Lysosomal pH rise during heat shock monitored by a lysosome-targeting near-infrared ratiometric fluorescent probe

Article abstract of DOI:10.1002/anie.201405742

Heat stroke is a life-threatening condition featuring a high body temperature and malfunction of many organ systems. The relationship between heat shock and lysosomes is poorly understood mainly because of the lack of a suitable research approach. Herein

Full text of DOI:10.1002/anie.201405742

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Chemie
DOI: 10.1002/anie.201405742
Analytical Methods
Lysosomal pH Rise during Heat Shock Monitored by a Lysosome-
Targeting Near-Infrared Ratiometric Fluorescent Probe**
Qiongqiong Wan, Suming Chen, Wen Shi, Lihong Li, and Huimin Ma*
Abstract: Heat stroke is a life-threatening condition, featuring
a high body temperature and malfunction of many organ
systems. The relationship between heat shock and lysosomes is
poorly understood, mainly because of the lack of a suitable
research approach. Herein, by incorporating morpholine into
a stable hemicyanine skeleton, we develop a new lysosome-
targeting near-infrared ratiometric pH probe. In combination
with fluorescence imaging, we show for the first time that the
lysosomal pH value increases but never decreases during heat
shock, which might result from lysosomal membrane perme-
abilization. We also demonstrate that this lysosomal pH rise is
irreversible in living cells. Moreover, the probe is easy to
synthesize, and shows superior overall analytical performance
as compared to the existing commercial ones. This enhanced
performance may enable it to be widely used in more
lysosomal models of living cells and in further revealing the
mechanisms underlying heat-related pathology.
temperature change and lysosomal pH value remains
unknown. Thus, accurate measurement of the acidity change
in lysosomes under heat stress such as heat shock is necessary
to gain insight into the mechanism of heat cytotoxicity.
In this respect, fluorescence spectroscopy has attracted
significant attention because of its high sensitivity and
unrivaled spatiotemporal resolution.[4–10] An excellent fluo-
rescent probe suited for monitoring the quantitative change
of lysosomal pH in real time should meet at least three
prerequisites: a) it should be lysosome-targeting, b) it should
have a long analytical wavelength (greater than l = 650 nm or
near-infrared) to minimize autofluorescence and biological
damage, and c) it should have a reversible pH-dependent
ratiometric response to avoid the influence of several variants,
such as concentration and optical path length.[7] To our
knowledge, a lysosome-targeting fluorescent pH probe with
these desired properties is still lacking. The most commonly
used ratiometric pH probe for lysosomes is the commercially
H
eat-related pathologies, such as heat
stroke, are one of the most serious causes of
mortality.[1] Despite the severity and increas-
ingly prevalent health risks associated with
heat-inflicted damage, the molecular and
organelle mechanisms responsible for the
direct cytotoxicity of heat are not well under-
stood.[2] As one of the vital organelles, the
lysosome usually has an acidic environment
of pH 3.8–5.0, and plays a key role in cellular
homeostasis and the mediation of a variety of
physiological processes, such as protein deg-
radation and plasma membrane repair.[3] The
minor pH fluctuation of lysosomes may influ-
ence these processes and even the fate of the
cells.[3a] However, the relationship between
Scheme 1. Synthesis of the lysosome-targeting near-infrared ratiometric fluorescent
pH probe Lyso-pH.
available probe DND-160, which unfortunately has a rather
short excitation wavelength of less than l = 400 nm.[8]
[*] Dr. Q. Q. Wan,[+] Prof. Dr. S. M. Chen,[+] Prof. Dr. W. Shi, L. H. Li,
Prof. Dr. H. M. Ma
With these criteria in mind, we have developed Lyso-pH
(Scheme 1 and Scheme S1 in the Supporting Information),
a new lysosome-targeting near-infrared ratiometric fluores-
cent pH probe, by incorporating a typical lysosome-targeting
moiety of morpholine[9] into a stable hemicyanine skeleton
with near-infrared spectroscopic features. We show that Lyso-
pH exhibits an accurate lysosome-targeting ability, excellent
photostability, and a good linear ratiometric response in the
pH range of 4–6 in living cells. Furthermore, using Lyso-pH-
based fluorescence imaging, we find that the lysosomal
pH value rises with the increase of temperature, and that
this rise in pH is irreversible in living cells. These results
demonstrate the relationship between the lysosomal pH value
and temperature during heat shock.
