A pH sensitive ratiometric fluorophore and its application for monitoring the intracellular and extracellular pHs simultaneously

Article abstract of DOI:10.1039/c2tb00179a

Intracellular and extracellular pH plays a key role in many cell biological processes. Probes for simultaneously monitoring the pH change inside and outside living cells are rarely available. In this paper, we describe a new ratiometric pH fluorophore that was synthesized by condensation of 4-bromine-1,8-naphthalimides and 3-amino-1,2,4-triazole. In the range of pH 5-8, the only N-H in the heterocycle-fused aromatic ring system of this fluorophore undergoes a reversible deprotonation-protonation process, which results in a large red shift of the absorption and emission spectra. In aqueous solution, this fluorophore exhibits good pH selectivity, high photostability, high tolerance to ionic strength, and high fluorescence quantum yield in both the acid and base forms. A long chain derivative of this fluorophore (HNNA) was designed for cellular pH sensing. HNNA was found to locate on the membrane structure of the cells, and was successfully used for mapping the pH change in both the extracellular microenvironment and the inner cells by confocal imaging. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2tb00179a

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Materials Chemistry B
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PAPER
A pH sensitive ratiometric fluorophore and its
application for monitoring the intracellular and
Cite this: J. Mater. Chem. B, 2013, 1,
661
extracellular pHs simultaneously†
a
a
Jin Zhou,ab Canliang Fang,ab Tianjun Chang,ab Xiangjun Liu and Dihua Shangguan
*
Intracellular and extracellular pH plays a key role in many cell biological processes. Probes for
simultaneously monitoring the pH change inside and outside living cells are rarely available. In this
paper, we describe a new ratiometric pH fluorophore that was synthesized by condensation of
4-bromine-1,8-naphthalimides and 3-amino-1,2,4-triazole. In the range of pH 5–8, the only N–H in the
heterocycle-fused aromatic ring system of this fluorophore undergoes a reversible deprotonation–
protonation process, which results in a large red shift of the absorption and emission spectra. In
aqueous solution, this fluorophore exhibits good pH selectivity, high photostability, high tolerance to
ionic strength, and high fluorescence quantum yield in both the acid and base forms. A long chain
derivative of this fluorophore (HNNA) was designed for cellular pH sensing. HNNA was found to locate
on the membrane structure of the cells, and was successfully used for mapping the pH change in both
the extracellular microenvironment and the inner cells by confocal imaging.
Received 28th September 2012
Accepted 13th November 2012
DOI: 10.1039/c2tb00179a
www.rsc.org/MaterialsB
The widely used small molecule indicators for intracellular
pH are pyrene dyes and xanthene dyes.6 However, some prob-
Introduction
It is well known that intracellular and extracellular pH plays a
key role in many physiological and pathological processes.1
Extracellular pH is known to become lower in tumor microen-
vironments2 and in several other physiological events.3 Sensors
that can reveal the pH difference between the intracellular and
extracellular regions would be helpful for studies of cancer and
cell biology.4 However, very few pH sensors reported so far can
simultaneously monitor the intracellular and extracellular pH
in biological environments.
Fluorescence-based pH sensors are very powerful tools for
monitoring pH change in biological samples. However, most of
the current pH sensors respond to pH change by increasing or
decreasing the uorescence intensity; this single-intensity-
based sensing is usually compromised by many non-pH factors,
such as concentration changes of the probes, the dri of the
light source or detector, as well as environmental effects in
complex samples. A ratiometric uorescent probe can avoid
these problems by measuring the ratio of the emission inten-
sities at two different wavelengths, which has attracted intense
research efforts recently.5
lems of these dyes, such as fast leakage, lack of membrane
permeability, poor photostability or sensitivity to ionic strength,
have to be considered in practical applications.1 To overcome
these problems, some ratiometric pH sensors based on nano-
scaffolds have been designed; their uorescent signal ratio
arises from a pH-sensitive dye and a pH-insensitive reference
dye, as well as uorescence resonance energy transfer (FRET).7
However, the cytotoxicity and the uncertain cellular location of
nanoparticles limit the application of these pH sensors in living
cells. Therefore, new small molecules for ratiometric pH
measurement are still necessary. Recently a ratiometric, near-
infrared uorescent pH probe has shown good potential for
intracellular pH sensing which is based on aminocyanine
bearing a diamine moiety.8 The pH change only causes the shi
of the excitation spectrum of this probe, resulting in the
changes of the uorescence ratio excited at different wave-
lengths. Additionally, the uorescence quantum yield of
cyanine dyes is low.
