Facile Synthesis of Red/NIR AIE Luminogens with Simple Structures, Bright Emissions, and High Photostabilities, and Their Applications for Specific Imaging of Lipid Droplets and Image-Guided Photodynamic Therapy

Article abstract of DOI:10.1002/adfm.201704039

Red/near-infrared (NIR) fluorescent molecules with aggregation-induced emission (AIE) characteristics are of great interest in bioimaging and therapeutic applications. However, their complicated synthetic approaches remain the major barrier to implementing these applications. Herein, a one-pot synthetic strategy to prepare a series of red/NIR-emissive AIE luminogens (AIEgens) by fine-tuning their molecular structures and substituents is reported. The obtained AIEgens possess simple structures, good solubilities, large Stokes shifts, and bright emissions, which enable their applications toward in vitro and in vivo imaging without any pre-encapsulation or -modification steps. Excellent targeting specificities to lipid droplets (LDs), remarkable photostabilities, high brightness, and low working concentrations in cell imaging application make them remarkably impressive and superior to commercially available LD-specific dyes. Interestingly, these AIEgens can efficiently generate reactive oxygen species upon visible light irradiation, endowing their effective application for photodynamic ablation of cancer cells. This study, thus, not only demonstrates a facile synthesis of red/NIR AIEgens for dual applications in simultaneous imaging and therapy, but also offers an ideal architecture for the construction of AIEgens with long emission wavelengths.

Full text of DOI:10.1002/adfm.201704039

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Facile Synthesis of Red/NIR AIE Luminogens with Simple
Structures, Bright Emissions, and High Photostabilities,
and Their Applications for Specific Imaging of Lipid
Droplets and Image-Guided Photodynamic Therapy
Dong Wang, Huifang Su, Ryan T. K. Kwok, Guogang Shan, Anakin C. S. Leung,
Michelle M. S. Lee, Herman H. Y. Sung, Ian D. Williams, Jacky W. Y. Lam,
and Ben Zhong Tang*
fluorescence imaging, direct visualization
Red/near-infrared (NIR) fluorescent molecules with aggregation-induced emis-
sion (AIE) characteristics are of great interest in bioimaging and therapeutic
applications. However, their complicated synthetic approaches remain the
major barrier to implementing these applications. Herein, a one-pot synthetic
strategy to prepare a series of red/NIR-emissive AIE luminogens (AIEgens)
by fine-tuning their molecular structures and substituents is reported. The
obtained AIEgens possess simple structures, good solubilities, large Stokes
shifts, and bright emissions, which enable their applications toward in
vitro and in vivo imaging without any pre-encapsulation or -modification
steps. Excellent targeting specificities to lipid droplets (LDs), remarkable
photostabilities, high brightness, and low working concentrations in cell
imaging application make them remarkably impressive and superior to com-
mercially available LD-specific dyes. Interestingly, these AIEgens can efficiently
generate reactive oxygen species upon visible light irradiation, endowing their
effective application for photodynamic ablation of cancer cells. This study,
thus, not only demonstrates a facile synthesis of red/NIR AIEgens for dual
applications in simultaneous imaging and therapy, but also offers an ideal
architecture for the construction of AIEgens with long emission wavelengths.
of bioanalytes could be achieved on site
and in time, and useful insights into com-
plex biological structures and processes
could be straightforwardly provided.[2] In
particular, fluorophores with intense emis-
sion in the red/near-infrared (NIR) region
are nowadays undergoing an explosive
development in biological applications,
owing to their capabilities of overcoming
the interference of optical absorption,
reducing light scattering, avoiding autoflu-
orescence of physiological environments,
as well as minimizing photodamage to bio-
logical structures.[3] Although many types
of red/NIR-emissive fluorophores have
been commercialized, current situation is
still far from ideal. Conventional red/NIR-
emissive fluorophores generally suffer
from a common photophysical phenom-
enon notoriously named as aggregation-
caused quenching (ACQ).[4] Conventional
fluorophores emit strongly in solution
phase; however, they experience emission
quenching upon aggregates formation due to intermolecular
π–π stacking and other nonradiative pathways. For examples,
some commercial bioimaging dyes including MitoTracker
Red, ER-Tracker Red, LysoTracker Deep Red, and Nile Red
are all brightly red emissive in diluted dimethyl sulfoxide
1. Introduction
Fluorescence imaging has been proven to be a highly sensitive
and noninvasive technology that offers researchers a very useful
tool for analytical sensing and optical imaging.[1] By utilizing
Dr. D. Wang, Dr. H. Su, Dr. R. T. K. Kwok, Dr. G. Shan, A. C. S. Leung,
M. M. S. Lee, Dr. H. H. Y. Sung, Prof. I. D. Williams, Dr. J. W. Y. Lam,
Prof. B. Z. Tang
Prof. B. Z. Tang
Guangdong Provincial Key Laboratory of Brain Science
Disease and Drug Development
Department of Chemistry
HKUST-Shenzhen Research Institute
No. 9 Yuexing 1st RD, South Area, Hi-tech Park, Nanshan
Shenzhen 518057, China
Hong Kong Branch of Chinese National Engineering Research
Center for Tissue Restoration and Reconstruction
Institute of Molecular Functional Materials
State Key Laboratory of Neuroscience
Division of Biomedical Engineering
and Division of Life Science
The Hong Kong University of Science and Technology
Clear Water Bay, Kowloon, Hong Kong, China
E-mail: tangbenz@ust.hk
Prof. B. Z. Tang
Guangdong Innovative Research Team
SCUT-HKUST Joint Research Laboratory
State Key Laboratory of Luminescent Materials and Devices
South China University of Technology
Guangzhou 510640, China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.201704039.
