Corannulene-Incorporated AIE Nanodots with Highly Suppressed Nonradiative Decay for Boosted Cancer Phototheranostics In Vivo

Article abstract of DOI:10.1002/adma.201801065

Fluorescent nanoparticles (NPs) based on luminogens with aggregation-induced emission characteristic (AIEgens), namely AIE dots, have received wide attention because of their antiquenching attitude in emission and reactive oxygen species (ROS) generation when aggregated. However, few reports are available on how to control and optimize their fluorescence and ROS generation ability. Herein, it is reported that enhancing the intraparticle confined microenvironment is an effective approach to advanced AIE dots, permitting boosted cancer phototheranostics in vivo. Formulation of a “rotor-rich” and inherently charged near-infrared (NIR) AIEgen with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] and corannulene-decorated PEG affords DSPE-AIE dots and Cor-AIE dots, respectively. Compared to DSPE-AIE dots, Cor-AIE dots show 4.0-fold amplified fluorescence quantum yield and 5.4-fold enhanced ROS production, because corannulene provides intraparticle rigidity and strong interactions with the AIEgen to restrict the intramolecular rotation of AIEgen to strongly suppress the nonradiative decay and significantly facilitate the fluorescence pathway and intersystem crossing. Thus, it tremendously promotes phototheranostic efficacies in terms of NIR image-guided cancer surgery and photodynamic therapy using a peritoneal carcinomatosis-bearing mouse model. Collectively, it not only provides a novel strategy to advanced AIE dots for cancer phototheranostics, but also brings new insights into the design of superior fluorescent NPs for biomedical applications.

Full text of DOI:10.1002/adma.201801065

CommuniCation
Cancer Theranostics
www.advmat.de
Corannulene-Incorporated AIE Nanodots with Highly
Suppressed Nonradiative Decay for Boosted Cancer
Phototheranostics In Vivo
Xinggui Gu, Xiaoyan Zhang, Huili Ma, Shaorui Jia, Pengfei Zhang, Yanjun Zhao, Qian Liu,
Jianguo Wang, Xiaoyan Zheng, Jacky W. Y. Lam, Dan Ding,* and Ben Zhong Tang*
Development of optical agents for cancer
Fluorescent nanoparticles (NPs) based on luminogens with aggregation-
induced emission characteristic (AIEgens), namely AIE dots, have received
wide attention because of their antiquenching attitude in emission and
reactive oxygen species (ROS) generation when aggregated. However, few
reports are available on how to control and optimize their fluorescence and
ROS generation ability. Herein, it is reported that enhancing the intrapar-
ticle confined microenvironment is an effective approach to advanced AIE
dots, permitting boosted cancer phototheranostics in vivo. Formulation
of a “rotor-rich” and inherently charged near-infrared (NIR) AIEgen with
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] and corannulene-decorated PEG affords DSPE-AIE dots and
Cor-AIE dots, respectively. Compared to DSPE-AIE dots, Cor-AIE dots
show 4.0-fold amplified fluorescence quantum yield and 5.4-fold enhanced
ROS production, because corannulene provides intraparticle rigidity and
strong interactions with the AIEgen to restrict the intramolecular rotation
of AIEgen to strongly suppress the nonradiative decay and significantly
facilitate the fluorescence pathway and intersystem crossing. Thus, it
tremendously promotes phototheranostic efficacies in terms of NIR
image-guided cancer surgery and photodynamic therapy using a peritoneal
carcinomatosis-bearing mouse model. Collectively, it not only provides
a novel strategy to advanced AIE dots for cancer phototheranostics, but
also brings new insights into the design of superior fluorescent NPs for
biomedical applications.
phototheranostics has recently attracted great
interest as they allow for real-time molecular
diagnosis and concurrent light-triggered treat-
ment.[1–8] Among various phototheranostic
agents, fluorescent nanoparticles (NPs) are
receiving broader attention due to their com-
bined merits in terms of high sensitivity and
temporal resolution of fluorescence imaging
technique, on-demand and in situ signature
of photodynamic therapy (PDT) as well as
unique enhanced permeability and retention
(EPR) effect of nanomaterials.[9–14] To meet
the requirements of ideal cancer photothera-
nostics, the fluorescent NPs must have sev-
eral necessary qualities: 1) sufficiently high
near-infrared (NIR) emission (>650 nm) and
reactive oxygen species (ROS) generation
efficiency of fluorescent component within
NPs; 2) strong resistance to photobleaching;
3) negligible cytotoxicity and in vivo toxicity;
and 4) suitable NP size and surface chemi-
stry,[15] permitting prominent EPR effect.
Compared with other extensively investi-
gated fluorescent NPs, organic fluorophore-
doped NPs hold the advantages of tunable
photophysical properties, flexible structural
tailoring, and good biocompatibility.[16] How-
ever, these π-conjugated fluorophores tend to
Prof. X. Gu, Dr. J. Wang, P. Zhang, Dr. X. Zheng, Dr. J. W. Y. Lam,
Prof. B. Z. Tang
X. Zhang, S. Jia, Prof. D. Ding
State Key Laboratory of Medicinal Chemical Biology
Department of Chemistry
Key Laboratory of Bioactive Materials
Ministry of Education
and College of Life Sciences
Nankai University
Hong Kong Branch of Chinese National Engineering Research Center
for Tissue Restoration and Reconstruction
Division of Life Science
State Key Laboratory of Molecular Neuroscience
Institute for Advanced Study
Tianjin 300071, China
E-mail: dingd@nankai.edu.cn
and Division of Biomedical Engineering
The Hong Kong University of Science and Technology
Clear Water Bay, Kowloon, Hong Kong, China
E-mail: tangbenz@ust.hk
Dr. H. Ma
Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced
Materials (IAM)
Nanjing Tech University (Nan-jingTech)
30 South Puzhu Road, Nanjing 211800, China
Prof. X. Gu
Beijing Advanced Innovation Center for Soft Matter Science and Engineering
Beijing University of Chemical Technology
Beijing 100029, China
Dr. Y. Zhao
School of Pharmaceutical Science & Technology
Tianjin Key Laboratory for Modern Drug Delivery & High Efficiency
and Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin)
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adma.201800403.
