A lysosomal probe for monitoring of pH in living cells and ovarian tumour

Article abstract of DOI:10.1016/j.dyepig.2016.12.043

Intracellular pH is important in regulating various cellular behaviour and pathological conditions. We synthesised a rhodamine-based lysosomal pH probe (hereafter referred to as RD) with a pKa of 4.10. The probe exhibited favourable “on-off” reversibility

Full text of DOI:10.1016/j.dyepig.2016.12.043

Accepted Manuscript
A lysosomal probe for monitoring of pH in living cells and ovarian tumour
Bo Li, Guang bo Ge, Lanlan Wen, Ye Yuan, Ran Zhang, Xiaojun Peng, Fengyu Liu,
Shiguo Sun
PII:
S0143-7208(16)31105-6
10.1016/j.dyepig.2016.12.043
DYPI 5668
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 3 November 2016
Revised Date: 18 December 2016
Accepted Date: 19 December 2016
Please cite this article as: Li B, Ge Gb, Wen L, Yuan Y, Zhang R, Peng X, Liu F, Sun S, A lysosomal
probe for monitoring of pH in living cells and ovarian tumour, Dyes and Pigments (2017), doi: 10.1016/
j.dyepig.2016.12.043.
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ACCEPTED MANUSCRIPT
A lysosomal probe for monitoring of pH in living cells
and ovarian tumour
Bo Lia,1, Guang bo Geb,1, Lanlan Wena,1, Ye Yuana, Ran Zhanga, Xiaojun Penga,
Fengyu Liua,* and Shiguo Sunc,*
a
State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of
Technology, No. 2, Linggong Road, Ganjingzi District, 116023 Dalian, China.
b Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
c
Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education,
School of Pharmacy, Shihezi University, Shihezi 832000, China
1 These authors contributed equally to this work.
ABSTRACT
Intracellular pH is important in regulating various cellular behaviour and
pathological conditions. We synthesised a rhodamine-based lysosomal pH probe
(hereafter referred to as RD) with a pKa of 4.10. The probe exhibited favourable
“on-off” reversibility between pH 3.25 and 7.20 for at least five cycles. RD can be
employed to distinguish cancer cells from normal cells on the basis of different
fluorescence response. RD performed well in the detection of lysosomal pH induced
by artesunate, chloroquine and dexamethasone in MCF-7 cells. The intense
fluorescence of RD in ovarian tumour indicated the higher acidity in tumour tissues
than that in normal tissues. Furthermore, RD worked well in the imaging of excised
mouse stomach and living mice under acidic stimulation. These data demonstrate the
applicability of RD in monitoring pH in complex biological systems.
Key words : monitoring pH, lysosome, fluorescent probe, cells, ovarian tumour
1. Introduction
Lysosome is an acidic organelle in all eukaryotic cells excluding erythrocytes [1],
which plays significant roles in many physiological activities, including maintaining
cholesterol homeostasis, repairing damaged cytoplasmic membrane, restoring bone
and tissue and defending against pathogens [2]. It is noted that lysosomal pH is
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closely related to its function [3, 4]. Malfunction of lysosomes can cause many
diseases, such as cancer, gout and neurodegenerative diseases [5]. Therefore, imaging
of lysosomes and monitoring of pH status is important.
To date, there are several methods had been developed to detect pH in vitro or vivo,
such as weak acid and base distribution [6], nuclear magnetic resonance spectroscopy
[7], microelectrodes [8], and fluorescent probes [9]. Compared with other techniques,
fluorescence detection using small molecular probes, has regarded as a promising
method in living cells due to its sensitivity, selectivity, simple operation, and
especially nondestructive characteristics. Some efforts have been directed toward the
development of fluorescent pH sensors [10-15], especially lysosomal pH sensors [2,
16-24]. For instance, Li [2] constructed a pH sensitive probe based on a 4-acylated
naphthalimide, Peng [16] synthesised a pH sensor by introducing a novel lysosomal
location group, Yu [17] developed two rhodamine-based pH probes via the click
reaction, Zhang [18] reported arhodamine-based pH probe, Zhao [19] exploited a
rhodamine B-based lysosomal pH probe, Liu [20] constructed three lysosomal pH
probes based on piperazine-modified BODIPY dyes and Wang [21] designed a
pH-activatable near-infrared (NIR) probe through combining a cyclic boronate with
Si-rhodamine. Some ratiometric fluorescent probes have been developed for tracking
lysosomal pH in vivo. Zhang [22] reported a new lysosome-targeted ratiometric
fluorescent probe by hybridizing morpholine with a xanthane derivative and an
o-hydroxybenzoxazole group, Zhao [23] reported a ratiometric lysosomal pH probe
based on the naphthalimide–rhodamine. Very recently, Lin [24] developed a novel
dual site-controlled and lysosome-targeted ratiometric fluorescent probe based on
coumarin and naphthalimide fluorophores. These probes showed general suitability of
specific labelling of acidic lysosomes and tracking their pH in living cells. However,
not much fluorescence difference can be observed in the case of normal and cancer
cells for these probes. A pyridineium-2-yl Darrow Red analogue with a pKa of 2.4
was adopted for the selective detection of cancer cells [25]. Unfortunately, the status
of normal cells cannot be visualised because of the absence of emission when the pH
exceeds 4.0. Cancer cells contain rich hydrogen ions [26], and the acidity of
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lysosomes in cancer cells is lower than that in normal cells [25]. A probe which can
work well both in cancer and normal cells, and which can easily distinguish different
cells on the basis of fluorescence response is highly desirable.