Beijing 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: mahm@iccas.ac.cn
[+] These authors contributed equally to this work.
[**] This work is supported by grants from the NSF of China (Nos.
21105104, 21275147, and 21321003), 973 Program (No.
2011CB935800), and the Chinese Academy of Science (KJCX2-EW-
N06-01 and CMS-PY-201301). We also thank X. Y. Jiang and Z.
Huang for help with the CLSM-710 (Carl Zeiss) confocal laser
scanning microscope.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201405742.
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Cyanines, a classic type of near-infrared fluorochromes,
have been frequently employed to develop different spectro-
scopic probes for imaging studies, but they are known to have
poor stability and high background fluorescence as a result of
ready autoxidation and photooxidation.[7a,11] However, hemi-
cyanines, formed from the decomposition of cyanines, usually
have high stability, and thus were our first choice to design the
stable near-infrared fluorescent pH probe, Lyso-pH. As
shown in Scheme 1, the probe Lyso-pH can be easily
synthesized by incorporating a lysosome-targeting moiety of
morpholine onto the hemicyanine skeleton of the commer-
cially available heptamethine dye, IR 780.
The spectroscopic properties of Lyso-pH were examined
in phosphate buffer solution (0.2m) at different pH values,
prepared by varying the relative amounts of Na2HPO4,
KH2PO4, and H3PO4. The probe displays sensitive absorption
(Figure 1a) and fluorescence (Figure 1b) spectroscopic
tion of the hydroxy group (Scheme S1). This indicates that the
ratiometric response of Lyso-pH matches well with the
physiological pH range (pH 3.8–5.0) of lysosomes, making it
promising as a near-infrared fluorescent probe for accurate
measurement of lysosomal pH values.
We next investigated the specificity of the probe for
pH detection over the detection of other biologically relevant
species, such as ions, protein, reactive oxygen species, amino
acids, and glucose. No obvious change in fluorescence signal
was detected in the presence of these species at their
physiological concentrations, as compared to the control
(Figure S7), indicating that Lyso-pH exhibits high selectivity
for pH detection. Moreover, the influence of temperature
from 318C to 458C was examined on the fluorescence of Lyso-
pH itself (Figure S8). It is found that changing the temper-
ature within this range does not significantly affect the
ratiometric fluorescence signal of Lyso-pH. Additionally,
Lyso-pH exhibits good biocompatibility (Figure S9). These
results indicate that Lyso-pH may detect changes in lysosomal
pH values with minimum interference from temperature and
other biologically relevant species.
To evaluate the lysosome-targeting performance of
Lyso-pH, HeLa or MCF-7 cells were co-stained with
Lyso-pH and LysoTracker Red DND-99 (a commercially
available lysosome-targeting dye). As DND-99 is not a ratio-
metric fluorescence probe, a single channel covering the
whole emission wavelength range of DND-99 is selected. For
comparison, the corresponding single channel covering the
whole emission wavelength range of Lyso-pH is used in this
experiment. As shown in Figure 2a, both DND-99 and Lyso-
pH display strong localized fluorescence within lysosomes.
The fluorescence images of DND-99 and Lyso-pH can be
merged rather well, confirming that Lyso-pH can specifically
target the lysosomes of living cells with good cell-membrane
permeability. Additionally, a high Pearson coefficient of 0.91
and an overlap coefficient of 0.90 are calculated from the
intensity correlation plots (Figure 2a, fourth image). Further-
more, there are relatively few changes in the emission
intensity profiles of the dyes Lyso-pH and DND-99 (Fig-
ure 2c) within the linear region of interest (ROI; given by
a yellow line in Figure 2a). Similar results are obtained for
HeLa cells (Figure S10a). These results demonstrate the
superior lysosome-targeting ability of Lyso-pH. Importantly,
this ability of Lyso-pH still holds at the higher temperatures of
418C and 458C (Figures S11 and S12 in the Supporting
Information).
