Aggregation enhanced responsiveness of rationally designed probes to hydrogen sulfide for targeted cancer imaging

Article abstract of DOI:10.1021/jacs.0c06533

Activatable molecular probes hold great promise for targeted cancer imaging. However, the hydrophobic nature of most conventional probes makes them generate precipitated agglomerate in aqueous media, thereby annihilating their responsiveness to analytes a

Full text of DOI:10.1021/jacs.0c06533

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Article
Aggregation Enhanced Responsiveness of Rationally Designed
Probes to Hydrogen Sulfide for Targeted Cancer Imaging
Rongchen Wang, Xianfeng Gu, Qizhao Li, Jie Gao, Ben Shi, Ge Xu, Tianli Zhu, He Tian, and Chunchang Zhao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c06533 • Publication Date (Web): 29 Jul 2020
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Aggregation Enhanced Responsiveness of Rationally Designed
Probes to Hydrogen Sulfide for Targeted Cancer Imaging
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Rongchen Wang,†,§ Xianfeng Gu,‡,§ Qizhao Li,† Jie Gao,‡ Ben Shi,† Ge Xu,† Tianli Zhu,† He Tian,†
Chunchang Zhao*,†
† Key Laboratory for Advanced Materials and Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine
Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai,
200237, P. R. China.
‡ Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, P. R. China.
ABSTRACT: Activatable molecular probes hold great promise for targeted cancer imaging. However, the hydrophobic nature of
most conventional probes makes them generate precipitated agglomerate in aqueous media, thereby annihilating their
responsiveness to analytes and precluding their practical applications for bioimaging. This study reports the development of two
small molecular probes with unprecedented aggregation enhanced responsiveness to H2S for in vivo imaging of H2S-rich cancers.
The subtle modulation of the equilibrium between hydrophilicity and lipophilicity by N-ethylpyridinium endows these designed
probes with the capability of spontaneously self-assembling into nanoprobes under physiological conditions. Such probes in an
aggregated state, rather than a molecular dissolved state, show NIR fluorescence light up and photoacoustic signals turn on upon
H2S specific activation, allowing in vivo visualization and differentiation of cancers based on differences in H2S content. Thus, our
study presents an effective design strategy which should pave the way to molecular design of optimized probes for precision cancer
diagnostics.
of endogenous H2S. Despite the advances, these nanoprobes
INTRODUCTION
are still subject to the limitations of complicated fabrication
Activatable molecular probes that change fluorescent
processes and poor preparation reproducibility, which is not
signals only in response to cancer-related biomarkers greatly
conducive to clinical translation. Therefore, it is of great
facilitate the advancement of targeted cancer imaging.1-3 In
scientific interest to develop new activatable small molecules
this context, various activatable fluorescent probes capable of
that are capable of spontaneous self-assembly to form
detection of diverse molecular targets in cancers have been
monodisperse
nanoparticles
with
improved
optical
successfully developed over the years.4-20 The prerequisite of a
practical system for in vivo imaging is the capability of a
molecular probe to function well under physiological
conditions. Although a large number of activatable small
molecule probes are now available, they naturally tend to
aggregate into precipitated agglomerate in aqueous media due
to the hydrophobic nature of most conventional organic probes;
this results in minimal responsiveness to analytes.21-26 Such
characteristics preclude their practical applications for
bioimaging.
responsiveness for cancer imaging.
Herein, we report two small molecular probes that show
unprecedented aggregation enhanced responsiveness (AER) to
H2S for in vivo imaging of H2S-rich cancers. Our designed
molecules are shown in Scheme 1, in which the hydrophilic N-
ethylpyridinium acts as the electron withdrawing group, while
the hydrophobic monochlorinated BODIPY core acts as the
H2S-responsive unit. Different from the traditional lipophilic
molecular probes (e.g. probes 3 and 4), which always have
poor or no response to analytes in pure aqueous solutions
because of their limited water solubility and the resultant
precipitation tendencies under physiological conditions,21-24
the designed amphiphilic probes 1 and 2 were able to
spontaneously self-assemble into nanoprobes with well-
defined nanostructure and showed surprising aggregation
enhanced responsiveness to H2S in buffers. High absorption
bands within NIR regions were activated upon introduction of
H2S, which gave rise to bright NIR emissions lighting up in
assembled 1 (the assembled form of probe 1) and strong
photoacoustic signals turning on in assembled 2 (the
assembled form of probe 2). However, the molecularly
dissolved probe 1 and 2 in the bulk solutions gave minimal
optical changes upon introduction of H2S. Undoubtedly, as
compared to traditional activatable probes, the unprecedented
Because increased H2S production gives rise to
tumorigenesis in many cancers, H2S-activatable molecular
probes should unambiguously provide valuable insight into
precise cancer imaging.27-32 However, the use of H2S-
responsive probes for cancer imaging is still in its infancy.
Although many H2S-responsive fluorescent probes have been
established and found widespread applications,33-41 most of
them are inapplicable for in vivo tumor location presumably
due to several intrinsic drawbacks including poor
hydrophilicity and slow reaction with H2S in aqueous solution.
