Imaging of Colorectal Cancers Using Activatable Nanoprobes with Second Near-Infrared Window Emission

Article abstract of DOI:10.1002/anie.201712528

Fluorescent probes in the second near-infrared window (NIR-II) allow high-resolution bioimaging with deep-tissue penetration. However, existing NIR-II materials often have poor signal-to-background ratios because of the lack of target specificity. Herein,

Full text of DOI:10.1002/anie.201712528

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Accepted Article
Title: Visualization of Colorectal Cancers Using Activatable
Nanoprobes with Second Near-Infrared Emissions
Authors: Ge Xu, Qinglong Yan, Xiaoguang Lv, Ying Zhu, Kai Xin, Ben
Shi, Rongchen Wang, Jian Chen, Wei Gao, Ping Shi, Chunhai
Fan, Chunchang Zhao, and He Tian
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201712528
Angew. Chem. 10.1002/ange.201712528
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http://dx.doi.org/10.1002/ange.201712528

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Visualization of Colorectal Cancers Using Activatable
Nanoprobes with Second Near-Infrared Emissions
†
†
Ge Xu,[a], Qinglong Yan,[b], Xiaoguang Lv,[c] Ying Zhu,*,[b] Kai Xin,[a] Ben Shi,[a] Rongchen Wang,[a] Jian
Chen,[a] Wei Gao,[a] Ping Shi,[c] Chunhai Fan,*,[b] Chunchang Zhao,*,[a] He Tian[a]
Abstract: Fluorescent probes in the second near-infrared window
(NIR-II) allow high-resolution bioimaging with deep-tissue
penetration. However, existing NIR-II materials often have poor
signal-to-background ratios due to the lack of target specificity.
Herein, we devised an activatable NIR-II nanoprobe for visualizing
colorectal cancers. This designed probe possessed H2S-activated
ratiometric fluorescence and light-up NIR-II emission of 900-1300
nm. By using this activatable and target specific probe for deep-
tissue imaging of H2S-rich colon cancer cells, we realized accurate
identification of colorectal tumors in animal models. We thus
anticipate that the development of activatable NIR-II probes will find
widespread applications in biological and clinical systems with high
precision and resolution.
traditional NIR-I imaging (750–900 nm).[4] Indeed, several
materials have been applied as NIR-II fluorophores non-
invasively to visualize biological events in a living subject,
including small-molecule dyes, metallic nanoparticles, carbon
nanotubes, quantum dots and rare earth nanoparticles.[5-9]
However, the NIR-II fluorescence signals are often not directly
associated with interactions of imaging agents with the targets of
interest, resulting in poor signal-to-background ratios. Thus
accurately measuring anatomical features or biological events of
interest with NIR-II probes remains challenging. As an
alternative strategy, activatable probes that undergo intrinsic
signal evolution only in response to specific biological targets or
events can amplify signals from the target and suppress
background.[10] However, few activatable materials with NIR-II
emissions have been explored for biological imaging
applications. Herein, we reported an activatable probe with
emission in the NIR-II region for visualizing colorectal cancers by
full utilization of the merits of NIR-II imaging at a depth and
spatial resolution.
Optical imaging is an essential tool for biological research and
biomedical applications due to the advantages of lower cost,
simple operation, noninvasive and real time capabilities.[1]
Specially, fluorescence imaging provides a powerful approach
for early detection and management of malignant tumors. For
example, fluorescent probes with emission at visible region can
selectively monitor intra- and extracellular cancer biomarkers by
readily coupling with the widely available biological imaging
instruments such as confocal and epifluorescence light
microscopy.[2] Although these great advances, conventional
fluorescence imaging still suffers from a lot of concerns
regarding the limited tissue penetration, poor spatial resolution
inside deep biological tissue and disturbance by strong
autofluorescence from living tissues, which have largely
restricted their applications in living animals.[3]
In this contribution, we demonstrated our design concept by
fabrication of nanoprobes through encapsulating
a H2S-
responsive fluorescent probe into the hydrophobic interior of
core–shell silica nanocomposites, considering that the
overexpressed H2S-producing enzyme cystathionine-β-synthase
(CBS) in colon cancer gives rise to increased H2S production.[11]
In fact, increased H2S production is closely linked to various
cancers, including ovarian, breast and colorectal cancers.[11b]
Thereby, H2S can be regard as a biomarker for cancer diagnosis
as well as a therapeutic potential on these tumors. Undoubtedly,
H2S-activated emissions in NIR-II window could provide valuable
insight into accurate location of colorectal cancers.
