NIR-II Chemiluminescence Molecular Sensor for In Vivo High-Contrast Inflammation Imaging

Article abstract of DOI:10.1002/anie.202007649

Chemiluminescence (CL) sensing without external excitation by light and autofluorescence interference has been applied to high-contrast in vitro immunoassays and in vivo inflammation and tumor microenvironment detection. However, conventional CL sensing u

Full text of DOI:10.1002/anie.202007649

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: NIR-II Chemiluminescence Molecular Sensor for In-Vivo High
Contrast Inflammation Imaging
Authors: Fan Zhang, Yanling Yang, Shangfeng Wang, Lingfei Lu,
Qisong Zhang, Peng Yu, and Yong Fan
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202007649
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NIR-II Chemiluminescence Molecular Sensor for In-Vivo High
Contrast Inflammation Imaging
Yanling Yang, Shangfeng Wang, Lingfei Lu, Qisong Zhang, Peng Yu, Yong Fan* and Fan Zhang*
Abstract: Chemiluminescence (CL) sensing without the external
excitation light and autofluorescence interference has been applied to
high-contrast in vitro immunoassays and in vivo inflammation and
tumor microenvironment detection. However, conventional CL
sensing usually operates in the range of 400-850 nm, which limits the
high-performance of in vivo imaging due to the serious light scattering
effect and signals attenuation in tissue. To address this challenge,
here we present a new type of CL sensor in the second near-infrared
window (NIR-II CLS) with deep penetration depth (~ 8 mm) by
employing successive CL resonance energy transfer (CRET) and
Förster resonance energy transfer (FRET) from the activated CL
substrate to two rationally designed donor-acceptor-donor
fluorophores BTD540 and BBTD700. NIR-II CLS can be selectively
activated by hydrogen peroxide over other reactive oxygen species
(ROSs). Moreover, NIR-II CLS is capable of detecting local
inflammation in mice with 4.5-fold higher signal-to-noise ratio (SNR)
than that under NIR-II fluorescence modality.
Optical sensing has become a vital technique for noninvasive
and real-time visualization of biological events with superior
spatio-temporal resolution.[1] Fluorescence (FL) is a common
modality for optical sensing that requires an excitation light source
to light up the fluorescent reporters. In recent years, researchers
took advantages of reduced photon scattering, and barely existed
tissue autofluorescence background in the second near-infrared
window (NIR-II, 1000-1700 nm) to develop various fluorescent
probes for in vivo NIR-II imaging.[2] Even though, the external
excitation light can still cause autofluorescence background from
endogenous biomolecules in FL sensing and imaging.[3] Moreover,
Figure 1. (a) The preparation scheme of NIR-II CLS. (b) The illustration of the
the major light absorbers like hemoglobins and water in biological
tissue can turn excitation photons into heat dissipation,[4] which
also hampers the long-time observation. In contrast,
chemiluminescence (CL) could conquer these limitations because
it eliminates the demand of excitation light source.[5] Classical
peroxyoxalate-based CL (POCL) is a nonradiative dipole-dipole
energy transfer process[6,7] from a CL donor (1,2-dioxetanedione
intermediate, DOD) to a suitable acceptor by the chemical
reaction between hydrogen peroxide (H2O2) and peroxyoxalate,
principle for generating NIR-II CL emission in the presence of H2O2. (c) The
structure of BTD540, BBTD700 and F127.
CL substrates and other fluorescent dyes,[10-13] or to increase the
conjugation of the Schaap’s adamantylidene-dioxetane system[14]
Due to external light-free luminescence, POCL has been widely
.
used
for
high-contrast
immunoassays[15]
and
tumor
microenvironment[9,10] detection. However, the penetration depth
and SNR for in vivo CL imaging in the reported wavelength region
remain to be improved. Developing CL in the NIR-II region is
promising to address this issue, which however remains a great
challenge.
