A red-emitting fluorescent probe imaging release of calcium ions from lysosome induced by chloroquine based on a photochromic crowned spirobenzopyran

Article abstract of DOI:10.1016/j.dyepig.2021.109467

Calcium ions play an important role in intracellular signaling, metabolism and a wide range of cellular processes. An abnormal calcium concentration may cause many adverse symptoms, such as physiological responses in obesity, immune responses and pathological responses in Alzheimer's disease. Therefore, the development of specific fluorescent probes for monitor Ca²⁺ in living organism is of great significance. Herein, a red-emitting probe SP-CE was successfully designed and synthesized based on spiropyran derivatives as the fluorophore and aza-15-crown-5 as the sensing moiety for the recognition of calcium ions. Under the ultraviolet light irradiation, SP-CE was transformed to merocyanine (MC) form, which releasing negative oxygen ions, thereby achieving the recognition of calcium ions with the collaboration of crown ethers. According to the literature, chloroquine (CQ) could stimulate the up-regulation of pH in the sub-organelle lysosome in cells, resulting in changes of the calcium ions abundance in the cytoplasm. Interestingly, SP-CE could be used to visualize the occurrence of the above mentioned physiological processes in living cells. In addition, the probe SP-CE has been successfully employed for labeling calcium ions in living zebrafish model.

Full text of DOI:10.1016/j.dyepig.2021.109467

Dyes and Pigments 193 (2021) 109467
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A red-emitting fluorescent probe imaging release of calcium ions from
lysosome induced by chloroquine based on a photochromic
crowned spirobenzopyran
,
,
,c,*
Yingchun Wua c, Caixia Yinb d, Weijie Zhangb, Jianbin Chaoa, Fangjun Huoa
a Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China
b Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China
c School of Chemistry and Chemical Engineering, Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China
d Shanxi Laboratory for Yellow River, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Calcium ions play an important role in intracellular signaling, metabolism and a wide range of cellular processes.
An abnormal calcium concentration may cause many adverse symptoms, such as physiological responses in
obesity, immune responses and pathological responses in Alzheimer’s disease. Therefore, the development of
specific fluorescent probes for monitor Ca2+ in living organism is of great significance. Herein, a red-emitting
probe SP-CE was successfully designed and synthesized based on spiropyran derivatives as the fluorophore
and aza-15-crown-5 as the sensing moiety for the recognition of calcium ions. Under the ultraviolet light irra-
diation, SP-CE was transformed to merocyanine (MC) form, which releasing negative oxygen ions, thereby
achieving the recognition of calcium ions with the collaboration of crown ethers. According to the literature,
chloroquine (CQ) could stimulate the up-regulation of pH in the sub-organelle lysosome in cells, resulting in
changes of the calcium ions abundance in the cytoplasm. Interestingly, SP-CE could be used to visualize the
occurrence of the above mentioned physiological processes in living cells. In addition, the probe SP-CE has been
successfully employed for labeling calcium ions in living zebrafish model.
Calcium ion
Spiropyran
Chloroquine
Aza-15-crown-5
1. Introduction
Fluorescent probes based on small organic molecules combined with
fluorescence imaging technology, using their unique advantages of good
Ca2+ plays a major role that transmits in intracellular signaling and a
variety of intracellular processes [1–4]. However, dysregulation of
intracellular Ca2+ signaling is related to physiological responses in
obesity, immune responses and pathological responses in Alzheimer’s
disease [5–11]. Therefore, it is important to track the dynamic changes
of calcium concentration in cytosolic and understand the Ca2+ signal
pathways in cells [12]. In addition, according to the literature reported
that chloroquine (CQ) could cause calcium ion release by increasing
lysosomal pH. Importantly, blockading lysosomal calcium ions release
disrupts CQ-induced M1 macrophages polarisation, suggesting that
lysosomal calcium release is necessary [13]. Therefore, a specific and
ultra-fast detection tool is urgently needed for visualizing the fluctuation
of calcium ion abundance in living cells and in vivo, which is essential for
preventing related diseases by formulating corresponding intervention
strategies.
specificity, high sensitivity, and in-situ monitoring, have become
important research methods in modern life sciences and disease diag-
nosis [14–17]. To date, great efforts have been made to monitor metal
ions based on crown ether recognition sites [18,19]. For example,
Heng’s research group designed a light-controllable spiropyran revers-
ible sensor optical probe for calcium ion detection [20]. Subsequently,
Yin’s research group designed a fluorescent probe based on photo-
chromic crown ether spiropyran for lithium ion detection [21]. How-
ever, these fluorescent probes for monitoring metal ions were rarely
exploited at the level of cellular imaging.