An ideal ratiometric pH probe should exhibit shied
absorption or emission spectra upon pH change, its uores-
cence quantum yields should be high in both acidic and basic
states, moreover, the change of absorption or emission spectra
should be reversible. For pH imaging in cells, this probe should
have good membrane permeability, clear cellular location and
low cytotoxicity. Herein, we report a novel pH-sensitive uo-
rophore, 3-amino-1,2,4-triazole fused 1,8-naphthalimide
(Scheme 1), which exhibits ratiometric uorescent response to
pH change in physiological range. A long-chain derivative of
aBeijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical
Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of
Sciences, Beijing, 100190, China. E-mail: sgdh@iccas.ac.cn
bGraduate School of the Chinese Academy of Sciences, Beijing 100049, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c2tb00179a
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Scheme 1 Synthetic processes for the pH probes.
this uorophore (HNNA) was designed and successfully applied
to monitor the pH change in the intracellular and extracellular
microenvironments. This is the rst time it has been reported
that a new naphthalimide derivative can simultaneously
monitor intracellular and extracellular pH by the ratio response.
Fig. 1 (a) Absorption spectra of 50 mM ENNA; all samples were measured in
HEPES buffer at pH 3.86, 4.47, 5.02, 5.52, 5.93, 6.23, 6.46, 6.69, 6.93, 7.33, 7.77,
8.28, 9.10, and 10.27. (b) Response curve of ratiometric absorbance (A480nm
/
Results and discussion
A
440nm) to pH. (c) Fluorescence spectra of 1.0 mM ENNA. All samples were
Synthesis
measured in 20 mM HEPES buffer at pH 3.86, 4.47, 5.02, 5.52, 5.93, 6.23, 6.46,
6.69, 6.93, 7.33, 7.77, 8.28, 9.10, and 10.27. (d) Response curve of ratiometric
fluorescence intensity (I480nm/I510nm, lex ¼ 455 nm) to pH.
4-Aminonaphthalimide derivatives have high uorescence
quantum yield, large Stoke's shi, good photostability, and
were widely used for the design of uorescent probes.9 Some
uorescent pH sensors based on naphthalimide have been
reported,10 but none of them exhibit ratiometric uorescence
response. The new pH-sensitive uorophore was synthesized by
condensation of 4-bromine-1,8-naphthalimides and 3-amino-
1,2,4-triazole (Scheme 1). The 4-bromine-1,8-naphthalimides
were prepared from 4-bromine-1,8-naphthalic anhydride and
primary amines. The nal products were 3-amino-1,2,4-triazole
fused naphthalimides; the fused ring structure was conrmed
Furthermore, the uorescence changes of ENNA and ANNA
were found to be reversible with the change of pH (see Fig. S3,
ESI†), suggesting that our probes are stable towards the pH
change. The signicant changes of both absorbance and emis-
sion spectra with the pH change in the physiological range
indicate that the 3-amino-1,2,4-triazole fused naphthalimide
can serve as a uorophore for colorimetric and uorescent
ratiometric pH measurements. This uorophore combines the
advantages of the sensitivity of uorescence with the conve-
nience and aesthetic appeal of a visual assay.
by NMR spectra (DEPT-135ꢀ, H,1H-COSY, HSQC and NOSEY),
and high-resolution mass spectrometry (see Supporting Infor-
1
Salt concentration has been reported to inuence the
absorbance and emission spectra of highly charged pH indi-
cators. The pKas of these dyes are dependent on the ionic
strength of the buffer.11 To examine the effect of ionic strength
mation for details†).
