Lysosome-specific sensing and imaging of pH variations in vitro and in vivo utilizing a near-infrared boron complex

Article abstract of DOI:10.1039/c8tb03353f

As a focus issue, the study of lysosomal pH has attracted much attention as it is closely associated with the state of lysosome, which plays a vital role in endocytosis and autophagy. In order to investigate the lysosomal pH, fluorescence bioimaging is one of the most widely-explored approaches. Unfortunately, the probes are insufficient to absorb or emit in the near-infrared (NIR) region, which could minimize photodamage to organisms and maximize tissue penetration in living systems. As a novel family of NIR dyes, hemicyanine has been selected for NIR bioimaging and biosensing owing to its excellent optical properties, easy preparation and good biocompatibility. Employing a classic rhodamine-hemicyanine hybrid, we first designed and synthesized a NIR boron complex (HCy-BIZ-BF₂) with lysosome-targeting and pH-sensing properties. It is worth mentioning that after HCy-BIZ was coordinated with boron fluoride, HCy-BIZ-BF₂ exhibited an improved photostability as well as an enlarged Stokes shift, and was subsequently applied to monitor the lysosomal pH in cells stimulated with chloroquine. Further investigation of pH changes in mice illustrated that HCy-BIZ-BF₂ performed well in detecting the pH in living organisms. Therefore, this concept of boron complex derived from hemicyanine is not only applicable in pH detection, but also conducive for preparing promising novel NIR bioprobes and obtaining precise measurements of physiological parameters in specific physiological processes.

Full text of DOI:10.1039/c8tb03353f

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DOI: 10.1039/C8TB03353F
Journal Name
ARTICLE
Lysosome-specific sensing and imaging of pH variations in vitro
and in vivo utilizing a near-infrared boron complex
a
a
a
a
a
a
a
Yuxiang Shi, Xiangchun Meng, Huiran Yang, Linna Song, Shujuan Liu,* Aqiang Xu, Zejing Chen,
Received 00th January 20xx,
Accepted 00th January 20xx
ab
a
Wei Huang,* and Qiang Zhao*
DOI: 10.1039/x0xx00000x
As a focus issue, the study of lysosomal pH has attracted much attention, as it is closely associated with the state of lysosome
which plays a vital role in endocytosis and autophagy. In order to investigate lysosomal pH, fluorescence bioimaging is one
of the most widely-explored approaches. Unforturnately, it is insufficient in near-infrared (NIR) region, which could minimize
photo damages of organisms and maximize tissue penetration in living systems. As a novel family of NIR dyes, hemicyanine
has been selected for NIR bioimaging and biosensing owing to its excellent optical properities, easy preparation and good
biocompatiability. Employing a classic rhodamine-hemicyanine hybrid, we first designed and synthesized a NIR boron
www.rsc.org/
complex (HCy-BIZ-BF
with boron fluoride, HCy-BIZ-BF
applied to monitoring lysosomal pH in cells stimulated with chloroquine. The further investigation of pH changes in mice
illustrated that HCy-BIZ-BF performed well in pH detection in living organisms. Therefore, this concept of boron complex
2
) with lysosome-targeting and pH-sensing properties. It is worth mentioning that after coordinated
2
exhibited improved photostability as well as enlarged Stokes shift, and was subsequently
2
derived from hemicynaine is not just in pH detection, but conductive to promising novel NIR bioprobes and precise
measurements of physiological parameters in specific physiological processes.
organisms29–32 and maximize tissue penetration to reduce the
3
3–35
Introduction
background interference in the living system.
However, it is
challengeable to develop this kind of probe due to the
widespread problems of low quantum efficiency, poor
photostability as well as small Stokes shift.
Among various biomarkers, pH is one of the vital parameters
closely concerned with cellular components and pathological
processes.1 In mammalian cells, organelles work in different
3
6,37
To develop
–4
excellent NIR probes, various fluorescence molecules, such as
5
,6
pH environments ranging from 4.5 to 8.0. Lysosome, filled
with numerous hydrolytic enzymes for cellular hydrolysis,
exhibits acidic environment (pH = 4.5–5.5) and plays a vital
role in endocytosis, autophagy and oxidative stress.
However, abnormal pH in lysosome would disrupt intracellular
pH homeostasis and cause many kinds of diseases, including
3
8
39–41
42,43
cyanine , rhodamine
and boron dipyrromethene
have
7
,8
been extensively investigated. Among these probes,
hemicyanine, deriving from cyanine family, is known for its
desirable optical properties including high fluorescence
quantum yield and good biocompatibility.
rhodamine-hemicyanine hybrid is one of the most effective
9
,10
1
1,12
4
4–46
Specifically,
1
3–
cancers, Alzheimer’s disease and cardiopulmonary problems.