DOI: 10.1002/adfm.201704039
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from their bulky structures; therefore, further
fabrications or modifications with surfactants,
proteins, or other materials are essentially
required before biological uses. For examples,
TTB[8] and TPE-TPA-DCM[9] (Scheme S1,
Supporting Information) are reputable in
biological applications because of their AIE
characteristics, bright emissions in red region
and high photostabilities, nonetheless, their
synthetic procedures respectively involve
seven- and six-step reactions with cumber-
some and iterative purifications. In addition,
efficient cellular uptake of TTB and TPE-TPA-
DCM requires pre-fabrications with poly-
ethylene glycol (PEG)-containing amphiphilic
polymers or bovine serum albumin. Indeed,
developing facile synthesis of red/NIR AIE-
gens having simple and processable structure
with bright emission remains an important
and challenging task with very limited suc-
cess achieved even though enthusiastic efforts
have been devoted by scientists.
Figure 1. ACQ versus AIE. ACQ phenomenon of conventional dyes: A) ER-Tracker Red,
B) MitoTracker Red, C) LysoTracker Deep Red, and D) Nile Red. E) AIE phenomenon of AIEgen
TPMN. F) Fluorescence photographs of Nile Red (left) and AIEgen TPMN (right) in the solid
state taken under 365 nm UV irradiation.
Lipid droplets (LDs),[10] which mainly
solutions, but the emissions are partially or totally quenched
upon the formation of aggregates after adding water
(Figure 1A–D, Table S1, Supporting Information). In par-
ticular, as illustrated in Figure 1F, Nile Red is almost nonemis-
sive in solid state. The fluorescence photographs in Figure 1
clearly demonstrate that ACQ phenomenon leads to low photo-
bleaching resistance; thus, this would greatly impede the
practical applications in the area of biomedical research, since
conventional fluorophores tend to form aggregates in physi-
ological environments or aqueous media due to the high hydro-
phobicity of their emitting centers with planar conformations.