pH responses of ENNA
An N-hydroxyethyl derivative, ENNA (2a) exhibited good solu- on the optical properties of ENNA, pH titration experiments
bility in aqueous solution, and showed a colour change from were performed in HEPES buffer containing different concen-
yellow to dark orange (Fig. 1b) and a uorescence change from trations of NaCl (0, 100, 150, 200 mM) (see Fig. S4 and 5, ESI†),
light blue to green (Fig. 1d) with the pH increase from 5.0–8.0. as well as in PBS buffer plus NaCl (0, 100, 150, 200 mM) (see
Therefore ENNA was used for investigation of the optical Fig. S6 and 7, ESI†). The emission spectra and the ratio of
properties of this pH-sensitive uorophore. The pH-dependent emission intensity at 480 and 510 nm (I480nm/I510nm) were
absorbance spectra (Fig. 1a) of ENNA show a 40 nm red-shi almost not inuenced by the change of ionic strength. The
from acidic state to basic state. Two isoabsorptive points at 395 negligible cross-sensitivity towards ionic strength may be due to
nm and 455 nm indicate that two distinct chromophores are the low number of charges of ENNA. These results suggest that
present at equilibrium. The absorbance ratio at 480 nm and 440 this uorophore is applicable to physiological ionic strength.
nm (A480nm/A440nm) (Fig. 1b) shows a signicant pH response in
The ground-state pKas of ENNA and ANNA were determined
the range of 5.0–8.0. The emission spectra of ENNA (Fig. 1c) to be 6.53 ꢁ 0.03 and 6.44 ꢁ 0.03 by absorption titration in
under excitation at the isoabsorptive point (455 nm) show a 30 HEPES buffer (containing 150 mM NaCl) (see Fig. S8, ESI†).
nm red-shi from acidic state to basic state. The ratiometric Both pKa values are within the physiological pH range, sug-
uorescence intensity at 480 and 510 nm (I480nm/I510nm
)
gesting that this uorophore could serve as a pH probe for use
(Fig. 1d) also shows a good pH response in the range of 5.0–7.5. in biological samples. The uorescence quantum yield (Ff) of
An N-carboxyhexyl derivative, ANNA (2b) showed almost ENNA was determined by using N-butyl-4-butylamine-1,8-
identical optical properties to ENNA (see Fig. S1 and 2†), indi- naphthalimide (Ff ¼ 0.7 in ethanol) as the standard.12 In HEPES
cating that the optical response of both molecules to pH change buffer at pH 7.44, the uorescence emission maximum of the
comes from the heterocycle-fused aromatic ring system and is deprotonated ENNA species is located at 510 nm with Ff ¼ 0.49.
not affected by the substituent at the imide N-atom. In HEPES buffer at pH 4.20, the emission maximum of ENNA
662 | J. Mater. Chem. B, 2013, 1, 661–667
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lies at 480 nm with Ff ¼ 0.95 (Ex: 430 nm; Em: 440–700 nm). The hand, the ENNA anion (base form) can be stabilized by reso-
high Ff in both acid and base forms in aqueous solution may be nance delocalization of the negative charge on to the whole
attributed to the large conjugated planar structure of this u- conjugated system, which can be proved by the considerable
orophore. Compared with in the acid form, the relatively lower upeld shi of the 1H NMR signals of all the aromatic protons
Ff in the base form might be attributed to the enhanced non- (H-(a)–(d) and (f)) (Fig. 2b). Both of the above reasons cause the
radiative decay resulted from the increased interaction of the lower pKa of this uorophore than that of other 4-amino-
anion form and water or counter ions in solution. The high Ff of naphthalimide derivatives. The negatively charged base form of
ENNA in both states indicates that this uorophore can serve as ENNA has higher electron donor strength than its neutral acid
a good ratiometric uorescent probe for pH measurement.
form, which enhances the extent of ICT and results in the red-
shied absorption and emission of the basic form (Fig. 1).
Mechanism of the pH response of ENNA
Stability of ENNA
The visible colour and strong uorescence of 4-amino-
naphthalimide derivatives are due to their outstanding internal
charge transfer (ICT) from the electron-rich amino group to the
electron-poor imide moiety.9 The above optical properties of
ENNA can also be attributed to the ICT mechanism (Fig. 2a).
The mechanism of the pH response of ENNA was investigated
by H NMR spectroscopy. Compared to the H NMR spectra of
ENNA in acidic state (LH), the N–H signal (peak e) of ENNA in
basic state (Lꢂ) disappeared (Fig. 2b), which suggests the
deprotonation of N–H (Fig. 2a). The electrospray ionization-
mass spectrometry (ESI-MS) analysis in negative ion mode only
exhibited a prominent peak at m/z 320.1 (Fig. S9†), corre-
sponding to the (M ꢂ H)ꢂ of ENNA, and no (M + H)+, (M + Na)+
or (M + K)+ ion peaks were found in positive ion mode, sug-
gesting that ENNA can be deprotonated easily.
