A lysosomal targeting fluorescent probe and its zinc imaging in SH-SY5Y human neuroblastoma cells

Article abstract of DOI:10.1016/j.cclet.2018.03.016

Zinc plays a significant role in oxidative balance and central nervous systems. Herein, we reported a highly sensitive fluorescent probe DR, bearing a morpholine group and a BPEN ligand in the naphthalimide fluorophore. Upon Zn²⁺ binding, DR ex

Full text of DOI:10.1016/j.cclet.2018.03.016

G Model
CCLET 4477 No. of Pages 3
Chinese Chemical Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
A lysosomal targeting fluorescent probe and its zinc imaging
in SH-SY5Y human neuroblastoma cells
a,
c
a
Houna Duana, Yu Dinga, Chusen Huangb, , Weiping Zhu , Rui Wang , Yufang Xu
*
*
a
State Key Laboratory of Bioreactor Engineerring, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science
and Technology, Shanghai 200237, China
The Education Ministry Key Laboratory of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Department of Chemistry,
b
College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234, China
c
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Zinc plays a significant role in oxidative balance and central nervous systems. Herein, we reported a
highly sensitive fluorescent probe DR, bearing a morpholine group and a BPEN ligand in the
naphthalimide fluorophore. Upon Zn2+ binding, DR exhibited remarkable fluorescence enhancement, and
showed high sensitivity to Zn2+ with the association constant of 4.9 Â108 L/mol, and the detection limit of
15 nmol/L. Confocal imaging experiments indicated that DR was able to localize to lysosomes in MCF-7
cells. Moreover, upon H2O2 stimulation in SH-SY5Y cells, endogenous release of Zn2+ was observed.
© 2018 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Received 27 January 2018
Received in revised form 9 March 2018
Accepted 12 March 2018
Available online xxx
Keywords:
Naphthalimide
Zinc ions
Cellular imaging
Lysosome
Fluorescent probe
Zinc, like calcium and iron, as an essential transition metal in
the human body, takes part in many important process of life. Zinc
is not only catalytic center and structural cofactor of many
biomacromolecules, such as enzymes and DNA-binding proteins,
but also a signaling messenger released by many central excitatory
synapses, it also plays an important role in central nervous system
[1]. It has been revealed that the origins and consequences of zinc
signalsare closely related with the incidence of certain diseases,
like prostate cancer [2], Alzheimer’s disease [3], epilepsy [4].
Lysosome degradation, as the final process of autophagy, is also
important in the process of protein degradation [5], and the
degradation process would producewaste protein and other waste
in cells. In general, it works slowly under normal conditions, but
can be influenced by oxidative stress [6]. Disordered lysosome
degradation will hurt normal proteins and organelles, and might
be involved in part of the pathogenesis of many cancers and
neurodegenerative diseases [7–9]. In recent years, it has been
investigated that zinc ions may be related with lysosome
membrane permeabilization (LMP) in oxidative process [10],
and LMP plays important roles in oxidative and zinc-induced
hippocampal neuronal death. In order to explore the relationship
between zinc and LMP, we need an efficient approach to further
study zinc ions in lysosome. Because of Zinc’s 3d104s0 electron
configuration, many of the existing methods are not suitableforthe
detection of zinc in cells [11], like UV spectrum, circular dichroism
spectrum, electron paramagnetic resonance, cyclic voltammetry
and atomic absorption spectrum (AAS).
Fluorescent probe has been provided as an efficiency approach
to detect metal ions in the past decades [12–17]. Roger Tsien first
reported a series of probes for calcium ions, which could imaging
Ca2+ in living cells [18]. From then on, many fluorescent chemical
probes and biosensors have been developed for different metal
ions. Frederickson designed the first fluorescent probe TSQ for Zn2
+, based on an aryl sulfonamide at 1987 [19]. After that, based on
different fluorophores, a large number of fluorescent probes have
been developed for sensing zinc ions. As a typical Zn2+ receptor, di-
2-picolylamine (DPA) has been widely used to construct Zn2+
probe. For example, Lippard et al. reported probes ZPs and ZSs to
detect zinc ions in different living cells, organelles or hippocampal
mossy fiber [20–25]. Wang et al. developed a highly sensitive zinc
probe WZS with the apparent dissociation constant Kd of
0.62 nmol/L [26]. Xu et al. reported a highly selective sensor ZTRS
for ratiometric Zn2+ sensing, which is cell permeable and can be
applied to trace zinc ions during the development of a living
organism [27]. Moreover, Yoon et al. had designed a zinc probe
based on naphthalimide fluorophore, which could image Zn2+
concentration changes in the vicinity of NMDA receptors in live
* Corresponding authors.
