Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH monitoring

Article abstract of DOI:10.1016/j.dyepig.2019.107612

Real-time monitoring of intracellular pH fluctuation is of great significance for diagnosis of diseases and study of related physiological and pathological processes. A novel near-infrared (NIR) pH-activated parent probe 4 was developed, and further functionalized to afford probes 6 and 7. With the decrease of pH from 11 to 2, they all exhibited a new absorption peak and strong fluorescence emission in NIR region, accompanied by rapid solution color change from pink to pale green. Moreover, the probes’ fluorescence responses toward pH fluctuations were reversible and stable, and the pseudo-linear relationship between the fluorescence intensity and pH value can be used to detect pH value in 2.76–6.45 range quantitatively. Finally, probe 6 and 7 were successfully applied to monitor the fluctuation of intracellular pH by a fluorescence turn-on mode, and the imaging results confirmed their low cytotoxicity and satisfactory cell-membrane permeability, as well as their application prospect in disease diagnosis (such as tumor specific imaging) or assist in study of pH-related biological processes in wide acidic environment.

Full text of DOI:10.1016/j.dyepig.2019.107612

Accepted Manuscript
Novel near-infrared pH-sensitive cyanine-based fluorescent probes for intracellular pH
monitoring
Di Jin, Bowei Wang, Yuqing Hou, Yuchao Du, Xue Li, Ligong Chen
PII:
S0143-7208(19)31085-X
DOI:
https://doi.org/10.1016/j.dyepig.2019.107612
Article Number: 107612
Reference:
DYPI 107612
To appear in: Dyes and Pigments
Received Date: 13 May 2019
Revised Date: 30 May 2019
Accepted Date: 1 June 2019
Please cite this article as: Jin D, Wang B, Hou Y, Du Y, Li X, Chen L, Novel near-infrared pH-sensitive
cyanine-based fluorescent probes for intracellular pH monitoring, Dyes and Pigments (2019), doi: https://
doi.org/10.1016/j.dyepig.2019.107612.
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ACCEPTED MANUSCRIPT

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Novel near-infrared pH-sensitive cyanine-based fluorescent
probes for intracellular pH monitoring
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Di Jina b, Bowei Wang*a b c, Yuqing Houa, Yuchao Dua, Xue Lia, Ligong Chen*a b c
a School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, P. R. China.
E-mail: bwwang@tju.edu.cn, lgchen@tju.edu.cn; Tel and Fax: +86 22 27406314.
b Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), P. R. China
c Tianjin Engineering Research Center of Functional Fine Chemicals, Tianjin, P. R. China
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Abstract
Real-time monitoring of intracellular pH fluctuation is of great significance for
diagnosis of diseases and study of related physiological and pathological processes. A
novel near-infrared (NIR) pH-activated parent probe 4 was developed, and further
functionalized to afford probes 6 and 7. With the decrease of pH from 11 to 2, they all
exhibited a new absorption peak and strong fluorescence emission in NIR region,
accompanied by rapid solution color change from pink to pale green. Moreover, the
probes’ fluorescence responses toward pH fluctuations were reversible and stable, and
the pseudo-linear relationship between the fluorescence intensity and pH value can be
used to detect pH value in 2.76-6.45 range quantitatively. Finally, probe 6 and 7 were
successfully applied to monitor the fluctuation of intracellular pH by a fluorescence
turn-on mode, and the imaging results confirmed their low cytotoxicity and
satisfactory cell-membrane permeability, as well as their application prospect in
disease diagnosis (such as tumor specific imaging) or assist in study of pH-related
biological processes in wide acidic environment.
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Keywords: Fluorescent probe; pH detection; Near-infrared; Cyanine; Cell imaging
24 1. Introduction
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pH is an important indicator to be monitored in the fields of industry,
agriculture, biochemistry, medicine, environment etc. [1]. Particularly, intracellular
pH is a key physiological parameter, which is closely related to various biological
processes such as cell proliferation and apoptosis, enzyme activity, ion transport and
immunization [2, 3]. The fluctuation of pH, especially elevated acidity level, is often
implicated with diverse systemic pathological problems, such as infection,
inflammation, metabolic disorders, and even Alzheimer’s disease and cancer [4, 5].