Low extracellular pH is a symbol of solid tumours [27]. This acidity mainly arises
from high lactic acid production due to enhanced glycolysis [28]. As a product of
aerobic respiration in vivo, CO2 may be another significant source of acidification of
tumour interstitium [29]. Some probes were reported for monitoring of tumour pH.
For instance, Kobayashi [30] designed four novel acidic pH–activatable probes by
conjugating the boron-dipyrromethene fluorophore with a cancer-targeting
monoclonal antibody. The next year, the group reported a dual-controlled activation
fluorescent imaging probe through combining pH-sensitive fluorophore pHrodo or
TAMRA with the cancer targeting molecules avidin and trastuzumab [31].
Shirmanova [32] used the novel genetically encoded indicator SypHer2 for pH
mapping in living cancer cells in vitro and in tumors in vivo. Achilefu [33] developed
a highly tumour-selective probe with a pKa of 4.7 to image acidic cell organelles and
detect primary and metastatic cancer. This professor introduced a cyclic
arginine-glycine-aspartic acid (cRGD) peptide to a Cy7 molecule to target Rvβ3
integrin (ABIR), which is highly overexpressed during tumour angiogenesis. In 2015,
Prof. Achilefu [34] found that the Cy7 molecule alone can detect tumour through
electrostatic changes produced by the extracellular acidic pH environment of diverse
solid tumors without tumor targeted groups. Thus, it inspires us to develop a single
molecule probe which can not only distinguish cancer cells from normal cells but also
detect tumours.
Scheme 1. Structure of Probe RD
Rhodamine dye, which features a spirocyclic structure, is an ideal OFF-ON
fluorescence switch candidate. In general, there are no fluorescence at basic and
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neutral pH because of the ‘‘ring-closed’’ state of spirocyclic structure. When the H+
increases within a certain range, the ring is open, leading to a significant fluorescence
enhancement [16, 35]. Accordingly, we developed a probe termed RD (Scheme 1) by
introducing 2, 4-dihydroxybenzoic acid to rhodamine B. RD, which features a pKa of
4.1, facilitates remarkable fluorescence intensity enhancement and significant colour
variation from colourless to pink when the pH is adjusted from 7.02 to 3.06. RD
performed well in the monitoring of lysosomal pH induced by artesunate (ART),
chloroquine and dexamethasone. The intense fluorescence of RD in ovarian tumour
indicated the higher acidity in tumour tissues than that in normal tissues. Furthermore,
RD worked well in the imaging of excised mouse stomach and living mice under
acidic stimulation.
2. Materials and methods
2.1 Materials and instruments
All solvents and reagents used were reagent grade and used without further
purification unless otherwise stated. The solution of RD was dissolved in DMSO at a
concentration of 1 mM as the stock solution and stored in a refrigerator (2 ºC) for use.
B-R-E (Britton-Robinson buffer/EtOH, v/v = 5 : 5) solution was mixed by 40 mM
acetic acid, boric acid, and phosphoric acid [36]. Sodium hydroxide (0.1 M) was
utilized for tuning pH values of B-R-E solution. The solution of metal ions was
prepared from nitrate salts. Deionized water was used throughout the experiment.
Cells were purchased from Institute of Basic Medical Sciences (IBMS) of the Chinese
Academy of Medical Sciences.