Tianjin University
Tianjin 300072, China
DOI: 10.1002/adma.201801065
©
1801065 (1 of 9)
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2018, 1801065

www.advancedsciencenews.com
www.advmat.de
aggregate within NPs. For conventional small-molecule fluores-
cent dyes with planner molecular structures, such aggregation
within NPs often causes significant quenching of light emis-
sion and ROS production owing to intermolecular interactions
such as π–π stacking and other nonradiative decays, which
tremendously limit their application in cancer phototheranos-
tics.[17,18] Much effort has been devoted to overcome the aggre-
gation-caused quenching (ACQ) effect within fluorescent NPs
through the introduction of bulky side groups and hydrophobic
counterions into fluorophores.[17] For example, it has been
recently reported that bulky tetrakis(pentafluorophenyl)borate
counterion could enable the preparation of Cy5 dye-loaded NPs
with high emission efficiency.[17b] However, the desired systems
with excellent properties in both emission and ROS generation
are still limited as a result of the difficulties to block the strong
π–π stacking. Thereby, it is urgently desirable to develop direct
and effective approaches to advanced fluorescent NPs with
both high fluorescence and ROS generation capacity for cancer
phototheranostics.
Aggregation-induced emission luminogens (AIEgens) have
recently emerged as an alternative fluorescent material to con-
struct fluorescent NPs, which perfectly address the challenge
of ACQ and show low in vivo side toxicities.[19–24] AIEgens
are often nonemissive in solution due to the consumption of
the excited state energy via nonradiative relaxation by intra-
molecular motion. Upon aggregated, such relaxation from
the lowest excited singlet state (S1) to the ground state (S0) is
largely restricted due to the steric hindrance, leading to the
energy of S1 going through the fluorescence pathway to S0.[21]
This uncommon feature makes AIEgens ideal for fabrication
of fluorescent NPs (usually named as AIE dots) with ultrahigh
brightness and photobleaching threshold. Besides, our and
several other groups have reported that for some AIEgens, the
intersystem crossing (ISC) process from S1 to the lowest excited
triplet state (T1) would facilely occur because of the designed
small S1-T1 energy gap, followed by ROS production via energy
transfer (ET) from T1 to ambient oxygen (O2), allowing for
photodynamic cancer ablation.[19,20,25–29] Despite of these pio-
neer studies, there has been nearly no report on how to control
and optimize the fluorescence and ROS generation ability of
AIE dots by tuning the photophysical property. As superior flu-
orescent NPs with extremely high efficacy of cancer photothera-
nostics are always in high pursuit, we are motivated to develop
a molecular guideline to simultaneously amplify the effective-
ness of both fluorescence imaging and PDT of AIE dots.
NIR emissive AIEgen (4-(2,2-bis(4-(diphenylamino)phenyl)-
1-(4-methoxyphenyl)vinyl)-1-methylpyridinium hexafluorophos-
phate, namely TPP-TPA; Figure 1A) with “rotor-rich” skeleton
and an inherent charge is designed and synthesized. Formula-
tion of TPP-TPA using 1,2-distearoyl-sn-glycero-3-phosphoeth-
anolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG)
and corannulene-decorated PEG (Cor-PEG) afforded two types
of AIE dots, DSPE-AIE dots and Cor-AIE dots, respectively. Due
to the large dipole moment of 2.1 D, superhydrophobicity, and
hyper-rigidity of bowl-shape corannulene,[30–32] Cor-AIE dots is
endowed with more intraparticle rigid microenvironments than
DSPE-AIE dots. Compared to DSPE-AIE dots with relatively low
NIR fluorescence and weak ROS generation capability, Cor-AIE
dots show 4.0-fold amplified fluorescence quantum yield and
1
5.4-fold enhanced ROS production. The results including H
NMR titration and theoretical calculation essentially demon-
strate that the corannulene providing intraparticle rigidity and
strong interactions with TPP-TPA plays a key role in forceful
restriction of intramolecular rotation of the encapsulated TPP-
TPA, leading to highly suppressed nonradiative decay. The
absorbed energy thus significantly flows to both the fluorescence
pathway and ISC process. Such highly amplified NIR emission
and ROS generation capacity are tremendously beneficial to the
phototheranostic efficacies in terms of NIR image-guided cancer
surgery and photodynamic cancer therapy using a peritoneal
carcinomatosis-bearing mouse model. Since to date, DSPE-PEG
is the most widely used encapsulation matrix for building AIE
dots, this study with comparison results not only provides a new
strategy and molecular guideline to prepare superior AIE dots,
but also brings new insights into the design of advanced fluores-
cent NPs for biomedical applications.
The chemical structure of inherently charged TPP-TPA was
shown in Figure 1A. To extend the emission spectrum to NIR
range, two electron-donating diphenylamine groups and one
electron-withdrawing 1-methylpyridinium unit were incorpo-
rated into the triphenylethene. Such strong electron donor–
acceptor interactions endow TPP-TPA with a large dipole
moment and the large number of rotatable aryl rings makes
the skeleton of TPP-TPA flexible. The whole synthetic route
of TPP-TPA was displayed in Figure 1A. Compound 1 was
obtained with a high yield of 95% according to the procedure
reported previously,[33] subsequently reacted with (4-methoxy-
phenyl)-pyridin-4-yl-methanone undergoing McMurry coupling
to yield compound 2 in 70%. Finally, reaction of compound
2 with iodomethane followed by ion-exchange reaction with
potassium hexafluorophosphate gave the desirable product
TPP-TPA in a high yield of 99%. All intermediates and product
were characterized by NMR and mass spectroscopies, from
which satisfactory data corresponding to their structures were
obtained (see Figures S1–S6, Supporting Information).