Therefore, real-time monitoring of intracellular pH is of great significance for
diagnosis of diseases and understanding of related physiological and pathological
processes.
Compared with the traditional pH detection methods (titration, nuclear
magnetic resonance and microelectrode method), fluorescence detection has been
widely used and studied, especially in intracellular pH detection, by virtue of its
simple operation, high sensitivity, good selectivity, low cost and rapid real-time
response [6, 7, 8, 9]. Up to date, diverse fluorescent probes based on fluorescein,
rhodamine, naphthalimide, BODIPY have been reported [10, 11]. Nevertheless, their
excitation and emission wavelengths are generally in the ultraviolet or visible region,
where background fluorescence is strong and tissue penetration is limited, which
affects their imaging sensitivity and further restricts their application in the detection
of intracellular pH. Cyanine-based probes, absorbing and emitting in the near-infrared
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(NIR) region, are ideal for biological imaging because NIR light can penetrate deeply
into tissue and cause almost no cell damage or background fluorescence interference
[12, 13, 14]. However, their photobleaching resistance, water solubility and
biocompatibility need to be improved to ensure their practical application. Moreover,
among the reported pH-sensitive cyanine-based probes, most focused on visualization
of minor pH changes in near-neutral range [15, 16], while fewer realized the
monitoring of pH fluctuations in a wide acidic range (pH=3.0-6.5). Thus, it still
remains challenge to develop new NIR pH-sensitive probes with ideal detection range,
sufficient water solubility and biocompatibility, high sensitivity and quick response
for real-time detection of pH variations in acidic environment.
Based on these, we designed and synthesized a NIR pH-sensitive indocyanine
probe (compound 4, Scheme 1), which contains a non-N-alkylated indolium moiety as
recognition site of proton, and bearing a carboxylic acid group as modification site for
achieving specific properties [17]. In order to prove its feasibility, we further
synthesized probe 6 and 7. For probe 6, a glucosamine was firstly introduced to
improve the probe’s solubility, biocompatibility as well as targeting ability to cancer
cells [18]. Probe 6 presented rapid and sensitive response to weakly acidic
environment, and its pH response range is suitable for specific imaging of cancer cells.
Besides, because a ratiometric fluorescent probe would reduce the instrumental and
environmental effects on a single fluorescent signal [19], we combined probe 4 with
another dye (naphthalimide as an example) to form a ratiometric probe 7. As expected,
the obtained probe exhibited strong fluorescence in acidic environment and can be
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used as a pH ratio probe in the intermediate acid range. Interestingly, probe 4, 6 and 7
all have different pH detection ranges, covered almost all acidic ranges, which make
them suitable for detecting intracellular pH fluctuations and studying physiological
and pathological processes under acidic conditions. Furthermore, probe 6 and 7 were
successfully applied to monitor the intracellular pH variations.
Br-
N
Br
COOH
O
CH3COOH
+
NH2
N
H
N
2
1
O
Cl
3
HOOC
OH
O
POCl3
+
DMF
+
Cl
3
Cl
4
C2H5OH
Br-
N
+
+
OH
N
O
N
N
1
2
HOOC
COOH
Cl
OH
O
N
N
HO
HO
OH
HCl
NH2
6
DIPEA, DMF
O
Cl
HN
HO
OH
OH
N
N
O
OH
EDC HCl
·
NH2
NHS, DMF
Cl
7
O
O
N
O
N
N
O
N
O
O
5
O
O
DIPEA, DMF
HN
N
O
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Scheme 1. Synthetic routes of the target probes.
