Wash-free 3D imaging and detection of glioma with a novel neuropotential targeted AIE probe

Article abstract of DOI:10.1039/d0cc07289c

We herein developed a novel tetraarylimidazole-based AIE probe TPIG-NP to selectively image and quantitatively detect glioma. Due to the distinct negatively charged glioma cells, TPIG-NP with an opposite charge could achieve wash-free imaging of glioma cells and 3D multicellular spheroids. This journal is

Full text of DOI:10.1039/d0cc07289c

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Wash-free 3D imaging and detection of glioma
with a novel neuropotential targeted AIE probe†
Cite this: Chem. Commun., 2021,
57, 801
Junyong Wu,‡a Anyao Bi,‡b Fan Zheng,b Shuai Huang,b Yongjiang Li,a
b
b
Daxiong Xiang*a and Wenbin Zeng
*
Received 4th November 2020,
Accepted 11th December 2020
Jipeng Ding,
DOI: 10.1039/d0cc07289c
rsc.li/chemcomm
We herein developed a novel tetraarylimidazole-based AIE probe concentration solution or in the aggregate state, most of the
TPIG-NP to selectively image and quantitatively detect glioma. Due conventional fluorophores are either weakly emissive or non-
to the distinct negatively charged glioma cells, TPIG-NP with an emissive, which has been known as the aggregation-caused
opposite charge could achieve wash-free imaging of glioma cells quenching (ACQ) effect. Tang et al., in Chem. Commun., 2001,
and 3D multicellular spheroids.
18, 1740, reported a novel class of fluorophores with aggregation-
induced emission (AIE) properties that solve the problems of the
Gliomas comprise a group of heterogeneous primary brain ACQ effect.12 AIE luminogens (AIEgens) show little fluorescence in
neoplasms, accounting for 81% of central nervous system the dissolved state and significantly enhanced fluorescence in
(CNS) malignancies.1,2 They can be further categorized into aggregates. It is based on the AIE characteristics that this type of
four grades based on the classification of the World Health probes is able to self-assemble into nanoparticles and can be used
Organization (WHO). The first two grades indicate low-grade for wash-free cell imaging as well.
glioma (LGG), while the latter two are high-grade glioma
Recently, our group has focused on the design of AIE probes
(HGG).3 Typically, a relatively high grade is related to a poor and built a tetraarylimidazole AIE parent nucleus TPI.13,14
prognosis. The median survival time of LGG is 11.6 years, Herein, in this study, we report a novel terpyridyl-based deri-
whereas glioblastoma (GBM), the most common type of grade vative nanoprobe TPIG-NP for the wash-free imaging of glioma
4 gliomas, has a median overall survival time of only 1.2 cells and tissues (Scheme 1). As the electrical potential of
years.4,5 Due to the diffuse dissemination of glioma cells, glioma cells is significantly lower than those of the other
complete excision of all tumour regions is almost impossible, tumour cells and normal brain cells, TPIG-NP with a positive
resulting in poor prognosis. Therefore, imaging is often charge can selectively be absorbed and enriched by glioma cells
required for the detection of glioma infiltration during surgery. through electrostatic attraction. High fluorescent signals with
Advances in various imaging technologies, such as ultra-
sound, computed tomography (CT), and magnetic resonance
imaging (MRI), have made great contributions in glioma
detection.6,7 Compared with these techniques, detection using
fluorescent probes possesses the ability of highly sensitive
imaging and real-time visualization so that it can precisely
visually distinguish glioma from the normal brain tissues more
easily.8–11 However, to achieve a high signal-to-background
ratio, a washing step is often required after incubation with
the probes to reduce the background fluorescence, which is not
suitable for monitoring glioma in situ. Meanwhile, with a high
a Department of Pharmacy, The Second Xiangya Hospital, Central South University,
Changsha, 410011, China. E-mail: xiangdaxiong@csu.edu.cn
b Xiangya School of Pharmaceutical Sciences, Central South University,
Changsha, 410013, P. R. China. E-mail: wbzeng@hotmail.com
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
Scheme 1 Schematic illustration of a self-assembled nanoprobe (TPIG-NP)
d0cc07289c
to exert imaging of glioma.
‡ These authors contributed equally to this work.