The stability of ENNA was tested by the time scan of its uo-
rescence intensity in 20 mM HEPES buffer at pH 5.85, 6.49 and
7.47 respectively under a constant 480 nm excitation over 1 h.
The uorescence intensities of ENNA at different pH remained
stable over the time range tested (Fig. S10†), which indicates
that ENNA solution is stable to the mediums, light and air.
1
1
pH selectivity of ENNA
For pH measurement in biological samples, a good probe
should not respond to potential interferents. A selectivity
experiment was carried out in 20 mM HEPES containing
different metal ions, proteins, and bioactive small molecules,
respectively. As shown in Fig. 3, no signicant change of the
uorescence intensities was observed at pH 5.33 (Ex/Em: 440/
480 nm), pH 6.25 (Ex/Em: 455/510 nm) and pH 7.67 (Ex/Em:
480/510 nm) in the presence of these additives, respectively,
indicating that ENNA is highly selective to pH.
Compared with other 4-aminonaphthalimide derivatives
that are insensitive to pH, the N–H of ENNA is integrated in a
large conjugated ring system. As shown in Fig. 2a, the electron-
withdrawing inductive effects of the imide moiety and triazole
moiety in this ring system weaken the N–H bond. On the other
Design, synthesis and uorescence properties of the probe for
cellular pH measurement
The above characteristics of this uorophore make it hold good
potential for ratiometric measurement of cellular pH. However,
maybe due to the good water-solubility of ENNA and ANNA,
both of them exhibit poor cell permeability. In order to increase
Fig. 3 Fluorescence intensities of 1.0 mM ENNA in 20 mM HEPES buffer at pH
5.33, 6.25, 7.67 in the presence of common metal ions and bioactive small
molecules. (1) Blank; (2) Na+ (100 mM); (3) K+ (100 mM); (4) Mg2+ (0.5 mM); (5)
Ca2+ (0.5 mM); (6) Zn2+ (0.5 mM); (7) Fe2+ (0.5 mM); (8) Fe3+ (0.5 mM); (9) Hg2+
(0.5 mM); (10) glucose (150 mM); (11) D-levulose (150 mM); (12) D-ribose (150 mM);
(13) arginine (150 mM); (14) lysine (150 mM); (15) tyrosine (150 mM); (16) tryp-
tophan (150 mM) and (17) bovine serum albumin (150 mM).
Fig. 2 (a) Proposed sensing process of probe ENNA and (b) 1H NMR spectra
taken in DMSO-d6 without (top) and with (bottom) NaOH.
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the cell permeability of this uorophore, we have to increase the suggest the different pH distribution in the cells. When
hydrophobicity by introducing hydrophobic groups.
replacing the PBS buffer (pH 7.4) with a low pH, high K+ HEPES-
Usually, substitution at the 9-position (imide N atom) of buffered solution (pH 5.5) and immediately imaging, the green
naphthalimides does not affect their uorescence properties.13 uorescence on the cell membrane (pointed out by arrows)
The pH response of ENNA and ANNA has been proved to come almost disappeared and the blue uorescence on the cell
from the reversible deprotonation of N–H in the heterocycle- membrane increased (Fig. 4b). Both blue and green uores-
fused-naphthalimide ring system. Therefore, we designed a new cence inside the cells did not show signicant changes in the
probe (HNNA) for cellular pH measurement by substitution rst minute aer the addition of the low pH buffer (Fig. 4b). The
with a hexadecyl at N-9 of naphthalimide. The long chain overlay image exhibited signicant colour difference between
substituent can rapidly insert into the bilayer membranes of the cell membrane and nuclear membrane (Fig. 4b), which
cells and increase the cell permeability of this uorophore by clearly illustrates the pH difference between extracellular and
the ip–op mechanism.14 The synthesized HNNA did not intracellular regions. A dividing cell was clearly observed as two
exhibit uorescence in water and phosphate buffered saline nuclei present in one cell membrane. However, a few minutes
(PBS) because of its low solubility, but exhibited strong uo- aer changing the buffer, the green uorescence in the cells
rescence in ethanol and in surfactant solutions (CTAB, SDS and also disappeared (Fig. S13†); this may be due to the pH change
Triton X-100) (see Fig. S11, ESI†), which suggests that HNNA inside the cells caused by the low pH high K+ solution. This set
may locate on the membrane structure of cells. The absorption of results suggests the rapid pH response of HNNA in cells.
