An Unsymmetrical Squaraine-Based Activatable Probe for Imaging Lymphatic Metastasis by Responding to Tumor Hypoxia with MSOT and Aggregation-Enhanced Fluorescent Imaging

Article abstract of DOI:10.1002/chem.201904675

Optoacoustic imaging has great potential for preclinical research and clinical practice, and designing robust activatable optoacoustic probes for specific diseases is beneficial for its further development. Herein, an activatable probe has been developed for tumor hypoxia imaging. For this probe, indole and quinoline were linked on each side of an oxocyclobutenolate core to form an unsymmetrical squaraine. A triarylamine group was incorporated to endow the molecule with the aggregation enhanced emission (AEE) properties. In aqueous media, the squaraine chromophore aggregates into the nanoprobe, which specifically responds to nitroreductase and produces strong optoacoustic signals due to its high extinction coefficient, as well as prominent fluorescence emission as a result of its AEE feature. The nanoprobe was used to image tumor metastasis via the lymphatic system both optoacoustically and fluorescently. Moreover, both the fluorescence signals and three-dimensional multispectral optoacoustic tomography signals from the activated nanoprobe allow us to locate the tumor site and to map the metastatic route.

Full text of DOI:10.1002/chem.201904675

A Journal of
Accepted Article
Title: An Unsymmetrical Squaraine-based Activatable Probe for
Imaging Lymphatic Metastasis through Responding to Tumor
Hypoxia by MSOT and Aggregation-enhanced Fluorescent
Imaging
Authors: Yi Lin, Lihe Sun, Fang Zeng, and Shuizhu Wu
This manuscript has been accepted after peer review and appears as an
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To be cited as: Chem. Eur. J. 10.1002/chem.201904675
Link to VoR: http://dx.doi.org/10.1002/chem.201904675
Supported by

10.1002/chem.201904675
Chemistry - A European Journal
An Unsymmetrical Squaraine-based Activatable Probe for Imaging Lymphatic
Metastasis through Responding to Tumor Hypoxia by MSOT and Aggregation-enhanced
Fluorescent Imaging
Yi Lin+, Lihe Sun+, Fang Zeng* and Shuizhu Wu*
Y. Lin+, L. Sun+, Prof. F. Zeng, Prof. S. Z. Wu
State Key Laboratory of Luminescent Materials and Devices, Key Laboratory of Luminescence
from Molecular Aggregates of Guangdong Province, College of Materials Science and
Engineering, South China University of Technology, Wushan Road 381, Guangzhou, 510640,
China
E-mail: mcfzeng@scut.edu.cn
shzhwu@scut.edu.cn
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for the author(s) of this article
can be found under http://doi.org/
1
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10.1002/chem.201904675
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Abstract: Optoacoustic imaging exhibits great potential for preclinical research and clinical
practice, and designing robust activatable optoacoustic probes for specific diseases is beneficial
for its further development. Herein, an activatable probe has been developed for tumor hypoxia
imaging. For this probe, indole and quinoline were linked on each side of oxocyclobutenolate
core to form an unsymmetrical squaraine. Triarylamine group was incorporated to afford the
aggregation enhanced emission (AEE) property. In aqueous media, the squaraine chromophore
aggregates into the nanoprobe which specifically responds to nitroreductase and produce strong
optoacoustic signals due to its high extinction coefficient, as well as prominent fluorescence
emission as a result of its AEE feature. The nanoprobe was used to image tumor metastasis via
lymphatic system optoacoustically and fluorescently. Moreover, both the fluorescence signals
and three-dimensional multispectral optoacoustic tomography signals from the activated
nanoprobe allow us to locate the tumor site and map the metastatic route.
Introduction
Lymphatic metastasis is one of the two major routes for spread of cancer cells, and
metastatic cells can migrate from a primary tumor to distant organs via lymphatics.[1,2]
Early detection of lymphatic metastasis can optimize disease management and improve
overall patient outcomes. However, identifying lymphatic metastasis in early stage
remains challenging despite of recent advances in metastasis detection, and one
impediment is that early metastases usually involve small-sized tumors and dispersed
distribution.[3] Histopathology, including recently developed histopathological whole
slide imaging (WSI), has been a commonly used approach for discriminating tumor cells
and localizing malignant lesions.[4] However, this method is invasive, and processing the
vast amount of data is tedious and time-consuming.