We have developed a novel class of fluorophores with twisted
conformations exhibiting extraordinary aggregation-induced
emission (AIE) feature,[5] which is completely opposite to ACQ
dyes. These compounds with AIE characteristics are nonemis-
sive or weakly emissive in solutions but are induced to flo-
rescent intensely in aggregates through a mechanism of the
restriction of intramolecular motions, resulting in intrinsic
capability to work perfectly at high concentrations or in aggre-
gation state with bright fluorescence (as shown in Figure 1E,F)
and a high photobleaching threshold. Therefore, AIE has
opened a venue to an array of possibilities with great potential
for high-tech innovations.[6]
Considering the great significances of both AIE and red/
NIR-emission, some red/NIR-emissive AIEgens have been
constructed and utilized in various biological applications.[6]
Nevertheless, in general, the preparation of red/NIR fluores-
cent molecules is extremely complicated regardless of either the
AIEgens or conventional dyes. Common synthetic strategies of
red/NIR fluorophores, including connection of strong electron-
donating (D) and -accepting (A) units by π-bridge(s), expansion
of π-conjugation, and combination of the two strategies,[7] usu-
ally require several-step reactions and inconvenient purifica-
tions, which are extremely time-, cost-, and energy-consuming,
tedious, and harmful to environment. Moreover, their applica-
tions are often restricted due to the inferior solubility resulting
locate in adipocytes, hepatocytes, adrenal cortex, and myocytes,
have been proven to be considerably important in various bio-
functions, such as regulations of the storage and metabolism
of neutral lipids, protein degradation, construction and main-
tenance of membrane, and signal transduction.[11] The abnor-
mality of LDs in cells is a critical biomarker for various diseases
including cancer, obesity, fatty liver disease, hyperlipidemia,
atherosclerosis, inflammation, virus infection, type II diabetes,
and neurodegeneration in Alzheimer’s disease.[12] Taking the
intrinsic advantages of fluorescence imaging, the development
of efficient fluorescent probes of LDs is highly desired. How-
ever, commercial fluorophores (such as BODIPY dyes, Nile
Red, and Oil Red O) for LDs imaging have their respective and
collective drawbacks. For instance, BODIPY dyes require rela-
tively long incubation time and their small Stokes shifts affect
the collection of imaging signal;[13] the specificity of Nile Red to
LDs is unsatisfactory;[14] prefixation of cells is necessary when
Oil Red O is employed.[15] Besides, those commercial fluo-
rophores exhibiting ACQ property show low photobleaching
resistance. In this context, a handful of LD-specific targeting
AIEgens have been exploited and proven to be powerful fluo-
rescent probes of LDs.[16] Nevertheless, both of those previously
reported AIEgens and commercial fluorophores can only be
used as LD-specific probes instead of further therapeutic appli-
cations. Indeed, LDs could be an ideal organelle for therapeutic
applications, thanks to their fluidity and relevance with various
biofunctions. Development of AIEgens with dual functions of
simultaneous LD-imaging and therapy is, therefore, attractive
and useful.
Herein, we report a one-pot synthetic approach to red/
NIR AIEgens with simple structures and bright emissions.
These AIEgens were successfully utilized as LD-specific bio-
probes in cell imaging and in vivo zebrafish imaging with
high photostabilities and brightness. Meanwhile, owing to
the high efficiencies of reactive oxygen species (ROSs) gen-
eration, they were proven to be effective in killing cancer cells
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Scheme 1. One-pot synthesis of AIEgens and the structures of TPMN, TTMN, MeTTMN, and MeOTTMN.
through photodynamic therapy (PDT) process, which is gaining
increasing attention on cancer treatment due to its distinct
advantages, such as precise controllability, minimal invasive
nature, and high spatiotemporal accuracy.[17]
molecular geometry, excluding the two phenyl rings at the end,
is relatively coplanar, thus allowing good electron delocalization
of the whole molecule and contributes. The ingenious combi-
nation of strong electron donor–acceptor (D-A) interaction and
extended π-conjugation with good electron delocalization in this
structure dose not only indicate a great potential of red emis-
sion, but also anticipate to result in an excellent push–pull
system and strong excited-state intramolecular charge transfer
(ICT), endowing large Stokes shift. In contrast, all the moieties
2. Results and Discussion
2.1. Synthesis and Single Crystal Analysis
Taking into account the interest of cascade-
reaction strategy,[18] a one-pot two-step three-
component synthetic protocol was carried
out through successive Suzuki–Miyaura
coupling/Knoevenagel condensation reac-
tions of substituted 4-bromo-N,N-dipheny-
laniline, 4-formyl aromatic boronic acid and
malononitrile, in the presence of palladium
catalyst using ethanol as solvent at 78 °C.
Consequently, compounds TPMN, TTMN,
MeTTMN, and MeOTTMN were isolated
with moderate yields ranging from 42–58%,
as depicted in Scheme 1.
Single crystals of TTMN were obtained by
the slow evaporation of its solution in mixed
solvents of CHCl3 and hexane, endowing
further study and deciphering of its optical
properties and AIE behavior. As illustrated
in Figure 2A,B, TTMN is comprised of tri-
phenylamine segment (D), thiophene frag-
ment (D and π-bridge), carbon–carbon double
bond (π-bridge), and two cyano units (A). The
Figure 2. A) Single crystal structure of TTMN. B) Side view of the crystal structure of TTMN.
C) Various inter- and intramolecular interactions in crystals of TTMN.