and emission spectra of HNNA in acidic and basic ethanol
In order to test the ratiometric uorescence response of
showed similar features to those in acidic and basic aqueous HNNA in cells, MCF-7 cells were rstly incubated with HNNA
solution (see Fig. S12, ESI†). The low uorescence of HNNA in (4 mM) in DMEM medium for two hours at 37 ꢀC. Aer removing
aqueous solution would reduce the background signal when the medium, cells were treated with nigericin (5 mg mLꢂ1) in high
used for live cell imaging.
K+ HEPES-buffered solution at different pH for a further 10 min
at 37 ꢀC. Nigericin is a potassium ionophore that allows for the
rapid equilibration of pH across biological membranes.15 As
shown in Fig. 5a, at pH 6.36 or lower pH, only the blue channel
exhibited uorescence. With the pH increasing, green uores-
cence increased and blue uorescence decreased gradually. The
curve of uorescenceratio (Fgreen/Fblue)versus pH was obtained in
the pH range of 6.4–8.1 (Fig. 5b), and it can be observed that a
small pH change caused a signicant change of uorescence
ratio. Based on this ratiometric uorescence curve, the average
pH value of the untreated MCF-7 cells was determined to be
pH imaging for living cells
In order to test whether the HNNA can be used for cellular
imaging, it was incubated with HeLa cells at 37 ꢀC for 2 hours in
DMEM medium. Prior to imaging, cells were washed with PBS
(pH 7.4) three times. Fluorescence images were acquired on a
laser-scanning confocal microscope (Olympus) with a blue
channel (Ex: 405 nm, Em: 425–475 nm) and green channel (Ex:
488 nm, Em: 500–600 nm). As shown in Fig. 4, the uorescence
from both channels could be observed in the cells. HNNA was
found to locate on/in the cell membrane, cytoplasm and nuclear
membrane, but did not appear in the nucleus. When cells were
maintained in PBS (pH 7.4), the green uorescence in most
areas was brighter than the blue uorescence, but the overlay
image clearly showed some blue areas (Fig. 4a), which may
Fig. 5 (a) Confocal fluorescence images of MCF-7 cells incubated in high K+
HEPES-buffered solution containing ionophores at different pH. (b) pH map (right)
calculation for untreated MCF-7 cells based on the calibration curve (left)
obtained using ionophores. Scale bar: 20 mm.
Fig. 4 The confocal images of HeLa cells. (a) Cells maintained in PBS (pH 7.4) and
(b) cells in high K+ HEPES buffer (pH 5.5), collected immediately after changing the
buffer. Scale bar: 20 mm, cell membrane was pointed out by arrows.
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7.41 ꢁ 0.06. The ratiometric uorescence response of HNNA was
also observed in HeLa cells, and the intracellular pH value of the
HeLa cells was determined to be 7.40 ꢁ 0.02 (see Fig. S14, ESI†).
Usually tumors exhibit a lower extracellular pH (6.2–6.9)
than normal tissues (7.2–7.4).2d,e,3 HNNA was found to locate on
the cell surface and inside cells (Fig. 6b). Therefore this ratio-
metric uorescent dye would be used to display the pH differ-
ence between extracellular and intracellular regions of tumor
cells. To mimic the environment of tumor cells, a PBS with a
slightly lower pH (6.7) was added to MCF-7 cells that had been
Fig. 7 Cytotoxicity of HNNA at varied concentrations (1 mM, 10 mM, 30 mM, 60
mM, 0.1 mM) on the viability of (a) MCF-7 cells and (b) HeLa cells.