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low background interference are then observed to image every indicating its potential of application in neural potential
single cell. In addition, the imaging capacity of the probe is also activity in biological events.21
approved in the 3D multicellular spheroids of glioma cells and
Before applying TPIG-NP to image living cells, for safety
in brain tissues as well.15–19 No washing steps are required to concerns, we first evaluated its cytotoxicity against brain-
remove any probes in this process, making TPIG-NP ideal for derived endothelial cells.3 (bEnd.3) and human glioma cells
glioma assays in vitro and in vivo. It is hoped that TPIG-NP can (U87-MG, HS683, and T98 cells). As depicted in Fig. 1, TPIG-NP
further provide a robust sensing platform for guiding the had little effect on the cell viability against the tested cell lines,
precise resection of glioma and improving the prognosis of making this nanoprobe a biocompatible probe for further cell
patients.
TPIG was synthesized via multiple steps as described in
studies (Fig. 2).
Encouraged by the right results in the MTT assay, we further
Scheme S1 (please see the ESI†). The AIE characteristic of TPIG explored its imaging capability towards different cells. Distin-
was measured as shown in Fig. S3 (see the ESI†). The formation guishing tumour cells from the surrounding normal cells is the
of monodisperse NPs was verified by transmission electron primary condition for precise surgical navigation. However, due
microscopy (TEM) analysis (see Fig. S5, ESI†). The optical to the infiltrative nature of malignant glioma, it was difficult to
properties of TPIG-NP are shown in Fig. S1–S3, ESI.†
precisely visually differentiate the boundary of glioma. Alter-
To fully evaluate the performance of different functionals in natively, in order to investigate the potential of TPIG-NP in
modelling the TICT formations, we calculated the S0 PbE0, the distinguishing glioma cells from the normal brain cells, bEnd.3
S1 de-excitation energies (vertical emissions) and resulting S1 and other three glioma cells HS683,T98 and U251 were treated
PbE0 in DMSO based on the optimized S1 molecular structures with 10 mM TPIG-NP for 2 h. As can be seen from Fig. 3, little
of TPIG. We also performed hole-electron analysis calculations signal can be recorded in bEnd.3 upon excitation at 430 nm,
on TPIG as a benchmark (Fig. 1a). Hole-electron analysis has while obvious yellow–green fluorescence was observed in
been proved to be a reliable approach to including electron– HS683 cells.
hole effects and intramolecular charge transfer excitations
Meanwhile, the imaging region was evaluated with a co-
(ICTs). It is clearly to find that the flow of electron in the localization experiment in HS683 cells, in which DAPI, a
exciting state of TPIG in holes is from the donor to the acceptor, commercially available nucleus tracker, was used as a refer-
demonstrating a typical ICT effect. It was reported that the ence. HS683 cells were incubated with TPIG-NP (10 mM) at 37 1C
crystal structure of the simple fluoresce in comprises a network for 2 h, followed by incubation with DAPI (2 mg mlÀ1) under the
of intermolecular avoid face-to-face p–p stacking.20 No effective same conditions. The merged image showed that the yellow–
face-to-face p–p stacking interactions can be observed due to green fluorescence from the probe hardly colocalized with the
the steric spatial repulsion of the benzene group which was blue signal from DAPI (Fig. 3), illustrating that TPIG-NP had a
conjuncted to imidazole. Structure analyses revealed that the good ability to stain the cell membrane and the cytoplasm of
restriction of the face-to-face p–p stacking interactions of TPIG glioblastoma cells. Next, another two glioma cell lines (T98 and
could be an effective approach to AIE. Electrostatic potential U87-MG) were also treated with TPIG-NP for 2 h, respectively.
(ESP) attracted ongoing studies in the biological and medicinal Their wash-free fluorescence images reflected the same
chemistry literature because it can unravel the charge distribu- phenomena as in HS683 cells (Fig. 3), proving TPIG-NP as an
tion of chemical molecules, which plays an important role in effective imaging probe for glioma cells.
biological cases. After calculation analysing to the ESP of TPIG
(Fig. 1b), the whole molecule possessed a positive charge,
Fig. 1 (a) Distribution of holes and electrons of states calculated at PBE0/
def2-TZVP in DMSO levels of theory in vacuo. (b). Electrostatic potential of
TPIG.
Fig. 2 (1). bEnd.3, (2). U87-MG cells, (3). U251 cells, (4). T98 cells, and (5).