The activatable nanoprobes are comprised of two organic
chromophores: a rational designed boron-dipyrromethene (ZX-
NIR) dye to generate the NIR-II emission only in the presence of
H2S, and an aza-BODIPY (aza-BOD) that is inert to H2S, serving
as the internal reference. Such a design is to construct probes
with multi-emission for ratiometric imaging, as multi-wavelength
Recent progresses have demonstrated that fluorescent
imaging in the second near-infrared window (NIR-II, 1000–1700
nm) can provide solutions for addressing the abovementioned
multiple issues, showing reduced autofluorescence, lower tissue
absorption, greatly improved tissue penetration depth and higher
in vivo spatial resolution compared to visible (450–750 nm) and
imaging
is
promising
in
acquiring
high
precision
[a]
G. Xu, K. Xin, B. Shi, R. Wang, J. Chen, W. Gao, Prof. C. Zhao,
Prof. H. Tian
Key Laboratory for Advanced Materials and Institute of Fine
Chemicals, School of Chemistry and Molecular Engineering
East China University of Science and Technology
Shanghai 200237, P. R. China
measurements.[12] As shown in Figure 1, the designed
nanoprobes were fabricated in two steps: 1) trapping the two
dyes ZX-NIR and aza-BOD into the hydrophobic interior of self-
assembled micellar aggregate based on 1,2-dimyristoyl-sn-
glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (mPEG-DSPE); 2) in situ shell cross-linking with
E-mail: zhaocchang@ecust.edu.cn
Q. Yan, Prof. Y. Zhu, Prof. C. Fan
[b]
Division of Physical Biology & Bioimaging Center, Shanghai Institute
of Applied Physics
Chinese Academy of Sciences
Shanghai 201800, P. R. China
E-mail: zhuying@sinap.ac.cn, fchh@sinap.ac.cn
X. Lv, Prof. P. Shi
State Key Laboratory of Bioreactor Engineering
East China University of Science and Technology
Shanghai 200237, P. R. China
(N-trimethoxysilylpropyl-N,N,N-tri-n-butylammonium
bromide
(TBNBr)) to produce water-dispersible core-shell silica
nanocomposites (NIR-II@Si) with a covalently cross-linked silica
shell. As a result, the silica cross-linkers realize the buildup a
shield for stable confinement of the two dyes within the same
cavity of these nanoparticles.[13] Furthermore, such silica
nanoparticles has the advantage of good water-solubility and
excellent biocompatibility as well as fast responsiveness.
[c]
†
These authors contributed equally.
We have employed the dye-screening approach for affording
probes with good responsiveness (Figure 1 and Scheme S1).
Supporting information for this article is given via a link at the end of
the document.
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The reactivity toward H2S was evaluated by fluorescence
analysis. As shown in Figure S1, compound 1 gave no
fluorescence response to H2S in CH3CN/PBS buffer mixtures
(1:1, v/v, 20 mM, pH 7.4, room temperature). As seen for
compound 3, enhancement of the electron deficiency of BODIPY
core by appending electron-withdrawing units increased the
reactivity toward H2S. Based on these preliminary studies, we
finally attached a better leaving group 4-nitrobenzenethiol
instead of p-thiocresol for affording the optimized model probe
ZX-NIR.
following our established method.[13] Aza-BODIPY was selected
as it has high absorption and emission at 670 nm and 700 nm,
respectively, which are well-resolved and mutually poor-
overlapping with those of ZX-NIR and the activated form NIRII-
HS, thus acting as an internal inference. The resulting water
dispersible nanocomposites were fully characterized by dynamic
light scattering (DLS) and transmission electron microscopy
(TEM) (Figure S4). NIR-II@Si was found to be stable in aqueous
solution. The particle diameters remained at 66 nm and no
aggregation was found in the suspension for days (Figure S4).
The loading contents of ZX-NIR and aza-BODIPY in NIR-II@Si
nanocomposites were determined to be 0.39 and 0.10 wt %,
respectively, according to the standard UV–vis–NIR absorbance
spectra.
Figure 1. (a) Schematic illustration of the construction of multi-wavelength
nanoprobes with activatable emissions in the second near-infrared window. (b)
The dye-screening approach for rational design of probes for H2S. EW:
electron withdrawing; LG: leaving group.