HeLa and MCF-7 cells were also co-stained with Lyso-pH
and Rhodamine 123 (a typical mitochondrial tracker). As
depicted in Figure 2b, Lyso-pH and Rhodamine 123 lead to
the production of bright localized fluorescence within lyso-
somes and mitochondria, respectively, and the merged image
of Lyso-pH and Rhodamine 123 exhibits a significantly differ-
ent fluorescence (the third image in Figure 2b), which reflects
the corresponding lysosomal and mitochondrial distributions.
Importantly, not only low values for the Pearson coefficient
(0.18) and the overlap coefficient (0.20) are obtained, but also
completely different changes in the intensity profile of ROI
are found (Figure 2d). Moreover, similar phenomena are
found for targeting lysosomes in HeLa cells (Figure S10b).
Figure 1. a) Absorption and b) fluorescence emission spectra of Lyso-
pH (10 mm) in phosphate buffer (0.2m) at different pH values. c) Plot
of I670/I708 (ratio of the fluorescence intensity of Lyso-pH at l=670 nm
and l=708 nm) versus pH values in the range pH 2.6–9.2. Inset: the
linear relationship between I670/I708 and pH values in the range pH 4.0–
6.0. d) pH reversibility study of Lyso-pH between pH 4.0 and 8.0.
lex =635 nm; data are expressed as the mean of three separate
measurements Æstandard deviation (SD).
responses to changes in pH values. As shown in Figure 1a,
the maximum absorption band at l = 598 nm of the probe in
pH 4.0 media is red-shifted to l = 681 nm in pH 7.4 media,
with a concomitant color change from blue to green (see
Figure S5 in the Supporting Information). Upon excitation at
l = 635 nm, the near-infrared emission intensity at l = 670 nm
of Lyso-pH decreases slightly with the change in pH values
from 4.0 to 7.4. This change in intensity is accompanied by
a large increase of fluorescence intensity at l = 708 nm
(Figure 1b), which provides the basis for achieving a ratio-
metric (I670/I708) detection. Most notably, the probe Lyso-pH,
with a pKa value of 5.00 Æ 0.01 and a quantum yield of Ff =
0.16 at pH 5.0, exhibits an excellent I670/I708 linearity in the
pH range 4.0–6.0 (Figure 1c). The probe shows also good
reversibility between pH 4.0 and pH 8.0 (Figure 1d and
Figure S6), which is attributed to the protonation/deprotona-
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tive response to pH change in the range of
pH 4.0–6.0 (Figure S16a). This can be de-
scribed by the linear equation for the
fluorescence intensity ratio, R = 4.41–
0.64pH (r= 0.995, where r is the correla-
tion coefficient). In MCF-7 cells, a good
linear relationship was also obtained,
which can be described by the equation:
R = 4.54–0.65pH (r= 0.998; Figures S15
and S16b).