74 2. Materials and methods
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2.1 Materials and apparatus
All reagents and solvents were purchased from commercial sources and used
without further purification unless otherwise noted. Deionized water was used
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throughout this work. H NMR and C NMR spectra were recorded on a Bruker
Avance III 400 MHz spectrometer, using tetramethylsilane (TMS) as an internal
standard. High-resolution mass spectra (HRMS) were carried out with a Bruker
Daltonics microTOF-Q II instrument (ESI). All pH measurements were performed on
a Sartorius basic pH-meter PB-10. UV-Vis absorption spectra were obtained using an
Evolution 300 UV-Vis spectrophotometer. Fluorescence spectra were measured on a
Jobin Yvon-Spex Fluorolog3 spectrophotometer. MTT assay was taken on a BioTek
Synergy HT microplate reader. The cell imaging was performed on a OLYMPUS
FV3000 confocal laser scanning microscopy. All measurements were performed under
ambient atmosphere at room temperature.
2.2 Synthesis
The synthetic routes of the probes were shown in Scheme 1.
2,3,3-Trimethyl-3H-indoline
(1)
and
2-chloro-1-formyl-3-(hydroxymethylene)-cyclohex-1-ene (3) were synthesized
according to our previously reported method [20, 21].
1-(5’-Carboxypentyl)-2,3,3-trimethylindolinium bromide (2): This compound was
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synthesized according to a reported method with some modifications [22]. A mixture
of compound 1 (6.37 g, 40 mmol) and 6-bromohexanoic acid (7.80 g, 40 mmol) was
stirred at 110 for 8 h. After cooled to 50 , the resultant purplish red viscous oil
was washed with ethyl acetate several times to afford a brown bulk solid. The solid
was triturated and washed with acetone several times at 50 . Then the resulting pink
powder was collected and washed with acetone to afford compound 2 as a light pink
powder (10.15 g, 71.63%).
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1H NMR (400 MHz, CD3OD) δ (ppm): 7.89-7.87 (m, 1H), 7.78-7.76 (m, 1H),
7.66-7.64 (m, 2H), 4.52 (t, 2H, J = 8.0 Hz), 2.34 (t, 2H, J = 7.2 Hz), 2.02-1.95 (m, 2H),
1.73-1.66 (m, 2H), 1.61 (s, 6H), 1.57-1.49 (m, 2H).
13C NMR (100 MHz, CD3OD) δ (ppm): 196.75, 175.96, 142.25, 141.34, 130.02,
129.36, 123.53, 115.44, 54.80, 48.09, 33.27, 27.44, 25.92, 24.30, 21.70.
HRMS (ESI Positive), m/z: calculated for [M-Br-] 274.1802, found 274.1805.
Compound 4: Compound 1 (0.79 g, 5 mmol), compound 2 (1.77 g, 5 mmol) and
compound 3 (0.86 g, 5 mmol) were added into 10 mL ethanol and the resulting
mixture was stirred at 70 under nitrogen atmosphere for 5 h to yield a dark purple
solution. After cooled to room temperature, the solvent was evaporated in vacuum.
The crude dark purple solid was purified by silica gel column chromatography with
gradient elution (dichloromethane to dichloromethane/methane of 40:1) to afford a
dark purple solid (0.94 g, 33.10%).
1H NMR (400 MHz, CDCl3) δ (ppm): 8.09 (d, 1H, J = 16.4 Hz), 7.63 (d, 1H, J = 7.6
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Hz), 7.54 (d, 1H, J = 12.4 Hz), 7.34-7.30 (m, 2H), 7.23-7.15 (m, 3H), 6.88 (t, 1H, J=
7.2 Hz), 6.71-6.63 (m, 2H), 5.48 (d, 1H, J = 12.4 Hz), 3.64 (t, 2H, J = 6.8 Hz),
2.60-2.57 (m, 4H), 2.40 (t, 2H, J = 6.8 Hz), 1.89-1.87 (m, 2H), 1.75-1.73 (m, 4H),
1.64 (s, 6H), 1.50 (s, 8H).