Visualizing the disease lesions and monitoring their onset, progression or regression
over time without tissue invasion is an attractive approach to evaluate the tumor metastasis. A
number of non-invasive imaging modalities, including magnetic resonance imaging,
ultrasound, positron-emission tomography, and optical imaging, have been employed to
visualize the disease evolution.[5] Recently, a newly developed imaging modality, known
as optoacoustic (photoacoustic) imaging, has demonstrated great potential in preclinical
research and clinical practice.[6-9] Using near-infrared (NIR)-absorbing materials as
contrast agents, and integrating laser excitation with ultrasound detection, the
optoacoustic (OA) imaging can achieve high-contrast and spectroscopic-based specificity
of optical imaging as well as the high spatial resolution of ultrasound imaging. As one of
the optoacoustic imaging techniques, the multispectral optoacoustic tomography
(MSOT),[10-12] which irradiates a sample with multiple wavelengths and detects ultrasound
waves from different photoabsorbing molecules in the tissue, can separately visualize
these different photoabsorbers (including endogenous substances such as melanin,
hemoglobin and myoglobin, as well as exogenous contrast agents such as optoacoustic
probes) in target tissue upon spectral unmixing process. In addition, MSOT can also afford
2
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three-dimensional (3D) images by rendering a stack of cross-sectional images as
maximum intensity projection (MIP) images.[13]
To afford high-quality optoacoustic images, a contrast agent should exhibit certain
optical properties, such as high molar extinction coefficient in near infrared (NIR)
region.[14] To date, a variety of inorganic and organic contrast agents have been designed
for optoacoustic imaging.[15-18] Compared to the inert contrast agents, the activatable
(responsive) ones are particularly advantageous because they can specifically respond to
the “abnormalities” in tissues and thereby produce changed optoacoustic signals, thus
achieving enhanced sensitivity for disease diagnosis.[19] Currently the activatable
optoacoustic probes are much less reported and need to be further sought after.[20] The
organic chromophores, with their molecular structures and electronic states more easily
tuned, are suitable for fabricating activatable probes. Compared to inorganic
chromophores, the organic ones generally exhibit much lower extinction coefficient.
Squaraine dyes, a family of four-membered ring chromophores featuring certain
optical properties such as sharp and strong absorption in NIR region (ε > 105 Lmol-1cm-1
which is very high among molecular dyes) and fluorescent emission with moderate
quantum yield,[21,22] have been widely utilized in photoelectric devices, photodynamic
therapy and NIR fluorescent sensing.[23] Recently, they also have been utilized as the
exogenous contrast agents and probes in optoacoustic imaging.[24] However, for achieving
optoacoustic imaging with enhanced specificity and accuracy, the activatable squaraine
probes which can respond to specific disease markers and exhibit distinct absorption
change within NIR range are highly desirable.
Squaraine dyes often show poor solubility in aqueous solutions due to their strong
structural rigidities and - stackings, which usually results in impairment of optical
properties such as aggregation-caused fluorescence quenching (ACQ), especially when it
comes to biodetection and imaging in vivo.[23,24] We suppose, one possible solution to this
issue is to endow the squaraine chromophore with aggregation induced (or enhanced)
emission (AIE/AEE) property[25-27] by introducing a suitable AIE/AEE-active group into
the squaraine structure. In that case, a squaraine dye may emit more strongly in the
aggregated state in aqueous media than in molecular state in good solvents, which allows
it to serve as a dual-mode (optoacoustic and fluorescent) imaging probe and realize mutual
verification between the two imaging modalities. NIR fluorescent imaging has the
advantage of high sensitivity.[28] Hence, the dual-mode imaging approach combining NIR
fluorescent imaging and MSOT imaging could have both high spatial resolution and
sensitivity. In addition, functioning as a nanoprobe in the aggregated state, the squaraine
chromophore may accumulate in tumor tissue owing to the enhanced permeation and
retention (EPR) effect and thereby achieve more sensitive and accurate imaging.