NMR spectra were recorded on a VARIAN INOVA 400 (or a Bruker Avance II 400
MHz) spectrometer. Chemical shifts (δ) were reported as ppm (in CDCl3 or DMSO-d6,
with TMS as the internal standard). Fluorescence spectra were performed on a
VAEIAN CARY Eclipse fluorescence spectrophotometer (Serial No. FL0812:M018).
Excitation and emission slit widths were modified to adjust the fluorescence intensity
to a suitable range. Absorption spectra were measured on a Perkin Elmer Lambda 35
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UV/VIS spectrophotometer (Perkin Elmer). Mass spectrometric data were achieved
with HP1100LC/MSD MS and an LC/Q-TOF-MS instruments. Imaging in vivo mice
was performed using a Night OWL II LB 983 system equipped with an NC 100 CCD
deep-cooled camera (Berthold Technologies, Bad Wildbad, Germany). All pH
measurements were performed using a Model PHS-3C meter. Flash column
chromatography was performed using silica gel (100-200 mesh) obtained from
Qingdao Ocean Chemicals. Lyso Tracker Green was purchased from Life
Technologies Co. (USA).
2.2 Synthesis of compound RD
RD was synthesised by two procedures (Scheme S1). The first step, RB (2.51 g, 5.2
mmol) was dissolved in ethanol (50 mL), then 5 mL ethylenediamine was added in
dropwise to the flask and the reaction mixture was stirred at 90ºC for 8 h. After the
solution was cooled to room temperature, the solution was concentrated under
reduced pressure and the residue was purified by column chromatography (DCM :
1
MeOH = 10 : 1, v/v) to afford a light yellow solid R2 in 75% yield. H NMR (400
MHz, CDCl3), δ 7.89 (m, 1H), 7.60-7.33 (m, 2H), 7.18-7.00 (m, 1H), 6.58-6.12 (m,
6H), 3.50-3.23 (m, 8H), 3.23 (t, J = 6.6 Hz, 2H), 2.45 (t, J = 6.6 Hz, 2H), 1.17 (t, J =
7.0 Hz, 12H). MS: (M+H)+, calcd: m/z = 485.29, found: m/z = 485.27.
The second step, 2,4-dihyoxybenzoicacid (308 mg, 2 mmol) was dissolved in
acetonitrile (20 mL), then 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCI, 384 mg, 2 mmol) and 4-dimethylaminopyridine (DMAP, drops)
were added into the flask and the reaction mixture was stirred at room temperature for
1 h. After, R2 (500 mg, 1 mmol) was added to the flask and the reaction mixture was
stirred at 90 ºC for 12 h. After cooled down to room temperature, the solution was
concentrated under reduced pressure and the residue was purified by column
chromatography (DCM : MeOH = 30 : 1, v/v) to afford a white solid RD in 65% yield.
1H NMR (400 MHz, CDCl3) , δ 12.91 (s, 1H), 8.31 (s, 1H), 7.93 (dd, J = 6.0, 2.4 Hz,
1H), 7.58 (d, J = 8.7 Hz, 1H), 7.55-7.40 (m, 2H), 7.11 (dd, J = 5.9, 2.1 Hz, 1H),
6.62-6.16 (m, 8H), 3.63-3.03 (m, 12H), 1.17 (t, J = 7.1 Hz, 12H). 13C NMR (100 MHz,
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CDCl3), δ 171.68, 170.72, 164.37, 162.66, 154.38, 153.84, 149.65, 133.76, 130.41,
129.02, 128.83, 128.46, 124.51, 123.70, 109.02, 107.94, 107.58, 104.53, 104.49,
98.29, 67.03, 46.38, 44.91, 41.89, 39.75, 13.10. HRMS: (M+H)+, calcd: m/z =
621.3071, found: m/z = 621.3067.
2.3 Fluorescence detection
Absorption and emission spectra were primarily measured in B-R-E solution. The
resulting solution was equilibrated for 15 min before measurement. The fluorescence
spectra were measured with excitation wavelength at 540 nm.
Photo-bleaching experiments were performed according to the literature [37].
Concentration of RD and commercialized dye Lyso Tracker Green were 10 ꢀM, at pH
4.25, 500 W halogen lamp as light source, saturated NaNO2 (50 g/L) solution as a
light filter (to cut off light of wavelength under 400 nm) and hot filters, keep light
source and the test samples at 30 cm distance respectively and record the fluorescence
intensity of probe RD and Lyso Tracker Green, and processing the biggest
fluorescence intensity values normalized.