ꢀ
incubated with HNNA (4 mM) for two hours at 37 C. Then the
cells were imaged aer 5 min at room temperature. In order to
enhance the contrast of the images, the blue pseudo colour was
changed to magenta. Compared to the images of cells in normal
PBS (pH 7.4), the magenta colour on the cell membrane
increased aer the addition of the low pH PBS (pH 6.7), but no
signicant change was observed inside the cytoplasm (see
Fig. 6a), which resulted in a great colour difference between the
cell membrane and cytoplasm in the overlay images. In another
experiment, a high K+ HEPES-buffered solution with lower pH
(5.5) was added to the MCF-7 cells and imaged immediately; the
images showed that the cell membrane (pointed out by yellow
arrows) and the membrane nanotubes between the cells
(pointed out by cyan arrows) exhibit a strong magenta color but
no green colour, and a signicant colour difference was
observed between the outer membrane and inner cell in the
overlay image (Fig. 6b). These results suggest that HNNA is a
good probe candidate for monitoring the pH uctuations, not
only in live cells, but also in the extracellular microenvironment
of cancer tissues.
Cytotoxicity assay of HNNA
The cytotoxicity of HNNA was investigated by the standard CCK-
8 assay. As shown in Fig. 7, no obvious toxicity toward MCF-7
and HeLa cells was found in the concentration range of 1–100
mM in 24 hours. For pH imaging in cells, 3–5 mM HNNA was
enough, suggesting that HNNA can be used to monitor the pH
change in living cells over a long period.
Conclusion
In summary, we have described a novel pH sensitive uo-
rophore that is synthesized by fusing 3-amino-1,2,4-triazole to
4-bromine-1,8-naphthalimides. Three derivatives (ENNA, ANNA
and HNNA) with different substituents at the imide N-atom of
this uorophore were synthesized and characterized. The pH
response is attributed to the reversible deprotonation of the one
and only N–H in the heterocycle-fused naphthalimide structure,
with a pKa of ꢃ6.5. The deprotonation of the N–H enhances the
ICT effect of this uorophore and results in a 30–40 nm red shi
of both the absorption and emission spectra. Because of the
large conjugated planar structure, this uorophore possesses
high uorescence quantum yield in both acid and base forms in
aqueous solution, which makes it serve as a colorimetric and
ratiometric uorescent probe for pH measurement. This uo-
rophore has high selectivity to pH, high tolerance to ionic
strength, and good stability, which are favourable for cellular
pH measurement. A long chain derivative, HNNA, can locate on
the membrane structure of the cells, and has been successfully
used to map the pH difference in both the extracellular micro-
environment and the inner cells by confocal imaging. Because
of the easy modication of the imide N-atom, this uorophore
holds great potential for the design of various of ratiometric pH
probes for application in biosystems or environmental systems.
Experimental section
Materials and reagents
4-Bromo-1,8-naphthalic anhydride was purchased from Liaon-
ing Liangang Dye Chemical Co. Ltd. 3-Amino-1,2,4-triazole,
6-aminocaproic acid, 1-hexadecylamine and nigericin (sodium
salt) were purchased from Alfa Aesar Inc. (Ward Hill, MA). Cell
Counting Kit-8 (CCK-8) was bought from Dojindo Molecular
Fig. 6 (a) The confocal images of MCF-7 cells maintained in PBS with pH 7.4 and
pH 6.7. (b) The confocal images of MCF-7 cells in high K+ HEPES-buffer (pH 5.5).
Scale bar: 20 mm, cell membranes were pointed out by yellow arrows, membrane
nanotubes were pointed out by cyan arrows.
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Technologies, Inc. Ethanolamine, ethanol, DMSO and other
reagents were purchased from Beijing Chemical Plant, China.
All chemical reagents were used without further purication.
The stock solutions of probes were prepared in DMSO. Metal
ions were prepared with analytical grade nitrate salts or chloride
salts. Stock solutions of metal ions and other molecules were
prepared in water. Distilled-deionized water was used
throughout this work and puried by a UPHW-III-90T upwater
purication system (Chengdu, China). All of the titration
experiments were performed at room temperature.
Confocal imaging
MCF-7 (hormone-dependent breast cancer) and HeLa (cervical
adenocarcinoma) cells were purchased from Cell Resource
Center of Shanghai Institute for Biological Sciences (Chinese
Academy of Sciences, Shanghai, China), and incubated in
Dulbecco's Modied Eagle Medium (DMEM, Gibco) supple-
mented with 10% fetal bovine serum (FBS, Gibco) and 1%
penicillin/streptomycin (Hyclone). Cells were cultured in a
humidied incubator at 37 ꢀC with 5% CO2. Cells were imaged
under an OLYMPUS FV1000-IX81 confocal microscope
(Olympus Corporation, Japan). Fluorescent images (512 ꢄ 512
pixels) were observed using a 100ꢄ objective lens and the
images were processed using the Olympus FV10-ASW 1.6 viewer
soware. The excitation wavelengths were 405 nm and 488 nm.
