Calixarene-Based Supramolecular AIE Dots with Highly Inhibited Nonradiative Decay and Intersystem Crossing for Ultrasensitive Fluorescence Image-Guided Cancer Surgery

Article abstract of DOI:10.1002/anie.201916430

Host–guest complexation between calix[5]arene and aggregation-induced emission luminogen (AIEgen) can significantly turn off both the energy dissipation pathways of intersystem crossing and thermal deactivation, enabling the absorbed excitation energy to mostly focus on fluorescence emission. The co-assembly of calix[5]arene amphiphiles and AIEgens affords highly emissive supramolecular AIE nanodots thanks to their interaction severely restricting the intramolecular motion of AIEgens, which also show negligible generation of cytotoxic reactive oxygen species. In vivo studies with a peritoneal carcinomatosis-bearing mouse model indicate that such supramolecular AIE dots have rather low in vivo side toxicity and can serve as a superior fluorescent bioprobe for ultrasensitive fluorescence image-guided cancer surgery.

Full text of DOI:10.1002/anie.201916430

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
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Accepted Article
Title: Calixarene-Based Supramolecular AIE Dots with Highly Inhibited
Nonradiative Decay and Intersystem Crossing for Ultrasensitive
Fluorescence Image-Guided Cancer Surgery
Authors: Chao Chen, Xiang Ni, Han-Wen Tian, Qian Liu, Dong-Sheng
Guo, and Dan Ding
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201916430
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10.1002/anie.201916430
Angewandte Chemie International Edition
COMMUNICATION
Calixarene-Based Supramolecular AIE Dots with Highly Inhibited
Nonradiative Decay and Intersystem Crossing for Ultrasensitive
Fluorescence Image-Guided Cancer Surgery
Chao Chen+, Xiang Ni+, Han-Wen Tian, Qian Liu, Dong-Sheng Guo, and Dan Ding*
Abstract: Molecular motion-associated organic molecules such as
aggregation-induced emission luminogens (AIEgens) for disease
phototheranostics are attracting increasing attention; however, how
to optimally harvest absorbed excitation energy for advanced
fluorescence imaging/diagnosis and concurrently avoid unnecessary
phototoxicity during bioimaging process is still challenging. To
address this issue, herein, we report that the host-guest
complexation between calix[5]arene and AIEgen can significantly
turn off both the energy dissipation pathways of intersystem crossing
and thermal deactivation, enabling the absorbed excitation energy
mostly focusing on fluorescence emission. The co-assembly of
calix[5]arene amphiphiles and AIEgens affords highly emissive
supramolecular AIE nanodots thanks to their interaction extremely
restricting the intramolecular motion of AIEgens, which also show
negligible cytotoxic reactive oxygen species generation. In vivo
studies with a peritoneal carcinomatosis-bearing mouse model
indicate that such supramolecular AIE dots have rather low in vivo
side toxicity and can serve as a superior fluorescent bioprobe for
ultrasensitive fluorescence image-guided cancer surgery.
imaging and/or photodynamic therapy through generation of
reactive oxygen species (ROS); and 3) thermal deactivation
pathway via nonradiative decay for photothermal therapy and
photoacoustic imaging.[3] As the three dissipation pathways of
absorbed excitation energy are always competing, the optimized
efficacy of the required phototheranostic function can be realized
by suppression of the other pathways. As for molecular motion-
associated organic molecules, such photophysical property
manipulation is usually determined by molecular motion.