4
7,48
approaches to design new hemicyanine probes.
Based on a
1
6
Therefore, it is critical to monitor pH changes in lysosome and
classical hybrid hemicyanine skeleton, numerous strategies
have been developed to further improve the photostability and
modulate the spectral range, like commonly-used extension
Besides, coordination
modification is a more convenient and effective method
through facile chelation between organic molecular and
living system to study cellular functions, which is beneficial to
the understanding of these pathological processes.
Fluorescence imaging17 based on chemical probes has been an
important approach for monitoring intracellular pH in the last few
decades, especially for its real-time,
temporal resolution imaging.
4
9
and and hetero-substitution.
1
8–21
high spatial and
2
2–28
Importantly, probes with
5
0–56
positive ion.
Unfortunately, the biosensing platform
near-infrared (NIR) absorption and emission are favourable in
bioimaging, since they could minimize photo damages of
composed of rhodamine-hemicyanine hybrid derivatives and
positive ions as coordination center has not yet been
established in the subcellular level, especially in the lysosome.
Herein, we designed and synthesized a novel boron complex
a. Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key
Laboratory for Biosensors, Institute of Advanced Materials (IAM), Nanjing
University of Posts and Telecommunications (NUPT), Nanjing 210023, P. R. China.
E-mail: iamqzhao@njupt.edu.cn; provost@nwpu.edu.cn; Fax: +86 25-85866396
2
HCy-BIZ-BF with NIR emission, selective pH-responsiveness
and high lysosome-targeting ability to monitor pH variations in
b.
Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical
University (NPU), Xi'an 710072, Shaanxi, China.
†
Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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2
changed slightly under 30-
absorption intensities of HCy-BIZ-BF
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2
DOI: 10.1039/C8TB03353F
minute continuous irradiation (660 nm, 250 mW/cm ), while
those of HCy-BIZ decreased obviously, demonstrating that HCy-
BIZ-BF
2
has a better photostability than HCy-BIZ. These results
exhibited outstanding
indicated that NIR probe HCy-BIZ-BF
2
photophysical properties, especially the enlarged Stokes shift
and high photostability.
pH-responsive properties of HCy-BIZ-BF
The pH-responsive properties of HCy-BIZ-BF
studied in PBS with various pH values. As demonstrated in Fig.
d and Fig. S1a, HCy-BIZ-BF was pH-sensitive in both emission
and absorption spectra. The emission intensity of HCy-BIZ-BF
2
2
were further
1
2
2
displayed obvious increase in acidic (pH = 3.29 to 6.24)
condition, while changed slightly in alkaline (pH = 7.40 to 9.07)
medium (Fig. 1d). In the meantime, a smaller red shift and slight
increase in absorption was observed, accompanied by the
colour of the solution changed from blue (pH = 3.29) to green
towards H+.
2
Scheme 1. Design concept and responsive mechanism of HCy-BIZ-BF
(
pH = 9.07) (Fig. S1b). This trend was in line with the calculated
absorption spectra of HCy-BIZ-BF (Fig S2b). Besides, a good
exhibited an enlarged Stokes shift (55 nm) and high linear relationship of the fluorescence intensity versus pH
photostability, achieving the real-time monitoring of lysosomal pH in values exhibited high sensitivity of HCy-BIZ-BF to pH changes,
Hela cells stimulated with chloroquine via confocal laser scanning especially in the range of 3.29 to 6.24 (Fig. 1e). According to the
lysosome. In particular, after coordinated with boron fluoride, HCy-
BIZ-BF
2
2
2
microscopy (CLSM). Meanwhile, pH changes in the living mice were
further investigated using this NIR probe with high signal-to-noise
ratio. It is expected that this strategy of hemicyanine skeleton
combined with boron fluoride could bring more opportunities to NIR
fluorescent probes.
Henderson-Hasselbalch equation (Fig. S2), the corresponding
pK was calculated to 5.03, illustrating that HCy-BIZ-BF could
determine slight pH variations in a range of 4.4–
a
2
Results and discussion
Design strategies and optical properties of HCy-BIZ-BF
2
To begin with, a novel hemicyanine derivative, HCy-BIZ, was
prepared by introducing the benzimidazole moiety into the
classical hemicyanine skeleton. Its π-conjugate system was
subsequently extended, resulting in an obvious red shift (20 nm)
3
6
in the emission spectrum compared with its original structure.