E-mail addresses: huangcs@shnu.edu.cn (C. Huang), wpzhu@ecust.edu.cn
(W. Zhu).
https://doi.org/10.1016/j.cclet.2018.03.016
1001-8417/© 2018 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: H. Duan, et al., A lysosomal targeting fluorescent probe and its zinc imaging in SH-SY5Y human
neuroblastoma cells, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.03.016

G Model
CCLET 4477 No. of Pages 3
2
H. Duan et al. / Chinese Chemical Letters xxx (2018) xxx–xxx
cells and tissues [28]. Based on these zinc probes, more and more
evidences showed that intracellular zinc ions are related with
lysosome, H2O2 and other active materials [29,30]. Although some
probes have been developed to detect Zn2+ in lysosome [31–33] to
explore the significance of Zn2+ in lysosomal membrane perme-
abilization, few of them were reported to study the relationship
between zinc ions and oxidative stress in neuroblastoma cells.
Since a protonated morpholine has a pKa value of ꢀ5, a
lysosomal pH, and N,N-bis(2-pyridylmethyl)ethylenediamine
(BPEN) is a good receptor for Zn(II). Herein, we developed a
fluorescent probe DR, which bearing a morpholine group and a
BPEN ligand. By attaching BPEN to the fluorophore through a rigid
benzene framework on the imide moiety, DR exhibited high
sensitivity to Zn(II). And the morpholine moiety in the 4-position
of naphthalimide fluorophore enabled the lysosomal targeting of
the probe (Scheme 1)
800
600
400
200
0
700
600
500
400
300
200
100
0
DR
DR + Zn2+
600
400
200
0
2 4 6 8 10
]
Zn2+ / 10-6
[
4
5
6
7
8
9
10
500 550 600 650
Fig. 1. (A) Changes of fluorescent intensity of DR (black) and DR-Zn2+ (red) under
different pH (adjusted by 1 mol/L HCl or 1 mol/L NaOH) in water (0.5% DMSO)
solution. (B) Fluorescence spectra of DR upon addition of Zn2+. Inset: Changes of
intensity plots of DR vs. different concentrations of Zn2+. Conditions: 0.1 mol/L Tris-
HCl buffer (0.5% DMSO, pH 5.0), [DR] = 5 m
mol/L, lex = 450 nm, lem = 550 nm, 25 ꢁC.
Probe DR can be readily synthesized from 4-nitro-1,8-naph-
thalic anhydride as we previously reported (Scheme S1 in
Supporting information) [34]. Based on the fluorescent recognition
mechanism of photo-induced electron transfer (PET), BPEN was
introduced into the imide moiety of the anphthalimide fluoro-
phore as the receptor of Zn2+, N-(2-aminoethyl)morpholine moiety
was introduced into the 4-position of naphthalimide as the
receptor of hydrogen proton. The introduction of morpholine not
only enables the lysosomal targeting of the probe, but also
improves the aqueous solubility of the probe. The detailed
synthesis procedures of the probe DR were present in Supporting
information.