In the absence of H2S, ZX-NIR shows intense absorption at
520 nm and emission band at 600 nm in CH3CN/PBS buffer
mixtures (1:1, v/v, 20 mM, pH 7.4, room temperature). When
exposed to 100 μM NaHS (a commonly employed H2S donor), a
new absorption band around 740 nm was built up and the
original absorption at 520 nm was reduced concomitantly
(Figure S2), showing a remarkable red-shift of 220 nm. Upon
excitation at 520 nm, the fluorescence at 600 nm showed a time-
dependent quenching. Fortunately, a new NIR-II emission, with
maximum emission peak of 900 nm, was activated in the
presence of H2S when the excitation wavelength was 740 nm.
Generally, the spectral range of fluorophores in NIR-II window is
considered to be between 1000 and 1700 nm. Although H2S-
triggered maximum emission wavelength lies at 900 nm, the
emission spectrum is quite broad and extends into the region
(1000-1300 nm) of NIR-II. These changes were ascribed to the
transformation of ZX-NIR into NIRII-HS through aromatic
nucleophilic substitution (SNAr), confirmed by HRMS analysis
(Figure S3).
According to reaction-time study, the reaction of 240 min
between ZX-NIR and H2S was found to be comparable with that
of most reported H2S probes (30−200 min).[14] However, such
reaction rate is not fast enough to image the transient H2S. To
address the issue of the limited solubility of ZX-NIR as well as its
relatively slow reaction with H2S in aqueous solution, ZX-NIR
and aza-BOD at the molar ratio of 3:1 were then incorporated
into the hydrophobic interior of water-dispersible NIR-II@Si
Figure 2. (a) Absorption and (b) fluorescence changes of NIR-II@Si (10 μM
ZX-NIR) upon addition with 100 μM NaHS in PBS buffer solution (pH 7.4). (c)
Time dependent NIR-II emission spectra of NIR-II@Si (10 μM ZX-NIR) in the
presence of 100 μM NaHS, λex = 780 nm. Inset is the photograph of the H2S-
activated NIR-II emission. (d) The comparison of NIR-I and NIR-II
fluorescence imaging of samples covered by pork tissues.
The optical response of NIR-II@Si toward H2S was studied
in PBS buffer solutions (pH 7.4). It was found that ZX-NIR
showed dramatic optical response to H2S within the water-
dispersible nanocomposites. Upon reaction with NaHS (100 μM),
the NIR absorption at 780 nm was generated, accompanied by
the reduction of the peak at 520 nm (Figure 2). Such treatment
with NaHS induced the completely quenching the emission at
600 nm (excitation at 520 nm) within 5 min (Figure S5).
Specifically, a robust increase in fluorescence intensity within
NIR-II window was observed when the excitation wavelength
was 780 nm, reaching a plateau within 5 min which allows for
monitoring of H2S-related biological processes in real-time. This
process triggered a bright NIR-II image with the relative quantum
yield of 0.37% under 810-nm laser irradiation (Figure 2c inset).
In contrast, the absorbance and fluorescence of aza-BOD,
different from that of ZX-NIR, are inert to H2S. Therefore, the
absorbance peak at 670 nm and fluorescence at 700 nm
showed no appreciable change in the presence of NaHS.
Interestingly, the fluorescence intensity ratio of I700/I600 increased
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with the gradual increase of NaHS concentrations, affording a
good linear relationship within the concentration range from 0 to
10 μM. The detection limit was measured to be 37 nM, indicating
the high sensitivity of NIR-II@Si for ratiometric detection of H2S.
The capability of NIR-II@Si to produce both NIR-I fluorescence
(emission of aza-BOD at 700 nm) and activatable NIR-II signals
(emission of NIRII-HS at 900 nm) enabled a direct comparison
of NIR-I and NIR-II fluorescence imaging in terms of NIR-II@Si
performance. The polyethylene tube was covered by pork
tissues with different thicknesses and subjected to imaging. As
shown in Figure 2d and Figure S5, the NIR-II fluorescence
signal of NIR-II@Si was detectable even through a 10 mm pork
tissue. In sharp contrast, the NIR-I fluorescence was attenuated
to the background level for samples covered by just a 3 mm pork
tissue. These results clearly demonstrate that NIR-II imaging is a
preferred technique over NIR-I fluorescence imaging for
NaHS induced a significant enhancement in the fluorescence
intensity ratio (I700/I600), while other analytes gave no obvious
ratiometric changes. In addition, experiments for the pH effect
on the response to H2S revealed that NIR-II@Si afforded good
optical response within a physiological range from pH 9 to
approximately 5 (Figure S7). Notably, the core-shell structured
NIR-II@Si showed good photostability in aqueous solution
(Figure S8). Minimal fluorescence spectra change was observed
after being stored for 24 h or under continuous irradiation for 2 h
with an Hg/Xe lamp.