13C NMR (100 MHz, CDCl3) δ (ppm): 184.00, 159.09, 152.97, 146.88, 144.41,
139.15, 138.26, 137.98, 128.21, 127.88, 127.81, 127.53, 125.56, 124.68, 121.74,
121.01, 120.12, 119.12, 106.45, 93.06, 52.34, 46.10, 42.33, 34.28, 29.73, 28.24, 26.80,
26.42, 26.07, 24.95, 24.73, 21.45.
HRMS (ESI Positive), m/z: calculated for [M+H+] 569.2929, found 569.2930.
N-(2-aminoethyl)-1,8-naphthalimide (5): This compound was synthesized according
to a reported method with some modifications [23]. 1,8-Naphthalenedicarboxylic
anhydride (0.59 g, 3 mmol) and ethylene diamine (0.90 g, 15 mmol) were added in 50
mL ethanol and the resulting mixture was stirred at reflux for 5 h to yield a yellow
suspension. After cooled to room temperature, the mixture was filtered and the filtrate
was evaporated under reduced pressure to afford a light yellow powder (0.65 g,
90.18%), which could be used in the next reaction without further purification.
Compound
6:
Compound
4
(0.14
g,
0.25
mmol),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 0.06 g,
0.30 mmol) and N-hydroxysuccinimide (NHS, 0.03 g, 0.30 mmol) were dissolved in
10 mL DMF and the resulting mixture was stirred at room temperature under nitrogen
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atmosphere for 24 h in the dark. Then D-glucosamine hydrochloride (0.07 g, 0.30
mmol) and N, N-diisopropylethylamine (DIPEA, 0.10 g, 125 µL, 0.75 mmol) were
added to the reaction solution and the mixture was stirred for an additional 6 h. After
cooled to room temperature, partial solvent was evaporated in vacuum, and then the
solution was poured into a large amount of water. The precipitate was collected and
purified by silica gel column chromatography with gradient elution (dichloromethane
/methane of 25:1 to 15:1) to afford a purplish red solid (0.07 g, 38.89%).
1H NMR (400 MHz, CD3OD) δ (ppm): 8.19-8.15 (m, 1H), 7.59-7.57 (m, 1H), 7.47 (d,
1H, J = 7.6 Hz), 7.36 (t, 1H, J = 8.0 Hz), 7.32-7.29 (m, 1H), 7.23-7.13 (m, 3H),
6.85-6.83 (m, 1H), 6.74 (t, 1H, J = 7.6 Hz), 6.56-6.54 (m, 1H), 5.53-5.48 (m, 1H),
5.12 (s, 1H), 3.87-3.79 (m, 3H), 3.71-3.69 (m, 4H), 3.42-3.34 (m, 1H), 2.68-2.44 (m,
4H), 2.27-2.24 (m, 2H), 1.88-1.80 (m, 2H), 1.74-1.65 (m, 4H), 1.58-1.56 (m, 6H),
1.44-1.42 (m, 8H).
HRMS (ESI Positive), m/z: calculated for [M+H+] 730.3617, found 730.3615.
Compound 7: The synthesis process is similar to that of compound 6, by using
compound 4 (0.17 g, 0.30 mmol), EDC·HCl (0.08 g, 0.40 mmol), NHS (0.05 g, 0.40
mmol), compound 5 (0.10 g, 0.40 mmol) and N, N-diisopropylethylamine (DIPEA,
0.16 g, 200 µL, 1.20 mmol). The resulting solid was purified by silica gel column
chromatography with gradient elution (dichloromethane /methane of 60:1 to 50:1) to
afford a purplish red solid (0.13 g, 54.85%).