To address these issues, great efforts have been devoted to
engineering water-soluble nanoprobes through encapsulation
of small activatable molecule probes into the hydrophobic
interiors of nanocomposites.42-46 These devised nanoprobes
realize accurate identification of cancers by real-time trapping
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aggregation enhanced responsiveness makes probes 1 and 2
conversion of probe 1 to 1-SH via thiol−halogen nucleophilic
aromatic substitution reaction (SNAr) (Scheme 2 and Figure
S2). The native fluorescent features of 1 and 1-SH
(fluorescence quantum yield = 0.88% for assembled state in
pure Tris buffer) afford no contributions to such aggregation
enhanced NIR emission due to the fact that probe 1 and 1-SH
show aggregation caused fluorescence quenching properties
(Table S1 and Figure S3).
1
2
3
4
5
6
7
8
ideal candidates for biosensing and imaging in vivo. As a
proof-of-concept example, using the assembled 1 that emitted
NIR fluorescence at 718 nm upon H2S activation, accurate
identification and differentiation of cancers were realized by
real-time trapping of endogenous H2S. This is the first
example in which a small molecular probe shows spontaneous
self-assembly in an aqueous medium with greatly enhanced
responsiveness to analytes, thus paving the way to molecular
design of optimized probes for precision cancer diagnostics.
Notably, after reaching the best performance at fw = 90%,
both the absorption and fluorescence responsiveness was
slightly attenuated upon further increasing the water fraction
fw. Nevertheless, self-assembled probe 1 produced highly
efficient responsiveness to H2S in 100% buffer, thus indicating
its suitability for visualization of H2S in living systems. The
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amphiphilic probe
1 spontaneously self-assembled into
nanoprobes and showed aggregation enhanced responsiveness
to H2S in buffers. This unprecedented AER was further
confirmed by the pseudo-first-order kinetics (kobs) at various
fw. As expected (Figure S4), assembled 1 at fw =100%
exhibited much faster kobs (7.13×10-3 S-1) than that of
molecular dissolved 1 at fw =50% (2.81×10-3 S-1).
Scheme 1. Chemical structures of 1, 2, 3, and 4. The amphiphilic
nature of 1 and 2 leads to spontaneous formation of positively
charged assemblies, while the lipophilic
3 and 4 forms
precipitated agglomerate in aqueous media. Such disparity
endows the probes with distinct responsiveness to H2S under
physiological conditions.
RESULTS AND DISCUSSION
As presented in Scheme 2 and Scheme S1, probes 1 and 2
were readily synthesized by the Knoevenagel condensation of
a
known aldehyde BODIPY47 with 2-methyl-N-
ethylpyridinium
and
4-methyl-N-ethylpyridinium,
respectively. The control probes 3 and 4 were prepared
following the similar synthesis procedure, wherein 4-
methylenepyridine and N-ethyl-2,3,3-trimethylindolenine
were linked to BODIPY core to elongate the conjugated
1
system. H and 13C NMR spectroscopy and HRMS identified
their structures and their high purity.
H2S
N
N
Figure 1. Normalized absorption (a) and fluorescence (b)
responses of probe 1 (10 μM) to NaHS (100 μM) in CH3CN/Tris
mixtures with various fw. Data were recorded 60 min after
addition of NaHS. Line 1 (black line) represents the absorption
and fluorescence of probe 1 without H2S. (c) DLS size profiles of
assembled 1 (10 μM) at various fw. Line 1-SH indicates the size
of assembled 1-SH at fw = 100%. (d) Fluorescence intensity
changes of assembled 1 upon addition of NaHS (100 μM) and
other competing analytes (1 mM) in Tris buffer (pH 7.4). 1) Free;
I
I
I
N
N
N
N
N
B
1
B
Cl
HS
F
F
F
F
OHC
N
N
1-SH
B
Cl
F
F
I
N
I
I
N
H2S
N
N
N
N
B
F
N
B
Cl
F
F
HS
F
2
2-SH
Scheme 2. Synthesis procedure and proposed SNAr reaction of
probe 1 and 2 with H2S.
-
-
-
2) F-; 3) Cl-; 4) Br-; 5) I-; 6) NO2 ; 7) N3 ; 8) HCO3 ; 9) SO42-; 10)
HPO42-; 11) ClO-; 12) H2O2; 13) OAc; 14) S2O32-; 15) GSH; 16)
-
Interestingly, the optical response of probes 1 and 2 to H2S
heavily depended on the volume fraction of water (fw) in the
CH3CN/Tris mixtures (pH 7.4, room temperature). For
example, the molecular dissolved probe 1 with 2-methylene-
N-ethylpyridinium function gave poor optical changes upon
introduction of 100 μM NaHS at fw = 50%. However, as the
fw value gradually increased from 50% to 90%, the H2S-
activated new absorption enhanced significantly and the
absorption peak hypsochromically shifted from 659 nm to 644
nm (Figure 1a and Figure S1). Similarly, the H2S-activated
NIR emission increased sharply and the emission peak shifted
from 751 to 725 nm upon increasing fw from 50% to 90%
(Figure 1b). These H2S-initiated optical features upon
increasing fw were ascribed to the aggregation enhanced
Hcy; 17) Cys; 18) NaHS. Data were recorded 60 min after
addition of NaHS.