In this contribution, we report for the first time that
enhancing the intraparticle confined microenvironment is an
effective new approach to advanced AIE dots, permitting boosted
cancer phototheranostics in vivo. As a proof-of-concept, a new
UV–vis absorption and photoluminescence (PL) spectra of
TPP-TPA were recorded in Figure 1B; and Figure S7, Supporting
Information. TPP-TPA absorbs at 440 nm with the absorption
tail extended to 600 nm in dimethylsulfoxide (DMSO), covering
most of visible light range. Such solution emits almost no light
even increasing the water fraction (fw) in DMSO-H2O mixture
up to 50%, ascribing to the active intramolecular rotation of the
aryl rings (Figure 1C). Then the emission of TPP-TPA enhanced
dramatically upon the fw is over 50%. Together with the plot of
Dr. Q. Liu
Department of Urology
Tianjin First Central Hospital
Tianjin 300192, P. R. China
Prof. B. Z. Tang
NSFC Center for Luminescence from Molecular Aggregates
SCUT-HKUST Joint Research Institute
State Key Laboratory of Luminescent Materials and Devices
South China University of Technology
Guangzhou 510640, China
©
Adv. Mater. 2018, 1801065
1801065 (2 of 9)
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.advmat.de
Figure 1. A) Synthetic route of NIR-emissive TPP-TPA. B) PL spectra of TPP-TPA in DMSO-H2O mixtures with different water fractions (fw). C) Plot of
the relative PL intensity (I/I0) at 680 nm versus fw of the DMSO-H2O mixture of TPP-TPA. Excitation wavelength: 440 nm. Insets show the fluorescent
photos of TPP-TPA in DMSO and DMSO-H2O mixture with the fw of 99% taken under 365 nm UV lamp. Concentration of TPP-TPA: 10 × 10−6 m.
D) Molecular orbital amplitude plots of HOMO and LUMO for TPP-TPA in ground states based on density functional theory (DFT) calculation under
the method of opt wB97XD/6-31g**.
emission intensity at 680 nm against fw and the inset fluorescent
photos of the red emission in the 99% aggregated solution com-
pared to the negligible emission in DMSO (Figure 1C), TPP-TPA
exhibited the typical AIE characteristic.[21] Notably, the emission
intensity slightly decreased after the fw of 80%, which mainly attrib-
utes to the serious twisted intramolecular charge transfer (TICT)
effect in the polar solvent water.[34] Such effect can be further
supported by the emission redshift of 10 nm from 80% to 99%
(Figure S8, Supporting Information) and can also be indicated by
the typical electron distribution of the highest occupied molecular
orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)
in both ground and excited states (Figure 1D; and Figure S9, Sup-
porting Information). Additionally, the Stocks shift for TPP-TPA
was evaluated to be 220 nm, which is much larger than the small
Stokes shifts less than 50 nm in most of commercial NIR fluoro-
phores,[19] avoiding the light contamination of excitation light and
self-absorption of emission during biomedical imaging.
The TPP-TPA-loaded NPs were prepared by a nanoprecipita-
tion method as shown in Figure 2A. TPP-TPA was formulated
using Cor-PEG with bowl-shaped corannulene and DSPE-PEG
with linear alkyl chain as the encapsulation matrix, respec-
tively, obtaining Cor-AIE dots and DSPE-AIE dots, which pos-
sess similar absorption to TPP-TPA itself in aqueous media
(Figure S10, Supporting Information). The sizes of Cor-AIE dots
and DSPE-AIE dots were recorded by dynamic light scattering
with the values of 46.9 and 49.1 nm, respectively (Figure S11A,B,
Supporting Information). Transmission electron microscopy
(TEM) was further used to confirm these nanoparticles bearing
the spherical shape (Figure S11C,D, Supporting Information).
As suggested in Figure 2B, Cor-AIE dots exhibited stronger emis-
sion with the quantum yield of 26.8%, which is four times larger
than 6.7% for DSPE-AIE dots. Besides, the average fluorescence
lifetime of Cor-AIE dots was measured to be 4.34 ns that is
about four times as large as that of DSPE-AIE dots (Figure 2C).
The fluorescence resonance energy transfer from corannulene
to TPP-TPA could be completely ruled out, because the whole
absorption spectrum of corannulene and its PEG derivatives
are located on the UV range and Cor-PEG exhibits negligible
fluorescence under the excitation of 500 nm of Cor-AIE dots
(Figure S12A,B, Supporting Information).[35] Therefore, such
enhanced emission and lengthened fluorescence lifetime were
originated from the enhanced radiative pathway of TPP-TPA.
Furthermore, 9,10-anthracenediyl-bis(methylene)dimalonic
acid (ABDA), a commercially available ROS indicator,[36] was
used to evaluate the ROS production capacities of Cor-AIE dots
and DSPE-AIE dots. Noteworthy, the absorbance of ABDA in
water was dramatically decreased in the presence of Cor-AIE
dots (Up in Figure 2D) under white light irradiation, whereas
faint decrease of ABDA absorbance was observed for DSPE-
AIE dots (Down in Figure 2D), suggesting that Cor-AIE dots
©
1801065 (3 of 9)
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2018, 1801065

www.advancedsciencenews.com
www.advmat.de
Figure 2. A) Scheme for the preparation of Cor-AIE dots and DSPE-AIE dots using nanoprecipitation method. B) PL and C) fluorescence lifetime spectra
of Cor-AIE dots and DSPE-AIE dots. Excitation wavelength: 500 nm. Inset shows the fluorescent photo of Cor-AIE dots taken under 365 nm UV lamp.
