keywords: water-soluble; mitochondrial targeting; viscosity sensing; benzothiazole
salt; zebrafish
1
. Introduction
Intracellular viscosity plays an important part in the process of cellular diffusion,
including bio-macromolecular interactions and diffusion of reactive metabolites [1-5].
Abnormal changes in the viscosity at cellular level accompany with a number of
diseases and malfunctions, such as atherosclerosis and Alzheimer’s disease [6].
Moreover, at the sub-organelle levels, mitochondrial viscosity could influence the
signaling interactions of chemicals and biomolecules. The abnormal viscosity in the
mitochondrial matrix could induce the change of mitochondrial network organization
and further influence metabolite diffusion, which is related to many diseases and
malfunctions in cellular aspects [7-8]. For instances, Lin et al. presented two new
mitochondrial-targeted fluorescent viscosity probes [9]. Very recently, Lin’s group
reported a ratiometric two-photon fluorescent probe to monitor the changes of
mitochondrial viscosity [10]. Tian and co-workers developed a water-soluble
two-photon fluorescence sensor, which is specifically responsive to the mitochondrial
viscosity in living cells [11]. Thus, it is significant to rationally design and synthesize
mitochondrial-targeted probes to monitor intracellular mitochondrial viscosity at
subcellular level.
In recent years, molecular rotor has been regarded as a sort of typical fluorophore,
in which the fluorescence relies on the rate of intramolecular rotation. In non-viscous
media, the probe coupled with a rotor moiety can give rise to a weak intrinsic
fluorescence, whereas in viscous media, the inhibited rotation reduces the probability
of non-radiative pathways and subsequently restores the fluorescence [12-17].
Therefore, such biocompatible molecular fluorophores containing molecular rotors
are potentially used as viscosity-sensitive fluorescent chemosensors in biosystem.

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In this work, a water-soluble molecule (MitoSN) with a rotor unit was designed
and synthesized (Scheme 1). Firstly, ethyl acetate group contributes the lipid
solubility and biocompatibility. Secondly, benzothiazole is an electron-withdrawing
building block with high photophysical stability. Thirdly, the benzothiazole cation
moiety not only works as targeting mitochondria moiety, but enhances the
water-solubility of the resulted MitoSN. As expected, MitoSN exhibits excellent
solubility and low cytotoxicity. The cellular studies demonstrate that MistoSN is able
to target mitochondria and exhibit obvious enhanced fluorescent response to
mitochondrial viscosity. Therefore, the “turn-on” fluorescent probe for mitochondrial
viscosity is achieved.
2
2
. Experiments
.1. Materials and apparatus
All chemicals were purchased as reagent grade and used without further
purification. The products were performed with a PerkineElmer 240 analyzer. IR
-1
spectra (4000-400 cm ), in KBr pressed pellets, were obtained on a Nicolet
1
13
FT-IR-870SX spectrophotometer. The H NMR and C NMR spectra were recorded
on at 25 ℃ using Bruker 400 Ultrashield spectrometer. Mass spectrum was recorded
on HRMS-LTQ Obritrap XL (ESI source).The UV-vis absorption spectra were
measured on a UV-1700 spectrophotomter. The one-photon excited fluorescence
spectra measurements were measured on Hitachi F-4600 fluorescence
spectrophotometer.
The fluorescence quantum yields (Φ) were determined by using fluorescein as the
reference according to the literature method [18].
2
.2 Synthetic of MitoSN
Preparation of M
2
-Methylbenzothiazole (1.49 g, 10 mmol) and ethyl 2-bromoacetate (1.67 g, 10
mmol) were mixed in 50 mL toluene. The solutions was refluxed for 24 h, then the
precipitate produced was filtered, washed with toluene and fried in vacuo to obtain