1H NMR (400 MHz, CDCl3) δ (ppm): 8.57 (d, 2H, J = 7.2 Hz), 8.18 (d, 2H, J = 8.0
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Hz), 8.08 (d, 1H, J = 16.0 Hz), 7.72 (t, 1H, J = 7.6 Hz), 7.60 (d, 1H, J = 7.2 Hz), 7.51
(d, 1H, J = 12.8 Hz), 7.33-7.29 (m, 2H), 7.22-7.12 (m, 3H), 6.86 (t, 1H, J = 7.2 Hz),
6.63-6.55 (m, 2H), 6.26 (s, 1H), 5.40 (d, 1H, J = 12.8 Hz), 4.41-4.38 (m, 2H),
3.69-3.66 (m, 2H), 3.51-3.48 (m, 2H), 2.56-2.53 (m, 4H), 2.13 (t, 2H, J = 6.8 Hz),
1.87-1.84 (m, 2H), 1.64-1.60 (m, 10H), 6.20 (s, 6H), 1.36-1.32 (m, 2H).
13C NMR (100 MHz, CDCl3) δ (ppm): 183.97, 173.01, 164.84, 158.74, 154.17,
147.16, 144.41, 139.10, 137.62, 136.94, 134.36, 131.59, 131.53, 128.19, 127.75,
127.11, 127.03, 125.32, 124.64, 122.26, 121.70, 120.97, 120.42, 119.99, 119.87,
106.38, 92.94, 52.43, 46.02, 42.18, 39.53, 36.49, 28.23, 26.84, 26.79, 26.38, 26.01,
25.31, 24.78, 21.47.
HRMS (ESI Positive), m/z: calculated for [M+H]+ 791.3722, found 791.3727.
2.3 Measurement of UV-Vis absorption and fluorescence spectra
The working solutions of probe 4, 6, 7 (1.0 × 10-5 M) were obtained by
dissolving appropriate amount of corresponding probe (1.0 × 10-6 mol) in 30 mL
ethanol and diluting them with water to the mark in 100 mL volumetric flask. The
working solution’s pH was adjusted by 1M or 0.1M NaOH and HCl, under the precise
monitoring of pH meter. For the fluorescence measurements, the excitation and
emission silt widths were both 3 nm (for probe 6 and 7) or 4 nm (for probe 4) and the
excitation wavelengths were 340/680 nm.
2.4 Evaluation of cytotoxicity
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The murine squamous cell carcinoma (SCC-7) cells were used for cytotoxicity
evaluation of probe 6 and 7 based on the standard cell viability protocols (MTT assay).
SCC-7 cells were seeded into a 96-well cell culture plate at a density of 1×104 cells
per well in 100 µL Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal
bovine serum (FBS) at 37
under 5% CO2 humidified atmosphere. After 24 h
incubation, the medium was replaced with fresh medium including different
concentrations of probe 6 and 7 (0.5, 1, 5 and 10 equiv. of testing concentration
(1×10-5 M), each concentration was done in triplicate). The cells incubated with fresh
medium in the absence of probe were used as the blank control. After further
incubation for 24 h, the medium was replaced with fresh medium containing
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (100 µL, 5
mg/mL in fresh medium) and incubated for another 4 h. Subsequently, the medium
was removed and rinsed with PBS buffer, then 200 µL DMSO was added to dissolve
the purple formazan crystals produced by living cells. After vibration for 20 min in
the dark, the absorbance at 570 nm of each well were recorded on a microplate reader.
The relative cell viability (%) was determined by the following formula: viability (%)
= (Ax / Acontrol) × 100, where Ax is the average absorbance of test group and Acontrol is
the average absorbance of the control group.
2.5 pH-dependent cell imaging and co-localization experiments
SCC-7 cells were seeded in a 6-well cell culture plate at a density of 5×105 cells
per well. After incubation for 24 h for cell attachment, the original medium was
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replaced by fresh medium containing probe 6/7 (1×10-5 M) and further incubated for
12 h at 37
under 5% CO2 humidified atmosphere. Then the culture medium was
removed and cells were washed twice with PBS buffer, fixed with 1 mL of 4%
formaldehyde for 15 min, rinsed twice with PBS buffer and stained with
4’,6-diamidino-2-phenylindole (DAPI) solution for 5 min. After thoroughly cleaning
with PBS buffer, the cells were soaked with PBS buffer at various pH values from 4.0
to 7.4, and then confocal fluorescence images were carried out on OLYMPUS
FV3000 confocal laser scanning microscopy with a 60×oil-immersion object lens.