2.4 Live cell imaging experiments
Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen)
supplemented with 10% fetal bovine serum (Invitrogen). The cells were seeded in
24-well flat-bottomed plates and then incubated for 24 h at 37ºC under 5% CO2.
Before imaging, the live cells were incubated with RD (5 µM) for different time and
then washed with phosphate-buffered saline (PBS) three times. Fluorescence imaging
was performed using an OLYMPUSFV-1000 inverted fluorescence microscope with a
60 × objective lens. Under the confocal fluorescence microscope, RD was excited at
559 nm and emission was collected at 565-620 nm. For the detection of Lysosomal
pH, MCF-7 cells were incubated with RD (5 µM) at 37 ºC for 1 h and then artesunate
(ART, 50 ꢀM), chloroquine (20 ꢀM), dexamethasone (100 ꢀM) were added
respectively to incubate with cells at 37 ºC for different time.
2.5 MTT assay
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Measurement of cell viability was evaluated by reducing of MTT (3-(4,
5)-dimethylthiahiazo(-2-yl)-3,5-diphenytetrazoliumromide) to formazan crystals
using mitochondrial dehydrogenases (Mosmann, 1983). HepG-2 cells were seeded in
96-well microplates (Nunc, Denmark) at a density of 1×105 cells/mL in 100 mL
medium containing 10% fetal bovine serum (FBS, invitrogen). After 24 h of cell
attachment, the plates were washed with 100 ꢀL/well PBS. The cells were then
cultured in medium with 0.5, 1, 2 and 5 ꢀM of RD for 12 h. Cells in culture medium
without RD were used as the control. Six replicate wells were used for each control
and test concentration10 ꢀL of MTT (5 mg/mL) prepared in PBS was added to each
well and the plates were incubated at 37 ºC for another 4 h in a 5% CO2 humidified
incubator. The medium was then carefully removed, and the purple crystals were
lysed in 200 ꢀL DMSO. Optical density was determined on a microplate reader
(Thermo Fisher Scientific) at 570 nm (OD) with subtraction of the absorbance of the
cell-free blank volume at 630 nm (ODK). Cell viability was expressed as a percent of
the control culture value, and it was calculated using the following equation [38]:Cells
viability (%) = (ODdye- ODKdye)/ (OD control- ODKcontrol) × 100.
2.6 Animal Models and in Vivo Animal Studies
All animal experiments were performed in compliance with the Guiding Principles
for the Care and Use of Laboratory Animals, Dalian Medical College, China. Before
animal imaging and analysis, SPF (Specific pathogen Free) Kunming mice
(7-week-old, 20-25 g) were starved 12 h because of possible food fluorescence
interference at the emission wavelength of the fluorescent dyes. The experiments were
performed using a Night OWL II LB 983 system equipped with an NC 100 CCD
deep-cooled camera (Berthold Technologies, Bad Wildbad, Germany), with an
excitation filter of 530 nm and an emission filter of 600 ± 20 nm.
3. Results and discussion
3.1 Design and synthesis of RD
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RD was readily synthesised by a two-step procedure (Scheme S1) and confirmed
1
by H NMR, 13C NMR and HRMS spectra (Fig. S1-S5). The proposed sensing
mechanism of RD for H+ is shown in Scheme S2. To provide evidence, the change
1
between RD and the protonated form RDH was characterized through H NMR
titration (Fig. S6). The addition of trifluoroacetic acid led to the ‘‘ring-opened’’ state
of spirocyclic structure RDH. Hence, a down-field shift can be observed for eight
protons of NEt2 and six protons of benzene ring in RB. After the addition of NaOH,
the spirocyclic ring was closed, and the corresponding proton shift was recovered.
3.2 Spectroscopic properties and optical responses to pH
The spectroscopic properties of RD were initially determined in a B-R-E solution.
As expected, the fluorescence intensity reached saturation within 15 min at pH 4.25
(Fig. S7a). As shown in Fig. 1a, the absorption spectra of RD in weak basic aqueous
solution (pH 7.02) displayed almost no absorption bands at 500–600 nm. The changes
of pH from 7.02 to 3.06 caused a gradual increase of the absorbance at 564 nm,
indicating that the spirolactam ring of rhodamine was opened. Moreover, the solution
changed from colourless to pink, which was visible by the naked-eye. The pH
response of RD is displayed in Fig. 1b. RD was non-fluorescent under neutral
conditions because of its “ring-closed” form. However, as the pH decreased from 7.02
to 3.06, a remarkable enhanced fluorescence signal appeared at 586 nm because of
spirocyclic ring opening. The fluorescence spectra of RD was obtained under pH 3.26
in Fig. S7b.