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l
white solid. Yield: 90%. H NMR (D
2
O,400 MHz) δ: 8.07 (d, J = 8.0 Hz, 1H), 7.90 (d,
J = 8.6 Hz, 1 H), 7.73 (d, J = 7.7 Hz, 1 H), 7.68 (m, 1 H), 5.58 (s, 2 H), 4.70 (s, 2 H),
4
.19 (q, J = 7.2 Hz, 2 H), 3.05 (m, 1 H), 1.16 (t, J = 7.2 Hz, 3 H).
Preparation of MitoSN
Using a 100 mL one-necked flask fitted with a stirrer and a condenser, 3.26 g (10
mmol) of M, 1.22 g (10 mmol) of p-Hydroxybenzaldehyde and 30 mL of absolute
ethanol were mixed. Three drops of piperidine were added to the solutions and
refluxed for 24 h. After cooling, the solution was filtered, and washed thrice with
-1
ether. Yellow solid product MitoSN was collected. Yield: 87 %. IR (KBr, cm )
selected bands: 3083, 2927, 1743, 1583, 1517, 1443, 1485, 1443, 1395, 1339, 1298,
1
d
8
2
273, 1214, 1170, 1050, 966, 828, 764, 635, 512, 490. 1H NMR (400 MHz,
-DMSO) δ : 10.71 (s, 1 H), 8.44 (d, J = 7.9 Hz, 1 H), 8.27 (d, J = 15.5 Hz, 1 H),
.15 (d, J = 8.4 Hz, 1 H), 7.97 (d, J = 8.7 Hz, 2 H), 7.90 (m, 3 H), 6.96 (d, J = 8.7 Hz,
6
1
3
H), 6.01 (s, 2 H), 4.25 (q, J = 7.1 Hz, 2 H), 1.25 (t, J = 7.1 Hz, 3 H). C NMR (100
-DMSO) δ: 173.9, 165.7, 162.6, 151.1, 141.1, 133.0, 129.5, 128.2, 126.9,
25.3, 124.4, 116.3, 109.4, 62.4, 49.2, 13.9. m/z : 340.09 ( [M] +).
MHz, d
6
1
3
3
. Results and Discussions
.1 Synthesis and spectral properties of MitoSN
The synthetic process of MitoSN was presented in Scheme 2. It can be easily
1
13
synthesized in two steps. MitoSN was fully characterized by H NMR, C NMR,
ESI-MS and IR spectra (ESI, Fig S1-S4). UV-vis absorption spectra of MitoSN in
different solvents were studied (Table S1). As shown in Fig S5, the absorption band
of MitoSN exhibits one band between 350 and 500 nm, which could be assigned to
the intramolecular charge transfer (ICT) mixed with π-π* transition, as confirmed by
TD-DFT calculations (Fig S6 and Table S2). The fluorescence emission peak of
MitoSN around 520 nm was observed when excited at 420 nm. The fluorescence
quantum yields of MitoSN are quite low in these solvents (Table S1), which is favor
of the bioimaging application due to its low background fluorescence. Moreover, a
weak solvatochromism was observed with the increasing solvent polarities, which is

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very important to the molecular rotor for sensing the intracellular viscosity [19-21].
Furthermore, the water solubility were also studied via UV-vis absorption spectra. As
displayed in Fig S7, the absorbance is linear to the concentration in the region of
1
0-100 μM, revealing MitoSN possessed good water solubility (100 μM), which is a
significant advantage for biological application.
3
.2 Response to viscosity by the naked eye
The viscosity-sensitive response of MitoSN was performed and presented in Fig 1.
MitoSN exhibited weak fluorescence and low quantum yields (QY < 0.01) in
non-viscous solution. In stark contrast, in high viscous solutions, a strong emission
peak at 530 nm was obviously observed (Fig 1a). The fluorescence intensity of
MitoSN enhanced about 35-fold (QY = 0.10) with the viscosity of the solutions
increasing from 1.0 cP (water) to approximately 950 cP (99% glycerol). Moreover,
the obvious “turn-on” fluorescence response of MitoSN could be observed by the
2
naked eye (inset of Fig 1a). In addition, a better linear relationship (R = 0.992, the
slope x = 0.495) was obtained between the logarithm of the fluorescence intensity
ratio (log(I/I )) and the logarithm of the viscosity (log η). These results revealed that
0
MitoSN could be worked as fluorescent probe to quantitatively detect the
environmental viscosity.
The mechanism of the light-switch effect of MitoSN can be explained by the
molecular rotor effect [22-23]. In non-viscous solution, MitoSN was non-emissive
due to that the free rotation of the vinyl bond induced a non-radiative process,
resulting in quenched fluorescence. However, in the viscous solutions, the rotation is
restricted, restoring the fluorescence enhancement [24-25]. Besides, the rotor
mechanism was further evidenced by the absorption spectra. As depicted in Fig S8, an
obvious red-shift was clearly observed from 418 nm to 433 nm, suggesting probe
MitoSN might take more plane shape to extend the degree of conjugation in viscous
media compared to non-viscous media.
3
.3. Biological imaging application

