Sensitive imaging of tumors using a nitroreductase-activated fluorescence probe in the NIR-II window

Article abstract of DOI:10.1039/d1cc03232a

A nitroreductase (NTR)-activated NIR-II fluorescence probe for tumor imaging is reported. The probe can emit fluorescence in the range of 900-1300 nm, and target hypoxic tumors with NTR overexpression, thus allowing for accurate delineation of tumor margi

Full text of DOI:10.1039/d1cc03232a

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Sensitive imaging of tumors using a
nitroreductase-activated fluorescence probe
in the NIR-II window†
Cite this: Chem. Commun., 2021,
57, 8174
Received 18th June 2021,
Accepted 16th July 2021
ab
ab
ab
Xiaofan Zhang,ab Xiaohua Li,
*
Wen Shi
and Huimin Ma
*
DOI: 10.1039/d1cc03232a
rsc.li/chemcomm
A nitroreductase (NTR)-activated NIR-II fluorescence probe for responses are more desirable because they produce light signals
tumor imaging is reported. The probe can emit fluorescence in only after the specific interaction with the tumor marker and
the range of 900–1300 nm, and target hypoxic tumors with NTR show lower background noise and clearer discrimination between
overexpression, thus allowing for accurate delineation of tumor the tumor and the surrounding normal tissue.7 Although signifi-
margins through deep penetration.
cant effort has been made for this purpose,8 the sensitivity of
most existing NTR fluorescent probes that emit beyond 900 nm is
Surgical resection of tumors is an effective method for treatment still inadequate for detecting trace levels of NTR in common
of cancer in clinical surgical oncology.1 However, the accurate biosystems. Thus, it is highly demanded to develop sensitive
delineation of tumor margins remains a challenge. Among the fluorescence off–on probes with the emission extended to the
methods employed, fluorescence imaging has become a promising NIR-II region for accurate delineation of tumor margins. In fact,
way due to its real-time, convenient and noninvasive optical high sensitivity has always been one of the most important
visualization with high sensitivity.2 Particularly, fluorescence scientific issues and research topics (including high selectivity
imaging in the second near-infrared window (NIR-II, 1000– and simple, inexpensive practicality) in the field of spectroscopic
1700 nm) exhibits reduced photon scattering and absorption as probes.9
well as negligible background auto-fluorescence in living tissues,
thus leading to deeper penetration and higher spatio-temporal is presented (Scheme 1) that is suitable for fluorescence imag-
resolution.3
ing in the NIR-II window. It provides a much lower detection
Herein, a new fluorescence off–on probe (RHC-NO2) for NTR
For some tumor tissues and cells, such as 4T1 and A549, one limit (5.9 ng mLÀ1) than the other current NTR-activated NIR-II
of the most common features is hypoxia. The hypoxic micro- probes.10 Probe RHC-NO2, with an emission peak at 921 nm,
environment endows cancer cells with higher resistance was designed by using the rhodamine hydrid polymethine
towards cancer chemotherapy and radiotherapy.4 Therefore, it framework. Meanwhile, a nitro group, serving as the NTR-
is of great clinical significance to evaluate the hypoxic degree in specific recognition moiety as well as the fluorescence
tumors, which is known to be directly associated with the quencher, was incorporated into the skeleton. Upon reaction
expression of nitroreductase (NTR), a tumor marker.5 So far, of the probe with NTR, the nitro group was reduced to an
many small molecular fluorescent probes have been reported amino group and the fluorescence at 921 nm was recovered
for NTR detection. However, most of them emit in the tradi- accordingly (Scheme 1). The fluorescence off–on response
tional visible and NIR-I (650–900 nm) spectral region, which provided a largely reduced background noise, thus leading to
may suffer from limited imaging depth.6 For hypoxic NTR detec- a high signal-to-background ratio in intravital imaging. Using
tion and in vivo imaging of tumors, fluorescence probes which not this probe, fluorescence imaging of tumors was successfully
only possess longer emission wavelengths (beyond 900 nm, better
extended to the NIR-II region) but also give off–on fluorescence
a Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical
Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190, China. E-mail: lixh@iccas.ac.cn, mahm@iccas.ac.cn
b University of Chinese Academy of Sciences, Beijing 100049, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
Scheme 1 Sensing mechanism of RHC-NO2 for NTR detection.
d1cc03232a
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achieved in two kinds of murine tumor models (A549 and HeLa
tumors).