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of TTMN could act as freely rotated molecular rotators that con-
sume the energy of the excited state upon photoexcitation, thus
ensuring that TTMN is weekly emissive in solution. Twisted con-
formation of triphenylamine segment extends the intermolec-
ular distance (3.484 Å) between two parallel planes (Figure 2C),
remarkably reducing the intermolecular π–π interaction, and
essentially preventing emission quenching in its aggregate
state. In addition, abundant inter- and intramolecular interac-
synthesized compounds exhibiting interesting features, such
as efficient excited-state intramolecular charge transfer, twisted
conformation, and high structural rigidity, may possess the dual
properties of high emission efficiency and large Stokes shift.
2.2. Photophysical Properties


tions (such as C H···π, C H···C, S···N, and S···C) in the
crystal lattice strongly rigidify the molecular conformation and
restrict molecular motions, resulting in the possibility of bright
emission in the crystal state. Interestingly, the existence of intra-
molecular S···N interaction may weaken undesirable twisted
intramolecular charge transfer (TICT) effect, which is one of the
major nonradiative pathways for the excited state to relax and
deactivate, thus eventually enhancing its emissive ability.
Compounds TPMN, TTMN, MeTTMN, and MeOTTMN pos-
sess good solubilities in common organic solvents, such
as toluene, dichloromethane, chloroform, tetrahydrofuran,
acetonitrile (ACN), methanol, and dimethyl sulfoxide. Their
UV–vis spectra measured in ACN are peaked at 441, 483, 492,
and 499 nm, respectively, as shown in Figure 3A. The absorp-
tion maximums of them locate in the range of visible light,
which endow less damage to biological system against UV
light. The gradually redshifted absorption wavelengths can be
attributed into the orderly enhanced D-A effect from TPMN to
MeOTTMN. On the other hand, as depicted in Figure S5 (Sup-
porting Information), the orderly decreased values of calculated
The combination of high emission efficiency with large Stokes
shift into one molecule is challenging and important for fluores-
cent materials. On the basis of above-mentioned results obtained
from single crystal analysis of TTMN, it is believed that those
Figure 3. A) Normalized absorption spectra of TPMN, TTMN, MeTTMN, and MeOTTMN in the ACN solution. B) PL spectra of TPMN (10 × 10−6 m)
in ACN/water mixtures with different water fractions (fw); λex: 441 nm. C) The plot of the emission maximum and the relative emission intensity (I/I0)
versus the composition of the aqueous mixture of TPMN, TTMN, MeTTMN, and MeOTTMN. Inset: fluorescence photographs of TPMN in the dilute
ACN solution and in ACN/water mixtures with 95% water fractions taken under 365 nm UV irradiation. D) Normalized PL spectra of TPMN
(λex: 441 nm), TTMN (λex: 483 nm), MeTTMN (λex: 492 nm), and MeOTTMN (λex: 499 nm) in the solid state. Inset: fluorescence photographs of
TPMN, TTMN, MeTTMN, and MeOTTMN in the solid state taken under 365 nm UV irradiation.
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energy gaps (2.565, 2.555, 2.498, and 2.405 eV) are in good
accordance with experimental data of absorption maximums.
The AIE property of TPMN, TTMN, MeTTMN, and
MeOTTMN was studied in ACN/water mixtures with different
water fractions (fw), which enabled certain extent of solute
aggregation. In ACN solution, TPMN, TTMN, and MeTTMN
weakly emit red photoluminescences (PLs) at 635, 664, and
673 nm, respectively with 0.1–0.32% of quantum yields, while
MeOTTMN is nonemissive in solution state (Table S2, Sup-
porting Information). All the four compounds show gradual
increase in PL intensity with raising fraction of water starting
from 70%, due to the formation of nanoaggregates that are
measured and confirmed by dynamic light scattering analysis.
The average hydrodynamic diameters of these nanoaggregates
that formed in corresponding suspensions containing 95%
fraction of water are ranging from 76.6 to 101 nm with polydis-
persity indexes from 0.1 to 0.22 (Figure S6, Supporting Infor-
mation). The strongest PL intensities were found with 90%
or 95% fraction of water upon aggregation, in which their PL
intensities were enhanced to about 266-, 12-, 68-, and 34-fold
comparing with those of ACN solutions (Figure 3C). Their
quantum yields (18.6%, 15.8%, 7.4%, and 1.1%) in solid state
also dramatically increased comparing with those in solu-
tion state. The remarkable enhancements of PL intensities
in both aggregated and solid states clearly demonstrate their
AIE characteristics. Their maximum emissions in aggrega-
tion state locate at 637, 672, 681, and 701 nm, indicating their
red-, far red-, and NIR-emissive properties. In addition, red-
shifts of emissions are observed from aggregate to solid state,
in which PL spectra are peaked at 648, 690, 719, and 715 nm
(Figure 3D). As illustrated in Table S2 (Supporting Informa-
tion), these AIEgens have extremely large Stokes shifts, even
more than 200 nm. Moreover, the fluorescence decay curves of
TPMN, TTMN, MeTTMN, and MeOTTMN in the solid state
reveal that their lifetimes range from 0.95 to 8.32 ns (Figure S7,
Supporting Information). Apparently, both long emission wave-
length and bright emission of AIEgen TTMN in aggregates
perfectly match the results and hypotheses that were obtained
from the analysis of its single crystal. The collected photophys-
ical data suggest that in this developed system, emissions in
red/NIR region are easily tunable by fine-varying their molec-
ular structures and substituents having different extent of D-A
effect, demonstrating excellent controllability of this system in
terms of emission.