A good linearity (R2 = 0.9867) between fluorescence intensity and pH (3.46-5.06)
was obtained by fluorescence titration (Fig. 2a). With fluorescence intensity (at 586
nm) as a function of pH, a pKa of 4.10 was calculated using the
Henderson-Hasselbalch equation [39], suggesting that RD may detect pH for acidic
organelles such as lysosomes in vivo. Furthermore, the reversibility of the probe in
the B-R-E solution was studied between pH 3.25 and 7.20 (Fig. 2b). Approximately
94% of the original signal was successfully restored after five cycles, providing a
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possibility for monitoring pH change repeated. The fluorescence quantum yield of RD
at pH 3.26 was determined to be 0.475 [40].
a
0.20
0.15
0.10
0.05
0.00
pH 3.06 7.02
3.06
7.02
450
500
550
600
650
Wavelength/nm
b
160
140
120
100
80
3.06
7.02
60
40
20
0
560
580
600
620
640
Wavelength (nm)
Fig. 1 (a) Absorption and (b) fluorescence spectra (λex = 540 nm) of RD (10 ꢀM) in B-R-E
solution with different pH 7.02, 6.06, 5.26, 4.86, 4.66, 4.46, 4.26, 4.06, 3.86, 3.66, 3.46, 3.26 and
3.06.
Nitrogen atom can bind with many metal ions, such as Pd2+ and Cu2+ in solution,
and the binding may cause ring-opening in rhodamine-based probes [41, 42]. Thus,
investigating whether other ions are potential interferents is necessary [43]. As shown
in Fig. S8a, RD did not exhibit any observable fluorescence change in the presence of
common cations, such as Na+, K+, Cr3+, Ag+, Cu2+, Cd2+, Hg2+, Fe3+, Pb2+, Ni2+, Co2+,
Fe2+, Zn2+, Ca2+, Mg2+, Mn2+and Al3+ at pH 7.02. While significant fluorescence
enhancement occurred in the case of pH 4.25 as shown in Fig. S8b. The high
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selectivity of RD over interfering species along with the above-mentioned excellent
reversibility indicates that RD demonstrates a significant potential as a pH probe for
practical applications.
160
120
a
160
80
40
120
0
3.6
4.0
4.4
4.8
5.2
pH
80
40
0
pKa=4.10
3
4
5
6
7
pH
b
80
pH=3.25
60
40
20
0
pH=7.20
1
0
2
4
5
3
Cycle Index
Fig. 2 (a) Sigmoidal fitting of the pH-dependent fluorescence intensity at 586 nm of RD (10 ꢀM)
in B-R-E solution, with different pH 7.02, 6.81, 6.36, 6.06, 5.86, 5.66, 5.46, 5.26, 5.06, 4.86, 4.66,
4.46, 4.26, 4.06, 3.86, 3.66, 3.46, 3.26 and 3.06. Inset: the good linearity in the pH range of
3.46-5.06, (b) The reversibility of the probe RD (10 ꢀM) between pH 3.25 and 7.20 in B-R-E
solution. Fluorescence intensity change of RD at 586 nm along with the addition of HCl (1 M) and
NaOH (1 M) to the solution for five times, respectively (λex = 540 nm).
3.3 Fluorescence imaging and distribution in living cells
To evaluate the cell membrane permeability of RD, imaging was monitored in the
red channel (565-620 nm) with a 559 nm laser under a confocal laser scanning
microscope. As shown in Fig. S9, the probe can stain the MCF-7 cell in 10 min,
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demonstrating its good membrane permeability. After 30 min, the staining can be
finished and focused mainly in the cytoplasm.
To study the subcellular distribution of RD, we used commercial Lyso Tracker
Green to co-stain with the probe (Fig. 3). After incubating with 5 ꢀM RD for 30 min,
MCF-7 cells were further stained with 1 ꢀM Lyso Tracker Green for 10 min. A
co-localisation coefficient (Pearson’s correlation) of 0.90 can be reached between the
red emission of RD and the green emission of Lyso Tracker Green. HeLa and HepG-2
cells were also tested with higher Pearson’s correlations of 0.98 and 0.94, respectively
(Fig. S10 and S11).
d
d
e
Fig. 3 Co-localization experiments of MCF-7 cells incubated with RD (5 ꢀM) for 30 min and then
incubated with Lyso Tracker Green (1.0 ꢀM) for 10 min at 37 ºC under 5% CO2. (a) Bright image,
(b) Confocal image from LysoTracker Green on green channel (λex = 488 nm), (c) Confocal image
from RD on red channel (λex = 559 nm), (d) Merged image of b and c, (e) Correlation plot of the
intensities of RD and Lyso Tracker Green (Rr = 0.90).