As shown in Fig. 1a, HCy-BIZ exhibited NIR absorption and
emission, with maximum peaks at 700 and 728 nm,
respectively. However, HCy-BIZ had a small Stokes shift (28 nm),
which could cause inevitable interference from the excitation
source in the fluorescence-collecting process.
Then, we resort to a facile coordination approach through the
chelation between HCy-BIZ and boron fluoride to form a novel
2
boron complex (HCy-BIZ-BF ). This boron coordination led to a
rigid hexatomic ring, thus the intramolecular rotations and
vibrations were restricted, contributing to higher stability. The
basic synthetic routes were shown in Scheme S1, and the
characterizations of all compounds mentioned were also
summarized in the Supporting Information.
Fig. 1 Optical characteristics of HCy-BIZ-BF
emission spectra of HCy-BIZ in methyl alcohol (10 μM). (b) Normalized absorption
and emission spectra of HCy-BIZ-BF (10 μM) in methyl alcohol. (c) Photostability
experiment of HCy-BIZ-BF (10 μM) and HCy-BIZ (10 μM) under continuous
) in PBS buffer (1% DMSO). (d) Emission spectra
(10 μM) in PBS buffer (1% DMSO, λex = 635 nm) with various pH
2
. (a) Normalized absorption and
2
Next, we evaluated the photophysical properties of HCy-BIZ-
2
BF
2
. After coordinated with BF
2
, HCy-BIZ-BF
2
has a maximum irradiation (660 nm, 250 mW/cm
2
of HCy-BIZ-BF
2
absorption at 680 nm and maximum fluorescence emission at
values (pH = 3.29, 3.97, 4.28, 5.12, 5.52, 5.77, 6.24, 7.40, 7.87, 9.07). (e) The plot
of fluorescence intensity (740 nm) over the pH range (3.29–9.07) and the curve
7
30 nm respectively, with the enlarged Stokes shift from 28 to
5
5 nm (Fig. 1b). Then, the photostability was investigated (insert) about emission intensity versus pH range (3.29–6.24). f) Reversibility
through UV-Vis absorption spectra. As exhibited in Fig. 1c, the
2
experiment of HCy-BIZ-BF (10 μM) in PBS buffer (1% DMSO, λex = 635 nm).
2
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6
.2, which covered lysosomal pH values in both normal and
abnormal situations.
According to the above-mentioned pH-responsive
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phenomena, the responsive mechanism of HCy-BIZ-BF
2
towards H+ was investigated by the 1H NMR titration
experiment, which was conducted by adding trifluoroacetic acid
2 6
(TFA) dropwise into the HCy-BIZ-BF solution (DMSO-d ). As
shown in Fig. 2a, upon reaction with TFA, proton peaks of low
field (7.2–7.5 ppm) gradually merged together, while the
characteristic peak corresponding to NH of HCy-BIZ-BF -H
2
appeared at d = 5.65 ppm, confirming the protonation process
and the existence of H atom. Thus, the pH-responsive
mechanism could be illustrated in Scheme 1. To be specific,
+
fluctuations of
H
concentration could influence
protonation/deprotonation processes of imine group, resulting
in the change of fluorescence signals.
Subsequently, the selectivity experiments were conducted
towards various biological species. As depicted in Fig. 2b and Fig.
2
+
2+
2+
2 2
S3, the presence of diverse ions (Ca , Mg , Cu , H O , etc.) and
biological relevant species L-glutathione (GSH), glycine (Gly) and
cysteine (Cys) did not cause any observable fluorescence
intensity changes. Importantly, HCy-BIZ-BF also exhibited
2
excellent reversibility between pH 3.5 and 9.2 (Fig. 1f), which
was beneficial to the precise and reversible measurements of
Fig. 3 (a) Autofluorescence experiments of Hela cells under different detection
conditions (λex = 405 nm, λem = 450–550 nm and λex = 635 nm, λem = 650–750 nm). (b) The
2
pH value. Generally speaking, HCy-BIZ-BF showed great
potentiality for NIR pH detection in complicated intracellular pixel intensity ratio (Icell/Iblank) from the cell regions (yellow circles) and non-cell regions
red circles) were applied to evaluate the relative autofluorescence in these ROIs. (c)
Fluorescence images (λem = 540–600 nm) of cells treated with DND-99 under different
time domains and HCy-BIZ-BF under different time domains. (d) The histograms about
the relative emission intensity of DND-99 and HCy-BIZ-BF in cells under different time
(
environments, especially in the acidic medium.