Table 1
Maximum of adsorption, emission and fluorescence quantum yield of DR and DR-
Zn2+ at different pH.
pH
lex (nm)
lem (nm)
FF
DR
7.4
7.4
5.0
5.0
450
450
450
450
550
550
535
535
0.008
0.049
0.022
0.121
DR-Zn2+
DR
DR-Zn2+
In order to investigate the probe DR’s potential for sensing
lysosomal zinc, we further performed the Zn(II) titration experi-
ments at pH 5.0. As shown in Fig. 1B, upon addition of Zn2+ to the
We first conducted the pH-titration of the probe DR and its Zn
(II) complex. The pH value of the test system was adjusted by HCl
and NaOH solution. As shown in Fig.1A and Table 1, probe DR emits
very weak fluorescence at pH 7.0–10.0 with a fluorescence
quantum yield of 0.009 (pH 7.0), which can be attributed to the
PET processes both from the aniline nitrogen and the morpholine
nitrogen atom. And 2-butyl-6-(butylamino)-1H-benzo[de]isoqui-
noline-1,3(2H)-dione with a fluorescence quantum yield of 0.66 in
ethanol was used as the standard reference in this work [35]. With
the decrease of the pH value from 7.0 to 4.0, the fluorescence
intensity of the probe DR had a little enhancement (FF = 0.022, pH
5.0). This can be explained by that the nitrogen-atom of morpho-
line moiety was protonated gradually, which inhibited the PET
process from the nitrogen-atom of morpholine to fluorophore.
probe’s solution (0–10 mmol/L, 0.1 mol/L Tris-HCl, 0.5% DMSO, pH
5), the fluorescence emission intensity of DR increased gradually
with the increase of the Zn(II) concentration, and reached a plateau
finally. The fluorescence intensity showed a linear enhancement
(correlation coefficient R2 = 0.998, Fig. S1 in Supporting informa-
tion) before the ratio of Zn(II):DR reached 1:1, and the Zn(II)-
binding titrations showed that DR forms a 1:1 complex with Zn(II)
in aqueous solution (Fig. S2 in Supporting information). The
association constant was calculated to be 4.9 Â108 L/mol, and the
detection limit was 15 nmol/L based on LOD = 3s/s. These results
revealed that the probe DR could sense trace lysosomal Zn2+ in
living cells.
When DR combined with Zn2+
, the fluorescence intensity
Next, we explored the specificity of DR (Fig. S3 in Supporting
information). In the presence of Na+, K+, Ca2+ and Mg2+, no
fluorescence enhancement was observed, while the fluorescence
increased remarkably with the further addition of Zn(II). Cu(II)
quenched the fluorescence of DR owing to the paramagnetic effect.
Similar to other Zn2+ probes containing DPA units, Cd2+, Hg2+, Pb2+
also induced fluorescence enhancement, but less than Zn2+. Since
there were few Cd2+, Hg2+, Pb2+ in normal human cells, the probe
DR should have the potential in imaging intracellular zinc ions.
Then we investigated the locating capability of DR in acidic
vesicle of living cells by performing a co-localization experiment
with a commercial marker for lysosomes (LysoTracker1 Red) in live
MCF-7 cells. MCF-7 cells were stained with LysoTracker1 Red and
DR and co-incubated for 1 h, then imaged with confocal
microscopy. As shown in Fig. 2, the images of green channel
(probe DR) and red channel (LysoTracker1 Red) overlapped very
well, suggesting that DR could be used as a lysosome-tracker probe
in living cells.
enhanced at pH 7.0–10.0 (FF = 0.049, pH 7.4). While in the range
of pH 7.5–4.0, the fluorescence intensity increased remarkably,
with 13.4 folds increase of the fluorescence quantum yield from
0.008 (probe DR, pH 7.4) to 0.121 (DR-Zn2+, pH 5.0), which can be
attributed to the suppressed PET processes both from the Zn(II)-
complexed BPEN site as well as from the protonated nitrogen of
morpholine moiety. These results suggested that the probe DR had
potential to stain lysosome in cells and sense free zinc in lysosome.
It is known that zinc(II) is related with oxidative stress in living
systems. For example, when Zn(II)-bound cysteine residues are
oxidized to form disulfide bonds, Zn2+ ions are released to result in
an increase of local Zn(II) concentrations. Such oxidation-effected
Zn(II) release may be linked to the formation of protein aggregates,
which are significant in neurodegenerative diseases such as
Scheme 1. Response mechanism of fluorescent probe DR.