Taking AIEgens with rich intramolecular motion (e.g.,
rotation, vibration and twisting) units for example, effective
restriction of intramolecular motion (RIM) often leads to the
promotion of both fluorescence and ROS production by blocking
the thermal deactivation route.[4] On the other hand, permitting
free intramolecular motions make AIEgens an advanced
photothermal conversion material due to the inhibition of
radiative and ISC pathways.[5] Based on the aforementioned
principle, AIEgens are particularly useful for construction of
highly fluorescent nanoparticle bioprobe (termed as AIE dot) due
to the formation of intraparticle aggregation astricting the
intramolecular motion.[6] Nevertheless, for an overwhelming
majority of currently available AIE dots, relatively loose
intraparticle packing of AIEgens significantly compromises their
fluorescent brightness. Liu, Tang, Zhao, and coworkers reported
that crystallization of AIEgens resulted in maximum fluorescence
because of the optimally compact packing of AIEgens in crystal
minimizing intramolecular motion.[7] However, the crystals even if
the nanocrystals that can suspend in water are not suitable for
biomedical applications owing to the critical concerns in terms of
long-term colloidal stability in biological environments, blood
circulation time and uptake efficiency by disease site.
Furthermore, in most cases, the red/near-infrared (NIR) emissive
AIE dots are able to produce light-activated ROS owing to the
electronic donor (D)-acceptor (A) structure usually decreasing
the energy gap between the lowest singlet excited state and
triplet excited state (ΔEST),[8] which is decidedly adverse to
maximize the emission and causes unnecessary phototoxicity
during bioimaging process. Thereby, alternative strategies to
build AIE dots with similar restriction degree of intramolecular
motion to that in crystal, highly inhibitory ISC and excellent
biological accessibility are extremely desirable for advancing the
biomedicine field, which however have never been reported yet.
Calixarene, that is a kind of macrocyclic host,[9] has been
recently emerged as a promising building block to construct
supramolecular nanomedicines/imaging agents,[10] showing the
merits of multiple stimuli-responsiveness, numerous modification
tunability, and low cytotoxicity.[11] In this work, we first report that
the host-guest complexation between calix[5]arene and AIEgen
can considerably turn off both the pathways of ISC and thermal
deactivation, making utmost absorbed excitation energy used for
fluorescence emission. We started with the design and synthesis
of a series of AIEgens with D-π-A structure, followed by
fabrication of calix[5]arene-based supramolecular AIE dots (S-
Recently, the classes of molecular motion-associated organic
molecules including molecular machines, molecular motors and
aggregation-induced emission luminogens (AIEgens) are
appealing increasing interest in the area of biomedicine, since
they provide a new opportunity to achieve optimal theranostic
function and efficacy for diseases via facilely regulating the
molecular motions.[1] Jablonski diagram that elaborates the
molecular photophysics can explain the relationship between
molecular motion and phototheranostic function/efficacy.[2]
According to the Jablonski diagram, after an organic molecule
absorbs the excitation light, there are generally three energy
dissipation pathways, which are correspondent to different
phototheranostic functions: 1) fluorescence emission pathway
for fluorescence imaging/diagnosis; 2) intersystem crossing
(ISC) to a triplet excited state for phosphorescence afterglow
[*]
Dr. C. Chen,[+] Prof. D. Ding
Tianjin Key Laboratory of Oral and Maxillofacial Function
Reconstruction, Tianjin Stomatological Hospital, The Affiliated
Stomatological Hospital of Nankai University, Tianjin 300041, China
E-mail: dingd@nankai.edu.cn
Dr. C. Chen,[+] Dr. X. Ni,[+] Prof. D. Ding
State Key Laboratory of Medicinal Chemical Biology, Key
Laboratory of Bioactive Materials, Ministry of Education, and College
of Life Sciences, Nankai University, Tianjin 300071, China
H. W. Tian, Prof. D. S. Guo
College of Chemistry, State Key Laboratory of Elemento-Organic
Chemistry, Key Laboratory of Functional Polymer Materials, Ministry
of Education, Nankai University, Tianjin 300071, China
Dr. Q. Liu
Department of Urology, Tianjin First Central Hospital, Tianjin
300192, China
These authors contributed equally to this work.