DAPI was excited at 405 nm and the blue emission was collected in the range of
430-470 nm. Probe 6/7 was excited at 633 nm and the red emission was collected in
the range of 700-800 nm.
217 3. Results and discussion
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3.1 Synthesis of probe 4, 6, 7
As shown in Scheme 1, probe 4, 6, 7 were obtained via simple synthetic routes.
Compound 1-3 and 5 were synthesized according to the reported method with some
modifications. Then the condensation of 1, 2 and 3 in ethanol provided probe 4. To
make it easier to conjugate with specific molecules, probe 4 was first converted into
reactive N-hydroxysuccinimidyl ester by using EDC·HCl and NHS. Then the active
ester further reacted with D-glucosamine/naphthalimide in the presence of DIPEA to
afford probe 6/7. The structures of intermediates and target probes were confirmed by
1H NMR, 13C NMR and HRMS analysis (Fig. S1-S11).
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3.2 Optical responses of probe 4, 6, 7 towards pH
To demonstrate the application prospect of the target compounds as
pH-sensitive probes, the spectral responses of 4, 6, 7 to pH fluctuations were initially
examined in ethanol-water (3/7, V/V) solution and their photophysical data were
shown in Table S1. As shown in their absorption spectra (Fig. 1a-3a), with the
decrease of pH value, they all presented similar change. Take probe 7 as an example,
in alkaline medium, there is only an absorption peak at 535 nm. As the pH decreased,
the absorption peak at 535 nm dropped gradually and a new absorption peak emerged
and gradually enhanced at 795 nm. A distinct isosbestic point existed at 640 nm,
attributing to the transition between acid form and base form. Meanwhile, with the
increase of acidity, the solution color changed from pink to purple, and further turned
green (as shown in Fig. 3f), which verified its potential application as a ‘naked-eye’
indicator for pH value. Probe 6 exhibited similar trend as probe 7, while probe 4
displayed another absorption peak in the pH range of 4.01-8.50. The peak suggested
the existence of a third form in the transformation, which may be attributed to the
effect of carboxyl group.
Fluorescence spectra (λex=680 nm) of the three probes upon pH variations were
shown in Fig. 1b-3b. All of them presented no fluorescence under neutral conditions,
and exhibited gradually enhanced fluorescence emission at ~805 nm with the increase
of acidity. However, they have different pH sensitive intervals and different pKa value.
In Fig. 1c-3c, the relative fluorescence intensity (I/Imax, where I is the fluorescence
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intensity at specific pH, Imax is the maximum fluorescence intensity of the probe)
towards pH were plotted. For probes 4, 6, 7, within the narrow range of 2.76-4.01,
3.65-4.70, 4.59-6.45, respectively, there is a good linear relationship between pH
value and relative fluorescence intensity, making them promising fluorescence probes
for quantitative monitoring of slight pH fluctuation. Their pKa values can be estimated
as 3.45, 4.05, 5.52 corresponding to 4, 6, 7 respectively, at 0.5 relative fluorescence
intensity [17].
Ratiometric fluorescent probes, introducing built-in correction through the ratio
of emission intensity at two different wavelengths, can provide more reliable
quantitative information [24]. Thus fluorescence spectra (λex=340 nm) of the probe 7
upon pH variations were performed to investigate whether the fluorescence of
naphthalimide is dependable on pH value (Fig. 3d). As expected, besides some
extreme conditions (pH<3 or pH>11), the fluorescence at 390 nm is relatively stable
at different pH values and can be used as a benchmark. Considering the two order
difference of fluorescence intensity at 390 nm and 805 nm, the relative fluorescence
intensity (RI805 and RI390) were used to calculate the ratio fluorescence intensity
(RI805/RI390) to obtain a more reliable result. As shown in Fig. 3e, the ratio
fluorescence intensity also has a good linear relationship with pH value in the range of
3.65-4.70, which makes it a promising pH-sensitive ratio fluorescence probe.