Please cite this article in press as: H. Duan, et al., A lysosomal targeting fluorescent probe and its zinc imaging in SH-SY5Y human
neuroblastoma cells, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.03.016

G Model
CCLET 4477 No. of Pages 3
H. Duan et al. / Chinese Chemical Letters xxx (2018) xxx–xxx
3
transfer from the aniline nitrogen and the morpholine nitrogen to
fluorophore at pH ꢀ7.4. Upon binding Zn2+, the fluorescence
quantum yield of DR enhanced about 13.4 folds at pH ꢀ5.0, which
exhibited high sensitivity to Zn2+ and the detection limit of was
calculated to be 15 nmol/L. Confocal imaging experiments indicat-
ed that DR was able to localize to lysosomes in MCF-7 cells.
Moreover, upon H2O2 stimulation in SH-SY5Y cells, endogenous
release of Zn2+ was observed, which was demonstrated by the
fluorescence changes after the cells were treated with H2O2 and
the membrane-permeable zinc chelator TPEN.
Acknowledgments
We thank the National Natural Science Foundation of China (No.
21476077) for financial support. We also very much appreciate
Prof. Xuhong Qian’s valuable suggestions in the preparation of this
manuscript.
Fig. 2. Confocal fluorescence images of MCF-7 cells: (A) Stained with DR (5 mmol/L,
lex = 488 nm, lem = 520–570 nm), (B) stained with LysoTracker Red (50 nmol/L,
lex = 561 nm, lem = 570–675 nm), (C) merge of (A) and (B), (D) the bright field image.
Appendix A. Supplementary data
All the cells were magnified 1560 times. Scale bar: 15 mm.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.cclet.2018.03.016.
References
[1] N. Roohani, R. Hurrell, R. Kelishadi, R. Schulin, J. Res. Med. Sci. 18 (2013) 144–
157.
[2] R.B. Franklin, L.C. Costello, Arch. Biochem. Biophys. 463 (2007) 211–217.
[3] O. Timofeeva, J.V. Nadler, Brain Res. 1078 (2006) 227–234.
[4] K. Golovine, R.G. Uzzo, P. Makhov, et al., Prostate 68 (2008) 1443–1449.
[5] G. Kroemer, M. Jäättelä, Nat. Rev. Cancer 5 (2005) 886–897.
[6] M. Kundu, C.B. Thompson, Annu. Rev. Pathol. Mech. Dis. 3 (2008) 427–455.
[7] Y. Uchiyama, M. Koike, M. Shibata, M. Sasaki, Methods Enzymol. 453 (2009)
33–51.
[8] B. Levine, Nature 446 (2007) 745–747.
[9] B. Levine, G. Kroemer, Cell 132 (2008) 27–42.
[10] J.J. Hwang, S.J. Lee, T.Y. Kim, et al., J. Neurosci. 28 (2008) 3114–3122.
[11] S.Y. Assaf, S.H. Chung, Nature 308 (1984) 734–736.
[12] K.P. Carter, A.M. Young, A.E. Palmer, Chem. Rev. 114 (2014) 4564–4601.
[13] Z. Xu, J. Tang, H. Tian, Chin. Chem. Lett. 19 (2008) 1353–1357.
[14] H.Y. Zhao, Y.M. Li, T.J. Gong, Q.X. Guo, Chin. Chem. Lett. 22 (2011) 1013–1016.
[15] C.X. Ding, C.H. He, Y.S. Xie, Chin. Chem. Lett. 24 (2013) 463–466.
[16] W. Feng, Q.L. Qiao, S. Leng, et al., Chin. Chem. Lett. 27 (2016) 1554–1558.
[17] M.N. Su, J. Ye, Q.W. Li, et al., Chin. Chem. Lett. 26 (2015) 1400–1402.
[18] R.Y. Tsien, Biochemistry 19 (1980) 2396–2404.
Fig. 3. Fluorescence images of DR (5
cultured with H2O2, (C) SH-SY5Y cells cultured with H2O2 and 10
F) bright field images of (A), (B) and (C), respectively. The excitation light was blue
light and cells were magnified 200 times.
m
mol/L) in (A) SH-SY5Y cells, (B) SH-SY5Y cells
m
mol/LTPEN, (D, E,
[19] C.J. Frederickson, E.J. Kasarskis, D. Ringo, R.E. Frederickson, J. Neurosci.