The aggregate formation of probe 1 could be confirmed by
dynamic light scattering (DLS) measurements (Figure 1c). At
fw = 50%, 60% and 70 %, probe 1 was found to be in the
molecularly dissolved state in CH3CN/Tris buffer mixtures;
thus, no DLS signals were detectable. However, probe 1 was
prone to form aggregates with average diameter of 29 ± 4 nm
at fw = 80%, 56 ± 5 nm at fw = 90% and 43 ± 5 nm at fw =
100%. Additional characterizations of the corresponding
morphology were also performed by scanning electron
microscopy (SEM) (Figure S5). Notably, assembled 1-SH
aggregated into much larger particles (124 ± 13 nm) at fw =
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100%, enabling a long residence time in the target cells to
emergence of NIR absorption at 744 nm could be ascribed to
the gradual evolution of aggregates from loose to compact,
which leads to a reduced energy gap between HOMO and
LUMO. According to H2S-initiated NIR absorption changes, a
detection limit of 760 nM was determined for assembled 2
(Figure S12).
achieve high imaging contrast.48-49 More importantly, both
assembled 1 and assembled 1-SH had good stability in buffers
(Figure S6).
1
2
3
4
5
6
7
8
In 100% Tris buffer, the fluorescence intensity of assembled
1 showed good linear correlation within the H2S concentration
range from 0 to 30 μM (Figure S7), thus affording a detection
limit of 60 nM which shows that assembled 1 could provide
sensitive detection of H2S. It was also found that assembled 1
exhibited a highly selective response to H2S. A distinct
enhancement of the fluorescence intensity at 718 nm was
triggered by H2S and not by other interfering species (Figure
1d and Figure S8). Importantly, such distinct responsiveness
of assembled 1 to H2S was maintained in a cell culture
medium (Figure S9), indicative of no interference from
biological contexts. Of note, assembled 1 was readily activated
to give good optical response within a physiological pH range
of 8.5 to approximately 5.5 (Figure S10).
The photoacoustic responsiveness of assembled 2 to H2S
was then evaluated in buffer solutions (pH 7.4). In the absence
of H2S, no detectable photoacoustic signals were initiated. In
contrast, when H2S was introduced to the solution, bright
photoacoustic signals were produced following excitation at
750 nm, which ultimately afforded a 13-fold turn-on response
(Figure 2b). The H2S-activataed PA intensities of assembled 2
showed a dose-dependent enhancement (Figure S12). This
H2S-initiated strong PA signal showed no interference from 1
mM GSH and 1 mM Cys, indicative of the probe’s potential
for imaging applications in complex living systems.
Furthermore, the good PA responsiveness to H2S was found to
be retained within a physiological range from pH 8.5 to
approximately 5.5 (Figure 2c).
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In order to acquire insight into the self-assembly process of
probe 2 that forms aggregates, the average sizes of formed
nanoparticles in CH3CN/Tris buffer mixtures with different fw
were monitored through DLS assay and SEM characterization.
The DLS results (Figure 2d) showed that the assembled 2 has
sizes of about 35 ± 5 nm at fw = 70%, 61 ± 8 nm at fw = 80%,
77 ± 7 nm at fw = 90% and 148 ± 12 nm at fw = 100%. No
DLS signals, however, could be obtained from the solution at
fw = 50% and 60% due to the fact that probe 2 is in the
molecularly dissolved state in these solutions. In addition, it
was found that assembled 2 and assembled 2-SH showed good
stability in buffers (Figure S13).
Figure 2. (a) Normalized absorption responses of probe 2 (10 μM)
to NaHS (100 μM) at various fw. Data were recorded 60 min after
addition of NaHS. Line 2 (black line) represents the absorption of
probe 2 without NaHS. (b) Photoacoustic amplitude changes of
assembled 2 (80 μM) upon addition of NaHS (100 μM) and other
competing analytes (1 mM) in pure Tris buffer (pH 7.4, room
temperature). Data were collected 60 min after addition of NaHS.
(c) The good PA responsiveness of assembled 2 to H2S within a
wide pH range. (d) DLS size profiles of assembled 2 (10 μM) at
various fw. Line 2-SH indicates the size of assembled 2-SH at fw
= 100%.
In the case of probe 2 with 4-methylene-N-ethylpyridinium
function, its amphiphilic nature enabled 2 to readily self-
assemble in physiological conditions. Owing to the
aggregation, probe 2 attained enhanced optical response to
H2S. Its H2S-initiated NIR absorption gradually enhanced with
gradually increasing fw from 50% to 80%, and the absorption
peak slightly shifted from 667 nm to 658 nm. Impressively,
further increasing fw from 80% to 100%, a new absorption
band around 744 nm was found to evolve sharply,
accompanied by the higher energy peak around 658 nm as a
shoulder (Figure 2a and Figure S11), indicative of self-
assembled 2 in 100% buffer being a H2S-activatable
photoacoustic probe. The H2S-initiated induction of NIR
absorption at 744 nm was not interrupted by biological
contexts such as a cell culture medium (Figure S9). Such an
Figure 3. (a) Mean zeta potential of probe 1 (10 μM) at various
fw. (b) Mean zeta potential of probe 2 (10 μM) at various fw.
(c) Normalized absorption responses of 1-SDS, 1-CTAB, 1-
PEG, and assembled 1 (probe 1, 10 μM) to NaHS (100 μM).
(d) Normalized absorption responses of 2-SDS, 2-CTAB, 2-
PEG, and assembled 2 (probe 2, 10 μM) to NaHS (100 μM).