D) The absorption spectra and E) the decomposition rate of ABDA for Cor-AIE dots (Up) and DSPE-AIE dots (Down) under white light irradiation
(60 mW cm−2, 400–1000 nm), where A0 and A are the absorbance at 378 nm before and after irradiation, respectively. Concentrations of nanoparticles
(Cor-AIE dots and DSPE-AIE dots) and ABDA are 0.01 mg mL−1 and 100 × 10−6m, respectively. F,G) Jablonski diagram showing the nonradiative, radia-
tive, and intersystem crossing (ISC) processes for AIEgens in flexible (DSPE-AIE dots) and rigid (Cor-AIE dots) matrixes. S0: the ground state, S1: the
lowest excited singlet state, T1: the lowest excited triplet state. knr, kr, and kISC are the rate constants of the nonradiative relaxation, the radiative decay
and the ISC process, respectively. FL: fluorescence.
were capable of producing more ROS to decompose ABDA
more quickly than DSPE-AIE dots under the same experi-
mental conditions. Plotting of the decomposition rate of ABDA
in Figure 2E quantitatively reveals that the ROS generation
ability of Cor-AIE dots is around 5.4 times better than that of
DSPE-AIE dots, indicative of the far higher ROS production
efficiency of Cor-AIE dots.
Meanwhile, the protons of methyl in 1-methylpyridinium (Hc)
and methoxyphenyl (Hd) moieties of TPP-TPA were also shifted
by around 0.07 and 0.02 ppm to the upfield, respectively. These
chemical shifts evidently imply the unique interactions and the
probably relative positions between corannulene and TPP-TPA,
with the positively charged 1-methylpyridinium of TPP-TPA
closed to the electronegative bottom of corannulene and the TPA
and methoxyphenyl units of TPP-TPA outside the bowl of coran-
nulene (Figure 3A; and Figure S13A, Supporting Information),
resulting in the shielding effect of corannulene for 1-methylpyri-
dinium of TPP-TPA. Density functional theory (DFT) calculation
was also used to study the ISC process (Figure S13B, Supporting
Information). The gaps between the S1 and T1 states (ΔEST) for
TPP-TPA upon the interaction with corannulene were signifi-
cantly decreased compared with that for TPP-TPA alone, and
the respective spin-orbital coupling (SOC) constants (ξ(S1,T1))
The underlying principle of such significantly enhanced fluo-
1
rescence and ROS production was studied. H NMR titration
experiment was first conducted to investigate the interactions
between corannulene and TPP-TPA (Figure 3A). Upon stepwise
adding corannulene into the CD2Cl2 solution of TPP-TPA, the sig-
nals of the aromatic protons in 1-methylpyridinium of TPP-TPA
gradually shifted upfield by 0.08 ppm (Ha in Figure 3B) and 0.05
ppm (Hb in Figure 3D), while those of the protons in corannulene
shifted by about 0.03 ppm (He) to the higher field (Figure 3C).
©
Adv. Mater. 2018, 1801065
1801065 (4 of 9)
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.advmat.de
Figure 3. 1H NMR titration experiment with corannulene gradually added into TPP-TPA solution. A) The structures of TPP-TPA and corannulene with
featured protons labeled with Ha, Hb, Hc, Hd, and He, and the calculated relative position between TPP-TPA and corannulene. B–E) The changes of
chemical shifts for TPP-TPA (the aromatic protons of 1-methylpyridinium B,D), the methyl protons in 1-methylpyridinium and anisole E)) and coran-
nulene C) were indicated with the dotted lines (red for TPP-TPA and blue for corannulene) and evaluated by Δδ. Concentrations of TPP-TPA was
10 × 10−3 m and corannulene were 10 × 10−3 m (1:1), 20 × 10−3 m (1:2), and 60 × 10−3 m (1:6) in CD2Cl2 solution.
between S1 and T1 were also increased, suggesting the more
accessible ISC process in the presence of corannulene.
can also provide a more isolated hydrophobic environment to
reduce the polar-solvent disruption (such as TICT) for TPP-TPA,
bringing about the further enhancement of emission efficiency
and ROS production. Indeed, the reduced TICT effect in Cor-AIE
dots was reflected by the slight blueshift of about 10 nm in the
emission spectrum compared to DSPE-AIE dots (Figure 2B).