Since overexpressed CBS promotes high levels of H2S in
human colon cancers, the ability to selective identification of
colon cancer cells was studied by tracking of increased H2S
production in dual-color imaging modality. Human hepatocellular
liver carcinoma cells (HepG2 cells) were used as a control due
to the minimized level of H2S in these cells. After validating low
providing better spatial resolution and deeper penetration depths. cytotoxicity of NIR-II@Si (Figure S9), human colorectal cancer
Combined, advantages including ratiometric fluorescence and
light-up NIR-II emission enable NIR-II@Si a promising tool for
preclinical applications in both living cells and animals with high
precision and resolution.
HCT116 cells were then loaded with NIR-II@Si for 30 minutes.
Bright and stable fluorescence signals were noted in the red
channel, while relatively weak fluorescence signals were
observed in the green channel (Figure 3). The ratio of the red
channel to green channel was used for quantification of images.
HCT116 cells incubated with NIR-II@Si showed a ratio of
approximately 3.0 (Figure S9). Such ratio of the red-to-green
signal was significantly attenuated by the addition of a CBS
inhibitor aminooxyacetic acid (AOAA) due to the inhibition effect
on the cellular H2S production. In contrast, stimulation of cells
with an allosteric CBS activator S-adenosyl-L-methionine (SAM)
resulted in dramatic elevation of red-to-green image ratio to 4.5.
These imaging results demonstrated the feasibility of NIR-II@Si
to specially image colon cancer cells by monitoring of the
elevated production of H2S. To further validate the obtained
assumption, control experiments with HepG2 cells were
performed. As shown in Figure S9, HepG2 cells showed strong
fluorescence in both green and red channels with the red-to-
green signal ratio of 1.4. Different from that in HCT116 cells,
almost no change of fluorescence ratio was observed when
HepG2 cells were pretreated with AOAA or SAM. All the results
indicate that NIR-II@Si can be used to differentiate types of
living cells, based on their difference in H2S contents.
NIR-II@Si was further explored for visualization of colorectal
cancers in vivo by full utilization of its promising light-up NIR-II
emission in response to H2S. In this study, HCT116
subcutaneous xenograft nude mice were established due to high
levels of H2S in HCT116 cells. HCT116 tumor-bearing mice were
administrated by intratumoral injection of NIR-II@Si. NIR-II@Si
was also injected into the normal muscle of a mouse to confirm
the advantage of activatable probes over “always-on” probes.
Then images were recorded at various times after the injection
(Figure 4 and Figure S10). Strong NIR-II emission was noted
specifically in the tumor region within 1 min post-injection of the
probe, while barely detectable fluorescence was observed in the
normal site injected with the probe. Such specific NIR-II signals
in the tumors increased with time and eventually leveled off in 30
min after probe injection. According to region of interest
measurements, the NIR-II signal ratio between the tumor and
normal site (T/N) was estimated to be 5.7 (20 min) (Figure S10).
Considering the “always-on” NIR-I fluorescence of NIR@Si from
aza-BOD, the NIR-I fluorescent signals were observed in both
Figure 3. Identification of colon cancer cells by confocal microscopy images in
dual-color imaging modality. (a) HCT116 cells incubated with NIR-II@Si (ZX-
NIR 10 μM) for 30 min. (b) HCT116 cells pretreated with 1 mM AOAA for 1h,
followed by incubation with NIR-II@Si for 30 min. (c) HCT116 cells pretreated
with SAM (3mM) for 1 h were loaded with NIR-II@Si for 30 min. (d) HepG2
cells incubated with NIR-II@Si for 30 min. The excitation wavelength was 561
nm, green channel at 600−630 nm, red channel at 700−750 nm; ratio images
generated from red channel to green channel. Scale bar: 20 μM.
NIR-II@Si exhibited high selectivity for H2S compared to a
panel of biologically related analytes, including reactive sulfur
(RSS), oxygen (ROS), and nitrogen species (RNS). As shown in
Figure S6, these inferring species caused negligible fluctuation
of fluorescence within 900-1300 nm, whereas the introduction of
NaHS triggered
a prominent enhancement of the NIR-II
fluorescence intensity. The ratiometric fluorescence mode
(I700/I600) also gave an accurate estimation of the selectivity. Only
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