The aforementioned unprecedented aggregation enhanced
responsiveness of probe 1 and 2 could be ascribed to an
important concern: the self-assembly of probe 1 and 2 in
aqueous solution affords aggregations with enhanced positive
charge density that enriches the local concentrations of HS-
(main forms of H2S in physiological pH) close to
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monochlorinated BODIPY,50-52 thus facilitating the SNAr
Page 4 of 8
model probe to further explore the potential applications in
living systems. To identify and differentiate cancer cells,
fluorescence light up imaging was performed with H2S-rich
HCT116 cells and H2S-deficient HepG2 cells. After
demonstration of a high biocompatibility in living cells
(Figure S18), cell imaging experiments were subsequently
conducted. As shown in Figure 4a, the incubation of HCT116
cells with assembled 1 for 1 h afforded bright red fluorescence
signals. To clarify that the obtained red fluorescence signal
was indeed activated by endogenous H2S, inhibitor and
activator assays were performed. The inhibitory production of
cellular H2S by cystathionine-β-synthase (CBS) inhibitor
aminooxyacetic acid (AOAA) attenuated the red fluorescence
in HCT116 cells, while the promotion of H2S production by
allosteric CBS activator S-adenosyl-l-methionine (SAM)
resulted in an increase of the red fluorescence. Due to the H2S-
deficiency in HepG2 cells, treatment of these living cells with
assembled 1 revealed no detectable red fluorescence signals
(Figure 4b). Furthermore, pretreatment of HepG2 cells with
AOAA or SAM also induced minimal red fluorescence images.
Collectively, assembled 1 can be used to selectively image
H2S-rich cancer cells by tracking endogenous H2S and
differentiate types of living cells based on H2S contents (rich
vs deficient). Also, we successfully established the
applicability of assembled 1 for tracking endogenous H2S
production by performing experiments with raw264.7
macrophages and Cardiomyocytes cells that express the H2S-
producing enzyme cystathionine γ-lyase (CSE) (Figure S19
and S20). Additionally, the spot-like cell images presented a
direct observation of the aggregation of probe 1 in cells
(Figure S21).56
1
2
3
4
5
6
7
8
reaction. In contrast, the molecularly dissolved state of probes
1 and 2 in the bulk solutions had minimal H2S enrichment
effect, thus resulting in poor responsiveness. The mean zeta
potential values of assembled probes in CH3CN/Tris buffer
mixtures with different fw stayed highly consistent with the
observed responsiveness to H2S (Figure 3a and Figure 3b),
inferring that positive charge density played a vital role in
regulating the reaction of the probes with H2S.
To elucidate the essential role of positive charge in
accelerating the responsive performance, we further
established nanoprobes 1/2-SDS, 1/2-CTAB and 1/2-PEG via
encapsulating probe 1 or 2 into the hydrophobic interiors of
micelles based on sodium dodecylsulfonate (SDS, anionic
surfactant), cetyltriethylammnonium bromide (CTAB, cationic
surfactant), and amphiphilic copolymer (mPEG-DSPE),
respectively. Experimental data showed that the highly
positive charged nature of 1/2-CTAB led to markedly rapid
optical responses to H2S in approximately 2 min, accompanied
by enhanced optical changes (Figure 3c-d and Figure S14-15).
In sharp contrast, 1/2-SDS gave minimal optical changes in
approximately 120 min since the negatively charged surface
prevented anion HS- from accessing probes 1 and 2 within the
hydrophobic cores. Interestingly, because of the neutral nature
of 1/2-PEG, mild responsiveness was observed. Within these
nanoprobes, small molecular probes 1 and 2 were maintained
at low local concentrations to minimize aggregation processes
in micelles.36 Accordingly, no aggregation induced red-shift
of the absorption of 2-SH to 744 nm was observed in 2-CTAB
and 2-PEG. The specific generation of NIR absorption at 744
nm thus indicated the superiority of self-assembled 2 as an
activatable photoacoustic probe.
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To further demonstrate the vital role of the positive charge
on the enrichment of HS- for inducing and accelerating the
responsiveness, probe 3 was synthesized; it had pyridine rather
than N-ethylpyridinium appending to the BODIPY core
through a vinylene unit. Since probe 3 had no cation function
and is lipophilic, this probe formed agglomerates that
precipitated in buffer. In consequence, negligible H2S-
activated absorption and emission could be observed with
increasing fw from 50% to 100% (Figure S16), indicative of
no aggregation enhanced responsiveness effect. We previously
reported an H2S-activatble probe 4,36 wherein electron-
withdrawing N-ethyl-2,3,3-trimethylindolenine was appended
to the BODIPY core through a vinylene unit. As indolium is a
lipophilic cation in nature, probe 4 naturally produces
precipitated agglomerates in aqueous media. Consequently,
probe 4 possessed good H2S-activated performance when it
was well dissolved in CH3CN/Tris media with fw = 50%
(Figure S17). In contrast, minimal optical changes were
initiated by H2S at fw = 100%. Evidently, probe 4 exhibited an
opposite performance compared to probe 1 and 2. Hence the
subtle variation of electron withdrawing groups appending to
monochlorinated BODIPY can generate great differences in
hydrophilicity and optical responsiveness to H2S under
physiological conditions. Undoubtedly, as compared to
traditional activatable probes, the unprecedented aggregation
enhanced responsiveness makes probes 1 and 2 good
candidates for biosensing and imaging in vivo.
Figure 4. Identification and differentiation of H2S-rich HCT116
cells (a) and H2S-deficient HepG2 cells (b) after incubation with
assembled 1 at 37 °C for 1 h. Inhibitor and activator assay were
performed by pretreatment of cells with AOAA (1 mM) and SAM
(3 mM), followed by further incubation with assembled 1 for 1h.