The absorption spectra of the probe with and without NTR
were studied in pH 7.4 phosphate buffer (PBS). As shown in
Fig. 1A, probe RHC-NO2 exhibits a maximum absorption at
677 nm. Addition of NTR results in the emergence of a new
absorption peak at 868 nm, which may be attributed to the
formation of the reaction product, RHC-NH2. The fluorescence
emission spectra of RHC-NO2 with and without NTR are depicted
in Fig. 1B. It is seen that the probe itself is almost non-fluorescent
(quantum yield F o 0.01%) due to the quenching effect of the
nitro group, but the reaction with NTR produces strong fluores-
cence at 921 nm (about 14-fold enhancement). Meanwhile, the
spectral properties of the synthesized RHC-NH2 (reaction product)
were examined. As expected, RHC-NH2 also shows maximal
absorption and fluorescence emission at 868 nm and 921 nm,
respectively, in PBS of pH 7.4 (Fig. S10, ESI†). These results
suggested the possible generation of RHC-NH2 upon reaction of
the probe with NTR.
Fig. 2 (A) Fluorescence response of RHC-NO2 (10 mM) to varied con-
centrations of NTR (0–10 mg mLÀ1). (B) Linear fitting curve of DF against the
concentration of NTR. (C) Fluorescence response of RHC-NO2 to various
species (from 1 to 18): blank, cysteine (1 mM), glutathione (1 mM), Na2S
(100 mM), vitamin C (1 mM), H2O2 (100 mM), NaOCl (100 mM), NaNO2
(100 mM), ꢀOH (100 mM), Ca2+ (2 mM), Cu2+ (100 mM), Fe2+ (100 mM),
glucose (10 mM), K+ (150 mM), Mg2+ (2 mM), Zn2+ (100 mM), ONOOÀ
(200 mM), NTR (10 mg mLÀ1). The results are the mean Æ standard deviation
of three separate measurements.
To further prove this, the reaction products of RHC-NO2
with NTR were subjected to ESI-MS and HPLC analysis (Fig. S11
and S12, ESI†), which indicated that the NTR-catalyzed
reduction of the probe indeed produced RHC-NH2. Moreover,
the stability of RHC-NO2 was investigated by measuring
its absorption and fluorescence spectra in PBS for different
periods of time (Fig. S13, ESI†). The results showed that the found to achieve the maximal fluorescence signal (Fig. S15, ESI†),
probe was rather stable at room temperature, which was which was roughly matchable to the basal level (about 30–168 mM)13
favourable for in vivo imaging.
of NADH in common biosystems. Moreover, the reaction conditions
In addition, the fluorescence intensity of RHC-NO2 and (such as time, pH and temperature) were also optimized. The time-
RHC-NH2 was compared in pH 7.4 phosphate buffer containing dependent fluorescence response of the probe toward NTR is shown
different fractions of FBS (Fig. S14, ESI†). It was found that the in Fig. S16 (ESI†), from which it can be seen that the fluorescence
probe itself exhibited very weak fluorescence either with FBS or intensity increases rapidly at 921 nm and reaches a plateau in about
without FBS, whereas RHC-NH2 displayed a largely enhanced 1 h. Meanwhile, pH and temperature fluctuations hardly affected
fluorescence with increasing the fraction of FBS in PBS, which the fluorescence of RHC-NO2, and the reaction of the probe with
may arise from the possible formation of the dye-protein NTR proceeded well at pH 7.4 and 37 1C (Fig. S17, ESI†), demon-
complex.11 Thus, in the following experiments, the reactions strating its outstanding biological applicability under the physio-
of RHC-NO2 with NTR were studied in the phosphate buffer logical conditions.
(20 mM, pH 7.4) containing 10% volume fraction of FBS, which
may be used to simulate the physiological environment.12
Under the optimized conditions (reaction at 37 1C for 1 h in
PBS of pH 7.4 containing 10% FBS in the presence of 200 mM
The influence of NADH (a cofactor of NTR) on the fluores- NADH), the fluorescence response of RHC-NO2 to NTR at varied
cence response was investigated, and 200 mM of NADH was concentrations is shown in Fig. 2A and B, revealing that the
fluorescence intensity at 921 nm gradually increases with
increasing NTR. There is a good linearity between DF and the
concentration of NTR in the range of 1–10 mg mLÀ1, with an
equation of DF = 1657 Â [C (mg mLÀ1)] À 730 (R = 0.994) and a
detection limit of as low as 5.9 ng mLÀ1, where DF is the
difference of fluorescence intensity of RHC-NO2 with and
without NTR at 921 nm. These observations suggested that
RHC-NO2 may be applied for in vivo imaging via the detection
of trace concentrations of NTR in the NIR-II window.
In order to get an insight into the selectivity of the probe,
fluorescence responses were tested in the presence of various
potential interfering species. As shown in Fig. 2C, no signifi-
cant fluorescence response is detected among the substances
Fig. 1 (A) Absorption and (B) fluorescence spectra of RHC-NO2 (10 mM)
before (a) and after (b) reaction with NTR (10 mg mLÀ1) in the presence of
NADH (200 mM) in pH 7.4 phosphate buffer containing 10% fetal bovine
serum (FBS). Inset: NIR-II fluorescence images of RHC-NO2 in test tubes
tested, including inorganic salts (Na2S, CaCl2, CuSO4, FeSO4,
KCl, MgCl2, ZnCl2), vitamin C, glucose, reactive oxygen species
before (a) and after (b) reaction with NTR, with an exposure time of 50 ms
(excited with 808 nm laser) and 1000 nm long-pass emission filter.