Due to the excellent NIR emission output and ROS produc-
tion, the utility and strength of Cor-AIE dots in cancer photo-
theranostics were investigated. After we demonstrated that
Cor-AIE dots could be internalized in cancer cells and generate
ROS within cells effectively (Figure S14, Supporting Infor-
mation), in vivo studies were carried out using a peritoneal
carcinomatosis-bearing mouse model, which was established
by intraperitoneal inoculation of murine 4T1 cancer cells. It
is worthy to note that the in vivo inoculated 4T1 cancer cells
express luciferase, which could emit bioluminescence when
the tumor-bearing mice were administrated with the substrate
of luciferase (D-luciferin), allowing for precise tracking of the
tumor nodules in the mouse peritoneal cavity.[38]
Recently, image-guided cancer surgery using NIR fluorescence
has been verified to be feasible during clinical cancer surgery,
which holds great promise to promote the outcomes of cancer
surgery in the clinic.[39] However, currently available NIR fluo-
rescent probes that are able to meet the necessary requirements
of image-guided cancer surgery are still limited. Accordingly, we
investigate whether the highly boosted NIR fluorescence in Cor-
AIE dots is beneficial to image-guided cancer surgery. In this
experiment, the peritoneal carcinomatosis-bearing mouse model
was selected, because there are a large number of tumor nodules
especially those with diameters <1 mm scattered in the mouse
Upon getting insight into the molecular structures of coran-
nulene and DSPE, bowl-shaped corannulene possesses a super-
hydrophobic skeleton and an ultrarigid curvature compared with
flexible alkyl-chained DSPE, which hence constructs a more
confined microenvironment in aqueous solution. On the other
hand, corannulene bears a large dipole moment and the bottom
of corannulene bowl is electronegative with the periphery being
electropositive, which attract inherent positively charged TPP-
TPA by dipole–dipole and electrostatic interactions. As a conse-
quence, when comparing the intraparticle microenvironments of
DSPE-AIE dots and Cor-AIE dots, the more confined and rigid
microcavity within Cor-AIE dots resulting from the uniqueness of
corannulene structure as well as the strong interactions between
corannulene and TPP-TPA benefits to restrict the intramolecular
rotation of the phenyl rings in TPP-TPA to a much greater extent,
which thus inhibits the nonradiative relaxation more effectively. As
the absorbed energy of TPP-TPA is fixed, the highly suppressed
nonradiative decay for Cor-AIE dots reasonably makes its absorbed
energy flow to both the fluorescence pathway and ISC process,
achieving significantly amplified emission and ROS generation
(Figure 2F,G). This is supported by the theoretical formulas of
ΦF = kr/(kr + knr + kISC) and ΦISC = kISC/(kr + knr + kISC),[37] where
the dramatic decrease in nonradiative rate knr undoubtedly
induces the extensive increase of the fluorescence emission effi-
ciency ΦF and the ISC efficiency ΦISC. In addition, as TPP-TPA
has strong electron donor–acceptor structures in the backbone, the
fast process of charge transfer induced fluorescence quenching in
aqueous media competes with ROS production.[36] Cor-AIE dots
©
1801065 (5 of 9)
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2018, 1801065

www.advancedsciencenews.com
www.advmat.de
peritoneal cavity. In the clinic, the submillimeter tumor nodules
are difficult to be spotted by surgeon during surgery; however,
these remaining small tumors are the major culprits for the in
situ cancer recurrence. So far, there have been rather finite NIR
fluorescent probes that are capable of accurately examining the
submillimeter tumors, mainly because of the low NIR emis-
sion output of these probes. This motivates us to assess whether
Cor-AIE dots can help surgeon find and remove the ultrasmall
tumor nodules with diameters <1 mm.
After the Cor-AIE dots were intravenously injected into the
peritoneal carcinomatosis-bearing mice for 24 h, the surgery
was performed by opening the mouse abdomen. In vivo NIR
fluorescence imaging during surgery indicates that bright
Cor-AIE dots fluorescence clearly lights up plenty of tissues as
well as their boundaries with rather high signal-to-background
ratio (Figure 4A; and Figure S15, Supporting Information). As
luciferase-expressed 4T1 tumors have bioluminescence, the
scattering intraperitoneal tumor nodules are able to be totally
and specifically displayed upon bioluminescence imaging
postinjection with D-luciferin. As shown in Figure 4A, the biolu-
minescence signal from luciferase and NIR fluorescence signal
from Cor-AIE dots are colocalized perfectly on the surface of the
intestines, suggesting that the Cor-AIE dots-visualized tissues
are indeed tumors, which are further confirmed by the hematox-
ylin and eosin (H&E) histological staining. Noteworthy, the fluo-
rescence intensity ratio of tumor to normal intestine is as high
as 5.2 for Cor-AIE dots, which exceeds the Rose criterion.[40]
Such ratio is significantly higher than those of many reported
Figure 4. A,B) Bright field, fluorescence, bioluminescence, and H&E staining images of the tumor nodules on the surface of the intraperitoneal intes-
tines A) and the peritoneum B) in peritoneal carcinomatosis-bearing mice after intravenous injection of Cor-AIE dots for 24 h. C–G) Tumor resection
with and without Cor-AIE dots fluorescence image-guided surgery. Representative fluorescence images C) before surgery, D) after unguided surgery,
and E) after reoperation with the aid of Cor-AIE dots image-guidance. F) The harvested nodules from unguided groups and Cor-AIE dots fluorescence
guided groups were examined with a fluorescence imaging system (Left) and a bioluminescence imaging system (Right). G) Histogram of nodule
diameters extracted from unguided and Cor-AIE dots fluorescence guided groups.
©
Adv. Mater. 2018, 1801065
1801065 (6 of 9)
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.advmat.de
NIR fluorescent probes including methylene blue and indocya-
nine green.[41,42] As depicted in Figure 4B, the tumor nodules on
the peritoneum are also roughed out with an even larger tumor-
to-peritoneum ratio of about 8.0. These results quantitatively
demonstrate that the Cor-AIE dots possess remarkable EPR
effect, permitting high tumor uptake via passive targeting and
thus leading to visualization of intraperitoneal tumor nodules
and their boundaries in a specific and high-contrast manner.
More importantly, it is also found that the Cor-AIE dots can dis-
tinctly delineate the tumor nodules with sizes <1 mm in the per-
itoneal cavity (indicated by the red arrows in Figure 4), revealing
that Cor-AIE dots are efficacious in sharply visualizing submil-
limeter tumors thanks to their highly boosted NIR emission.
Then the application of the amplified NIR emission of Cor-
AIE dots in guidance for surgical tumor removal was studied.