Scale bar = 10 μm. (c) Relative fluorescence intensities quantified
from images in (a) and (b). (d) Representative fluorescence
images of tumor-bearing mice at different time post subcutaneous
injection of assembled 1 (30 nmol) into tumor-bearing mouse
models for identification and differentiation of cancers based on
H2S-specific activation. (e) Fluorescence intensities of tumors
quantified from images in (d).
The widely available instruments for NIR fluorescence
imaging and activatable fluorescent probes are valuable tools
in medicine,53-55 so we used assembled 1 as a proof-of-concept
Next, assembled 1 was further explored to identify and
differentiate types of cancers based on H2S contents through
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subcutaneous injection into H2S-rich HCT116 tumor-bearing
REFERENCES
(1) Zhang, J.; Chai, X.; He, X.-P.; Kim, H.-J.; Yoon, J.; Tian, H.
Fluorogenic probes for disease-relevant enzymes. Chem. Soc. Rev.
2019, 48, 683.
(2) Liu, H.-W.; Chen, L.; Xu, C.; Li, Z.; Zhang, H.; Zhang, X.-B.;
Tan, W. Recent progresses in small-molecule enzymatic fluorescent
probes for cancer imaging. Chem. Soc. Rev. 2018, 47, 7140.
(3) Razgulin, A.; Ma, N.; Rao, J. Strategies for in vivo imaging of
enzyme activity: an overview and recent advances. Chem. Soc. Rev.
2011, 40, 4186.
(4) Wu, X.; Shi, W.; Li, X.; Ma, H. A Strategy for Specific
Fluorescence Imaging of Monoamine OxidaseꢀA in Living Cells.
Angew. Chem. Int. Ed. 2017, 56, 15319.
(5) Chen, W.; Matsunaga, T.; Neill, D. L.; Yang. C.-T.; Akaike, T.;
Xian, M. Rational Design of a Dual-Reactivity-Based Fluorescent
Probe for Visualizing Intracellular HSNO. Angew. Chem. Int. Ed.
2019, 58,16067.
(6) Cheng, H.-B.; Zhang, Y.-M.; Liu, Y.; Yoon, J. Turn-On
Supramolecular Host-Guest Nanosystems as Theranostics for Cancer.
Chem. 2019, 5, 553.
(7) Zhang, H.; Fan, J.; Wang, J.; Zhang, S.; Dou, B.; Peng, X. An
Off–On COX-2-Specific Fluorescent Probe: Targeting the Golgi
Apparatus of Cancer Cells. J. Am. Chem. Soc. 2013, 135, 11663.
(8) Miao, Q.; Yeo, D. C.; Wiraja, C.; Zhang, J.; Ning, X.; Xu, C.;
Pu, K. Near-Infrared Fluorescent Molecular Probe for Sensitive
Imaging of Keloid. Angew. Chem. Int. Ed. 2018, 57, 1256.
(9) Zhou, Z.; Wang, F.; Yang, G.; Lu, C.; Nie, J.; Chen, Z.; Ren, J.;
Sun, Q.; Zhao, C.; Zhu, W.-H. A Ratiometric Fluorescent Probe for
Monitoring Leucine Aminopeptidase in Living Cells and Zebrafish
Model. Anal. Chem. 2017, 89, 11576.
(10) Li, H.; Li, Y.; Yao, Q.; Fan, J.; Sun, W.; Long, S.; Shao, K.;
Du, J.; Wang, J.; Peng, X. In situ imaging of aminopeptidase N
activity in hepatocellular carcinoma: a migration model for tumour
using an activatable two-photon NIR fluorescent probe. Chem. Sci.
2019, 10, 1619.
(11) Gu, K.; Xu, Y.; Li, H.; Guo, Z.; Zhu, S.; Zhu, S.; Shi, P.;
James, T. D.; Tian, H.; Zhu, W.-H. Real-Time Tracking and In Vivo
Visualization of β-Galactosidase Activity in Colorectal Tumor with a
Ratiometric Near-Infrared Fluorescent Probe. J. Am. Chem. Soc. 2016,
138, 5334.
(12) Li, Y.; Sun, Y.; Li, J.; Su, Q.; Yuan, W.; Dai, Y.; Han, C.;
Wang, Q.; Feng, W.; Li, F. Ultrasensitive Near-Infrared
Fluorescence-Enhanced Probe for in Vivo Nitroreductase Imaging. J.
Am. Chem. Soc. 2015, 137, 6407.
(13) Wang, R.; Chen, J.; Gao, J.; Chen, J.-A.; Xu, G.; Zhu, T.; Gu,
X.; Guo, Z.; Zhu, W.; Zhao, C. A molecular design strategy toward
enzyme-activated probes with near-infrared I and II fluorescence for
targeted cancer imaging. Chem. Sci. 2019, 10, 7222.
(14) Zheng, X.; Wang, X.; Mao, H.; Wu, W.; Liu, B.; Jiang, X.
Hypoxia-specific ultrasensitive detection of tumours and cancer cells
in vivo. Nat. Commun. 2015, 6, 5834.
(15) Wang, F.; Zhu, Y.; Zhou, L.; Pan, L.; Cui, Z.; Fei, Q.; Luo, S.;
Pan, D.; Huang, Q.; Wang, R.; Zhao, C.; Tian, H.; Fan, C. Fluorescent
InꢀSitu Targeting Probes for Rapid Imaging of Ovarian-Cancer-
Specific γ-Glutamyltranspeptidase. Angew. Chem. Int. Ed. 2015, 54,
7349.