2
Evaluations of HCy-BIZ-BF
Before the in vivo and in vitro experiments, the cytotoxicity of
HCy-BIZ-BF towards Hela cells was evaluated using 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT)
assay. After 24-hour incubation, the viabilities of Hela cells
retained around 90% at various incubating concentrations of
2
in living cells
2
domains. These data were exhibited as the mean ± SD. Scale bar: 30 µm.
2
These data showed that NIR-emission probe HCy-BIZ-BF
showed superb anti-interference performance, which could
effectively reduce the interferences from background.
Photostability is another important property, especially for
small-molecule fluorescence probes. To compare the
2
photostability between HCy-BIZ-BF and DND-99 (an available
lysosome tracker), the related experiment in Hela cells was
conducted. Cells were simultaneously excited by the continuous
lasers (532 nm and 660 nm), and the fluorescence images were
collected through different channels (green channel, 540–600
nm and red channel, 670–750 nm). As exhibited in Fig. 3c, the
fluorescence intensity of green channel (DND-99) was bleached
quickly over time with a greater decline to around 60% of the
initial intensity after 40 min. On the contrary, tiny emission
2
2 2
HCy-BIZ-BF from 0 to 50 µM, indicating that HCy-BIZ-BF is
biocompatible in living cells (Fig. S3). Then, the merits of its NIR
emission were depicted in Fig. 3. While observing cells without
any fluorescent probe, nearly no autofluorescence signals was
obtained under the irritation at 635 nm, while an obvious
background fluorescence could be observed excited by the 405
laser (Fig. 3a). These differences were further analysed by the
statistic data collected from the regions of interests (ROIs) in the
cell imaging results, where the mean value (Icell/Iblank) of the NIR
signals upon excitation at 635 nm was only a half of that under
the irritation at 405 nm (Fig. 3b).
decrease (less than 10%, Fig. 3d) of HCy-BIZ-BF
after 40-minute irradiation, verifying good photostability in
living cells. These results illustrated that HCy-BIZ-BF has
2
was observed
2
excellent anti-interference property and high intracellular
photostability, which reveals the promising application for pH-
related bioimaging.
Detection of lysosomal pH variations in the living cells
Based on the excellent NIR emission, good biocompatibility,
high anti-interference property and photostability, probe
1
Fig. 2 (a) H NMR spectra of HCy-BIZ-BF
2
in DMSO-d
6
without or with TFA. (b)
Emission spectra of HCy-BIZ-BF
2
(10 μM) with different ions, ROS and bioactive
molecules. λex = 635 nm, λem = 740 nm.
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Fig. 5 (a) CLSM images of Hela cells incubated with HCy-BIZ-BF
em = 650–750 nm) at different pH buffer solutions. (b) Intracellular pH calibration curve
of HCy-BIZ-BF in Hela cells from (a) △F/F (F: stands for fluorescence intensity at
different pH values; F : means fluorescence intensity at pH = 4.14). Scale bar: 40 µm..
2
(100 nM, λex = 635 nm,
λ
2
0
0
2
Subsequently, HCy-BIZ-BF was used to evaluate the
variations of pH values in living cells. The experiment was
+
conducted using high K buffer and nigericin in bath solutions of
various pH (4.14, 4.97, 6.23, 7.16, 8.34). As shown in Fig. 5a, the
fluorescence intensity of HCy-BIZ-BF increased proportionally
2
with the pH varying from 4.14 to 8.34. Accordingly, the
intracellular pH standard curve was obtained (Fig. 5b), with the
mode. (b) Enlarged views of fluorescence image and DIC (λex = 635 nm, λem = 650-750 regression equation Y = 0.2685x – 1.116 (R = 0.98853).
2
Fig. 4 (a) CLSM images of Hela cells incubated with HCy-BIZ-BF (100 nM) through Z-scan
2
nm). (c) Colocalization images of Hela cells incubated with HCy-BIZ-BF
nm, λem = 650–750 nm) and DND-99 (50 nM, λex = 515 nm, λem = 540–600 nm), along with
fluorescence intensity correlation plot of HCy-BIZ-BF
of Hela cells incubated with HCy-BIZ-BF (50 nM, λex = 635 nm, λem = 650–750 nm) and
2
(50 nM, λex = 635
2
Then, HCy-BIZ-BF was applied to monitor the pH fluctuations
and DND-99. Colocalization images in lysosome. It is reported that lysosome is the terminal of
2
2
endocytosis pathway, and it has a highly acid lumen with pH
Mito-Tracker Green (200 nM, λex = 488 nm, λem = 500–560 nm) along with their
fluorescence intensity correlation plot. (d) Intensity profiles within the ROI 1 (DND-99
5
7
values ranging from 4.5 to 5.5. Its pH values, however, can
experiment) and ROI 2 (Mito-Tracker Green experiment) across Hela cells. Scale bar: 30 grow gradually under the stimulation of chloroquine (a kind of
µm.
antimalarial drug). Under the same conditions, the related
fluorescence images of Hela cells were captured (Fig. 6a).