To investigate the influence of temper-
ature on lysosomal acidity, the dynamic
changes of lysosomal pH values in HeLa
and MCF-7 cells during heat shock were
monitored by confocal fluorescence imag-
ing using Lyso-pH. In this experiment,
418C and 458C were used as the heat
shock temperatures acting on the cells. As
shown in Figures 3a,b (see also Figure S17
for more details), when compared to the
control group (378C) the merged images of
the heat-treated cells exhibit obvious color
changes (more yellow in color) with the
temperature increase, which is attributed
to the small increase of the lysosomal
pH value. From the constructed pH cali-
bration curves (Figure S16), these pH alt-
erations in lysosomes can be quantitatively
determined (Figure 3c). It is found that the
normal lysosomal pH values of HeLa and
MCF-7 cells (control groups at 378C) are
4.58 Æ 0.09 and 4.55 Æ 0.08, and heat shock
at 418C causes the increase of their lyso-
somal pH values to 4.80 Æ 0.06 and 4.81 Æ
Figure 2. Lysosome-targeting properties of Lyso-pH in MCF-7 cells. a) Colocalization images
of MCF-7 cells stained with DND-99 (50 nm, red channel, lex =559 nm, lem =570–670 nm)
and Lyso-pH (50 nm, blue channel, lex =635 nm, lem =650–750 nm), and the correlation of
Lyso-pH and DND-99 intensities. b) Colocalization imaging of MCF-7 cells stained with
Rhodamine 123 (50 nm, green channel, lex =488 nm, lem =500–600 nm) and Lyso-pH (as in
(a)), and the correlation of Lyso-pH and Rhodamine 123 intensities. c) Intensity profiles
within the ROI (yellow line in Figure 2a) of Lyso-pH and DND-99 across MCF-7 cells.
d) Intensity profiles within the ROI (yellow line in Figure 2b) of Lyso-pH and Rhodamine 123
across MCF-cells. Scale bars=20 mm.
The intracellular photostability of Lyso-pH was also
evaluated by comparison with that of DND-99. In this
experiment, HeLa cells were successively incubated with
DND-99 and Lyso-pH, and the photostability of the two
dyes in the cells was compared under a simultaneous con-
tinuous excitation by l = 559 nm and 635 nm lasers.[12] As
shown in Figure S13, the fluorescence of DND-99 is bleached
quickly in the first 20 minutes, and decreases to about 60% of
the initial intensity after 40 minutes. In contrast, the fluores-
cence of Lyso-pH is rather stable, remaining almost constant
(the fluorescence intensity decreases less than 10%) even
after 40 minutes of continuous intensive irradiation. This
excellent intracellular photostability of Lyso-pH offers great
potential for bioimaging applications.
To quantitatively determine lysosomal pH values, the
intracellular pH calibration curves of Lyso-pH in both
HeLa cells and MCF-7 cells were first made with the anti-
biotic nigericin as a H+/K+ ionophore.[4o,7c] As depicted in
Figure S14 for HeLa cells, the fluorescence intensity of the
first row (red channel) decreases with a change in the
pH value from 4.0 to 6.0, whereas that of the second row
(green channel) gradually increases. Additionally, the merged
images of red and green channels exhibit distinct pH-
dependent patterns. Importantly, the fluorescence intensity
ratio (R = I650-680/I690-720) from the two channels shows a sensi-
0.07, respectively. Higher temperature treatment at 458C
further raises their lysosomal pH value to 4.89 Æ 0.07 and
4.91 Æ 0.08. Although the lysosomal pH differences between
418C and 458C have no statistical significance, those between
378C and 418C (or 458C) are significant (Figure 3c). Addi-
tional control experiments were carried out by first heating
the cells to 458C and then adding the probe. These experi-
ments show negligible differences from those carried out by
adding Lyso-pH to the cells at 378C and then heating the cells
to 458C, illustrated by similar ratio values in both cases (see
Figure S18). This also suggests that the probe is stable in cells
during the heat treatment from 378C to 458C. Surprisingly, no
decrease in the lysosomal pH value is detected during the
heat treatments. This phenomenon may be called unidirec-
tional change (that is, the increase rather than the decrease of
the lysosomal pH value induced by heat shock), which is
similar to the fact that some diseases lead to an increase
rather than a decrease in human body temperature. More-
over, this finding is supported by a comparative study with
DND-160 (a commercially available lysosomal pH probe), as
shown in Figures S19 and S22. These results clearly indicate
that heat shock can lead to an increase of lysosomal pH. The
reason for this may be rather complex, but a possible
speculation may be made that heat shock might cause the
permeabilization of the lysosomal membrane.[13] As a conse-
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Figure 3. Relationship between lysosomal pH and heat shock in HeLa and MCF-7 cells monitored by Lyso-pH. a),b) The fluorescence images
(top, merged; bottom, differential interference contrast) of Lyso-pH-loaded HeLa cells (a) and MCF-7 cells (b) under heat shock at 378C, 418C,
and 458C (left to right) for 20 minutes. The fluorescence images are the merged images from both red (lem =650–680 nm) and green (lem =690–
720 nm) channels at lex =635 nm. See also Supporting Information for more details. c) Lysosomal pH changes with temperature in HeLa and
MCF-7 cells. The lysosomal pH values in control and heat-treated cells were quantified by image analysis with 100–200 cells counted per
condition. Results are expressed as the mean of three separate measurements ÆSD. Statistical analyses are performed using the Student’s t-test:
*P<0.001. d),e) Reversibility of the lysosomal pH change in HeLa cells (d) and MCF-7 cells (e) with temperature. Results are expressed as the
mean of three separate measurements ÆSD. Scale bars=20 mm.