[+]
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201916430
Angewandte Chemie International Edition
COMMUNICATION
AIE dots) using carboxylic acid-modified calix[5]arene
pentadodecyl ether (CC5A-12C).[12] It is demonstrated that in the
presence of CC5A-12C, both the intramolecular motion and ISC
transition (ROS generation) of AIEgens can be tremendously
restricted, thus leading to ultrabright S-AIE dots in water. Finally,
we assessed the in vivo toxicity of such S-AIE dots and studied
their feasibility and advantage in practical biomedical application
in terms of fluorescence image-guided cancer surgery.
distribution are helpful in decreasing their ΔEST and thus
boosting ISC and ROS generation efficiency.[8,14]
Next, as illustrated in Figure 1C and Figure S10, pegylated
calix[5]arene-based S-AIE dots were prepared by
nanoprecipitation method using CC5A-12C and
a
4-
(dodecyloxy)benzamido-terminated methoxy poly(ethyleneglycol)
(PEG-12C) as the matrix of nanocarriers. The AIEgens, 1~4,
respectively, were loaded via the host-guest complexation
between calix[5]arene receptor and each AIEgen. The
hydrophilic PEG chains can extend to the water, serving as the
protecting outer layer to stabilize the S-AIE dots. The dynamic
light scattering (DLS) and transmission electron microscopy
(TEM) data depict that the 1~4-loaded S-AIE dots are all
spherical in shape with similar mean hydrodynamic diameters of
~150 nm (Figure 1D and Figure S11).
A
B
LUMO
1
3
4
HOMO
2
C
D
100
CC5A-12C
80
60
40
20
Co-assembly
200 nm
extremely
effective
S-AIE dot
RIM of
AIEgen
100 200 300 400 500 600
to some extent
Diameter (nm)
AIEgen
Brightness
PEG-12C
Co-assembly
DSPE-PEG
DSPE-PEG-AIE dot
Figure 1. (A) Chemical structures of the AIEgens. (B) The electron density
distribution of frontier orbitals of 1. (C) Schematic of S-AIE dots and DSPE-
PEG-AIE dots and chemical structure of CC5A-12C. (D) DLS profile and TEM
image (inset) of 1-loaded S-AIE dots.
Four positively charged AIEgens were synthesized and used
to assess the generality of our approach (Figure 1A).
Compounds 2~4 were synthesized according to the previous
literature.[13,14] Figure S1 shows the synthetic route to compound
1 mainly through Knoevenagel condensation and methylation
with CH3I. The intermediates and final compound were
characterized by standard spectroscopic techniques (Figure S2-
Figure 2. (A) Photoluminescence (PL) spectra of 1-loaded S-AIE dots and
DSPE-PEG-AIE dots in water (λex: 445 nm). (B) Plot of ln(A0/A) against light
irradiation time. A0 and A represent the ABDA absorbance at 378 nm without
and with exposure to white light, respectively. (C) The steady-state PL spectra
of 1-loaded S-AIE dots at room temperature and frozen in liquid nitrogen. (D)
The phosphorescence (Phos) spectra, acquired after 10 ms delay, of 3-loaded
DSPE-PEG-AIE dots and S-AIE dots frozen in liquid nitrogen.
S7).
In
our
molecular
design,
cyanostilbene/cyanostyrylthiophene acts as the AIE skeleton
and π-bridge to build D-π-A structure (pyridinium salt as A). All 4
compounds exhibit prominent AIE effect (Figure S8).[14]
The photophysical properties of the as-prepared S-AIE dots
were investigated. As a positive control, the most widely used
AIE dot doping matrix, 1,2-distearoyl-sn-glycero-3-phosphoeth-
As shown in Figure 1B and Figure S9, density functional
theory (DFT) calculations reveal that compared with 2 and 4, 1
and 3 with crowded TPE unit have much more twisted molecular
structures, which are beneficial to reduced intermolecular
interactions such as π-π stacking, contributing to stronger AIE
activity. The study on electronic structures in the ground state
(S0) at the level of B3LYP/6-31G (d,p) indicates that all the
molecules exhibit charge separation characteristic, with the