Furthermore, confocal fluorescent imaging was performed in Human hepatoma
carcinoma cells (HepG-2) and Human normal liver cells (HL-7702) using RD. Probe
RD gave very strong red emission in HepG-2 cells (Fig. S11), while only slightly
weaker emission was observed in HL-7702 cells (Fig S12g-i). To provide further
evidence, normal cells (COS-7, BEAS-2B) and cancer cell lines (MCF-7, HeLa) were
used to repeat imaging. The results indicate that for normal cells, such as COS-7,
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BEAS-2B and HL-7702, only weak emission was found after incubation for 60 (Fig.
S12) and 120 min (Fig. S13), while, intense red emission can be observed for the
above mentioned cancer cells in 30 min (Fig. 3, S10 and S11). This result
demonstrates that the lysosomal pH in cancer cells is lower than that in normal cells
as reported [25]. The different fluorescence responses of RD can be employed to
separate cancer cells from normal cells.
The cytotoxicity of RD was evaluated using MTT assay (Fig. S14). Negligible
toxicity existed under 5 ꢀM. Especially, RD exhibited higher photostability than
commercial Lyso Tracker Green at pH 4.25 (Fig. S15). The fluorescence intensity of
RD only decreased to 94% after irradiation under a 500 W halogen lamp for 4 h,
whereas that of Lyso Tracker Green significantly decreased to 54%. This result
demonstrates the higher photostability of RD than Lyso Tracker Green. Thus, RD may
be used for long-term monitoring of pH in the future.
3.4 Monitoring lysosomal pH in cells
RD
RD/ART 5h
a
c
b
2000
1600
1200
800
400
0
e
RD
RD/ART 5h
d
1 h
6 h
Fig. 4 Confocal images of RD (5.0 ꢀM) in MCF-7 cells. (a, b) RD incubated with cells for 1 h, (c)
RD incubated with cells for 6 h, (d) Incubated with RD for 1 h then exposed to 50 ꢀM ART for 5
h, (e*) Quantification of fluorescence intensity from RD and RD/ART. Scale bar: 20 ꢀm, λex = 559
nm, λem = 565-620 nm. (*: The values obtained were averaged from six arbitrarily
fluorescent areas in a, b, c, d image.
)
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Artesunate (ART) [44], a water-soluble derivative of artemisinin, has been widely
used to cure malaria. It preferably accumulates in the lysosomes and decreases
lysosomal pH in a time-dependent manner [45]. Thus, ART-induced lysosomal pH
changes were studied on RD-stained MCF-7 cells (Fig. 4). To compare the intensity
on amounts, six arbitrarily fluorescent areas in every image were chosen to represent
the lysosomal regions of the MCF-7 cells. The values obtained were subsequently
averaged for each case (Fig. 4e) [16]. After treatment with 50 ꢀM ART for 5 h, RD
exhibited a stronger red fluorescence compared with the control. To provide further
evidence (Fig. S16), chloroquine [46] was also used to induce lysosomal pH changes
as reported in the literature [47, 48]. However, chloroquine led to the alkalisation of
the lysosome, and this effect was opposite to that of ART. Compared with the
fluorescence intensity in the cells only stained with RD, that in the cells stained with
RD and chloroquine significantly reduced. These results indicate that RD can be a
lysosomal pH-responsive sensor in cell biology.
Dexamethasone is a chemotherapeutic and anti-inflammatory agent [49], typically
used to induce cell apoptosis [16]. RD detected lysosomal pH changes during
apoptosis induced by dexamethasone (Fig. S17 and S19) in MCF-7 cells. The addition
of dexamethasone decreased the fluorescence intensity in the cells in a
time-dependent manner because of the increase in pH. However, without incubation
with dexamethasone, the fluorescence intensity of RD showed no significant change
even when the incubation time was prolonged (Fig. S18 and S19). Furthermore,
apoptosis-related morphological changes were achieved 2.5 h after dexamethasone
treatment (Fig. S17). This observation agrees with the literature [16, 50].