HS683 cells were separately treated with TPIG-NP for 4 h initially, and then
cultured with a fresh medium for 72 h.
802 | Chem. Commun., 2021, 57, 801--804
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cells. These results intuitively reflected a significant difference
of fluorescence between bEnd.3 and glioma tumour cells
through the use of TPIG-NP. This further revealed that TPIG-
NP could specifically image glioma cells among the brain cells
which made it possible to apply TPIG-NP in auxiliary surgical
resection of glioma. We hypothesized that it was the electro-
static attraction between the glioma cells and the probe that
caused the highly selective imaging of TPIG-NP towards
glioma cells.
Considering the results obtained from cell staining and flow
cytometry assays, we then investigated the potential of the
brain cells to find out the reason for the difference between
the fluorescence imaging in the treatment groups. As depicted
in Fig. 4b, the glioma cells (U87-MG, T98, U251, and HS683
cells) obviously possessed more negative cell potentials than
bEnd.3 cell. These results implied that the higher electrostatic
attraction of TPIG-NP with glioma induced highly selective
imaging of the probe.
As oxygen concentration gradients always exist in the
350–500 mm 3D multicellular spheroids (MCSs), it makes the
3D MCSs the ideal models for the simulation of solid tumours
at early stages. In this work, 10 mM of TPIG-NP was used for the
MCSs of U87-MG cells which were developed for further eval-
uating the penetrative effect of the probe. After 2 h of incuba-
tion, strong yellow–green fluorescence signals were detected
from the U87-MG MCSs (Fig. 5), indicating that TPIG-NP could
easily penetrate into the sphere. Since the higher amount of the
extracellular matrix components hampered the extracellular
flow of the probe, the fluorescence of the outer layers of the
spheroids was brighter, while lower signals were collected from
the core part. These results confirmed that TPIG-NP had the
potential to image glioma tumours.
Fig. 3 Images of bEnd.3, U87-MG, T98 and HS683, respectively, treated
with 10 mM TPIG-NP and DAPI (2 ug mlÀ1) for 2 h by collecting the
emissions at 530 nm upon excitation at 430 nm. Scale bar: 30 mm.
In order to further accurately identify and quantitatively
detect the cells stained by TPIG-NP, we decided to combine
flow cytometry (FCM) with fluorescence imaging which would
be a more sensitive approach to characterize the probe in
different cells. This could also eliminate some of the limita-
tions demonstrated by conventional FCM, also providing mor-
phological confirmation and distinguishing ability on brain
cells as well. The bEnd.3 and glioma tumour cells were all
treated with 10 mM TPIG-NP for 2 h, before FCM was per-
formed. As shown in Fig. 5a, obviously, the mean fluorescence
intensity (MFI) of bEnd.3 cell is 242, indicating that it had
almost no fluorescence signal. On the other hand, as shown in
Fig. 4a, all glioma cells (2, 3, 4, and 5) had nearly 10 fold (about
2 Â 103 of MFI from FCM), in which high fluorescence signals
were observed indicating the show-up of a great deal of glioma
To further evaluate the imaging performance of TPIG-NP
in vivo, an orthotopic glioma mouse model was established
through the injection of U87-MG cells into the brain (Fig. 6).
T1-weighted magnetic resonance imaging (MRI) was applied to
monitor the growth of glioma tumours. After 7 days of tumour
Fig. 4 (a) Accurate quantitative cell staining by flow cytometry, and each
cell population was gated based on the intensity of SSC. ((1). bEnd.3, (2).
U87-MG, (3). U251, (4). T98, (5). HS683). (b). The fluorescence intensity of
various brain cell potentials. p o0.001.
Fig. 5 Imaging of TPIG-NP in 3D glioma cell multicellular spheroids.
((a): U87-MG, (b): U251, (c): T98, (d): HS683.) Scale bar: 50 mm.
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Hunan Provincial Science and Technology Plan (2016TP2002),
the Science and Technology Foundation of Hunan Province
(2020SK3019, 2019SK2211 and 2019GK5012), and the Funda-
mental Research Funds for the Central Universities of Central
South University (2018zzts041 and 2018dcyj067).
Conflicts of interest
The authors declare no competing financial interest.
Notes and references
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804 | Chem. Commun., 2021, 57, 801--804
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