highest occupied molecular orbital (HOMO) electron density
majorly distributed on the electron rich segments and the lowest
unoccupied molecular orbital (LUMO) electron density mainly
distributed on the electron poor moieties. Noteworthy is that for
both 1 and 3, the more effective separations of HOMO-LUMO
anolamine-N-[methoxy(polyethylene
glycol)-2000]
(DSPE-
PEG)[4,6] were also employed to formulate each AIEgen into
DSPE-PEG-AIE dots (Figure 1C). The absorption spectra as
displayed in Figure S12 indicate that the 1~4-loaded S-AIE dots
have an absorption maximum at 442, 440, 478, and 462 nm,
respectively, whereas the 1~4-loaded DSPE-PEG-AIE dots
show an around 10 nm red-shifted absorption when compared
with each corresponding S-AIE dots. Figure S13 displays the
emission spectra of 1~4-loaded S-AIE dots in water, respectively,
at different mass ratios of CC5A-12C to each AIEgen. When
compared with each AIEgen itself in water (mass ratio = 0:1), the
fluorescence intensity significantly increases with the increase of
CC5A-12C amount, which nearly reaches to the plateau at mass
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10.1002/anie.201916430
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ratio = 9:1. As shown in Figure 2A and Figure S14, at the same
experimental condition, the maximum fluorescent intensities of
1~4-loaded S-AIE dots in aqueous solution are ~31.5, ~19.3,
~10.6, and ~12.5 times higher than the corresponding 1~4-
loaded DSPE-PEG-AIE dots, respectively, accompanied with
obvious blueshifts of emission peaks. These results reveal that
the host-guest complexation can largely boost the emission
(mainly ranging from 500 to 750 nm) of AIEgens. As a
intramolecular twisting between D and A inhibiting twisted
intramolecular charge transfer (TICT) process. We next
optimized the structures of 1 and CC5A-12C in the S0 state,
respectively, at the ωB97xD/6-31G(d,p) level in Gaussian 09
package, which indicate that 1 locates on the outside of
calix[5]arene while 1 and CC5A-12C align through a quasi-
antiparallel mode. The distance between 1 and CC5A-12C is
quite close which helps rigidify the intramolecular motion of 1
(Figure S16). These results together reasonably verify the
extremely efficient RIM in S-AIE dots, leading to vitally inhibitory
thermal deactivation pathway.
consequence,
using
4-(dicyanomethylene)-2-methyl-6-(p-
dimethylaminostyryl)-4H-pyran in methanol (quantum yield (QY)
= 0.43) as the standard, the photoluminescence (PL) QYs of
1~4-loaded S-AIE dots in aqueous solution are determined to be
0.72, 0.50, 0.23, and 0.16, respectively. To our knowledge, QY
of 0.72 in water represents one of the highest values among
various red-fluorescent materials.[6,15]
The ROS generation capacities of 1~4-loaded S-AIE dots
and DSPE-PEG-AIE dots in aqueous media were studied and
compared, as all of them are not phosphorescent at room
temperature.
Using
9,10-anthracenediyl-bis(methylene)
dimalonic acid (ABDA) as the ROS indicator,[14] it is found that
CC5A-12C greatly diminishes the light-activated ROS
generation abilities of all 4 AIEgens (Figure 2B and Figure S15).
For instance, upon white light irradiation, the ABDA
decomposition rate constant for 1-loaded DSPE-PEG-AIE dots
is as high as 0.00557 s-1, determined by the slope of the plot in
Figure 2B, while such constant for 1-loaded S-AIE dots is
negligible and close to zero. This result suggests that the host-
guest complexation between calix[5]arene and AIEgen would
significantly suppress the ISC pathway.
To deeply understand the difference in photophysical
property between S-AIE dots and DSPE-PEG-AIE dots,
fluorescence anisotropy study was firstly performed with the
widely used 1,6-diphenyl-1,3,5-hexatriene (DPH) as the probe.