(16) Liu, Y.; Tan, J.; Zhang, Y.; Zhuang, J.; Ge, M.; Sh, B.; Li, J.;
Xu, G.; Xu, S.; Fan, C.; Zhao, C. Visualizing glioma margins by real-
time tracking of γ-glutamyltranspeptidase activity. Biomaterials 2018,
173, 1.
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mouse models and H2S-deficient HepG2 tumor-bearing mouse
models (Figure 4). The NIR fluorescence images of H2S-rich
HCT116 tumor indicated that obvious NIR emission was
initiated within 1 min post-injection of assembled 1, indicative
of rapid activation in H2S-rich regions. This fluorescence
intensity gradually increased over time and leveled off at 60
min post-injection. The HCT116 tumor visualization based on
H2S-activation of assembled 1 was further confirmed by in
vivo inhibition assay. It was found that attenuation of NIR
fluorescence emerged when the HCT 116 tumor was
pretreated by AOAA. Note that at 1 h after injection, the NIR
fluorescence signal in the HCT 116 tumor region was triple
that in AOAA-pretreated tumors. Owing to the low level of
H2S in HepG2 tumors, the NIR fluorescence signals slightly
increased (Figure 4d), and thus it was hard to delineate the
tumor. These imaging data demonstrated that the increased
NIR fluorescence signals in the tumor region came from H2S-
specific activation of assembled 1, which enables the
discrimination of H2S-rich cancers from those that possess low
H2S level.
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CONCLUSION
In summary, we designed and synthesized two small
molecular probes that possess unprecedented aggregation
enhanced responsiveness to H2S for in vivo imaging of H2S-
rich cancers. Traditional activatable molecular probes readily
aggregate to precipitated agglomerates in aqueous media and,
in that form, provide minimal responsiveness to analytes. In
contrast, our designed probes displayed H2S specific activation
in the aggregated state rather than in the molecularly dissolved
state, giving rise to bright NIR emissions lighting up and
strong photoacoustic signals turning on responses which make
these new probes ideal candidates for in vivo imaging. Such
unique characteristics allow identifying and differentiating
types of cancers based on H2S contents. The new probes are
the first examples that show aggregation enhanced
responsiveness to analytes in an aqueous medium. This work
presents a promising approach to the design of optimized
probes for precisely targeted cancer imaging.
ASSOCIATED CONTENT
Supporting Information. Detailed synthesis and experimental
procedures, supplementary figures, and characterizations are
provided. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* E-mail: zhaocchang@ecust.edu.cn
Author Contributions
R. W., X. G. contributed equally.
Notes
The authors declare no competing financial interest.
(17) Urano, Y.; Sakabe, M.; Kosaka, N.; Ogawa, M.; Mitsunaga,
M.; Asanuma, D.; Kamiya, M.; Young, M. R.; Nagano, T.; Choyke, P.
L. Rapid Cancer Detection by Topically Spraying
Glutamyltranspeptidase–Activated Fluorescent Probe. Sci. Transl.
Med. 2011, 3, 110ra119.
(18) Gu, K.; Qiu, W.; Guo, Z.; Yan, C.; Zhu, S.; Yao, D.; Shi, P.;
Tian, H.; and Zhu, W.-H. An enzyme-activatable probe liberating
AIEgens: on-site sensing and long-term tracking of β-galactosidase in
ovarian cancer cells. Chem. Sci. 2019, 10, 398.
a
γ-
ACKNOWLEDGMENT
We gratefully acknowledge financial support by the National
Natural Science Foundation of China (21672062, 21874043,
21572039, 21788102) and Shanghai Municipal Science and
Technology Major Project (Grant No.2018SHZDZX03).
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(19) Li, X.; Kim, C.-Y.; Lee, S.; Lee, D.; Chung, H.-M.; Kim, G.;
Generation via Fluvastatin-Stimulated Upregulation. J. Am. Chem.
Heo, S.-H.; Kim, C.; Hong, K.-S.; Yoon, J. Nanostructured
Phthalocyanine Assemblies with Protein-Driven Switchable
Photoactivities for Biophotonic Imaging and Therapy. J. Am. Chem.
Soc. 2017, 139, 10880.
Soc. 2015, 137, 8490.
1
2
3
4
5
6
7
8
(37) Chen, W.; Pacheco, A.; Takano, Y.; Day, J. J.; Hanaoka, K.;
Xian, M. A Single Fluorescent Probe to Visualize Hydrogen Sulfide
and Hydrogen Polysulfides with Different Fluorescence Signals.
Angew .Chem. Int. Ed. 2016, 55,9993.
(38) Lippert, A. R.; New, E. J.; Chang, C. J. Reaction-Based
Fluorescent Probes for Selective Imaging of Hydrogen Sulfide in
Living Cells. J. Am. Chem. Soc. 2011, 133, 10078.
(39) Henthorn, H. A.; Pluth, M. D. Mechanistic Insights into the
H2S-Mediated Reduction of Aryl Azides Commonly Used in H2S
Detection. J. Am. Chem. Soc. 2015, 137, 15330.
(40) Xu, G.; Guo, W.; Gu, X.; Wang, Z.; Wang, R.; Zhu, T.; Tian,
H.; Zhao, C. Hydrogen Sulfide-Specific and NIR-Light-Controllable
Synergistic Activation of Fluorescent Theranostic Prodrugs for
Imaging-Guided Chemo-Photothermal Cancer Therapy. CCS Chem.