Aiming to develop an activatable dual-mode probe for visualizing and tracking
lymphatic metastasis of solid tumor, we designed an unsymmetrical squaraine-based NIR
chromophore (SPT-NO2) for specifically responding to nitroreductase (NTR, a biomarker
highly expressed in solid tumor hypoxia), as shown in Scheme 1. For this chromophore,
a triethyleneglycol-modified indole and a quinoline are linked to each side of the
oxocyclobutenolate core to form a squaraine, and a dicyanomethylene group is also linked
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onto the core to enhance its chemical stability and improve the electron deficiency of the
core so as to further shift the absorption band of the chromophore to longer wavelength.
Moreover, triarylamine group is incorporated into the structure to afford AIE/AEE
property so as to realize dual-mode imaging. The resultant molecular dye readily forms
the nanoprobe (NP-SPT-NO2) in aqueous media due to its amphiphilic structure and -
stacking. The nanoprobe is non-fluorescent and shows an absorption band at 742 nm. The
presence of nitroreductase converts the electron-withdrawing nitro group to the electron-
donating amino group, which leads to shifting of the absorption peak to 690 nm and strong
fluorescence at 762 nm for the nanoprobe solution. Upon injection into the tumor-bearing
mice, the nanoprobe quickly responds to the highly-expressed nitroreductase in tumor
hypoxia and produces strong MSOT and fluorescent signals for imaging primary tumor
and lymphatic metastasis. Moreover, the 3D MSOT images obtained by rendering a stack
of 2D cross-sectional images allow us to track the lymphatic metastasis process and
visualize the whole metastatic route from the primary tumor at footpad to the sentinel
lymph node in a spatiotemporal manner.
Scheme 1. Schematic illustration of the nanoprobe’s response toward nitroreductase
(tumor hypoxia) (a) and lymphatic metastasis imaging (b). (I) Injection of tumor cells into
footpad at rear left paw. (II) Primary tumor (PT) developed in footpad. (III) Tumor cells
spread to sentinel lymph node (SLN) via lymphatic vessel, which was imaged and mapped
optoacoustically and fluorescently by using the nanoprobe.
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Results and discussion
The molecular probe SPT-NO2 (compound 5) was synthesized according to the route shown
in Scheme S1 and the structure of the product and intermediates were well characterized by NMR
and mass spectrometry (Figures S1-10). For comparison, SPT-NH2 (compound 6) was also
synthesized and characterized (Figures S11-12), which in theory is the product of the reaction
between SPT-NO2 and nitroreductase (NTR). As the DMSO solution of molecular SPT-NO2 was
dispersed into pH 7.4 PBS, it readily formed into nanosized aggregates (the nanoprobe NP-SPT-
NO2), which was verified by the size distribution curves (from dynamic light scattering
measurement) and transmission electronic microscopy (TEM) images, as shown in Figure S13.
The molar extinction coefficient for SPT-NH2 has been determined as 3.6  10 5 M-1cm-1 (Figure
S14) which is very high among organic dyes[10-12], priming SPT-NH2 for optoacoustic imaging.
The spectral properties of the nanoprobe NP-SPT-NO2 before and after incubation with 0.2
U mL-1 nitroreductase (NTR) under 37 °C for 70 min were determined. As shown in Figure S14a
and S14c, NP-SPT-NO2 (5 μM) in PBS (with 5 % DMSO) displays an absorption band at around
740 nm and almost no emission when excited at 690 nm. While after incubation with NTR, its
absorption peak shifts to around 690 nm and a prominent emission band can be observed at 762
nm, which are almost identical to those for NP-SPT-NH2 in PBS (Figure S14c and S14d),
suggesting that NP-SPT-NO2 probably turns into NP-SPT-NH2 upon incubation with NTR. The
significant change in spectral properties is due to the transformation from the strong electron-
withdrawing nitro group to the electron-donating amino group in the probe structure and the
subsequent variation of the electronic state.