Fluorescence signals were collected from 425–475 nm in
channel I and 500–600 nm in channel II. Ratio data of the
images were processed using Image-Pro Plus soware.
Cells were seeded in a 35 mm ꢄ 12 mm style cell culture dish
(F20 mm glass bottom) and cultured for 24 h. Aer removing
the medium, cells were incubated with 1 mL of fresh medium
containing HNNA (4 mM) for 2 h. Prior to imaging, cells were
washed with PBS (pH 7.4) three times, and then treated with
nigericin (5 mg mLꢂ1) in 1 mL of high K+ HEPES-buffered
solution (different pH) for 10 min. The high K+ HEPES-buffered
solution contained 125 mM KCl, 20 mM NaCl, 0.5 mM CaCl2,
0.5 mM MgCl2, 5 mM glucose and 20 mM HEPES. The different
pH was adjusted by adding 0.2 M NaOH. PBS were prepared
with 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM
K2HPO4.
Instruments
1H NMR spectra were recorded at 300 MHz, and 13C NMR
spectra and DEPT-135ꢀ were recorded at 75 MHz on a Brucker
AM 300 spectrometer with tetramethylsilane (TMS) as the
internal standard. J values were given in Hertz. H, H-COSY,
NOESY and HSQC were measured on a Brucker AM 600 spec-
trometer. Low resolution mass spectra (MS) were recorded on a
LC-MS 2010A (Shimadzu) instrument using standard condi-
tions. High-resolution MS were obtained on a Bruker Daltonics
Flex-Analysis. UV-visible absorption spectra were recorded on a
Hitachi U-2550 UV/vis spectrophotometer (Kyoto, Japan). Fluo-
rescence emission spectra were recorded on a Hitachi F-4600
uorescence spectrouorometer (Kyoto, Japan) with the exci-
tation and emission slit widths set at 2.5 nm.
Optical measurements
The absorption and uorescence spectra were recorded in
HEPES buffer in a 1.0 cm quartz cuvette. Buffers with different
pH were prepared by adding appropriate volumes of 0.2 M NaOH
solution or 0.2 M HCl solution to 10 mL of the 20 mM HEPES
solution. The uorescence quantum yield, reversibility, stability
and selectivity were measured in HEPES buffer. For UV-vis
spectra measurement, 4–5 mL of stock solutions (10 mM) of
ENNA or ANNA were diluted in 1 mL of HEPES buffer (at different
pH). For emission spectra measurements, 1 mL of stock solutions
(1 mM) of ENNA or ANNA were diluted in 1 mL of HEPES (at
different pH). The uorescence emission spectra were collected
withexcitation atthe isoabsorptive point 455 nm. Tomaintain an
ionic strength as the physiological environment, all perfor-
mances were carried out in the presence of 150 mM NaCl.
Cytotoxicity assay
MCF-7 and HeLa cells were seeded in 96-well plates (5 ꢄ 103
cells per well) and grown for 8 h. Then different concentrations
of HNNA (100 mM, 60 mM, 30 mM, 10 mM, 1 mM) were added to
the cells and they were further incubated for 24 h. Then the
medium was replaced with 100 mL of fresh medium containing
10 mL of CCK-8 reagent (without FBS and penicillin/strepto-
mycin). Aer incubation at 37 ꢀC/5% CO2 for 1 h, the absor-
bance at 450 nm was measured on a SpectraMax M5 (Molecular
Devices, CA, USA). The cell viability rate (VR) was calculated
according to the equation: VR ¼ (A ꢂ A0)/(AS ꢂ A0) ꢄ 100%,
where A is the absorbance of the experimental group, AS is the
absorbance of the control group and A0 is the absorbance of the
blank group (no cells).