Methods 20 (1987) 91–103.
Alzheimer’s and Parkinson’s [36]. Hence, we finally used the probe
DR to investigate the effect of oxidative stress in SH-SY5Y cells. In
this experiment, we treated SH-SY5Y cells in three different ways:
(A) SH-SY5Y cells were stained with DR; (B) SH-SY5Y cells were co-
incubation with H2O2 for 1 h, then stained with DR; (C) SH-SY5Y
cells were co-incubation with H2O2 for 1 h, followed by the additon
of TPEN (one kind of chelating agent for zinc) and stained with DR.
As shown in Fig. 3, after SH-SY5Y cells were treated with H2O2, the
green fluorescence enhanced significantly (Fig. 3B) compared with
those non-treated cells (Fig. 3A), which revealed the release of Zn2+
after stimulating by H2O2. When cells were simultaneously treated
with H2O2 and TPEN, the fluorescence got very dark (Fig. 3C),
which further indicated that such fluorescence changes were
related to the changes of Zn(II) concentrations in SH-SY5Y cells.
These results demonstrate that DR has the potential in imaging free
zinc in human neuroblastoma cells.
[20] S.C. Burdette, G.K. Walkup, B. Spingler, R.Y. Tsien, S.J. Lippard, J. Am. Chem. Soc.
123 (2001) 7831–7841.
[21] X.A. Zhang, D. Hayes, S.J. Smith, S. Friedle, S.J. Lippard, J. Am. Chem. Soc. 130
(2008) 15788–15789.
[22] C.C. Woodroofe, S.J. Lippard, J. Am. Chem. Soc. 125 (2003) 11458–11459.
[23] B.A. Wong, S. Friedle, S.J. Lippard, J. Am. Chem. Soc. 131 (2009) 7142–7152.
[24] R.J. Radford, W. Chyan, S.J. Lippard, Chem. Sci. 4 (2013) 3080–3084.
[25] M. Khan, C.R. Goldsmith, H. Zhen, et al., Proc. Natl. Acad. Sci. U. S. A. 111 (2014)
6786–6791.
[26] J. Wang, Y. Xiao, Z. Zhang, et al., J. Mater. Chem. 15 (2005) 2836.
[27] Z. Xu, K.H. Baek, H.N. Kim, et al., J. Am. Chem. Soc. 132 (2010) 601–610.
[28] X. Chen, C.S. Lim, D. Lee, et al., Biosens. Bioelectron. 91 (2017) 770–779.
[29] S.J. Lee, J.Y. Koh, Mol. Brain 3 (2010) 30–38.
[30] L.M. Hyman, K.J. Franz, Coord. Chem. Rev. 256 (2012) 2333–2356.
[31] H. Zhu, J. Fan, S. Zhang, et al., Biomater. Sci. 2 (2014) 89–97.
[32] S.L. Kelleher, N.H. McCormick, V. Velasquez, V. Lopez, Adv. Nutr. 2 (2011) 101–
111.
[33] L. Xue, G. Li, D. Zhu, Q. Liu, H. Jiang, Inorg. Chem. 51 (2012) 10842–10849.
[34] X. Qian, Y. Xiao, Y. Xu, et al., Chem. Commun. 46 (2010) 6418–6436.
[35] D. Yuan, R.G. Brown, J.D. Hepworth, M.S. Alexiou, J.H.P. Tyman, J. Heterocycl.
Chem. 45 (2008) 397–404.
In this work, we had designed a fluorescent sensor DR based on
BPEN ligand and the target group morpholine. DR had a low
fluorescence quantum yield (FF = 0.008) owing to the electron
[36] L. Zhu, Z. Yuan, J.T. Simmons, K. Sreenath, RSC Adv. 4 (2014) 20398–20440.
Please cite this article in press as: H. Duan, et al., A lysosomal targeting fluorescent probe and its zinc imaging in SH-SY5Y human
neuroblastoma cells, Chin. Chem. Lett. (2018), https://doi.org/10.1016/j.cclet.2018.03.016