With a triarylamine moiety in its structure, the activated probe SPT-NH2 is potentially
AIE/AEE active. To investigate the AIE/AEE behaviour of SPT-NH2, SPT-NH2 was dissolved
in DMSO/water mixtures with different water fractions, and the fluorescence spectra were then
recorded and are shown in Figure S15. It is clear that, the fluorescence intensity of SPT-NH2
increased as water ratio rised, revealing that SPT-NH2 is AEE active. Water is a poor solvent of
SPT-NH2, and continuous addition of water induces the gradual aggregation of SPT-NH2 in the
mixture, thereby inhibiting intramolecular rotation of triarylamine moieties and reducing the non-
radiative dissipation of excited energy. Correspondingly, the fluorescence intensity enhances.
Additionally, incorporation of the triarylamine moiety into the molecular structure of the probe
may prevent from forming closely packed structure, which is beneficial for the NTR-
mediated reaction and fluorescence emission
.
The absorption and fluorescence spectra for the nanoprobe NP-SPT-NO2 treated with NTR
of different levels are presented in Figure 1a-1b and Figure S16. With increasing NTR levels, the
absorbance of the nanoprobe at 800 nm decreased, while that at 690 nm increased; and the
fluorescence intensity at 762 nm enhanced simultaneously. The absorbance ratio (A690/A800) also
increased steadily with the increasing NTR level (Figure S17). The time-dependent spectral
response of the nanoprobe toward NTR is shown in Figure 1c and Figure S18, which indicates
that the longer incubation time causes greater fluorescence intensity increment until fluorescence
reaches its maximum. Moreover, the relative optoacoustic intensity of the nanoprobe dispersion
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upon NTR treatment also increased with the increasing NTR levels (Figure 1d). Taken together,
the nanoprobe is capable of responding to NTR in aqueous media and generating strong
fluorescence and optoacoustic signals. Moreover, after NTR-mediated reaction, the activated
probe molecules (SPT-NH2) remain insoluble in PBS and assume the aggregated state (Figure
S13d); and due to its AEE-active nature, the NP-SPT-NH2 exhibits even stronger fluorescence
rather than being quenched (Figure S14a and S14d). Some other groups also observed the similar
phenomenon that a nanosized AIE-based probe turned into another nanostructure upon
incubation with a specific analyte.[29]
Figure 1. Optical and optoacoustic response of the nanoprobe NP-SPT-NO2 (5 μM) toward NTR.
(a) Absorption spectra of NP-SPT-NO2 incubated with NTR of varied activities. (b) Fluorescence
spectra of NP-SPT-NO2 incubated with NTR of varied levels. (c) Fluorescence spectra of NP-
SPT-NO2 incubated with 0.2 U mL-1 NTR for different time. (d) Relative optoacoustic intensity
[(I-I0)/I0] of NP-SPT-NO2 incubated with NTR of varied activities (n = 3), the inset displays
representative optoacoustic images of the NP-SPT-NO2 in phantom upon incubation with NTR
of five different activities. Excitation wavelength: 690 nm.
High resolution mass spectrometry (HRMS) and high performance liquid chromatography
(HPLC) were performed to verify the generation of SPT-NH2 upon incubation of NTR with NP-
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SPT-NO2 under 37 °C for 20 min. One can find in the HRMS spectrum shown in Figure S19, the
mass peak at 815.3558 corresponding to the molecular probe SPT-NO2 and that at 785.3813
corresponding to SPT-NH2 could be observed clearly. Moreover, HPLC results (Figure S20)
indicate that, the retention time for SPT-NO2 (1.72 min) and for SPT-NH2 (1.34 min) can all be
observed after incomplete reaction between NTR and NP-SPT-NO2. These results confirm that
SPT-NH2 is produced after the NTR-mediated reaction. The pH- and photo-stability of NP-SPT-
NO2 and the activated nanoprobe NP-SPT-NH2 were then tested to evaluate its suitability for
fluorescence and optoacoustic imaging, and the results are presented in Figures S21-22, which
indicate that NP-SPT-NH2 and NP-SPT-NO2 display good spectral stability at varied pHs or upon
light irradiation. In addition, the nanoprobe also exhibits quite good selectivity towards NTR in
the presence of various biologically relevant substances including ions, amino acids and some
common enzymes (Figure S23).