3.5 Imaging of ovarian tumour
Considering that most of the tumour tissues are partially acidic [27, 34, 51, 52], we
focused on the detection of tumour in vivo using RD. Ovarian tumour model mice
(7-week-old, 20-25 g) were bought from Dalian Medical College, China. Before
animal imaging and analysis, the mice were starved for 12 h because of possible food
fluorescence interference at the emission wavelength of the fluorescent dyes. Then,
the ovarian tumour-bearing mouse was in-situ injected with RD (50 ꢀL, 500 ꢀM, in
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DMSO) and observed using an in vivo imaging system. After injection of RD for 20
min, a strong fluorescence signal was detected in the tumour, and the fluorescence
intensity became stronger along with the prolongation of time (Fig. 5).
Fig. 5 Representative fluorescence images (pseudocolor) of mice given an in-situ injection of RD
(50 ꢀL, 500 ꢀM, in DMSO ) in tumour and blank respectively. (a) Blank, (b) in-situ injection of
RD in tumour and blank for 10 min, (c) for 20 min, (d) for 40 min, (e) for 60 min, (f) for 80 min,
(g) for 100 min, (h) for 120 min, (i) for 150 min. Images were taken with an excitation laser of
530 nm and an emission filter of 600 ± 20 nm. The inserted figure was the enlarged fluorescent
position.
To provide further evidence, we dissected tumour and some organs from another
group of ovarian tumour-bearing mice first, and then injected RD (50 ꢀL, 100 ꢀM, in
DMSO) on the corresponding tumour and organs. As shown in Fig. 6, an exclusive
emission signal was observed in the tumour 20 min after the injection. The fluorescent
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signal in the tumour indicates that the acidity in ovarian tumour tissues is higher than
that in normal tissues.
Fig. 6 Fluorescence images (pseudocolor) of tumour and organs excised from mice. Images were
taken after injection 20 min with an excitation laser of 530 nm and an emission filter of 600 ± 20
nm.
3.6 Imaging in vivo mice
Hence, SPF mice starved 12 h were sacrificed. Then, the stomach and other organs
were excised. As shown in Fig. S20, no fluorescence intensity was found for other
organs. However, the fluorescence intensity in the stomach sharply increased after
injection with RD for 30 min due to the pH of the stomach is less than 4 [53].
The Living mice administered with a skin-pop injection of RD (50 ꢀL, 200 ꢀM)
and a mixture (100 ꢀL) of phosphate buffer (10 mM) and EtOH (5/5, v/v) at pH 4.09
(Fig. 7) and 2.94 (Fig. S21) were also imaged using a Night OWL II LB 983 system
equipped with an NC 100 CCD deep-cooled camera.
The fluorescence intensity became strongest after injection for 20 min (Fig. 7f), and
then decreased along with time extension at pH 4.09 (Fig. 7g, h, i). The same trend
can be observed in the case of pH 2.94 (Fig. S21), while the strongest fluorescent
emission happened after injection for 15 min (Fig. S21e). The fluorescence intensity
15

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was stronger and the response time was shorter at pH 2.94 than that at pH 4.09. In
comparison, not any fluorescence can be observed in the corresponding blank control
group. These results demonstrate that RD is an efficient fluorescent pH probe in vivo
imaging.
a
d
g
c
b
Injection
e
f
h
i
Fig. 7 Representative fluorescence images (pseudocolor) of mice given a skin-pop injection of RD
(50 ꢀL, 200 ꢀM ) and a mixture (100 ꢀL) of phosphate buffer (pH 4.09, 10 mM) and EtOH (5/5,
v/v). (a) Blank, (b) Skin-pop injection of a mixture of phosphate buffer (pH 4.09, 10 mM) and
EtOH (5/5, v/v), (c) 10 min later, skin-pop injection of RD in the mouse in b for 5 min, (d) for 10
min, (e) for 15 min, (f) for 20 min, (g) for 25 min, (h) for 30 min, (i) for 40 min. Images were
taken with an excitation laser of 530 nm and an emission filter of 600 ± 20 nm. The inserted figure
was the enlarged fluorescent position.
16

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4. Conclusions
In summary, a rhodamine B-based lysosomal pH probe termed RD with a pKa of
4.10 was developed. The probe exhibited high selectivity, good photostability and
high reversible response to pH, as well as low cytotoxicity. RD can be an efficient pH
probe for distinguishing cancer cells from normal cells and detecting tumours. The
fluorescence signal in the ovarian tumour indicates that the acidity in tumor tissues is
higher than that in normal tissues. Furthermore, RD performed well in complex
biological systems, such as excised mouse stomach and living mice.