HCy-BIZ-BF
cells. After incubation with HCy-BIZ-BF
cytoplasm exhibited obvious NIR fluorescence (Fig. 4a).As
shown in the Z-scan imaging results, HCy-BIZ-BF distributed
extensively from top to bottom, which can be detailly
demonstrated by the enlarged images of cells’ cross section (X-
2
was applied to evaluating its distribution in living
According to pH standard curve, the determination of time-
dependent changes of pH value was plotted (Fig. 6b).
Specifically, the initial pH value in lysosomes under normal
incubations was calculated to be 4.67. After cells were
stimulated with chloroquine for 10 min, the pH increased to
2
for 15 min, the cell
2
5
.25 with enhanced fluorescence intensity, and was finally
Z) and the longitudinal section (Y-Z), verifying that HCy-BIZ-BF
2
varied to 6.58 after 30-minute stimulation. These results
can quickly enter and sufficiently permeate cells. Interestingly,
by studying the co-stained experiment with a commercial
lysosome tracker (DND-99), it was found that HCy-BIZ-BF
tended to accumulate in lysosomes. As depicted in Fig. 4c, the
fluorescence images of HCy-BIZ-BF merged rather well with
that of DND-99, with high Pearson’s Correlation Coefficient
0.92). In addition, the emission intensity profiles across cells of
2
2
(
ROIs were selected to study the overlap images, and a relatively
high overlap was found in Fig. 4d. To further demonstrate this
specificity, mitochondrion-related experiments were also
conducted by co-incubation with HCy-BIZ-BF
Green. In contrast, cell images of HCy-BIZ-BF
2
and Mito-Tracker
and Mito-Tracker
2
Green displayed totally different distribution with a lower
Pearson’s Correlation coefficient (0.56), as well as the distinct
emission intensity plot of ROIs (Fig. 4d). Besides, the experiment
2
results of HCy-BIZ-BF co-stained with commercial tracker of
endoplasmic reticulum and Golgi apparatus also exhibited low
Pearson’s correlation coefficient (Fig. S6). Therefore, the NIR
Fig. 6 Time courses of pH responses in lysosomes caused by chloroquine. (a) Hela
cells were incubated with HCy-BIZ-BF (100 nM) for 15 min, and then treated with
2
probe HCy-BIZ-BF
2
is an excellent lysosome-targeting probe chloroquine (100 µM). The fluorescence images were recorded at different times.
with high selectivity.
(
b) The plot of time-dependent pH values. Scale bar: 30 µm.
4
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Conclusions
View Article Online
DOI: 10.1039/C8TB03353F
In summary, we first introduced a novel strategy of boron
complex on the basis of a classical rhodamine-hemicyanine
hybrid, and designed a novel pH-sensing lysosome probe HCy-
BIZ-BF
and biological applications with recent reported lysosomal
probes in Table S1. It is concluded that HCy-BIZ-BF was
2
. Meanwhile, we have compared its primary properties
2
characterized with NIR emission, lineal pH responsibility and
lysosome targetability. Most importantly, after the chelation
with boron fluoride, HCy-BIZ-BF
shift and improved photostability, which is superior to its
precursor. Subsequently, HCy-BIZ-BF was applied to
2
possesses enlarged Stokes
2
monitoring pH in lysosome stimulated with chloroquine, and
further successfully investigating the changes of pH in living
mice with high signal-to-noise ratio. It is accordingly believed
that this concept of boron complex combined with tunable
classic hemicyanine would offer more chances to construct
novel NIR fluorescent probes.
Experimental section
The detailed information of instruments, materials, synthesis
and characterization of all compounds, cell cultivation, in
vitro/in vivo imaging were provided in the supporting
information.