Prior to cell imaging experiments, the cytotoxicities of the nanoprobe NP-SPT-NO2 against
30
]
4T1 cell line (murine mammary carcinoma cell line overexpressing NTR under hypoxia [
)
and LO2 cell line (normal human hepatic cell line which has no overexpression of NTR) were
evaluated (Figure S24), and the result verifies that the nanoprobe is of little cytotoxicity. The
intracellular imaging of NTR by NP-SPT-NO2 was investigated in 4T1 cells by fluorescent
imaging. In the experiment, the cells were incubated in hypoxic atmosphere for 3 hours (oxygen
level was maintained at 0.1% O2 in hypoxic incubator chamber) or under normal condition
(normoxia), and the fluorescence images are shown in Figure S25. Under hypoxia, the cells
displayed obvious fluorescence, which is in great contrast to the situation in normoxia where
only negligible intracellular fluorescence could be observed. Moreover, the imaging of the
cells in culture dishes on an Ami small animal imaging system for 4T1 treated with NP-
SPT-NO2 of varied concentrations for 3 hours in hypoxia with or without being pretreated
with dicoumarin of varied concentrations for 1 hour is also illustrated in Figure S25, which
further confirm the response to NTR of the probe. In addition, optoacoustic (OA) imaging
for NTR in 4T1 cells by the nanoprobe are shown in Figure S26. Clearly, after incubation with
NP-SPT-NO2 under hypoxia, strong OA signals as a result of irradiation at 690 nm could be
30
observed in 4T1 cancer cells;[ ] while the OA signals (irradiated at 690 nm) were insignificant
in the cells under normoxia. These results indicate that the NP-SPT-NO2 can respond to the
intracellular NTR, thereby producing strong fluorescent and optoacoustic signals.
The biosafety of NP-SPT-NO2 was evaluated by performing histological analysis (H&E
staining) for the sections from major organs (including lung, heart, spleen, liver and kidney) of
healthy mice upon treatment with the nanoprobe. As displayed in Figure S27, no obvious
difference between the group treated with NP-SPT-NO2 (1 mg kg-1) via i.v. injection and that
injected with PBS could be found, confirming that NP-SPT-NO2 is of good biosafety.
To prove the feasibility of using NP-SPT-NO2 to image tumor hypoxia, we first
explored the capability of the nanoprobe in fluorescent imaging and multispectral optoacoustic
tomography (MSOT) imaging of subcutaneous tumor in mice. As shown in Figure 2a, at 30 min
after i.v. injection of NP-SPT-NO2, weak fluorescence could be observed in abdominal region.
At 4 h post injection, the fluorescence in the tumor site (at right rear flank) was visible and then
reached maximum at around 24 h. The fluorescence was still visible in tumor site at 48 h post
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injection (Figure 2a and Figure S28). Figure 2a and Figure S29 show that, the activated probe
might mainly be cleared out of the body mainly via kidneys.
Figure 2. Fluorescence and MSOT imaging of mice bearing subcutaneous tumor. (a) Typical
fluorescence images of a tumor-bearing mouse at varied time upon intravenous injection of NP-
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SPT-NO2. Excitation filter: 675 nm; Emission Filter: 735 nm. (b) Cross sectional MSOT
images at tumor site for the same mouse at varied time upon injection of NP-SPT-NO2. Upper
panels (probe): multispectral resolved signals from the activated probe; Lower panels (merge):
over lay of activated probe’s signal with the grayscale single-wavelength (800 nm) background
image. (c) 3D MIP MSOT images covering the tumor region of the mice upon intravenous
injection of NP-SPT-NO2 for varied time. Scale bar: 5 mm.