which involved
a chemically initiated electron exchange
luminescence mechanism.[8] POCL systems commonly emit in
the visible region, and it is readily absorbed and scattered by the
molecules and cells in biological matrices[9]. Currently, the CL
wavelengths have been successfully extended to red light to
improve the potential bioapplication by CRET between activated
Herein, we report the NIR-II CL sensor (NIR-II CLS) for high
contrast in vivo inflammation imaging based on a classical POCL
system. In our design, two donor-acceptor-donor (D-A-D)
fluorescent dyes (BTD540 and BBTD700) are used to transduce
the high energy of CL donor (DOD) to NIR-II photons by
integrating successive CRET (DOD-to-BTD540) and FRET
(BTD540-to-BBTD700) processes (Figure 1). The large Stokes
shift (> 100 nm) of both BTD540 and BBTD700 and the extremely
high FRET efficiency (94.12%) between them play crucial roles
for the efficient NIR-II CL. Furthermore, NIR-II CLS can be
selectively activated by H2O2 over other reactive oxygen species
Y. Yang, S. Wang, L. Lu, Q. Zhang, P. Yu, Prof. Y. Fan, and Prof. F.
Zhang
Department of Chemistry, State Key Laboratory of Molecular
Engineering of Polymers and iChem, Shanghai Key Laboratory of
Molecular Catalysis and Innovative Materials
Fudan University
Shanghai 200433, China.
E-mail: fan_yong@fudan.edu.cn, zhang_fan@fudan.edu.cn
Supporting information for this article is given via a link at the end of
the document.
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(ROSs) under physiological conditions with long duration (60 min)
and deeper penetration depth (~ 8 mm), allowing for in vivo
detection of the H2O2-induced local inflammation in lymph node
and arthrosis region of the mice. Compared with NIR-II FL sensing
in the same system, 4.5-fold enhancement in SNR was achieved
for NIR-II CL sensing, showing a great promise for in vivo
biosensing.
The synthesis procedure of NIR-II CLS is shown in the Figure
1a.
The
CL
substrate
bis[3,4,6-trichloro-2-
(pentyloxycarbonyl)phenyl]oxalate (CPPO) is oxidized by H2O2 to
generate the unstable 1,2-dioxetanedione (DOD) intermediate,
which then transfers chemical energy to BTD540 through CRET
process, and subsequently to BBTD700 through FRET to provide
CL signals in the NIR-II region (Figure 1b). BTD540 and BBTD700
were synthesized by simple one-step Suzuki coupling reaction
under a nitrogen atmosphere (Scheme S4 and S5) with the
absorption/emission peaks at 540/680 nm and 700/985 nm
(Figure S1a and S1b, Figure S2a and S2b), respectively. The
Stokes shift of BTD540 and BBTD700 are up to 140 and 285 nm,
respectively. The two dyes compose a potentially efficient FRET
pair due to the large spectral overlapping integrals (Figure 2a).
To verify our design for the NIR-II CL molecular sensor, we fisrt
incorporated the CPPO, BTD540 (CRET acceptor) into polymeric
surfactant F127 (termed as BTD540 micelle) to validate the CRET
process. The optimal conditions were screened by tuning the
amount of BTD540, which were obtained at 1.6% (Figure S3).
Under this circumstance, BTD540 micelle showed the average
diameter and hydrodynamic size of 21 ± 2.6 nm (TEM, Figure
S4b) and ~ 18 nm (DLS, Figure S4a), respectively. Compared
with BTD540 solution dissolved in CH2Cl2, BTD540 micelle
dispersed in H2O showed similar absorption spectra (Figure S1a),
and the slight blue-shift of the FL signal was due to the self-
aggregation of BTD540 (Figure S1b). Upon the addition of H2O2
to BTD540 micelle, an apparent CL signal was observed with
emission peak shape similar to the FL signal (Figure 2b) ，
indicating the successful occurance of CRET process.