Fig. 7 (a) Photoluminescence images of mouse labeled with HCy-BIZ-BF
and pH solution (60 μL, pH = 5.0) was injected into two adjacent spots on back of the
nude mouse. Then, HCy-BIZ-BF (60 μL, 50 µM) was subcutaneously injected into these
2
. Saline (60 μL)
2
two different locations, respectively (λex = 640 nm, λem = 710 ± 40 nm). (b) The
photoluminescence intensity of issues injected with saline (red circle) and pH solution
Animal models
(green circle) (c) A sketch of photoluminescence imaging in labeled living mouse.
All the nude mice were purchased from the Comparative
Medicine Centre of Yangzhou University. All the animal-based
experiments were performed in compliance with the criteria of
the National Regulation of China for Care and Use of Laboratory
Animals and approved by the Jiangsu Administration of
Experimental Animals.
2
demonstrated that HCy-BIZ-BF is an efficient NIR probe that
can monitor the lysosomal pH variations in cells treated with
chloroquine in real time.
Mapping of pH changes in living mouse model
Excellent photophysical properties and robust pH-sensitive
2
response in cells provided the basis that HCy-BIZ-BF can be
Conflicts of interest
There are no conflicts to declare.
used for pH detection in living mice. First, saline (pH = 6.9) and
PBS (pH = 5.0) were injected subcutaneously into two sides on
the back of the nude mouse. Under the excitation at 640 nm,
the NIR emission signals of HCy-BIZ-BF
monitored by the in vivo imaging system. As depicted in Fig. 7a,
without HCy-BIZ-BF there was no
photoluminescence in the right back (saline) and the left back Distinguished Young Scientists (61825503), National Natural
PBS, pH = 5.0). After treated for 5 min, the same volume HCy- Science Foundation of China (51473078 and 21671108),
BIZ-BF
solution (60 μL, 50 μM) was subcutaneously injected National Program for Support of Top-Notch Young
2
in the mouse could be
Acknowledgements
2
,
observable This work was supported by the National Funds for
(
2
into two different pH areas. After 10-minute incubation, the Professionals, Scientific and Technological Innovation Teams of
emission intensity of the right back was much stronger than that Colleges and Universities in Jiangsu Province (TJ215006),
of the left back (Fig. 7b). The statistic data further explained this Priority Academic Program Development of Jiangsu Higher
trend that the fluorescence intensity of HCy-BIZ-BF
2
in saline Education Institutions (YX03001), China Postdoctoral Science
spot was about 1.3 times that in acidic site. According to the Foundation funded project (2016M601789), and Jiangsu
above-described experiments, this evident fluorescence signal Province Postdoctoral Science Foundation funded project
enhancements could be caused by the deprotonation of HCy- (1701034A).
2 2
BIZ-BF in the saline locations. In short, HCy-BIZ-BF is
characteristic with superb properties as a NIR fluorescence
probe for pH detection in the living system.
Notes and references
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3
4
J. R. Casey, S. Grinstein and J. Orlowski, Nat. Rev. Mol. Cell Biol., 31 D. Aigner, S. M. Borisov, P. Petritsch and I. KlVimiewaAnrtti,cleCOhnelmine.
010, 11, 50-61. Commun., 2013, 49, 2139-2141.
J. P. Luzio, P. R. Pryor and N. A. Bright, Nat. Rev. Mol. Cell Biol., 32 H. Lee, W. Akers, K. Bhushan, S. Bloch, G. Sudlow, R. Tang and S.
007, 8, 622-632. Achilefu, Bioconjugate Chem., 2011, 22, 777-784.
B. Tang, F. Yu, P. Li, L. Tong, X. Duan, T. Xie and X. Wang, J. Am. 33 J. Madsen, I. Canton, N. J. Warren, E. Themistou, A. Blanazs, B.
2
DOI: 10.1039/C8TB03353F
2
Chem. Soc., 2009, 131, 3016-3023.
S. Wu, Z. Li, J. Han and S. Han, Chem. Commun., 2011, 47, 11276-
Ustbas, X. Tian, R. Pearson, G. Battaglia, A. L. Lewis and S. P.
Armes, J. Am. Chem. Soc., 2013, 135, 14863-14870.
1
1278.
34 P. Li, X. Duan, Z. Z. Chen, Y. Liu, T. Xie, L. B. Fang, X. R. Li, M. Yin
and B. Tang, Chem. Commun., 2011, 47, 7755-7757.
5
6
J. Y. Han and K. Burgess, Chem. Rev., 2010, 110, 2709-2728.
J. T. Hou, W. X. Ren, K. Li, J. Seo, A. Sharma, X. Q. Yu and J. S. Kim, 35 F. Yu, P. Li, P. Song, B. Wang, J. Zhao and K. Han, Chem. Commun.,
Chem. Soc. Rev., 2017, 46, 2076-2090. 2012, 48, 4980-4982.