quence, neutralization might occur to some extent between
the cytosol, with a higher pH value, and lysosomes, thereby
increasing the lysosomal pH.
Furthermore, the analytical performances of Lyso-pH and
DND-160 were also compared, which reveals that Lyso-pH
has a longer excitation wavelength (lex = 635 nm), lower
autofluorescence (Figure S21), and higher photostability
(comparing Figures S20 and S13) than DND-160 (lex =
375 nm). This clearly demonstrates that Lyso-pH is more
suitable for lysosomal pH studies.
Finally, the reversibility of the lysosomal pH change in
HeLa and MCF-7 cells with temperature was examined in
real-time by fluorescence imaging. As depicted in Figure 3d
and Figure S23, after the heat-treated HeLa cells at 418C and
458C are cooled to 378C for 20 minutes, their lysosomal
pH values are found to be 4.95 Æ 0.09 and 5.07 Æ 0.10,
respectively. These values are much higher than the average
value of approximately pH 4.6 from the control groups at
378C. For MCF-7 cells, similar results were obtained (Fig-
ure 3e and Figure S24). Furthermore, a longer incubation
time of 1 hour at 378C does not contribute to the recovery of
the lysosomal pH (Figure S25). These observations suggest
that the lysosomal pH of the heat-treated cells cannot be
restored to its initial status, indicating that the rising of
lysosomal pH during heat shock is an irreversible process.
In conclusion, on the basis of the relatively stable
semicyanine skeleton that is formed from the degradation
of IR 780, we have developed a near-infrared ratiometric
fluorescent probe, Lyso-pH, for lysosomal pH measurements.
The probe can be readily prepared and shows excellent
lysosome-targeting ability and high photostability as com-
pared to the existing commercially available probes. More
importantly, using our probe Lyso-pH the change of lysoso-
mal pH with temperature is investigated, revealing for the
first time the increase of the lysosomal pH during heat shock
and the irreversibility of this process. We believe that
Lyso-pH may find more applications in detecting pH values
in vivo and exploring the function of intracellular acidic
lysosomes.
Received: May 29, 2014
Revised: July 29, 2014
Published online: && &&, &&&&
Keywords: dyes/pigments · fluorescent probes · heat shock ·
.
imaging agents · lysosomal pH
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Communications
Analytical Methods
Feeling hot, hot, hot: A near-infrared
ratiometric pH probe with a stable semi-
cyanine skeleton is developed for lysoso-
mal pH measurements. The readily pre-
parable probe shows an excellent lyso-
some-targeting ability and high photo-
stability. The probe is used to investigate
the change of lysosomal pH with tem-
perature, revealing for the first time that
lysosomal pH values rise during heat
shock and the process is irreversible.
Q. Q. Wan, S. M. Chen, W. Shi, L. H. Li,
H. M. Ma*
&&&& — &&&&
Lysosomal pH Rise during Heat Shock
Monitored by a Lysosome-Targeting
Near-Infrared Ratiometric Fluorescent
Probe
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