Figure 2. (a) Normalized absorbance (dash dot line) and emission (solid line)
spectra of BTD540 (black line) and BBTD700 (red line) in CH2Cl2. (b) FL (black
curve, λex = 540 nm) and CL spectra (red curve) of micelles containing only
BTD540. (c) Emission spectra of BTD540 and BBTD700 mixtures in CH2Cl2 (λex
= 540 nm). The molar ratio of BBTD700/BTD540 is 0 (dark red curve), 1:1
(orange curve), 2:1 (green curve), 5:1 (blue curve), 10:1 (purple curve),
respectively. (d) TEM image of NIR-II CLS. Insert: DLS of NIR-II CLS. (e) FL
(black curve, λex = 540 nm) and CL (red curve) spectra of NIR-II CLS in the
presence of H2O2 (19 mM). (f) Time-course of fluorescence intensities of
micelles containing only BTD540 and BBTD700 with emissions at 662 nm and
935 nm in the presence of H2O2 (19 mM) under 540 nm and 700 nm excitation,
respectively. (g) CL signals of NIR-II CLS in 96-well plate through stacked slices
of chicken ham at varied depth in different detection windows. (h)
Corresponding CL intensity of NIR-II CLS with stacked slices of chicken ham in
(g). (i) Intensity profiles along the dashed lines with the same color in a (insert)
Gaussian point spread function facula. (j) Optical images of capillaries filled with
NIR-II CLS for NIR-II FL imaging with inhomogeneous illumination (top) and
NIR-II CL imaging with homogeneous illumination (bottom). Scale bar, 5 mm.
(k) Corresponding intensity profiles along the capillary in (j).
We then incorporated the BTD540 and BBTD700 into polymeric
surfactant F127 to further verify the efficiency of the secondary
FRET process. By increasing BBTD700/BTD540 mole ratio from
0:1 to 10:1 in micelle, the FL intensity ratio of BBTD700 to
BTD540 was increased by 16-fold under 540 nm exciation,
suggesting that efficient FRET process occurred between
BTD540 and BBTD700 (Figure 2c). The highest FRET efficiency
between BTD540 and BBTD700 was determined to be 94.12%
with the BBTD700/BTD540 mole ratio of 5:1[16]. The Förster
distances (R0) of these two dyes were also calculated to be 1.6
nm, which guarantee the efficient FRET between the dyes and
bridge the chemical energy to the NIR-II region (see
supplementary information).
Based on the above analysis, we prepared the biocompatible
NIR-II CLS by incorporating optimal amount of BTD540,
BBTD700 and CPPO molecules into the hydrophobic interior of
F127 micelles (Table S1). The diameter and the hydrodynamic
size of the as-prepared NIR-II CLS are 23 ± 3.0 nm (TEM, Figure
2d) and ~ 21 nm (DLS, Figure 2d insert), respectively. In the NIR-
maximum fluorescence and CL emssion peak of the NIR-II CLS
both blue-shift to 935 nm upon 540 nm excitaion or by adding
H2O2, respectively, which may due to the self-aggregation of D-A-
D dyes in the micelles (Figure 2e and Figure S2b). This result
clearly indicated the successful occurence of the effective DOD-
to-BTD540 CRET and BTD540-to-BBTD700 FRET processes.
Moreover, no obvious size alteration of NIR-II CLS was observed
after storage in water for 10 days (Figure S5), indicating the
excellent stability of NIR-II CLS. Thanks to the D-A-D structure
and F127 shells, the as-prepared BTD540-BBTD700 micelle
(without CPPO) showed tolerant antioxidant stability in the
presence of H2O2, with more than 65% fluoresence intenisty
remained after 30 min (Figure 2f and Figure S6). To evaluate the
advantage of CL in the NIR-II region, we compared the 1/e tissue
penetration depth[17] in two detection windows (600-800 nm and >
850 nm, separated by corresponding optical filters) using chicken
ham as mimic tissue. As shown in Figure 2g and 2h, the visibility
of the CL signals deteriorated as the tissue depth increased from
0 to 8 mm for all the two detection windows. The deeper
penetration depth (~ 8 mm) was observed in NIR-II region than
II CLS, the absorbance of BTD540 (λabs,
= 540 nm) and
max
BBTD700 (λabs,
= 700 nm) were simultaneously observed
max
(Figure S2a). Compared to the BBTD700 molecule in CH2Cl2, the
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10.1002/anie.202007649
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that in visible region (~ 1 mm), suggesting that NIR-II region is the
optimal window for deep tissue imaging. It is worth noting that the
penetration depth in our system is still shallower than that of other
excitation-free luminescence[18], which may be caused by the
relatively small molar extinction coefficient of the D-A-D structure
inhomogeneous illumination of the excitation light (Figure 2j, top)
in NIR-II FL imaging. Even near the far end of the capillary tube,
the NIR-II FL signal was attenuated to almost zero (Figure 2k).