P. Saftig and J. Klumperman, Nat. Rev. Mol. Cell Biol., 2009, 10, 36 W. Qin, T. Rohand, M. Baruah, A. Stefan, M. V. der Auweraer, W.
7
6
23-635.
Dehaen and N. Boens, Chem. Phys. Lett., 2006, 420, 562-568.
37 U. Balijapalli and S. K. Iyer, Eur. J. Org. Chem., 2015, 2015, 5089-
8
9
S. J. Lee, K. S. Cho and J. Y. Koh, Glia, 2009, 57, 1351-1361.
Y. Yue, F. Huo, S. Lee, C. Yin and J. Yoon, Analyst, 2016, 142, 30-
5098.
4
1.
38 W. Sun, S. Guo, C. Hu, J. Fan and X. Peng, Chem. Rev., 2016, 116,
1
0 S. Chen, Y. Hong, Y. Liu, J. Liu, C. W. T. Leung, M. Li, R. T. K. Kwok,
7768-7817.
E. Zhao, J. W. Y. Lam, Y. Yu and B. Z. Tang, J. Am. Chem. Soc., 2014, 39 M. Beija, C. A. Afonso and J. M. Martinho, Chem. Soc. Rev., 2009,
1
36, 11196-11196.
38, 2410-2433.
1
1
1 C. Watts, Biochim. biophys. Acta., 2012, 1824, 14-21.
2 K. A. Christensen, J. T. Myers and J. A. Swanson, J. Cell Sci., 2002,
1
40 J. Wang, S. Xia, J. Bi, Y. Zhang, M. Fang, R. L. Luck, Y. Zeng, T.-H.
Chen, H.-M. Lee and H. Liu, J. Mater. Chem. B, 2019, 7, 198-209.
41 G. K. Vegesna, J. Janjanam, J. Bi, F.-T. Luo, J. Zhang, C. Olds, A.
Tiwari and H. Liu, J. Mater. Chem. B, 2014, 2, 4500-4508.
15, 599-607.
1
1
3 D. Ellis and R. C. Thomas, Nature, 1976, 262, 224-225.
4 R. Zhang, S. G. Kelsen and J. C. Lamanna, J. Appl. Physiol., 1990, 42 S. Kolemen and E. U. Akkaya, Coord. Chem. Rev., 2018, 354, 121-
8, 1101-1106. 134.
5 H. Izumi, T. Torigoe, H. Ishiguchi, H. Uramoto, Y. Yoshida, M. 43 M. Zhu, P. Xing, Y. Zhou, L. Gong, J. Zhang, D. Qi, Y. Bian, H. Du
6
1
Tanabe, T. Ise, T. Murakami, T. Yoshida, M. Nomoto and K.
Kohno, Cancer Treat. Rev., 2003, 29, 541-549.
6 H. Zhu, J. L. Fan, Q. L. Xu, H. L. Li, J. Y. Wang, P. Gao and X. J. Peng,
Chem. Commun., 2012, 48, 11766-11768.
and J. Jiang, J. Mater. Chem. B, 2018, 6, 4422-4426.
44 L. Yuan, W. Lin, S. Zhao, W. Gao, B. Chen, L. He and S. Zhu, J. Am.
Chem. Soc., 2012, 134, 13510-13523.
45 J. Zhang, C. Li, R. Zhang, F. Zhang, W. Liu, X. Liu, S. M. Lee and H.
Zhang, Chem. Commun., 2016, 52, 2679-2682.
1
1
1
7 Ettinger, A.; Wittmann, T. Methods Cell Biol., 2014, 123, 77-94.
8 Q. Wan, S. Chen, W. Shi, L. Li and H. Ma, Angew. Chem. Int. Ed., 46 C. Han, H. Yang, M. Chen, Q. Su, W. Feng and F. Li, ACS Appl.
014, 53, 10916-10920. Mater. Interfaces, 2015, 7, 27968-27975.
9 K.-K. Yu, K. Li, J.-T. Hou, H.-H. Qin, Y.-M. Xie, C.-H. Qian and X.-Q. 47 J. Wang, S. Xia, J. Bi, M. Fang, W. Mazi, Y. Zhang, N. Conner, F. T.
2
1
2
Yu, RSC Adv., 2014, 4, 33975-33980.