2020, 2, 527.
(41) Sasakura, K.; Hanaoka, K.; Shibuya, N.; Mikami, Y.; Kimura,
Y.; Komatsu, T.; Ueno, T.; Terai, T.; Kimura, H.; Nagano, T.
Development of a Highly Selective Fluorescence Probe for Hydrogen
Sulfide. J. Am. Chem. Soc. 2011, 133, 18003.
(42) Shi, B.; Gu, X.; Fei, Q.; Zhao, C. Photoacoustic probes for
real-time tracking of endogenous H2S in living mice. Chem. Sci. 2017,
8, 2150.
(43) Wang, F.; Xu, G.; Gu, X.; Wang, Z.; Wang, Z.; Shi, B.; Lu, C.;
Gong, X.; Zhao, C. Realizing highly chemoselective detection of H2S
in vitro and in vivo with fluorescent probes inside core-shell silica
nanoparticles. Biomaterials 2018, 159, 82.
(44) Xu, G.; Yan, Q.; Lv, X.; Zhu, Y.; Xin, K.; Shi, B.; Wang, R.;
Chen, J.; Gao, W.; Shi, P.; Fan, C.; Zhao, C.; Tian, H. Imaging of
Colorectal Cancers Using Activatable Nanoprobes with Second Near-
Infrared Window Emission. Angew. Chem. Int. Ed. 2018, 57, 3626.
(45) Wang, R.; Dong, K.; Xu, G.; Shi, B.; Zhu, T.; Shi, P.; Guo, Z.;
Zhu, W.-H.; Zhao, C. Activatable near-infrared emission-guided on-
demand administration of photodynamic anticancer therapy with a
theranostic nanoprobe. Chem. Sci. 2019, 10, 2785.
(46) Shi, B.; Gu, X.; Wang, Z.; Xu, G.; Fei, Q.; Tang, J.; Zhao, C.
Fine Regulation of Porous Architectures of Core–Shell Silica
Nanocomposites Offers Robust Nanoprobes with Accelerated
Responsiveness. ACS Appl. Mater. Interfaces 2017, 9, 35588.
(47) Zhao, C. C.; Zhang, J. X.; Wang, X. Z.; Zhang, Y. F. Pyridone
fused boron-dipyrromethenes: synthesis and properties. Org. Biomol.
Chem. 2013, 11, 372.
(48) Ye, D.; Shuhendler, A. J.; Cui, L.; Tong, L.; Tee, S. S.;
Tikhomirov, G.; Felsher, D. W.; Rao, J. Bioorthogonal cyclization-
mediated in situ self-assembly of small-molecule probes for imaging
caspase activity in vivo. Nat. Chem. 2014, 6, 519.
(49) Zhou, L.; Lv, F.; Liu, L.; Wang, S. In Situ-Induced
Multivalent Anticancer Drug Clusters in Cancer Cells for Enhancing
Drug Efficacy. CCS Chem. 2019, 1, 97.
(50) Wu, Z.; Liang, D.; Tang, X. Visualizing Hydrogen Sulfide in
Mitochondria and Lysosome of Living Cells and in Tumors of Living
Mice with Positively Charged Fluorescent Chemosensors. Anal. Chem.
2016, 88, 9213.
(51) Tian, H.; Qian, J.; Bai, H.; Sun, Q.; Zhang, L.; Zhang, W.
Micelle-induced multiple performance improvement of fluorescent
probes for H2S detection. Anal. Chim. Acta 2013, 768, 136.
(52) Uchiyama, S.; Iwai, K.; de Silva, A. P. Multiplexing Sensory
Molecules Map Protons Near Micellar Membranes. Angew. Chem. Int.
Ed. 2008, 47, 4667.
(53) Huang, J.; Li, J.; Lyu, Y.; Miao, Q.; Pu, K. Molecular optical
imaging probes for early diagnosis of drug-induced acute kidney
injury. Nat. Mater. 2019, 18, 1133.
(54) Cheng, P.; Miao, Q.; Li, J.; Huang, J.; Xie, C.; Pu, K.
Unimolecular Chemo-fluoro-luminescent Reporter for Crosstalk-Free
Duplex Imaging of Hepatotoxicity. J. Am. Chem. Soc. 2019, 141,
10581.
(55) Miao, Q.; Xie, C.; Zhen, X.; Lyu, Y.; Duan, H.; Liu, X.;
Jokerst, J. V.; Pu, K. Molecular afterglow imaging with bright,
biodegradable polymer nanoparticles. Nat. Biotech. 2017, 35, 1102.
(20) Dong, Z.; Feng, L.; Hao, Y.; Chen, M.; Gao, M.; Chao, Y.;
Zhao, H.; Zhu, W.; Liu, J.; Liang, C.; Zhang, Q.; Liu, Z. Synthesis of
Hollow Biomineralized CaCO3-Polydopamine Nanoparticles for
Multimodal Imaging-Guided Cancer Photodynamic Therapy with
Reduced Skin Photosensitivity. J. Am. Chem. Soc., 2018, 140, 2165.
(21) Yuan, Y.; Zhang, C.-J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu,
B. Specific Light-Up Bioprobe with Aggregation-Induced Emission
and Activatable Photoactivity for the Targeted and Image-Guided
Photodynamic Ablation of Cancer Cells. Angew. Chem. Int. Ed. 2015,
54, 1780.