These results demonstrated that NIR-II CL without external light
can be the better alternative for in vivo imaging.
dye molecule itself (ε ≈ 103-104 M−1 cm−1, λabs < 900 nm) [19]
.
It’s worth noting that the appropriate dye type with efficient
match of energy levels and large Stokes shift for the two energy
transfer processes (CRET and FRET) are critical to realize
efficient CL emission in NIR-II region (Figure 3). To realize
efficient CRET, a proper energy gap between the LUMO level of
DOD intermediate and the HOMO level of the acceptors is
neseccary. We summarize most commonly used CRET acceptors
(Figure S7–S9), including semiconducting polymers and organic
dyes[12f,13], and caluculated the energy gaps by performing density
functional theory (DFT) (Figure 3a). A reasonable energy gap
range between 1.20 eV and 5.01 eV is concluded out with the
feasible CRET. In this work, the energy gap between the HOMO
level of BTD540 and the LUMO level of DOD intermediate is 1.52
eV, which is within the above reasonable range, suggesting
BTD540 is a proper CRET acceptor. On one hand, due to the
energy match limitations, direct CRET process is unable to realize
long wavelength CL in NIR-II region (Figure 3b). For example, we
adopted three typical dyes (Rh6G, Cy5 or CS-2) with gruadually
prolonged absorption wavlengths. When encapsulated into the
micelles separately with CPPO, the CL intensities of them
decreased rapidly due to the gruadually lowered CRET
efficiencies (Figure S10). On the other hand, introducing FRET
processes after CRET may extend the emission wavelength of CL
to NIR-II region. However, the efficient energy transfer between
the the acceptor and donor is the key point to guarantee the
strong CL emission. Taking the CPPO-Rh6G-Cy5-Cy7.5 micelle
as a example (Figure 3c), although CL signal at 810 nm was
detected by the multi-step FRET with small Stokes shift, it was
very weak due to the accumulated energy loss in multiple energy
transfer process (Figure S11). By contrast, the specially designed
BTD540/BBTD700 as one-step FRET couple with excellent
energy match as well as large Stokes shifts can gurantee the
efficient energy transfer and the previously unreached remarkable
NIR-II CL (Figure 3d).
Next, we investigated the effects of H2O2 concentration,
temperature and reaction time on the efficiency of NIR-II CL. With
increasing of H2O2 concentration (3.5~13 mM) and temperature
(15~45°C), the NIR-II CL signals were both increased gradually
(Figure 4a-4h). At 15°C and 25°C, a sustained increase of CL
intensity were observed with increasing H2O2 concentration and
time (Figure 4a and 4b, 4e and 4f). Further elevating the
temperature to 37°C made CL signals continue to increase, with
the strongest CL signal presented after 20 min upon the addition
of H2O2 (13 mM) (Figure 4g and 4j), and the corresponding
intensity was 4.2-fold higher than that at 25°C (Figure 4i). The
half-life of NIR-II CL was found to be 146 min, 136 min, 40 min
and 40 min at 15°C, 25°C, 37°C and 45°C, respectively (Figure
S12). When the temperature is increased up to 45°C, the CL
signals were further increased 2.0 fold compared to that at 37oC
and the maximum value was detected at 15 min (Figure 4d, 4h
and 4i), which should be attributed to the promoted chemical
reaction rate between CPPO and H2O2, which shortened reaction
time with the temperature increasing. After reaching the emission
peaks, the signal gradually decreased due to the decreasing
Figure 3. (a) The energy levels of most commonly adopted CRET acceptors
and the active DOD intermediate. It should be noted that the energy gap
between the HOMO level of BTD540 and the LUMO level of DOD intermediate
is 1.52 eV, within the reasonable energy gap of the commonly CRET acceptors.
The CL schemes of the direct CRET in (b) Rhodamine and Cyanine dyes, (c)
CRET+Multistep FRET in Rhodamine and Cyanine dyes, (d) CRET+FRET
processes in D-A-D dyes.