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Fig. 1. (a) Absorption and (b) Fluorescence spectra (λex=680 nm) of probe 4 (1.0×10-5
M) upon pH variations in ethanol-water (3/7, V/V) solution; (c) Changes of relative
fluorescence intensity upon pH.
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Fig. 2. (a) Absorption and (b) Fluorescence spectra (λex=680 nm) of probe 6 (1.0×10-5
M) upon pH variations in ethanol-water (3/7, V/V) solution; (c) Changes of relative
fluorescence intensity upon pH.
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Fig. 3. (a) Absorption, (b) Fluorescence spectra (λex=680 nm) and (d) Fluorescence
spectra (λex=340 nm) of probe 7 (1.0×10-5 M) upon pH variations in ethanol-water
(3/7, V/V) solution; Changes of (c) Relative fluorescence intensity and (e) Ratio
fluorescence intensity upon pH; (f) Visual color changes of probe 7 upon pH change.
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3.3 Proposed mechanism
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We speculated that the pH-sensitive spectral changes were attributed to the
protonation and deprotonation of the non-alkylated indole nitrogen atom (as shown in
Scheme 2 with probe 7 as representative). In acidic environments, protonation of the
indole nitrogen atom will enhance the ICT process, which further leads to the
bathochromic shift in absorption spectra and strong fluorescence emission in NIR
region. Conversely, deprotonation of the nitrogen atoms will restore the probe to its
base form, which results in a hypsochromic shift in absorption spectra and no
observable NIR fluorescence emission.
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Scheme 2. Proposed mechanism of probe 7 to pH.
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3.4 Reversibility and stability of probe 4, 6, 7
To investigate its practical application performance, the reversibility and stability
of the three probes were examined. As shown in Fig. 4a-4b (probe 4 as representative),
both its absorption and fluorescence exhibited desirable reversibility when the pH of
working solution were adjusted cyclically between 3 and 11. Their photostability were
examined under 254/365 nm UV lamp and daylight irradiation. As shown in Fig. 4c
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and Fig. S13 (probe 4 as representative), during 60 minutes exposure, their
fluorescence intensity barely decreased, indicating that these probes are stable even
under acidic conditions. Furthermore, after stored at room temperature for a week,
there was no obvious impurity produced by thin layer chromatography (TLC)
detection, which demonstrated their good chemical stability. Based on all above, all
the probes displayed reversible and sensitive response to pH, possessing good stability
and near-infrared absorption and emission suitable for imaging applications. Among
them, the pH linear detection intervals of probe 6 and 7 are more appropriate for study
of physiological and pathological processes under acidic conditions (pH=4-7), which
enabled them as promising probes to monitor intracellular pH fluctuations.
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Fig. 4. Reversible changes of (a) absorption spectra and (b) fluorescence intensity
(λex=680 nm, λem=805 nm) of probe 4 (1.0×10-5 M) toward cyclically adjusted pH
between 3 and 11; (c) Time-dependent changes of fluorescence intensity (λex=680 nm,
λem=805 nm) of probe 4 (1.0×10-5 M) at pH=3 under 254 nm irradiation.
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3.5 Cell cytotoxicity and cell imaging application of probe 6 and 7
With the encouragement of the above results, the applicability of probe 6 and 7
in the monitoring of intracellular pH fluctuations was investigated. Firstly, their
cytotoxicity towards SCC-7 cells were evaluated by the standard MTT assay. As
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shown in Fig. S12, probe 6 displayed very low cytotoxicity, which may be attributed
to the introduction of glucosamine, and the cell viabilities were just slightly affected
when the probes’ concentration is less than 10 µM (working concentration). Although
cell viability decreased with the increase of the probes’ concentration, it was still
higher than 70% when incubated with high concentration of both probes (5 equiv. of
the working concentration), indicating that the two probes are both suitable for cell
imaging.