We then examined MSOT imaging of the subcutaneous tumor using NP-SPT-NO2. Figure
2b shows the cross-sectional MSOT images at the tumor site at varied time upon i.v. injection of
NP-SPT-NO2(1 mg kg-1). The upper row in Figure 2b shows the multispectrally resolved MSOT
signal from the activated nanoprobe (NP-SPT-NH2); while the lower row displays the overlay of
the MOST signals (in color) from NP-SPT-NH2 with the single wavelength background image
(800 nm) serving as the anatomical reference (in grayscale). At 0 h, almost no MSOT signal of
the activated nanoprobe could be seen. At longer time the MSOT signal began to emerge and
steadily enhanced at the tumor site (Figure 2b and Figure S30). It can be seen that, the MSOT
signal at the tumor site was evident and remained visible at 48 h. With the MSOT system, the
cross-sectional images can be rendered as maximum intensity projection (MIP) images with
three-dimensional (3D) information. Figure 2c displays the 3D MIP images at different time after
i.v. injection of NP-SPT-NO2. Owing to the advantageous features of MSOT imaging such as
higher spatial resolution in depth, computed tomography, multispectral imaging and unmixing,
we could not only precisely locate the tumor in the body, but also obtain three dimensional and
detailed information of the tumor hypoxia. Compared to the fluorescent imaging, MSOT
produces images with much higher resolution and can reflect the tumor heterogeneity (Figure
2b and 2c). The above results indicate that the NP-SPT-NO2 can be used as an activatable
fluorescent/MSOT dual-mode probe for imaging tumor hypoxia by responding to NTR in vivo.
Furthermore, both the healthy and tumor-bearing mice were sacrificed at 12 h post
injection of the nanoprobe and their tumour and some organs were dissected for ex vivo
fluorescent and MSOT imaging (Figures S31 and S32). For the healthy mice, only weak
fluorescent or MSOT signals could be observed in metabolic organs such as kidneys and liver.
While for the tumor-bearing mice, strong fluorescent signal was found in the tumor, and weaker
fluorescence signals in kidneys and very weak signals in liver. The MSOT images show similar
pattern.
Lymphatic metastasis is one of the most common routes for the spread of cancer and form
initial metastasis at the regional lymph node, which is the first lymphatic node that tumor drains
and also known as sentinel lymphatic node (SLN). The lymphatic metastasis mouse model was
established via injection of 4T1 cells into the left rear footpad of the mice, followed by formation
of primary tumor at the footpad and spread of tumor cells to sentinel lymphatic node at popliteal
region (Scheme 1). This process was recorded by MSOT imaging and fluorescent imaging, as
shown in Figure 3. The cross-sectional MSOT images at footpad (primary tumor site) and
popliteal lymphatic node (the metastasis site) have been obtained and presented in Figure 3a and
3c, respectively. Figure 3b and Figure 3d show the cryosection images at the footpad site and the
popliteal position respectively, as the references for the locations of organs/tissues. At footpad
section, MSOT signal began to emerge at about day 10 post injection of tumor cells, and became
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prominent at day 21. While at the lymphatic node section, MSOT signal could be seen ca. 21
days post injection. This observation indicates that the tumor cells have spread to the popliteal
lymph node from the primary tumor at the footpad, which is supported by the recorded MSOT
intensity data shown in Figure 3e. The fluorescence images (Figure 3f) and intensity data (Figure
3g) also clearly indicate that, 21 days post injection of cancer cells at the footpad, the tumor cells
have spread into the popliteal lymph node via the lymphatic vessel. In addition, with 3D MIP
MSOT images (Figure 3h), we can observe the whole picture of lymphatic metastasis route from
the primary site at the footpad to the sentinel lymphatic node via a lymphatic vessel.
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Figure 3. MSOT and fluorescent imaging of tumor lymphatic metastasis by using the nanoprobe.
(a) Cross sectional MSOT images at paw position for the control (Day 0) and the mice after 10
or 21 days upon 4T1 cell injection at footpad. Probe: multispectral resolved signal from the
activated nanoprobe. Merge: overlay of the activated probe’s signal with the background signal
(grayscale). Scale bar: 5 mm. (b) A cryosection image of a female mouse with the cross section’s
location comparable to those shown in a. (c) Cross sectional MSOT images at popliteal position
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for the control and the mice after 10 or 21 days upon 4T1 cell injection. (d) A cryosection image
of a mouse with the cross section’s location comparable to that in c. (e) Mean MSOT intensities
at ROI (circled area) at different time. n = 5. Gray column: footpad, red column: lymph node.