Spiropyran (SP) is one of the most common photochromic com-
pounds, under UV/visible light irradiation, which could be reversibly
converted from SP form to MC form [22–24]. “Hard” metal ions could be
chelated with crown ethers by using lone pairs from oxygen effectively
[25]. Due to these favourable characteristics, we designed and
* Corresponding author. Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China.
E-mail address: huofj@sxu.edu.cn (F. Huo).
https://doi.org/10.1016/j.dyepig.2021.109467
Received 11 March 2021; Received in revised form 6 May 2021; Accepted 7 May 2021
Available online 2 June 2021
0143-7208/© 2021 Elsevier Ltd. All rights reserved.

Y. Wu et al.
Dyes and Pigments 193 (2021) 109467
Fig. 1. Spectroscopic analysis of photochromic SP-CE. Fig. 1(a) and (b) show that the UV–Vis absorption spectrum change of SP-CE (0.1 mM) under the ultraviolet
light irradiation and visible light irradiation in H2O:CH3CN = 4:1 (v/v). Fig. 1(c) and (d) shown the fluorescence spectral change of SP-CE (0.1 mM) under ultraviolet
light irradiation and visible light irradiation.
synthesized a red-emitting fluorescent probe (SP-CE), using spiropyran
derivatives as the fluorophore and aza-15-crown-5 as the recognition
site for calcium ions. Upon adding calcium ions into H2O:CH3CN = 4:1
(v/v), the crown ether partly chelated with calcium ions to form cor-
responding complexe, and induced the hydroxyl in the MC to negative
oxygen ions, which enhanced the coordination ability with calcium ions.
Further cells and zebrafish experiments clearly revealed that SP-CE
could be used to monitor calcium ions in biological systems. Addition-
ally, the probe could detect the calcium ions released from lysosome to
cytoplasm due to chloroquine-induced pH changes in the lysosome.
in vacuum. Finally, a dark gray solid was obtained (0.30 g, 52.3%). A
similar substance was reported in this patent. We referred to the syn-
thesis method of SP-CE in this patent [26]. mp, 103 ◦C–106 ◦C. IR
(cmꢀ 1): 2970, 2865, 2362, 1706, 1641, 1609, 1508, 1484, 1460, 1331,
1270, 1126, 1081, 1029, 940, 912, 804, 747, 679.1H NMR (600 MHz,
CD3CN) δ 8.10 (s, 1H), 8.03 (d, J = 8.9 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H),
7.14 (d, J = 7.2 Hz, 1H), 7.07 (d, J = 10.4 Hz, 1H), 6.86 (t, J = 7.4 Hz,
1H), 6.76 (d, J = 9.0 Hz, 1H), 6.68 (d, J = 7.7 Hz, 1H), 5.97 (d, J = 10.4
Hz, 1H), 3.66–3.34 (m, 22H), 2.72 (dt, J = 15.0, 7.4 Hz, 1H), 2.58–2.52
(m, 1H), 1.27 (s, 3H), 1.15 (s, 3H) (Fig. S3). 13C NMR (151 MHz, CD3CN)
δ 171.24, 159.32, 146.59, 141.14, 136.18, 128.10, 127.77, 125.64,
122.76, 122.01, 121.81, 119.41, 119.06, 115.37, 107.01, 106.83, 70.54,
70.44, 69.93, 69.75, 69.57, 69.49, 68.83, 52.56, 50.07, 48.67, 39.73,
31.65, 25.26, 18.95. ESI-MS of probe SP-CE m/z: [SP-CE]+ calcd for
582.28129; [SP-CE + Na]+ alcd for 604.26308 (Fig. S3).
2. Experiments
2.1. Synthesis of SP-CE
2.1.1. Compound 1
2.2. Materials
Compound 1 was prepared according to literature [22]. mp,
176 ◦C–178 ◦C. IR (cmꢀ 1): 3309, 2973, 2529, 1720, 1594, 1469, 1400,
1360, 1349, 1270, 1207, 1030, 939, 757. ESI-MS m/z: [C + H]+ Calcd
for C14H18NO+2 232.13321, Found (Fig. S1).
Related solvents and apparatus are described in the support
information.