Thus, the real-time responsive properties of the two probes for intracellular pH
fluctuations were investigated. The cells were treated with probe 6 and 7, respectively,
and further stained with DAPI (a commercial nucleus-targeting fluorescent probe) to
determine the distribution of probe 6 and 7 by colocalization. After rinsed by fresh
medium, the intracellular pH was set to different values (4.0, 5.0, 6.0 and 7.4) with
PBS buffer solutions and confocal fluorescence imaging was performed subsequently
(Fig. 5 and 6). Compared with the distribution of DAPI, it was found that the two
probes were mainly dispersed in cytoplasm, and the imaging results manifested that
both probes had favorable membrane permeability. To probe 6, as shown in Fig. 5, no
red fluorescence was observed at pH 7.4 (physiological environment of normal tissue)
while weak red fluorescence emerged at pH 6.0 (physiological environment of tumor
tissue) and evidently increased with the decrease of pH value. The turned-on
fluorescence and rapid response endowed probe 6 promising prospect as a switchable
sensor for real-time monitoring of intracellular pH fluctuation and study of pH-related
physiological and pathological processes. Moreover, it’s also a promising probe for
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tumor specific imaging. Compared with probe 6, the switch-on response interval of
probe 7 is more acidic, which is consistent with its spectra response towards pH
variations. As shown in Fig. 6, it displayed no fluorescence at pH 6.0 and exhibited
weak fluorescence at pH 5.0 as well as strong fluorescence at 4.0, which demonstrated
that it is also a promising fluorescence indicator for monitoring slight pH fluctuation
or aiding the research of physiological processes in acidic organelles such as
lysosome.
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Fig. 5. Confocal fluorescence imaging for DAPI (blue channel, 430-470 nm) and
probe 6 (red channel, 700-800 nm) in SCC-7 cells at different pH values. Scale bar:
50 µm.
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Fig. 6. Confocal fluorescence imaging for DAPI (blue channel, 430-470 nm) and
probe 7 (red channel, 700-800 nm) in SCC-7 cells at different pH values. Scale bar:
50 µm.
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4. Conclusion
In summary, three novel NIR pH-sensitive cyanine-based probes, bearing a
non-N-alkylated indolium moiety as recognition site of proton, have been designed
and synthesized. They all displayed rapid and sensitive response to pH fluctuation
through both fluorescence emission and visual color change, providing both
spectroscopic and naked-eye detection methods to pH fluctuations. Based on the
linearly fluorescence response to pH value, the probes could realize the quantitative
detect of pH value within the narrow range of 2.76-4.01, 3.65-4.70, 4.59-6.45,
respectively. Furthermore, their favorable properties such as desirable reversibility
and stability, low cytotoxicity as well as absorption and emission in the NIR region
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guaranteed their application in biological systems. Finally, the cellular imaging results
of probe 6 and 7 illustrated that they exhibited satisfactory cell-membrane
permeability and desirable performance in monitoring of intracellular pH fluctuations.
Furthermore, it demonstrated that probe 6 could achieve tumor specific imaging and
they both have potential to assist the research of pH-related physiological and
pathological processes, both in normal tissues and acidic organelles.
Acknowledgement
We are grateful to professor Xiuping Yan and associate professor Xu Zhao
(Jiangnan University) for their helpful suggestions and assistance with the cell
imaging experiments.
Funding
This work was supported by the National Natural Science Foundation of China
(Grant 21576194).
Conflict of Interest
The authors declare that they have no conflict of interest.
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Hightlights
ó
Novel pH-sensitive NIR cyanine-based fluorescent probes (4, 6, 7) were
developed.
ó
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ó
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All probes exhibited colorimetric and fluorescence responses to pH fluctuations.
The three probes could achieve quantitative detection of pH value in 2.76-6.45.
Probe 7 is a ratio fluorescent probe for pH.
Probe 6 and 7 were successfully applied to monitor intracellular pH fluctuations.