Due to the elimination of the external exciation light, CL would
not suffer from the distorted signals in wide-field imaging as
commonly occured in FL imaging. To verify this, we studied the
the intensity distribution of a NIR-II CLS contained capillary in both
CL (without excitation) and FL imaging modes (excited by 808 nm
laser). As shown in Figure 2i, the laser beam followed a Gaussian
point spread function (PSF) and the intensity distribution within
the illumination site decreased from the center to the periphery
with the maximum localized at the center. In NIR-II CL imaging,
the uniform intensity was observed along the capillary tube
(Figure 2j, bottom). However, we observed an incongruous
intensity distribution along the capillary tube due to the
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concentration of CPPO and H2O2 with the progress of reaction.
Therefore, the following in vivo CL imaging were conducted at
physical temperature (37oC) within 60 min. In addition, NIR-II CLS
exhibited excellent linear relationship to H2O2 concentration and
the CL limit of detection (LOD) was determined to be 17.4 μM
(Figure S13). Moreover, no CL signals were observed when H2O2
imaging in the same mouse by adopted micelle containing only
BBTD700 as the reference.
The lymphatic inflammation model of mice was established by
intradermal injection into the rear paw of 34 mM (75 μL) hydrogen
peroxide. Then, BBTD700 micelle was injected intradermally at
the rear paw of mouse, and NIR-II FL signal was detected in the
lymph node region with the highest SNR of only 1.40 after 40 min
post-injection under 808 nm laser excitation (Figure 5b and S17).
In comparision, the NIR-II CL signal was quickly observed in the
lymph node region at 1 min after intradermal injection of NIR-II
CLS at the rear paw of mouse, and then the signal decreased due
to the fast flow of H2O2 in mouse (Figure S18). Even though, 3.4-
fold enhancement of SNR was obtained in the CL imaigng (4.74)
compared to that of NIR-II FL (1.40) due to the ultralow tissue
autofluorscence background in the absence of exciation light
(Figure 5b). In addtion to the elimination of background noise, the
absence of external light may also avoid the risk of overheating
when performing CL imaging. In comparison, a 808 nm laser,
which can excite NIR-II CLS used in conventional FL imaging,
indeed increased the skin temperature of a nude mouse by ~ 5 oC
within 60 min and it continued to rise (Figure 5c). This might be
destructive to the living body.
-
was displaced by other reactive oxygen species (ClO-, O2 ,
ONOO- and HO•) at 37°C , even prolonging the reaction time
(Figure S14), indicating the good selectivity of NIR-II CLS to H2O2
(Figure 4k).
The reliable in vitro spectral properties and selectivity test for
H2O2 with NIR-II CLS encouraged us to further explore its
potential for in vivo NIR-II CL imaging. First, the potential
cytotoxicity of NIR-II CLS was evaluated in human umbilical vein
endothelial cells (HUVEC), which showed over 87% viability after
incubation with 6.2 mg mL-1 nanosensor for 24 h, indicating the
low cytotoxicity of NIR-II CLS (Figure S15). We next carried out
lymphatic and arthrosis inflammation imaging models (Figure 5a)
for NIR-II CL bioimaging using the home-built small animal system
Figure 5. (a) Schematic illustration of executing CL and FL imaging carrying on
mice lymph node and arthrosis. For FL imaging, 808 nm excitation light with 800
ms exposure time (P = 32 mW cm-2) was adopted. (b) The comparison of SNR
between the NIR-II CL imaging (1 min post-injection of 0.98 mg NIR-II CLS) and
the NIR-II FL imaging (40 min post-injection of 1.2 mg BBTD700 micelle) of
lymphatic inflammation, *P < 0.05. Insert: corresponding images of lymphatic
node (n = 3). (c) Temperature variation curves of nude mouse skin recorded
with and without laser irradiation (808 nm, 94 mW cm-2). Insert: Skin
temperature of nude mouse irradiated at 60 min. Scale bar, 5 mm, n = 3. (d) In
vivo NIR-II FL imaging (top panel) of BBTD700 micelle (1.2 mg) and NIR-II CL
imaging (middle panel) of arthrosis inflammation with NIR-II CLS (0.92 mg) at
different post-injection time, respectively. Control experiments with healthy mice
for NIR-II CL imaging of arthrosis with NIR-II CLS (0.92 mg) were also carried
out for comparison (bottom panel). The corresponding NIR-II CL/FL signals
intensity (e) and SNRs (f) in (d) as a function of post-injection time, *P < 0.05.