The DPH emission is polarized upon the excitation with
polarized light. The extent of polarization depends on the
orientation of DPH relative to the direction of the polarized
excitation, while the rotational motion of DPH causes
depolarization. The more viscous the microenvironment is, the
larger restriction extent of the rotational motion will be provided,
Figure 3. Optimized molecular structures and calculated energy diagrams of
(A)
1 and (B) "1+CC5A-12C" complex. (C) Schematic of 3 dissipation
pathways of the absorbed excitation energy for different AIE dots, which are
likened to 3 water taps. FE: fluorescence emission; TD: thermal deactivation.
which leads to a less depolarization and thus
a higher
fluorescence anisotropy value.[16] It is found that for 1-loaded
DSPE-PEG-AIE dots, the fluorescence anisotropy value of DPH
is determined to be 0.219. Interestingly, for 1-loaded S-AIE dots,
the fluorescence anisotropy value reaches up to 0.392, which is
nearly equal to the intrinsic anisotropy of DPH (0.395).[17] The
extremely high polarization of DPH fluorescence indicates that
the intramolecular motion of 1 in the presence of CC5A-12C can
be thoroughly restricted. Furthermore, the steady-state PL
spectra of 1-loaded S-AIE dots at room temperature and frozen
in liquid nitrogen were measured. As displayed in Figure 2C,
although the freezing effect of liquid nitrogen is able to further
restrict the intramolecular motion of AIEgen in theory, the
maximum fluorescence intensity is hardly changed before and
after decreasing the temperature of AIE dot solution, implying
that the intramolecular motion of 1 has already been suppressed
effectively by CC5A-12C even at room temperature. In addition,
the emission peaks of S-AIE dots are significantly blue-shifted
when compared with those of DSPE-PEG-AIE dots (Figure 2A
and Figure S14), revealing the effectively restricted
Besides ROS generation, the influence of CC5A-12C on ISC
transition was also studied through measurement of low
temperature phosphorescence of both S-AIE dots and DSPE-
PEG-AIE dots. As depicted in Figure 2D and Figure S17, 3- and
4-loaded AIE dots possess low temperature phosphorescence
when frozen in liquid nitrogen. Noteworthy is that the
phosphorescence intensities of S-AIE dots are much lower than
those of DSPE-PEG-AIE dots, indicating the reduced ISC
transition by CC5A-12C. Since ΔEST correlates closely with the
ISC process,[8] we calculated the energy levels of singlet and
triplet excited states based on the structures at S0,min of 1 and
"1+CC5A-12C" complex. The results in Figure 3A,B demonstrate
that the ΔEST of the complex is much larger than that of 1 itself.
The increased ΔEST value is probably attributed to the structure
rigidification of 1 by the multiple intermolecular interactions
between 1 and CC5A-12C. As a result, if we liken the three
dissipation pathways of the absorbed excitation energy to three
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10.1002/anie.201916430
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water taps, the host-guest complexation between calix[5]arene
and AIEgen significantly turns off the taps of ISC and thermal
deactivation, enabling the "water" (absorbed excitation energy)
mostly flow out as the fluorescence (Figure 3C).
(Figure S23). Encouragingly, the SBR[21] (tumor-to-surrounding
normal intestine) for S-AIE dot-treated mice is determined to be
48.5 ± 5.6, which represents the highest among various
fluorescent probes for FIGCS via intravenous injection, to the
best of our knowledge. In comparison, another group of tumor-
bearing mice were intravenously injected with 100 μL of 1-
loaded DSPE-PEG-AIE dots with the same concentration of 1 as
that of S-AIE dots. Although DSPE-PEG-AIE dots are also able
to accurately outline the intraperitoneal metastatic tumors by
fluorescence, the SBR value (8.4 ± 2.1) is far lower than that
achieved by S-AIE dots (Figure 4D). Furthermore, the data in
Figure S24 and S25 prove that the S-AIE dots give superb
performance in FIGCS during surgery, considerably improving
the outcome of tumor removal operation.
In summary, we have introduced an alternative class of
fluorescent probes with ultrahigh brightness by taking advantage
of AIE property and host-guest complexation between
calix[5]arene and AIEgen. When compared with currently
available fluorescent bioprobes including AIEgen-based ones,
the uniqueness of calix[5]arene-based S-AIE dots includes: 1)
providing one of the very limited examples of AIEgen-based
bioprobes with the intramolecular motion of AIEgen close to
complete restriction; 2) both the pathways of ISC and thermal
deactivation are concurrently inhibited by calix[5]arene, leading
to the absorbed excitation energy mostly focusing on
fluorescence emission. This is not achievable by previous
reported AIEgen-based probes; 3) as a result, the 1-loaded S-
AIE dots have an ultrahigh PL QY of 0.72, which is one of the
highest values among various red-fluorescent materials in water;
and 4) the 1-loaded S-AIE dots can realize in vivo SBR (tumor-
to-normal tissue) of ~48.5, which represents the highest among
various fluorescent probes for FIGCS via intravenous injection.