2.1.2. Compound 2
2.3. Fluorescence imaging
Related synthetic steps for compound 2 were described in the sup-
port information.
2.3.1. Cell imaging
HeLa cells and HepG2 cells were cultured in DMEM medium con-
taining 12% fetal bovine serum and 1% double antibody at 37 ◦C and 5%
carbon dioxide incubator. When the cells were in the logarithmic growth
phase, HeLa cells and HepG2 cells were inoculated in a special laser
confocal culture dish, and continue culturing for another 24 h until the
cells adhered to the wall.
2.1.3. Compound 3
Compound 2 (0.581 g, 1 mmol), EDCl (0.767 g, 4 mmol) and 1-
hydroxybenzotriazole (0.54 g, 4 mmol) were dissolved in 15 mL of
anhydrous N,N-dimethylformamide solution, the mixture was reacted
for 30 min under the protection of argon at 0 ◦C. Then 1-aza-15-crown-5-
ether (0.58 g, 1 mmol) and triethylamine (0.3 mL) were added to the
reaction solution, and the mixture continued to react at room temper-
ature for 24 h. After the reaction was completed, the reactants were
poured into distilled water (100 mL) to continue stirring, and the
resulting precipitate was filtered, washed with distilled water and dried
The probe SP-CE (10 μM) was dissolved in phosphate buffered saline
(PBS) (2 mL), and then added to a confocal culture dish inoculated with
◦
HeLa cells and HepG2 cells and incubated at 37 C for 20 min. In the
experiment for detecting exogenous calcium ions, the HeLa cells and
HepG2 cells loaded with SP-CE were incubated in PBS at 37 ◦C for 20
2

Y. Wu et al.
Dyes and Pigments 193 (2021) 109467
Fig. 3. Fluorescence response of probe SP-CE (100 μM) towards various bio-
logically relevant species (500
μ
M) at 622 nm, (1) Zn2+ (2) Cu2+, (3) Na+, (4)
Ag+, (5) Mg2+, (6) Ca2+, (7) Fe2+, (8) Li+, (9) K+.
Fig. 2. Fluorescence spectrum responses of SP-CE (10
μM) to calcium ions
(0–200 μM), in H2O:CH3CN = 4:1 (v/v) λex = 535 nm, λem = 622 nm, slit: 10
nm/10 nm; Inserted: The working curve of SP-CE (10
μ
M) in the presence of
various concentrations of Ca2+ (0–100
μM), λex = 535 nm, slit: 10 nm/10 nm.
min, then incubated with calcium ions (100 μM) for 15 min, washed
twice with PBS (2 mL), and then imaged. In the experiment for detecting
endogenous calcium ions, HeLa cells and HepG2 cells loaded with SP-CE
and CQ (100 μ
M) were incubated in PBS at 37 ◦C for 40 min, washed
with PBS (2 mL) twice, and then imaged. The incubated cells were
imaged under a UV lamp irradiation for 4 min.
2.3.2. Zebrafish culture and imaging
The probe SP-CE (20 μM) was added to E3 embryo medium con-
taining zebrafish (5 days old) and incubated at 29 ◦C for 20 min and then
imaged. In the experiment to detect exogenous calcium ions, the
zebrafish was incubated with SP-CE (20 μM) for 20 min, then incubated
Scheme 1. Synthesis route for SP-CE.
with calcium ions (100 μM) for 20 min, then imaged. After incubation,
the zebrafish was irradiated under UV light (365 nm) for 4 min.
3. Results and discussion
3.1. Study on the photochromic properties of probe SP-CE
We studied the photochromic properties of the SP-CE. The probe SP-
CE (0.1 mM) showed a strong UV absorption band at 530 nm under the
UV lamp 365 nm irradiation (0–4 min). However, when the SP-CE was
irradiated with visible light (0–2 min), the UV absorption spectrum
signal returned to the initial state, indicating that the MC form was
converted into SP form. At the same time, when irradiated with 365 nm
ultraviolet lamp, the fluorescence intensity of the SP-CE (0.1 mM) at
622 nm also increased with time (0–4 min). Nevertheless, it dropped to
its original state under visible light irradiation, and the progress sup-
ported the UV–Vis spectrum analysis results.
Scheme 2. Mechanism diagram of the SP-CE response Ca2+
.