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To further investigate the potential application of probe MitoSN in biological
imaging, the cytotoxicity test was firstly evaluated by MTT assay. As shown in Fig
S9, after over 24 hours of incubation with probe MitoSN, high cell viability was
obtained at the concentrations between 5 μM and 25 μM. The results elucidated that
MitoSN in such concentration range exhibits low toxicity in living cells.
Cell-staining experiment was carried out under confocal microscopy. As displayed
in Fig 2, MitoSN could penetrate the cell cytosol within a short incubation period (30
min) and exhibited intense luminescence. To demonstrate the intracellular localization
of probe MitoSN, co-staining experiments within Mitotracker deep red (MTDR) were
performed. The fluorescence of probe MitoSN overlapped well with that of MTDR at
a Pearson’s coefficient Rr = 0.92 (Fig 2a and 2b). Fig 2c showed that the intensity
profiles of probe MitoSN and MTDR are nearly overlapping, which indicated that
probe MitoSN precisely targeted mitochondria in live cells.
Mitochondrial membrane potential (ΔΨm) is an important parameter related to
mitochondrial metabolic function [26]. CCCP, one of the representatives uncouples
for mitochondria-related pathology and pharmacology was chosen to examine
whether ΔΨm could influence the fluorescence signals of MitoSN. As shown in Fig 3,
after treatment with CCCP, the fluorescence intensity of MitoSN exhibited distinct
weakening, which implied that MitoSN was localized in mitochondria that may be
membrane potential dependent.
Comparing to the relatively aqueous cytoplasm, mitochondrial matrix with various
fundamental materials possesses higher viscosity, including DNA, enzymes,
ribosomes, and nucleotide cofactors. To investigate the potential application of probe
MitoSN as a mitochondrial-viscosity probe, confocal imaging was performed. Hela
cells were pretreated with nystatin, a well-known ionophore which could induce
mitochondrial viscosity alterations [24], and then incubated with probe MitoSN, Fig 3
indicated that the fluorescence of MitoSN increased after treatment with nystatin. The
results indicated that MitoSN possessed an appropriate mitochondrial targeting ability,
which is in accord with mitochondrial membrane potential and it can be applied to
detect abnormal mitochondrial-viscosity changes induced by nystatin.