To this end, a surgeon from Tianjin First Central Hospital
(Tianjin, China) was invited to conduct the operation. As
shown in Figure 4C,D, when the surgeon was blinded to the
NIR fluorescence imaging by Cor-AIE dots, he removed a lot of
intraperitoneal tumors with relatively large diameters (>1 mm)
according to his experience. However, after unguided surgery,
there are a number of residual tumor nodules remaining in the
peritoneal cavity indicated by Cor-AIE dots, which are mainly
the ones with diameters <1 mm (Figure 4D). The surgeon then
performed a second operation under the guidance of Cor-AIE
dots fluorescence, which realizes almost complete removal of
the remaining small tumors (Figure 4E–G), confirmed by the
negligible intraperitoneal bioluminescence signal. It is noted
that all the harvested tumor nodules (mainly submillimeter
ones) in the second operation have bioluminescence signals
(Figure 4F), validating the precise cancer surgery assisted by
Cor-AIE dots. After unguided and Cor-AIE dots fluorescence
image-guided surgery, respectively, the survival of the mice
was monitored over time with each group containing ten mice.
Due to the high malignancy and fast growth of intraperitoneal
4T1 tumors, all the ten mice in unguided surgery group died
within 2 weeks. Encouragingly, seven of ten mice in the cohort
with Cor-AIE dots fluorescence image-guided surgery are able
to survive during 2 week-monitoring duration (Figure S16,
Supporting Information). These results together illustrate that
Cor-AIE dots greatly promote the cancer surgery outcome by
accurately lighting up the submillimeter tumor nodules, con-
siderably prolonging the lifetimes of tumor-bearing mice
postoperation.
of the Supporting Information. The tumor size and growth
were monitored as the time elapsed through bioluminescence
imaging benefitting from the luciferase-expressed 4T1 cancer
cells. Figure 5A; and Figure S17 (Supporting Information)
exhibit the time-dependent bioluminescence imaging of the
tumor-bearing mice in each group. It is apparent that tumors
with intense bioluminescence signal exist in the abdomen of
mice before different treatments on day 0. Dramatically, after
receiving the treatment of “Cor-AIE dots + L,” the intraperitoneal
tumor growth of mice is considerably suppressed, as evidenced
by the similar average bioluminescence intensity of intraperito-
neal tumors on day 9 to that on day 0 (Figure 5B). As controls,
both the treatments of “Cor-AIE dots” and “Light (L)” fail to slow
down the growth of intraperitoneal tumors, compared with that
of “Saline” (Figure 5B; and Figure S17, Supporting Informa-
tion), revealing that the highly efficacious anticancer activity in
“Cor-AIE dots + L” group roots in the PDT via Cor-AIE dots gen-
erating ROS in tumors. It is important to note that the PDT of
DSPE-AIE dots does not have any inhibitory effects on tumor
growth (Figure 5A,B). This result not only indicates the high
malignancy of intraperitoneal tumors, but also implies that the
ROS production ability of DSPE-AIE dots is not strong enough
to work in this tumor-bearing animal model. The treatment of
“Cor-AIE dots + L” leads to greatly prolonged lifetimes of mice
and the median survival time for “Cor-AIE dots + L” group is
far longer than that for “DSPE-AIE dots + L” group (Figure 5C).
The sharp comparison in the antitumor efficacy between the
PDT of Cor-AIE dots and DSPE-AIE dots reasonably highlights
the necessity and importance of our new approach to superior
AIE dots.
In summary, we have first reported that enhancing the intra-
particle confined microenvironment could serve as an effective
approach to guiding the design of superior AIE dots with both
highly amplified fluorescence and ROS production, benefit-
ting to greatly improved cancer phototheranostics in vivo. With
facile introduction of two electron-donating diphenylamine
groups and one electron-withdrawing 1-methylpyridinium unit
into the triphenylethene, a new NIR emissive AIEgens, namely
TPP-TPA, was simply synthesized, featured with “rotor-rich”
skeleton and an inherent charge. Then, encapsulation of TPP-
TPA with DSPE-PEG or Cor-PEG afforded DSPE-AIE dots or
Cor-AIE dots with different intraparticle rigid microenviron-
ments. Compared to DSPE-AIE dots, Cor-AIE dots exhibited
4.0-fold amplified fluorescence quantum yield and 5.4-fold
enhanced ROS production. As corannulene possesses large
dipole moment of 2.1 D, superhydrophobicity, hyper rigidity,
and uneven electron distribution, it provides larger intrapar-
ticle rigidity. Together with the strong interactions between
corannulene and TPP-TPA indicated by 1H NMR titration,
the intraparticle confined microenvironment in Cor-AIE dots
forcefully restricts the intramolecular rotation of the encapsu-
lated TPP-TPA and highly suppresses its nonradiative decay
leading to the flow of excited-state energy to both the fluores-
cence pathway and ISC process, which were also supported
by theoretical calculation. Such high NIR emission and ROS
production tremendously promoted the phototheranostic effi-
cacies in terms of NIR image-guided cancer surgery and PDT
with a peritoneal carcinomatosis-bearing mouse model. This
study not only brings in a new category of corannulene-based
In many cases in the clinic, the surgeon cannot perform the
tumor-removal operation after opening the abdomen, as there
are so many small tumors that are hard to be excised completely.
As a result, the surgeon has to close the abdominal wall, give
up surgery, and choose another treatment strategy. Since in
addition to high NIR emission, strong ROS generation ability
is the other signature of Cor-AIE dots by virtue of the impor-
tant contribution of corannulene, we investigated the feasibility
of Cor-AIE dots in photodynamic tumor therapy in the afore-
mentioned cases that surgical resection was not suggested after
opening the abdomen. To this end, peritoneal carcinomatosis-
bearing mice were randomly assigned into five groups, which
were designated as “Saline,” “Light (L),” “DSPE-AIE dots + L,”
“Cor-AIE dots + L,” and “Cor-AIE dots” with the corresponding
treatments described in details in the Experimental Section
©
1801065 (7 of 9)
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2018, 1801065

www.advancedsciencenews.com
www.advmat.de
Figure 5. A) The time-dependent bioluminescence imaging of the peritoneal carcinomatosis-bearing mice in “Saline,” “DSPE-AIE dots + L,” and
“Cor-AIE dots + L” groups. B) The average bioluminescence (BL) intensities of intraperitoneal tumors on days 0, 1, 3, 5, and 9 in various groups indi-
cated. **represents P < 0.01, in comparison between the average BL intensity of intraperitoneal tumors in “Cor-AIE dots + L” cohort and other groups.