3.3. Selectivity of SP-CE
We then tested the selection recognition of the SP-CE to calcium ions
under H2O:CH3CN = 4:1 (v/v). As shown in Fig. 3, after adding calcium
ions, the emission intensity increased significantly at 622 nm. Whereas
3.2. SP-CE fluorescence spectrometric titration responds to calcium ions
The spectral properties of SP-CE and its response to calcium ions
were obtained in H2O:CH3CN = 4:1 (v/v) (see Fig. 1). Upon adding
upon the addition of other ions (500
μ
M) (Zn2+, Cu2+, Na+, Ag+, Mg2+
,
Fe2+, Li+, K+), the emission intensity did not increase remarkably.
Therefore, the experiment proved that SP-CE has good selectivity to
calcium ions. In addition, We tested the effect of different pH on the
fluorescence responses of SP-CE to calcium ions. Notably, the fluores-
cence changes of SP-CE to Ca2+ reached a maximum at around pH = 7. It
was further confirmed that SP-CE could recognize Ca2+ in physiological
environment (Fig. S5).
(0–200
CE (10
μM) calcium ions to the detection solution containing probe SP-
μ
M), the solution changed from light pink to dark pink and the
corresponding spectrum mainly showed an upward trend at near 620
nm, as depicted in Fig. 2. We plotted the maximum emission intensity of
SP-CE at λ = 535 as a function of calcium ions concentration. The
ex
probe exhibited a good linear range and low detection limit (0.570
μ
M)
for calcium ions (0–100 μM) (Fig. 2 Inserted). The detection limit of the
probe was calculated according to the definition of IUPAC (CDL = 3σ/k)
[27,28].
3

Y. Wu et al.
Dyes and Pigments 193 (2021) 109467
Fig. 5. Confocal images of the SP-CE responds to exogenous Ca2+and endog-
enous Ca2+ in HeLa cells. (a1) HeLa cells were incubated with probe SP-CE (10
μ
M) for 20 min (a2) HeLa cells were incubated with SP-CE (10 μM) for 20 min,
then treated with exogenous calcium ions (0.1 mM) for 15 min (b1) HeLa cells
were incubated with SP-CE (10 M) for 20 min (b2) Hela cells were incubated
Fig. 4. Confocal images of the SP-CE responds to exogenous Ca2+and endog-
enous Ca2+ in HepG2 cells. (a1) HepG2 cells were incubated with SP-CE (10
μ
with SP-CE (10
μM) and CQ (100 μM) for 40 min. Red channel: emission
μ
M) for 20 min (a2) HepG2 cells were incubated with SP-CE (10 μM) for 20
wavelength 622 ± 20 nm (excitation wavelength 535 nm) Scale bar: 20 μM.
min, and then incubated with exogenous calcium ions (0.1 mM) for 15 min (b1)
HepG2 cells were incubated with SP-CE (10 M) for 20 min (b2) HepG2 cells
were incubated with SP-CE (10 M) and CQ (100 M) for 40 min. Red channel:
emission wavelength 622 ± 20 nm (excitation wavelength 535 nm) Scale bar:
20 M.
μ
size of alkali metal ions and crown ethers. (see Scheme 1)
μ
μ
3.5. Cellular imaging
μ
We evaluated the SP-CE response to calcium ions in cell imaging
3.4. Proposed mechanism
experiments. After HepG 2 cells with SP-CE (10 μM) were incubated for
20 min and then irradiated with UV light (4 min), a weak red fluores-
cence signal appeared in the red channel (Fig. 4a1). Furthermore, we
also tracked to the exogenous calcium ions by fluorescence imaging.
The response mechanism of SP-CE for detecting calcium ions was
described in Scheme 2. Besides, as shown in Scheme 2, the SP-CE
structure was easily transformed into MC-CE structure under UV irra-
diation, and the presence of calcium ions would further promoted the
transformation of SP-CE structure into MC-CE through the combined
action of negative oxygen anions and crown ether. Therefore, the fluo-
rescence intensity was significantly enhanced after adding calcium ions.