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Encouraged by the above results, we further explored the capability of MitoSN for
mapping microviscosity in vivo. A five-day-old zebrafish was exposed to 10 μM
MitoSN at different time. As displayed in Fig 4a, when a three-day-old zebrafish was
prepared with MitoSN for 20 min, a bright green fluorescence was detected.
Moreover, the merge images revealed that the bright fluorescence was distributed
over almost the whole body of the zebrafish. After 1 hour, a significant luminescence
was observed in the liver and the whole body (Fig 4b). Moreover, the fluorescent
probe MitoSN was primarily on the liver of the zerafish after 2 hours (Fig 4c). We
hypothesized that the probe MitoSN might slowly be transferred to the liver, and the
probe may eventually be metabolized by the liver. These results revealed that MitoSN
could act as a potential candidate to visualize the micro-viscosity in vivo.
4
. Conclusions
In summary, a novel water-soluble MitoSN as a fluorescent viscosity probe was
developed. MitoSN exhibits a remarkable turn-on” fluorescent response (ca. 35-fold
enhancement) towards the increasing viscosity in the water-glycerol system. Cellular
studies demonstrated that MitoSN was capable of imaging intracellular mitochondria
viscosity. Furthermore, MitoSN was also successfully employed to visualize the
microviscosity of live zebrafish larva in vivo. The valuable findings related to the
rational design in this work could be a useful source to develop new
mitochondrial-targeting viscosity probes for the mitochondria microenvironment.
Acknowledgements
This work was supported by grants from the Doctoral Startup Foundation of
Wannan Medical College (wYRCQD201715), Natural Science Foundation of Anhui
Province (1508085SMB208). The Science Foundation of Department Education of
Anhui Province (KJ2017ZD49), and the Talent Foundation of the Education
Department of Anhui (gxbjZD2016111).

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References:
[
1] Y. Baek, S. J. Park, X. Zhou, G. Kim, H. M. Kim, J. Yoon. Biosens. Bioelectron.,
6 (2016) 885-891.
2] P. Ning, P. Y. Dong, Q. Geng, L. Bai, Y. Q. Ding, X. H. Tian, R. Shao, L. Li, X.
M. Meng. J. Mater. Chem. B., 5 (2017) 2743-2749.
3] D. D. Li, X. H. Tian, A. D. Wang, L. J. Guan, J. Zheng, F. Li, S. L. Li, H. P. Zhou,
J. Y. Wu, Y. P. Tian. Chem. Sci., 3 (2016) 2257-2263.
4] M. G. Ren, B. B. Deng, K. Zhou, X. Q. Kong, J. Y. Wang, W. Y. Lin. Anal.
Chem., 89 (2017) 552-555.
5] P. S. Sherin, I. Lopez-Duarte, M. R. Dent, M. Kubankova, A. Vysniauskas, J. A.
8
[
[
[
[
Bull, E. S. Reshetnikova, A. S. Klymchenko, Y. P. Tsentalovich, M. K. Kuimov.
Chem. Sci., 8 (2017) 3523-3528.
[
6] G. Bosman, I. G. P. Bartholomeus, W. J. De Grip. Gerontology., 37 (1991)
5-112.
7] Deliconstantinos, George, V. Villiotou, J. C. Stavrides. Biochemical.
Pharmacology., 49 (1995)1589-1600.
9
[
[
[
8] S. J. Singer, G. L. Nicolson. Science., 175 (1972) 720-731.
9] K. Zhou, M. G. Ren, B. B. Deng, W. Y. Lin. New. J. Chem., 41 (2017)
1
1507-11511.
10] M. G. Ren, K. Zhou, L. Wang, K. Y. Liu, W. Y. Lin. Sens Actuat B., 262 (2018)
52-459.
11] M. Zhao, Y. Z. Zhu, J. Su, Q. Geng, X. H. Tian, J. Zhang, H. P. Zhou, S.Y.
Zhang, J. Y. Wu, Y. P. Tian. J. Mater. Chem. B., 35 (2016) 5907-5912.
12] X. Dai, B. L. Dong, M. G. Ren, W. Y. Lin. J. Mater. Chem. B., 6 (2018)
81-385.
[
4
[
[
3
[
[
13] X. Song, N. Li, C. Wang, Y. Xiao. J. Mater. Chem. B., 5 (2017) 360-368.
14] M. H. Lee, N. Park, C. Yi, J. H. Han, J. H. Hong, K. P. Kim, D. H. Kang, J. L.
Sessler, C. Kang, J. S. Kim. J. Am. Chem. Soc., 136 (2014)14136-14142.