0 Beatty, K. E.; Fisk, J. D.; Smart, B. P.; Lu, Y. Y.; Szychowski, J.;
Luo, H. P. Lu and H. Liu, Bioconjugate Chem., 2018, 29, 1406-
1418.
Hangauer, M. J.; Baskin, J. M.; Bertozzi, C. R.; Tirrell, D. A. 48 L. Yuan, W. Lin, Y. Yang and H. Chen, J. Am. Chem. Soc., 2012, 134,
Chembiochem., 2010, 11, 2092-2095. 1200-1211.
1 M. Grossi, M. Morgunova, S. Cheung, D. Scholz, E. Conroy, M. 49 Y. Li, Y. Wang, S. Yang, Y. Zhao, L. Yuan, J. Zheng and R. Yang, Anal.
2
Terrile, A. Panarella, J. C. Simpson, W. M. Gallagher and D. F.
O'Shea, Nat. Commun., 2016, 7, 10855.
2 W. Luo, H. Jiang, X. Tang and W. Liu, J. Mater. Chem. B, 2017, 5,
Chem., 2015, 87, 2495-2503.
50 P. Tao, Y. Miao, H. Wang, B. Xu and Q. Zhao, Chem. Rec., 2018,
DOI: 10.1002/tcr.201800139.
51 H. Mtiraoui, R. Gharbi, M. Msaddek, Y. Bretonnière, C. Andraud,
P.-Y. Renard and C. Sabot, RSC Adv., 2016, 6, 86352-86360.
52 W. Duan, Q. Liu, Y. Huo, J. Cui, S. Gong and Z. Liu, Org Biomol.
Chem., 2018, 16, 4977-4984.
53 H. Xiao, P. Li, W. Zhang and B. Tang, Chem. Sci., 2016, 7, 1588-
1593.
2
2
2
2
4
768-4773.
3 B. Dong, X. Song, C. Wang, X. Kong, Y. Tang and W. Lin, Anal.
Chem., 2016, 88, 4085-4091.
4 J. Liu, Y. Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y.
Shi and W. Guo, J. Am. Chem. Soc., 2014, 136, 574-577.
5 A. Becker, C. Hessenius, K. Licha, B. Ebert, U. Sukowski, W.
Semmler, B. Wiedenmann and C. Grotzinger, Nature biotechnol., 54 Ma, D. L.; Wu, C.; Li, G.; Leung, C. H. J. Anal. Test., 2018, 2, 77-89.
2
001, 19, 327-331.
55 Ma, D.-L.; Che, C.-M.; Yan, S.-C. J. Am. Chem. Soc., 2009, 131,
2
6 Vellaisamy, K.; Li, G.; Ko, C. N.; Zhong, H. J.; Fatima, S.; Kwan, H.
1835-1846.
Y.; Wong, C. Y.; Kwong, W. J.; Tan, W.; Leung, C. H.; Ma, D. L. 56 Sun, H.; Li, W.; Dong, Z. Z.; Hu, C.; Leung, C. H.; Ma, D. L.; Ren, K.
Chem. Sci., 2018, 9, 1119-1125. Biosens. Bioelectron., 2018, 99, 361-367.
7 Z. Dong, Q. Han, Z. Mou, G. Li and W. Liu, J. Mater. Chem. B, 2018, 57 C. Settembre, A. Fraldi, D. L. Medina and A. Ballabio, Nat.
, 1322-1327. Rev. Mol. Cell Biol., 2013, 14, 283-296.
8 S. Xia, J. Wang, J. Bi, X. Wang, M. Fang, T. Phillips, A. May, N.
Conner, M. Tanasova, F. T. Luo and H. Liu, Sensor. Actuat. B-
Chem., 2018, 265, 699-708.
2
2
6
2
3
9 S. O. McDonnell and D. F. O'Shea, Org. Lett., 2006, 8, 3493-3496.
0 L. Fan, Y. J. Fu, Q. L. Liu, D. T. Lu, C. Dong and S. M. Shuang, Chem.
Commun., 2012, 48, 11202-11204.
6
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Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C8TB03353F
Lysosome-specific sensing and imaging of pH variations in vitro and in vivo utilizing
a near-infrared boron complex
a
a
a
a
a
a
Yuxiang Shi, Xiangchun Meng, Huiran Yang, Linna Song, Shujuan Liu,* Aqiang Xu,
a
ab
a
Zejing Chen, Wei Huang,* and Qiang Zhao*
A NIR lysosome-targetable boron complex has been developed based on
hemicyanine for monitoring pH variations in vitro and in vivo.