The corresponding reaction product was characterized by ESI-MS. The
SP-CE acetonitrile solution (1 mL, 1 mM) was mixed with CaCl2 aqueous
solution under the ultraviolet light irradiation for 4 min, and then
sample were measured by a mass spectrometer. As shown in Fig. S4, an
obvious strong peak indicates that MC-CE is stably complexed with
calcium ions. Due to the influence of the negative oxygen ion of spi-
ropyran on the binding efficiency of calcium ions, we used Prism-8
software to perform a non-linear regression fitting for calcium ions, as
shown in Fig. S6, the stability constant of the SP-CE combining with
calcium ions was 1.12 × 104 Mꢀ 1 in H2O: CH3CN = 4:1. Therefore, the
selective binding of MC-CE isomer for calcium ions was attributed to the
electrostatic interaction with p-nitrophenolate and the suitability of the
HepG2 cells were incubated with SP-CE (10 μM) for 20 min and then
incubated with calcium ions (0.1 mM) for 15 min and irradiated with UV
light (4 min). A similar fluorescence phenomenon appeared in the red
fluorescence signal, and it was greatly enhanced. (Fig. 4a2). Meanwhile,
we also investigated the sensing performance of probe SP-CE for
endogenous calcium ions in living cells. We used CQ drugs that change
the lysosomal pH value, causing calcium ions to be released into the
cytoplasm. As shown in Fig. 4b2, HepG2 cells were cultured with the SP-
CE (10 μM) and CQ (100 μM) for 40 min, a significant red fluorescence
signal was enhanced in the red fluorescence channel. At the same time,
SP-CE was studied in HeLa cells (Fig. 5). These results implied that SP-
CE could be considered as a cell-permeable molecular tool to examine
and visualize dynamic equilibrium of calcium ions in cells.
4

Y. Wu et al.
Dyes and Pigments 193 (2021) 109467
Fig. 6. Confocal images of probe SP-CE response to exogenous Ca2+ in zebrafish. (a) The zebrafish was incubated with the SP-CE (20
μ
M) for 20 min, and then
M) for 20 min, and then
M.
irradiated with UV (365 nm) light for 4 min. (b) The SP-CE-loaded (20
μ
M) zebrafish incubated for 20 min then incubated with Ca2+ (100
μ
irradiated with UV (365 nm) light for 4 min. Red channel: emission wavelength 622 ± 20 nm (excitation wavelength 535 nm) Scale bar: 500
μ
3.6. Zebrafish imaging
Comprehensive Utilization Science and Technology Project
(2018XYSDJS-05), the Shanxi Province Foundation for Selected (2019),
Key R&D Program of Shanxi Province (201903D421069), Natural Sci-
ence Foundation of Shanxi Province of China (201901D111015), Shanxi
Province Foundation for Returness (2017–026), Teaching reform project
of colleges and universities in Shanxi Province (2019), Innovative Tal-
ents of Higher Education Institutions of Shanxi (2019), Scientific and
Technological Innovation Programs of Higher Education Institutions in
Shanxi (2019L0031), Shanxi Collaborative Innovation Center of High
Value-added Utilization of Coal-related Wastes (2015-10-B3) and Sci-
entific Instrument Center of Shanxi University (201512).
Next, we explored the feasibility of using the probe SP-CE (20 μM)
for imaging low concentration of calcium in zebrafish. As shown in
Fig. 6a, the zebrafish was incubated with the probe SP-CE (20 μM) for
20 min and then irradiated with UV lamp at 365 nm for 4 min, a weak
red fluorescence signal appeared in the red channel. Simultaneously,
when the zebrafish was incubated with SP-CE (20 μM) for 20 min, then
incubated with calcium ions (100 μM) for 20 min, under the ultraviolet
light irradiation for 4 min, the red fluorescence signal was enhanced in
the red channel (Fig. 6b). Therefore, these imaging findings demonstrate
the probe can effectively image calcium ions in vivo.
Appendix A. Supplementary data
4. Conclusions
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109467.
In summary, a red-emitting fluorescent probe SP-CE was explored to
detect the endogenous and exogenous calcium ions in cells. SP-CE
response mechanism was based on calcium ion induce the isomerized
spiropyran MC to release negative oxygen ions, which could be specif-
ically combined with crown ethers to achieve specific detection for
calcium ions. Besides, further cells and zebrafish experiments clearly
revealed that SP-CE could be used for monitoring calcium ions in bio-
logical system. Moreover, we applied CQ drugs to promote the release of
calcium ions into the cytoplasm and to detect the level of calcium ion in
the cytoplasm.
Author contributions
Yingchun Wu: Investigation.
Fangjun Huo: Conceptualization, Writing-original draft.
Weijie Zhang: Validation.
Jianbin Chao: Project administration.
Caixia Yin: Funding acquisition, Writing-review & editing.
Declaration of interest statement
Declaration of competing interest
We have no any interest conflict.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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