C) The curve of survival rate after different treatments. All the experiment groups are “Saline,” “Cor-AIE dots,” “Light (L),” “DSPE-AIE dots + L,” and
“Cor-AIE dots + L.” “L” is under the white light (0.4 W cm−2) for 10 min. The concentrations of DSPE-AIE dots and Cor-AIE dots injected are 1 mg mL−1
based on TPP-TPA. The volume of injection is 150 µL.
studies were performed in compliance with the guidelines set by Tianjin
Committee of Use and Care of Laboratory Animals and the overall
project protocols were approved by the Animal Ethics Committee of
Nankai University.
AIE dots with superior biomedical performances, but also rea-
sonably demonstrates that enhancing the intraparticle confined
microenvironment that further inhibits the nonradiative decay
of encapsulated AIEgens is a unique and new strategy to pre-
pare AIE dots with both highly enhanced fluorescence and ROS
generation capacity. This underlying principle to highly boost
the phototheranostic efficacy should also be beneficial to other
fluorescent NPs systems.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
Supporting Information
aggregation-induced emission, AIE nanodots, cancer phototheranostics,
near-infrared fluorescence image-guided surgery, photodynamic therapy
Supporting Information is available from the Wiley Online Library or
from the author.
Received: February 13, 2018
Revised: March 19, 2018
Published online:
Acknowledgements
X.G. and X.Z. contributed equally to this work. This work was partially
supported by the National Natural Science Foundation of China Grant
(Nos. 21502022, 21788102, 51622305, and 21702016), the National
Basic Research Program of China (Nos. 973 Program, 2015CB856503,
2013CB834701, and 2013CB834702), the Innovation and Technology
Commission (No. ITC-CNERC14SC01), the Research Grants Council of
Hong Kong (Nos. 16308016, 16305015, C2014-15G, N_HKUST604/14,
and A-HKUST605/16), and the Science and Technology Plan of
Shenzhen (No. JCYJ20160229205601482). D.D. thanks the PCSIRT
(No. IRT13023), the Science & Technology Project of Tianjin of China
(No. 15JCYBJC29800), and the 111 project (No. B08011). All animal
[1] S. Kunjachan, J. Ehling, G. Storm, F. Kiessling, T. Lammers, Chem.
Rev. 2015, 115, 10907.
[2] C. Chu, H. Lin, H. Liu, X. Wang, J. Wang, P. Zhang, H. Gao,
C. Huang, Y. Zeng, Y. Tan, G. Liu, X. Chen, Adv. Mater. 2017, 29,
1605928.
[3] H. Fan, G. Yan, Z. Zhao, X. Hu, W. Zhang, H. Liu, X. Fu,
T. Fu, X. B. Zhang, W. Tan, Angew. Chem., Int. Ed. 2016, 55,
5477.
[4] G. Tian, X. Zhang, Z. Gu, Y. Zhao, Adv. Mater. 2015, 27, 7692.
©
Adv. Mater. 2018, 1801065
1801065 (8 of 9)
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
www.advmat.de
[5] X. Chen, S. S. Gambhir, J. Cheon, Acc. Chem. Res. 2011, 44,
841.
[6] K. K. Ng, G. Zheng, Chem. Rev. 2015, 115, 11012.
[7] X. Ai, J. Mu, B. Xing, Theranostics 2016, 6, 2439.
[8] C. Wang, L. Xu, C. Liang, J. Xiang, R. Peng, Z. Liu, Adv. Mater. 2014,
26, 8154.
[9] M. P. Melancon, M. Zhou, C. Li, Acc. Chem. Res. 2011, 44, 947.
[10] Y. Li, T. Lin, Y. Luo, Q. Liu, W. Xiao, W. Guo, D. Lac, H. Zhang, C. Feng,
S. Wachsmann-Hogiu, J. H. Walton, S. R. Cherry, D. J. Rowland,
D. Kukis, C. Pan, K. S. Lam, Nat. Commun. 2014, 5, 4712.
[11] H. Wang, P. Agarwal, S. Zhao, J. Yu, X. Lu, X. He, Nat. Commun.
2014, 6, 10081.
[26] X. Wang, J. Hu, G. Zhang, S. Liu, J. Am. Chem. Soc. 2014, 136, 9890.
[27] a) W. Wu, D. Mao, F. Hu, S. Xu, C. Chen, C. J. Zhang, X. Cheng,
Y. Yuan, D. Ding, D. Kong, B. Liu, Adv. Mater. 2017, 29, 1700548;
b) X. Zhen, Y. Tao, Z. An, P. Chen, C. Xu, R. Chen, W. Huang, K. Pu,
Adv. Mater. http://doi.org/10.1002/adma.201606665.
[28] F. Hu, Y. Huang, G. Zhang, R. Zhao, H. Yang, D. Zhang,
Anal. Chem. 2014, 86, 7987.
[29] G. Feng, B. Liu, Small 2016, 12, 6528.
[30] K. Shi, T. Lei, X. Y. Wang, J. Y. Wang, J. Pei, Chem. Sci. 2014, 5,
1041.