(g) Intensity profiles along the arthrosis position of a mouse (the white line in
5d), Scale bar, 5 mm. Bars represent mean±s.d. (n = 3). (850 nm long-pass
filter).
Figure 4. The CL imaging of NIR-II CLS (8.2 mg ml-1) upon addition of different
concentration of H2O2 at (a) 15°C , (b) 25°C , (c) 37°C and (d) 45°C . (e-h)
Corresponding NIR-II CL signals upon addition of different concentration H2O2
in (a-d), respectively. (i) Time-dependent NIR-II CL signal intensities upon
addition of H2O2 (13 mM) at 15~45°C. (j) Time-dependent CL signal intensities
upon addition of different concentration H2O2 at 37°C . (k) NIR-II CL signal
intensities of NIR-II CLS (8.2 mg ml-1) upon addition of various ROS (13 mM) at
37°C . Insert: Corresponding NIR-II CL imaging upon addition of various ROS.
(Figure S16). To show the advantages of NIR-II CL imaging in our
designed system based on the D-A-D dyes, we took NIR-II FL
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The arthrosis inflammation model of mice was established by
intra-articular injection of 33 mM (75 μL) hydrogen peroxide, then
NIR-II CLS were used by sequentially intra-articular injection into
a mouse. With the progress of reaction with H2O2, the CL signals
and SNRs in the arthrosis area were increased over time (Figure
5d-f). The maximum SNR of 5.85 in the arthrosis area was
observed at 30 min. We also aquired the NIR-II FL signals with
BBTD700 micelle (Figure 5d). The FL signal was gradually
increased to a maximum at 5 min post-injection and dropped 44%
at 60 min (Figure 5e). During this whole period, the maximum FL
SNR of 1.24 in the arthrosis area was obtained at 5 min, which
was still 4.5-fold lower than that with CL imaiging (Figure 5f).
Besides, the smaller full width at half maxima (FWHM = 6.73 mm)
of the arthrosis position tracing in CL imaging over NIR-II FL
imaging (FWHM = 7.38 mm) also showed superior accuracy
(Figure 5g). For comparison, we also carried out control
experiment on the lymphatic and arthrosis imaging with healthy
mice. No CL signals were observed in the above two regions due
to the lack of H2O2. Based on the above results, NIR-II CLS can
achieve higher SNR for in vivo inflammation imaging.
In summary, we have developed a NIR-II CL sensor for
overcoming the drawbacks of short-wavelength CL emission and
lower penetration depth through engineering the cascade CRET
and FRET processes.The as-prepared biocompatible NIR-II CLS
have deeper penetration depth and higher SNR for in vivo CL
inflammation imaging. To reduce the energy loss during multi-
step energy transfer, we rationally designed the dyes with
excellent energy match and the large Stokes shift characteristics
to effeciently transfer the chemical energy, which made a better
foundation for the development of in vivo CL NIR-II imaging. It
provides a promising perspective and strategy for constructing
probes with extended emission wavelength in classical CL for
higher constrast imaging. Meanwhile, it also offers the possibility
of sensing various analytes by changing the chemiluminescent
substrate in the furture. Although the sensitivity and brightness of
the current NIR-II CLS shown here is still not ideal, the present
results indicated that the NIR-II CLS is promising for NIR-II CL in
vivo imaging with higher SNR compared to the NIR-II
fluorescence signals in the same system. Besides, the NIR-II CLS
may be further improved by rationally designing probes with
higher quantum yield (for example, the introduction of substituted
thiophene and shielding units[19]), larger molar extinction
coefficient, and better oxidation resistance. Given CL imaging
immune to background interference and light damage from the
external excitation light, it could motivate more pre-clinical and
clinical translation applications.
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Keywords: NIR-II imaging• chemiluminescence imaging •
bioimaging • inflammation tracing •
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Novel NIR-II chemiluminescence
sensor was prepared by cascade
CRET and one-step FRET for in
vivo high contrast inflammation
imaging.
Yanling Yang, Shangfeng Wang,
Lingfei Lu, Qisong Zhang, Peng Yu,
Yong Fan*, Fan Zhang*
Page No. – Page No.
NIR-II Chemiluminescence
Molecular Sensors for In-Vivo High
Contrast Inflammation Imaging
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