ACCEPTED MANUSCRIPT
[
(
[
15] A. C. Benniston, A. Harriman, V. L. Whittle, M. Zelzer. Eur. J. Org. Chem., 3
2010) 523-530.
16] J. Sutharsan, D. Lichlyter, N. E. Wright, M. Dakanali, M. A. Haidekker, E. A.
Theodorakis. Tetrahedron., 66 (2010) 2582-2588.
[
(
[
(
[
17] X. D. Yin, Y. J. Li, Y. L. Zhu, X. Jing, Y. L. Li, D. B. Zhu. Dalton. Trans., 39
2010) 9929-9935.
18] X. D. Yin, Y. J. Li, Y. L. Zhu, X. Jing, Y. L. Li, D. B. Zhu. Dalton. Trans., 41
2010) 9929-9935.
19] D. Miguel, S. P. Morcillo, A. Martin-Lasanta, N. Fuentes, L. Martinez-Fernandez,
I. Corral, M. J. Ruedas-Rama, D. J. Cardenas, L. A. D. Cienfuegos, A. Orte, J. M.
Cuerva. Org. lett., 17 (2015) 2844-2847.
[
20] M. A. Haidekker, T. P. Brady, D. Lichlyter, E. A. Theodorakis. J. Am. Chem.
Soc., 128 (2006) 398-399.
21] L. Wang, Y. Xiao, W. M. Tian, L. Z. Deng. J. Am. Chem. Soc., 135 (2013)
903-2906.
22] M. A. Haidekker, T. P. Brady, D. Lichlyter, E. A. Theodorakis. Bioorg. Chem.,
3 (2005) 415-425.
23] F. Liu, T. Wu, J. F. Cao, S. Cui, Z. G. Yang, X. X. Qiang, S. G. Sun, F. L. Song,
J. L. Fan, J. Y. Wang, X. J. Peng. Chem. Eur. J., 19 (2013) 1548-1553.
24] Z. G. Yang, J. F. Cao, Y. X. He, J. H. Yang, T. Kim, X. J. Peng, J. S. Kim. Chem.
Soc. Rev., 13 (2014) 4563-4601.
25] Z. G. Yang, Y. X. He, J. H. Lee, N. Park, M. Suh, W. S. Chae, J. F. Cao, X. J.
Peng, H. Jung, C. Kang, J. S. Kim. J. Am. Chem. Soc., 135 (2013) 9181-9185.
26] F. Miao, W. J. Zhang, Y. M. Sun, R. Y. Zhang, Y. Liu, F. Q. Guo, G. F. Song, M.
G. Tian, X. Q. Yu. Biosens. Bioelectron., 55 (2014) 423-429.
[
2
[
3
[
[
[
[

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Scheme 1. The molecular structure and proposed mechanism of MitoSN for viscosity
response.
Scheme 2. The synthesis routes of MitoSN.
Figure 1. (a) Changes of fluorescence spectra with variation of solution viscosity in
water–glycerol system. (b) The fluorescence quantum yields of MitoSN with increasing
solution viscosity. (c) The linear response between log (I /I
0
) for MitoSN and log(viscosity) in
the water/glycerol solvent.
Figure 2. (a) Colocalization fluorescence microscopy images of Hela cells treated with 10 µM
MitoSN and Mito-Tracker Red. (b) Correlation plot of Mito-Tracker deep Red and MitoSN
intensities; (c) Intensity profile of ROIs across Hela cells. Scale bar =20 µm.
Figure 3. Confocal microscopy images of Hela cells incubated with 10 μM MitoSN before
and after treatment by CCCP and nystatin, respectively. Scale bar =20 µm.
Figure 4. Confocal fluorescence images of zebrafish (3-day-old) after being treated with
MitoSN (10 μM) in water for 20 min (a), 1h (b) and 2 h (c). The scale bars: 100 μm.

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Highlights
MitoSN possesses good hydrosoluble.
MitoSN exhibited ca. 35-fold fluorescence enhancement with the increasing
viscosity.
MitoSN was sensitive to local viscosity and selectively stained mitochondria in living
cells.
MitoSN displayed a significant luminescence in the whole body of zebrafish.

Graphics Abstract

Figure 1

Figure 2

Figure 3

Figure 4