[31] B. M. Schmidt, T. Osuga, T. Sawada, M. Hoshino, M. Fujita,
Angew. Chem., Int. Ed. 2016, 55, 1561.
[12] Q. Fan, K. Cheng, Z. Yang, R. Zhang, M. Yang, X. Hu, X. Ma, L. Bu,
X. Lu, X. Xiong, W. Huang, H. Zhao, Z. Cheng, Adv. Mater. 2015,
27, 843.
[32] M. Jurícˇek, N. L. Strutt, J. C. Barnes, A. M. Butterfield, E. J. Dale,
J. J. Baldridge, J. Fraser Stoddart, J. S. Siegel, Nat. Chem. 2014,
6, 222.
[13] T. L. Doanea, C. Burda, Chem. Soc. Rev. 2012, 41, 2885.
[14] J. A. Barreto, W. O’Malley, M. Kubeil, B. Graham, H. Stephan,
L. Spiccia, Adv. Mater. 2011, 23, H18.
[15] Y. Liu, T. Chen, C. Wu, L. Qiu, R. Hu, J. Li, S. Cansiz, L. Zhang,
C. Cui, G. Zhu, M. You, T. Zhang, W. Tan, J. Am. Chem. Soc. 2014,
136, 12552.
[16] G. Chen, I. Roy, C. Yang, P. N. Prasad, Chem. Rev. 2016, 116, 2826.
[17] a) A. Reisch, A. S. Klymchenko, Small 2016, 12, 1968;
b) K. Trofymchuk, A. Reisch, P. Didier, F. Fras, P. Gilliot, Y. Mely,
A. S. Klymchenko, Nat. Photonics 2017, 11, 657.
[18] A. Master, M. Livingston, A. Sen Gupta, J. Controlled Release 2013,
168, 88.
[33] a) X. Gu, E. Zhao, J. W. Y. Lam, Q. Peng, Y. Xie, Y. Zhang, K. S. Wong,
H. H. Y. Sung, I. D. Williams, B. Z. Tang, Adv. Mater. 2015, 27, 7093;
b) X. Gu, E. Zhao, T. Zhao, M. Kang, C. Gui, J. W. Y. Lam, S. Du,
M. M. T. Loy, B. Z. Tang, Adv. Mater. 2016, 28, 5064.
[34] Z. R. Grabowski, K. Rotkiewicz, Chem. Rev. 2003, 103, 3899.
[35] P. Bachawala, T. Ratterman, N. Kaval, J. Mack, Tetrahedron 2017, 73,
3831.
[36] W. Wu, G. Feng, S. Xu, B. Liu, Macromolecules 2016, 49, 5017.
[37] Q. Peng, Y. Niu, Q. Wu, X. Gao, Z. Shuai, Aggregation-Induced
Emission: Fundamentals and Applications, Vols. 1 and 2, John Wiley
and Sons Ltd, Hoboken, NJ, USA 2013, Ch. 17.
[38] J. Liu, C. Chen, S. Ji, Q. Liu, D. Ding, D. Zhao, B. Liu, Chem. Sci.
2017, 8, 2782.
[19] X. Gu, R. T. K. Kwok, J. W. Y. Lam, B. Z. Tang, Biomaterials 2017,
146, 115.
[39] A. L. Vahrmeijer, M. Hutteman, J. R. van der Vorst, C. J. H. van de
Velde, J. V. Frangioni, Nat. Rev. Clin. Oncol. 2013, 10, 507.
[40] A. L. Antaris, H. Chen, K. Cheng, Y. Sun, G. Hong, C. Qu,
S. Diao, Z. Deng, X. Hu, B. Zhang, X. Zhang, O. K. Yaghi,
Z. R. Alamparambil, X. Hong, Z. Cheng, H. Dai, Nat. Mater. 2016,
15, 235.
[41] C. T. Zeng, W. T. Shang, K. Wang, C. W. Chi, X. H. Jia, C. Fang,
D. Yang, J. Z. Ye, C. H. Fang, J. Tian, Sci. Rep. 2016, 6,
21959.
[42] O. T. Okusanya, D. Holt, D. Heitjan, C. Deshpande, O. Venegas,
J. Jiang, R. Judy, E. DeJesus, B. Madajewski, K. Oh, M. Wang,
S. M. Albelda, S. Nie, S. Singhal, Ann. Thorac. Surg. 2014,
98, 1223.
[20] D. Ding, K. Li, B. Liu, B. Z. Tang, Acc. Chem. Res. 2013, 46, 2441.
[21] a) J. Mei, N. L. C. Leung, R. T. K. Kwok, J. W. Y. Lam, B. Z. Tang,
Chem. Rev. 2015, 115, 11718; b) J. Mei, Y. Hong, J. W. Y. Lam,
A. Qin, Y. Tang, B. Z. Tang, Adv. Mater. 2014, 26, 5429.
[22] X. Wu, X. Sun, Z. Guo, J. Tang, Y. Shen, T. D. James, H. Tian,
W. H. Zhu, J. Am. Chem. Soc. 2014, 136, 3579.
[23] A. Shao, Y. Xie, S. Zhu, Z. Guo, S. Zhu, J. Guo, P. Shi, T. D. James,
H. Tian, W. H. Zhu, Angew. Chem., Int. Ed. 2015, 54, 7275.
[24] K. Gu, Y. Xu, H. Li, Z. Guo, S. Zhu, P. Shi, T. D. James, H. Tian,
W. H. Zhu, J. Am. Chem. Soc. 2016, 138, 5334.
[25] M. Li, Y. Gao, Y. Yuan, Y. Wu, Z. Song, B. Z. Tang, B. Liu,
Q. C. Zheng, ACS Nano 2017, 11, 3922.
©
1801065 (9 of 9)
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2018, 1801065