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(H2O2, NaOCl, NaNO2, ꢀOH, ONOOÀ) and biothiols (cysteine,
glutathione). By contrast, the addition of NTR causes remarkable
fluorescence increase, revealing that RHC-NO2 exhibits high
selectivity for NTR even in complex biological environment.
Next, enzyme inhibition experiments were performed to
further verify that the fluorescence response was generated by
the NTR-catalyzed reaction. As seen from Fig. S18 (ESI†), the
fluorescence intensity with 0.1 mM dicoumarin (curve d) is
much weaker than that (curve c) without the inhibitor, and
more dicoumarin (0.2 mM) results in a greater decrease in
fluorescence intensity (curve e). These results suggest that the
fluorescence off–on response indeed arises from the enzyme
catalyzed reaction.
Besides, the spectral properties of RHC-NH2 were explored
in different solvents (Fig. S19 and Table S1, ESI†), and similar
spectral properties, i.e., with the maximum absorption/emis-
sion at about 850/920 nm but an increased quantum yield up to
about 0.1%, were observed, though the absorption or fluores-
cence intensities were affected by the variation of solvents. The
pH effects on the spectral properties are displayed in Fig. S20
(ESI†), disclosing that the absorption at 868 nm hardly changes
and strong NIR fluorescence beyond 900 nm is observed in a
wide span of pH from 4 to 10, though the fluorescence becomes
stronger in an alkaline medium. This suggests that RHC-NH2,
with bright emission in the NIR-II region, may act as a useful
platform for designing activatable long wavelength fluorescent
probes through further modification of its amino group.
The cytotoxicity of probe RHC-NO2 was assessed by the
standard MTT assay before its application for in vivo imaging
(Fig. S21, ESI†), and no significant toxicity was observed in the
case of A549 or HeLa cell line, suggesting the good biocompat-
ibility of the probe.
Fig. 3 Representative NIR-II images of A549 tumor-bearing mice (n = 3)
after tail vein injection of RHC-NO2 (200 mL, 500 mM) with 808 nm
excitation, 1000 nm long-pass emission filter and exposure time of
50 ms. The full images of the mouse at different time points (0–96 h)
are given in Fig. S22 (ESI†). Scale bar: 1 cm.
Bearing in mind that the probe displayed the merits of
bright fluorescence beyond 900 nm, excellent stability and
biocompatibility, we applied it to NIR-II fluorescence imaging
of tumor in living animals. In this experiment, female BALB/c
nude mice bearing the subcutaneous A549 tumor were chosen
as a model because of the elevated level of NTR under hypoxic
conditions. Probe RHC-NO2 was intravenously injected into
A549 tumor-bearing mice. As is seen from Fig. 3 and Fig. S22
(ESI†), the fluorescence intensity in the tumor gradually
increases and the maximal NIR-II signal is detected after about
12 h of injection. Then, the fluorescence decreases with time.
Most importantly, the tumor becomes distinguishable from the
surrounding normal tissue after 4 h of injection, and the tumor
boundary remains clear until about 24 h. As mentioned above,
the fluorescence enhancement could be ascribed to the reac-
tion of RHC-NO2 with the overexpressed NTR in A549 tumors. It
is noticed that upon injection of the probe, the fluorescence is
also observed in other parts of the mouse body besides the
tumor region, which is probably attributed to the metabolism
of the probe via the liver and kidney (such metabolic pathways
are quite common in previously reported literature14). More-
over, after 24 h of injection, the fluorescence in the tumor
remains bright, while that in other organs becomes quite weak.
Therefore, 24 h may be selected as the time point for
Fig. 4 Ex vivo biodistribution of RHC-NO2 in A549 tumor-bearing mice
after a tail vein injection of RHC-NO2 (200 mL, 500 mM). (A) NIR-II
fluorescence imaging of dissected organs and tumor after 48 h injection
of RHC-NO2 with 808 nm excitation and 1000 nm LP emission filter and
exposure time of 50 ms. (B) Quantified fluorescence intensity in A549
tumor-bearing mice (n = 3). Scale bar: 5 mm.
fluorescence imaging and identification of A549 tumors.
Besides, similar phenomena were observed in HeLa tumor-
bearing mice (Fig. S23, ESI†), with a maximal signal detected
at about 6 h. These results demonstrated the capability of
RHC-NO2 for the delineation of tumor margins in bioimaging.
Furthermore, the mice were anatomized to analyze ex vivo
biodistribution of the probe in major organs and tumor. It was
found that the NIR-II fluorescence was mostly observed in the
dissected tumor after 48 h of intravenous injection of RHC-NO2
to the A549 tumor-bearing mice (Fig. 4), with some seen in the
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Conflicts of interest
There are no conflicts to declare.
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