Determination of pKa
The ground-state pKa values of ENNA and ANNA were deter-
mined by collecting their absorption spectra from 350 nm to
550 nm in HEPES buffers (containing 150 mM NaCl) with
different pH. The absorbance at 480 nm was plotted versus pH
value of buffered solution; the data were t to a Growth/Sia-
moidal DoseResp curve (see Fig. S8, ESI†). pKa values were
calculated according to the following equation:
Synthesis of compound 1a, N-hydroxyethyl-4-bromine-
1,8-naphthalimide
2.0 g (7.22 mmol) 4-bromine-1,8-naphthalic anhydride was
dissolved in ethanol (160 mL) and heated to reux. Then 440 mL
of ethanolamine (7.3 mmol) was added to the mixture slowly
aer the temperature was cooled down to 50 C. The resulting
mixture was heated to reux with stirring for 1 h. The reaction
ꢀ
pKa ¼ pH + log[(AHB ꢂ A)/(A ꢂ ABꢂ)].
where, AHB, A and ABꢂ represent the absorbance of absolute acid was over when the solution became clear. When the solution
form, the absorbance at the pH chosen and the absorbance of was cooled to room temperature, a precipitate emerged. The
absolute base form respectively.
precipitate was collected by vacuum ltration, and then washed
666 | J. Mater. Chem. B, 2013, 1, 661–667
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with water and ethanol three times, respectively, and nally
dried by vacuum (yield 90%).
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1H NMR (CDCl3, 300 MHz) d (ppm): 8.65 (d, J ¼ 9.0 Hz, 1H),
8.57 (d, J ¼ 9.0 Hz, 1H), 8.40 (d, J ¼ 9.0 Hz, 1H), 8.03 (d, J ¼ 9.0
Hz, 1H), 7.84 (t, J ¼ 7.5 Hz, 1H) 4.44 (t, J ¼ 6.0 Hz, 2H), 3.98 (t,
J ¼ 6.0 Hz, 2H), 2.09 (s, 1H); 13C NMR (CDCl3, 75 MHz) d (ppm):
162.9, 162.9, 132.5, 131.5, 131.3, 130.8, 129.7, 128.9, 128.7,
128.2, 122.8, 122.0, 57.7, 41.9; MS (ESI): m/z 320.1 (M + H)+.
Synthesis of compound 2a (ENNA)
430 mg (1.3 mmol) compound 1a N-hydroxyethyl-4-bromime-
1,8-naphthalimide, 200 mg (2.4 mmol) 3-amino-1,2,4-triazole
and 120 mg (3 mmol) NaOH were dissolved in DMSO (8 mL).
The resulting mixture was heated to 150 ꢀC for 12 h with stirring
under an atmosphere of nitrogen. Aer cooling to room
temperature, the solution was evaporated under reduced pres-
sure. The residue was puried by column chromatography
using silica-gel (100–200 mesh) and 8% methanol in dichloro-
methane as eluent to give a salmon pink solid compound
(128 mg, 30.7%).
1H NMR (DMSO-d6, 300 MHz) d (ppm): 13.28 (s, 1H), 8.54 (d,
J ¼ 9 Hz, 1H), 8.43 (d, J ¼ 9 Hz, 1H), 8.31 (s, 1H), 7.62 (d, J ¼ 6
Hz, 1H), 7.17 (d, J ¼ 9 Hz, 1H), 4.15 (t, J ¼ 6 Hz, 2H), 3.59 (t, J ¼
7.5 Hz, 2H); 1H NMR (DMSO-d6 + NaOH solid, 300 MHz) d
(ppm): 8.37 (d, J ¼ 9 Hz, 1H), 8.22 (s, 1H), 8.11 (d, J ¼ 9 Hz, 1H),
7.33 (d, J ¼ 6 Hz, 1H), 6.89 (d, J ¼ 9 Hz, 1H), 4.18 (t, J ¼ 7.5 Hz,
2H), 3.55 (t, J ¼ 7.5 Hz, 2H); 13C NMR (DMSO-d6 + NaOH solid,
75 MHz) d (ppm): 163.2, 162.7, 155.6, 154.6, 154.3, 139.7, 133.7,
132.7, 132.2, 113.7, 113.3, 111.3, 103.3, 101.5, 58.2, 41.4; MS
(ESI): m/z 320.1 (M ꢂ H)ꢂ; HRMS (ESI): m/z calcd for C16H10N5O3
(M ꢂ H)ꢂ 320.078903, found, 320.078494.
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The synthesis and characterization of other compounds can
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Acknowledgements
We gratefully acknowledge the nancial support from Grant 973
Program (2011CB935800 and 2011CB911000) and NSF of China
(21075124).
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