(22) Niu, L.-Y.; Chen, Y.-Z.; Zheng, H.-R.; Wu, L.-Z.; Tung, C.-H.;
Yang, Q.-Z. Design strategies of fluorescent probes for selective
detection among biothiols. Chem. Soc. Rev. 2015, 44, 6143.
(23) Li, X.; Lee, D.; Huang, J.-D.; Yoon, J. Phthalocyanine-
Assembled Nanodots as Photosensitizers for Highly Efficient TypeꢀI
Photoreactions in Photodynamic Therapy. Angew. Chem. Int. Ed.
2018, 57, 9885.
(24) Zhen, X.; Zhang, J.; Huang, J.; Xie, C.; Miao, Q.; Pu, K.
Macrotheranostic Probe with Disease-Activated Near-Infrared
Fluorescence, Photoacoustic, and Photothermal Signals for Imaging-
Guided Therapy. Angew. Chem. Int. Ed. 2018, 57, 7804.
(25) Peng, H.-Q.; Niu, L.-Y.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.;
Yang, Q.-Z. Biological Applications of Supramolecular Assemblies
Designed for Excitation Energy Transfer. Chem. Rev. 2015, 115, 7502.
(26) Feng, L.; Betzer, O.; Tao, D.; Sadan, T.; Popovtzer, R.; Liu, Z.
Oxygen Nanoshuttles for Tumor Oxygenation and Enhanced Cancer
Treatment. CCS Chem. 2019, 1, 239.
(27) Ma, Y.; Li, X.; Li, A.; Yang, P.; Zhang, C.; Tang, B. H2S-
Activable MOF Nanoparticle Photosensitizer for Effective
Photodynamic Therapy against Cancer with Controllable Singlet-
Oxygen Release. Angew. Chem. Int. Ed. 2017, 56, 13752.
(28) Zhang, K.; Zhang, J.; Xi, Z.; Li, L.-Y.; Gu, X.; Zhang, Q.-Z.;
Yi, L. A new H2S-specific near-infrared fluorescence-enhanced probe
that can visualize the H2S level in colorectal cancer cells in mice.
Chem. Sci. 2017, 8, 2776.
(29) Shi, B.; Yan, Q. L.; Tang, J.; Xin, K.; Zhang, J. C.; Zhu, Y.;
Xu, G.; Wang, R. C.; Chen, J.; Gao, W.; Zhu, T. L.; Shi, J. Y.; Fan, C.
H.; Zhao, C. C.; Tian, H. Hydrogen Sulfide-Activatable Second Near-
Infrared Fluorescent Nanoassemblies for Targeted Photothermal
Cancer Therapy. Nano Lett. 2018, 18, 6411.
(30) Wu, L.; Sun, Y.; Sugimoto, K.; Luo, Z.; Ishigaki, Y.; Pu, K.;
Suzuki, T.; Chen, H.-Y.; Ye, D. Engineering of Electrochromic
Materials as Activatable Probes for Molecular Imaging and
Photodynamic Therapy. J. Am. Chem. Soc. 2018, 140, 16340.
(31) Shi, B.; Ren, N.; Gu, L.; Xu, G.; Wang, R.; Zhu, T.; Zhu, Y.;
Fan, C.; Zhao, C.; Tian, H. Theranostic Nanoplatform with Hydrogen
Sulfide Activatable NIR Responsiveness for Imaging-Guided On-
Demand Drug Release. Angew. Chem. Int. Ed. 2019, 58, 16826.
(32) Szabo, C.; Coletta, C.; Chao, C.; Módis, K.; Szczesny, B.;
Papapetropoulos, A.; Hellmich, M. R. Tumor-derived hydrogen
9
10
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13
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15
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17
18
19
20
21
22
23
24
25
26
27
28
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
sulfide,
produced
by
cystathionine-β-synthase,
stimulates
bioenergetics, cell proliferation, and angiogenesis in colon cancer.
Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 12474.
(33) Lin, V. S.; Chen, W.; Xian, M.; Chang, C. J. Chemical probes
for molecular imaging and detection of hydrogen sulfide and reactive
sulfur species in biological systems. Chem. Soc. Rev. 2015, 44, 4596.
(34) Hartle, M. D.; Pluth, M. D. A practical guide to working with
H2S at the interface of chemistry and biology. Chem. Soc. Rev. 2016,
45, 6108;
(35) Yi, L.; Xi, Z. Thiolysis of NBD-based dyes for colorimetric
and fluorescence detection of H2S and biothiols: design and biological
applications. Org. Biomol. Chem. 2017, 15, 3828.
(36) Zhao, C.; Zhang, X.; Li, K.; Zhu, S.; Guo, Z.; Zhang, L.;
Wang, F.; Fei, Q.; Luo, S.; Shi, P.; Tian, H.; Zhu, W.-H. Förster
Resonance Energy Transfer Switchable Self-Assembled Micellar
Nanoprobe: Ratiometric Fluorescent Trapping of Endogenous H2S
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(56) Shao, A.; Xie, Y.; Zhu, S.; Guo, Z.; Zhu, S.; Guo, J.; Shi, P.;
James, T. D.; Tian, H.; Zhu, W.-H. Far-Red and Near-IR AIE-Active
Fluorescent Organic Nanoprobes with Enhanced Tumor-Targeting
Efficacy: Shape-Specific Effects. Angew. Chem. Int. Ed. 2015, 54,
7275.
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SYNOPSIS TOC.
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