Columns represent mean ± SD. The p-values (***p<0.001) were determined using two-sided
Student’s t-test. (f) Fluorescent images of the control and the mice at day 0, 10 and 21 after 4T1
cell injection. (g) Mean fluorescent intensity at ROI at different time. n = 5. Gray column: footpad,
red column: lymph node. Columns represent mean ± SD. The p-values (***p<0.001, **p<0.01)
were determined using two-sided Student’s t-test. (h) 3D MIP MSOT images for the control (Day
0) and for the mouse after 10 or 21 days upon injection of 4T1 cells into the footpad. Organ
labeling: 1: Primary tumor at the footpad; 2: Sentinel lymph node at the popliteal position; 3:
Lymphatic vessel. Scale bar: 5 mm. (i) Photographs of the mice showing the footpad and the
popliteal lymph node for the control and the mouse after 21 days upon injection of 4T1 cells into
the footpad. (j) Immunohistochemical images for CD206 in the popliteal lymph node section
from the control (Day 0) and the mouse after 21 days upon injection of 4T1 cells. Scale bar: 20
m.
After imaging, the mice were humanely sacrificed and their skin from paw to the popliteal
position was removed for photographing. As revealed in Figure 3i, the control (at day 0) shows
no sign of tumor invasion, while the tumor-bearing mouse at day 21 has swollen footpad and
popliteal lymph node. This observation verifies our imaging results. In addition, the CD206
levels at the popliteal lymphatic node for the control and the tumor-bearing mice were evaluated
by immunohistochemical analysis (IHC). As a macrophages mannose receptor, CD206 plays key
roles in tumor proliferation, invasion and metastasis, and high level of CD 206 is associated with
31
tumor invasion.[ ] As shown in Figure 3j, for the control (day 0), CD206 was normally expressed
in the section of the popliteal lymphatic node; while for the lymphatic metastasis model, CD206
is obviously over-expressed as evidenced by the large stained area in the tissue slice, indicating
the tumor cells have metastasized to the mouse’s popliteal lymph node and thus further proving
our imaging results.
Conclusion
In summary, an activatable dual-mode nanoprobe based on unsymmetrical squaraine has been
successfully designed and synthesized for imaging lymphatic metastasis in mouse model. This
nanoprobe can be activated by highly-expressed nitroreductase in tumor hypoxia, and generates
strong optoacoustic and fluorescent signals for imaging. Compared to other organic and
activatable optoacoustic probes, this unsymmetrical squaraine-based one exhibits much higher
molar extinction coefficient and produces strong optoacoustic signals. Moreover, the nanoprobe
formed by squaraine aggregates exhibited enhanced (rather than quenched) fluorescence
emission due to its AEE feature. The MSOT images have higher resolution than fluorescent ones
and can reflect the tumor heterogeneity. Through spectral unmixing and computed tomography
treatment by MSOT system, three-dimensional optoacoustic signals from the activated probe
were obtained for locating the tumor site and mapping the metastatic route. This approach may
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provide insights for designing other optoacoustic probes with AIE or AEE feature for diagnosing
and locating diseases, as well as offer new preclinical opportunity to assess disease evolution and
progression.
Acknowledgements
This work was supported by NSFC (21788102, 21875069, 51673066 and 21574044), the Natural
Science Foundation of Guangdong Province (2016A030312002) and the Fund of the Key
Laboratory of Luminescence from Molecular Aggregates of Guangdong Province
(2019B030301003).
Conflict of interest
The authors declare no conflict of interest.
Keywords: Unsymmetrical squaraine, aggregation-enhanced emission, activatable probe,
lymphatic metastasis imaging, multispectral optoacoustic tomography
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TOC graphical abstract:
An Unsymmetrical Squaraine-based Activatable Probe for Imaging Lymphatic
Metastasis through Responding to Tumor Hypoxia by Multispectral Optoacoustic
Tomography and Aggregation-enhanced Fluorescent Imaging
Yi Lin+, Lihe Sun+, Fang Zeng* and Shuizhu Wu*
An activatable nanoprobe based on unsymmetrical-squaraine has been developed for tumor
hypoxia imaging, which specifically responds to nitroreductase and produce strong optoacoustic
signals due to high extinction coefficient and prominent fluorescence emission because of AEE
feature. 3D MSOT signals could be obtained for locating tumor site and mapping metastatic route.
17
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