Moreover, the compromised ROS production of S-AIE dots is
beneficial to avoid unnecessary phototoxicity during bioimaging.
Thereby, this study demonstrates that calixarene is an ideal
platform to develop advanced AIE bioprobes.
Figure 4. (A) Typical fluorescence imaging (FLI) and (B) bioluminescence
imaging of the intraperitoneal tumor nodules from mice after intravenous
injection of 1-loaded S-AIE dots for 24 h. (C) Typical FLI of 1-loaded DSPE-
PEG-AIE dot-treated mice at 24 h post-injection. (D) SBR based on the FLI in
(A) and (C). ** P < 0.01, in comparison between two types of AIE dots.
The 1-loaded S-AIE dots were selected for the next
biomedical application due to the ultrahigh PL QY. After
validating that the S-AIE dots have rather low cytotoxicity and in
vivo side toxicity (Figure S18-S21), their application in
fluorescence image-guided cancer surgery (FIGCS) was
performed. Very recently, FIGCS has received great attention
and applied in the clinic, as it can essentially help the surgeon
improve the outcome of tumor removal operation.[18]
Development of bright fluorescent probes with rather high signal-
to-background ratio (SBR) is in urgent pursuit.[19] To estimate the
utility of our S-AIE dots in FIGCS, a peritoneal carcinomatosis-
bearing mouse model was established by intraperitoneal
injection of luciferase-expressed murine 4T1 cancer cells. Such
tumor-bearing mouse model has two characters: 1) there are
many tumor nodules including tiny ones with diameters < 1 mm
dispersed in the abdominal cavity; and 2) the tumor
nodules/cells can emit bioluminescence upon injection of D-
luciferin by virtue of the interaction between luciferase and D-
luciferin, allowing for precise tumor localization.
Acknowledgements
This work was supported by the NSFC (51873092 and
51873090), and the Fundamental Research Funds for the
Central Universities, Nankai University.
Keywords: aggregation-induced emission • calixarene •
supramolecular nanoparticle • photophysical property •
fluorescence image-guided cancer surgery
The tumor-bearing mice were intravenously injected with 1-
loaded S-AIE dots (100 μL, 0.25 mg mL-1 based on 1), which
was followed by opening the mouse abdomen at 24 h post-
administration. The mice were then imaged during operation
using IVIS® in vivo fluorescence imaging system. As displayed in
Figure 4A and Figure S22, the S-AIE dots are capable of
significantly accumulating into and lighting up intraperitoneal
tumor nodules through remarkable enhanced permeability and
retention (EPR) effect,[20] confirmed by the good colocalization
with tumor bioluminescence post D-luciferin injection (Figure 4B)
and the hematoxylin and eosin (H&E) histological analysis
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This article is protected by copyright. All rights reserved.

10.1002/anie.201916430
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
A series of calix[5]arene -based
supramolecular AIE nandots were
synthesized with rather high quantum
yields in water by virtue of the host-
guest complexation making the
absorbed excitation energy mostly
focusing on fluorescence emission,
which thus realized ultrahigh signal-to-
background ratio in fluorescence
image-guided cancer surgery.
Chao Chen+, Xiang Ni+, Han-Wen Tian,
Qian Liu, Dong-Sheng Guo, and Dan
Ding*
Page No. – Page No.
Calixarene-Based Supramolecular
AIE Dots with Highly Inhibited
Nonradiative Decay and Intersystem
Crossing for Ultrasensitive
Fluorescence Image-Guided Cancer
Surgery
This article is protected by copyright. All rights reserved.