Acknowledgments
This work was financially supported by National Natural Science Foundation of
China (Nos. 21272030, 21472016, 21306019, 21576042, 21421005).
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Supplementary data (Fig. S1–S18)
A lysosomal probe for monitoring of pH in living cells
and ovarian tumour
Bo Lia,1, Guangbo Geb,1,Lanlan Wena,1,Ye Yuana, Ran Zhanga, Xiaojun Penga,
Fengyu Liua,* and Shiguo Sunc,*
a
State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of
Technology, No. 2, Linggong Road, Ganjingzi District, 116023 Dalian, China.
b Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
c
Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education,
School of Pharmacy, Shihezi University, Shihezi 832000, China
1 These authors contributed equally to this work.
*Correspondence should be addressed to:
School of Chemistry, Dalian University of Technology,
No.2, Linggong Road, Ganjingzi District
Email: liufengyu@dlut.edu.cn
And Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education,
School of Pharmacy, Shihezi University, Shihezi 832000, China
Phone: +86-29-87092226
Fax: +86-29-87082832
Email: sunsg@nwsuaf.edu.cn
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Scheme S1. Synthesis of compound RD
RDH
RD
Scheme S2. Proposed Sensing Mechanism for H+
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Fig. S1 1H NMR spectrum of R2 in CDCl3
YY-M485 #40-45 RT: 0.11-0.12 AV: 6 SB: 12 0.92-0.95 NL: 3.64E4
T: ITMS + c ESI Full ms [200.00-1100.00]
485.27
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
507.23
10
274.24
318.27
5
338.27
302.31
457.22
437.18
591.94
362.32
218.41
675.00 690.21
246.26
250
565.08
390.41 409.19
400
536.27
605.02
780.05
758.89
290.06
649.82
650
712.75
806.23
800
739.69
0
300
350
450
500
m/z
550
600
700
750
Fig. S2 MS spectrum of R2
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Fig. S3 1H NMR spectrum of RD in CDCl3
Fig. S4 13C NMR spectrum of RD in CDCl3
25

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YY-M620-HR #11-12 RT: 0.11-0.12 AV: 2 SB: 3 0.01-0.02 NL: 4.90E5
T: FTMS + p ESIFull ms [300.00-1300.00]
621.3067
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
643.2885
659.2629
666.3644
313.2019
5
353.2660
532.3543
550
728.2678
415.1894
600.4672
816.2160
800
453.2101
450
763.2667
869.2573
850
489.6859
500
0
350
400
600
m/z
650
700
750
900
Fig. S5 HRMS spectrum of RD
(c)
(b)
(a)
N(CH2CH3)2
Fig. S6 Partial 1H NMR spectra of (a) RD in DMSO-d6/D2O (9 : 1, v/v, adjusting pH to 7), (b) RD
in DMSO-d6/D2O (9 : 1, v/v) in the presence of CF3COOH (adjusting pH to 4), (c) RD
inDMSO-d6/D2O (9 : 1, v/v) in the presence of CF3COOH and then NaOH (adjusting pH to 7).The
NaOH was dissolved in D2O and made into 200 mM solution.
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a
80
70
60
50
40
30
20
10
0
5
10
15
20
25
30
Time (min)
b
250
200
150
100
50
0
560
580
600
620
640
Wavelength/nm
Fig. S7 (a) Time-dependent fluorescent intensities at 586 nm of RD (10 ꢀM) in B-R-E solution
when pH is 4.25. (b) Fluorescence spectra of RD (10 ꢀM) in B-R-E solution with pH 3.26. (λex =
540 nm).
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a
80
70
60
50
40
30
20
10
0
b
80
70
60
50
40
30
20
10
0
Fig. S8 Relative fluorescence intensity at 586 nm of RD (10 ꢀM) in the presence of different ions
in B-R-E solution, concentration of the other ions is 200 ꢀM (a) pH = 7.02, (b) pH = 4.25 (λex =
540 nm).
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Fig. S9 Confocal fluorescence images of MCF-7 cells with RD (5 ꢀM) after the cells were
incubated for 10, 20, 30 and 45 min at 37 ºC under 5% CO2, respectively. Scale bar: 20 ꢀm, λex =
559 nm, λem = 565-620 nm.
29