the PL intensity; nevertheless, the enhanced emission feature
of AIEgens in aggregates reveals a stronger AIE feature than
the TICT effect in this system, reasonably benefitting from
both the rigidified molecular conformation caused by powerful
inter- and intramolecular interactions and restricted molecular
motions in aggregates.
2.3. Bioimaging and Therapy
To evaluate the cytotoxicities of AIEgens TPMN, TTMN,
MeTTMN, and MeOTTMN in living cells, 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was
used with different concentrations of AIEgens. As illustrated in
Figure S10 (Supporting Information), no significant decrease of
the cell viability is observed even when the HeLa cells were cul-
tured in the presence of 10 × 10−6 m of AIEgens for 24 h, dem-
onstrating good biocompatibilities of these AIEgens to HeLa
cells.
Cell imaging study further proceeded by employing HeLa
cells as a cell model, in which the cells were pretreated with
oleic acid aiming to induce considerable amount of neutral
lipids. The influence of AIEgen concentration was investigated
by the uses of 2 × 10−6, 1 × 10−6, 500 × 10−9, and 200 × 10−9
m
of TPMN. As shown in Figure S11 (Supporting Information),
TPMN was strongly emissive in cells, in which the brightness
did not significantly decline even the concentration was down
to 200 × 10−9 m, suggesting a high brightness of TPMN in cell
imaging. Colocalization experiment was then conducted by
incubating HeLa cells with each presented AIEgen for 30 min
and BODIPY493/503 Green for 10 min. The cell imaging of
AIEgens and BODIPY493/503 Green overlapped perfectly, indi-
cating their powerful LD-specific targeting capability (Figure 4).
Pearson’s correlation coefficients that are commonly used to
determine the linear association of two variables were calcu-
lated to be up to 97% (Figure S12, Supporting Information),
solidly demonstrating the high specificities of these AIEgens
for staining LDs. The excellent LDs-staining specificity can be
attributed to the efficiently accumulating of these lipophilic
AIEgens in the hydrophobic spherical LDs which contain
mainly diverse neutral lipids, such as triacylglycerol and cho-
lesteryl ester, due to the “like–like” interactions. Moreover, as
illustrated in Figure 4 and Figure S11 (Supporting Informa-
tion), LDs are clearly visualized with a very high signal-to-
noise ratio when relatively low concentrations (200 × 10−9 m for
TPMN, TTMN, and MeTTMN, and 2 × 10−6 m for MeOTTMN)
of these AIEgens were utilized. Comparing with other AIEgens
used for LDs-imaging, the presented AIEgens hold the lowest
working concentrations,[16] which is almost comparative to that
of BODIPY493/503 Green. Interestingly, efficient cell uptake of
the presented AIEgens can be achieved without any encapsula-
tion or modification steps.
To investigate the TICT effect, TTMN was chosen as an
example. It was found that emission maximum of TTMN
slightly redshifts from 664 to 671 nm and the emission
intensity remarkably decreases with the increase of water
faction at low water content in mixed ACN/water solutions
(Figure S8, Supporting Information), indicating the existence
of TICT effect. In fact, ACN/water is not an ideal system to
study TICT effect, due to the small polarity difference. Thus,
PL spectra of TTMN were recorded in different solvents with
varied polarities. As depicted in Figure S9 (Supporting Infor-
mation), when the solvent is changed from nonpolar toluene to
polar dimethyl sulfoxide, the emission maximum largely red-
shifts from 573 to 665 nm, while emission intensity was con-
siderably reduced, suggesting a strong TICT effect. Indeed, AIE
properties and the TICT effect are competitive in determining
As one of the key criteria for evaluating a fluorescent bio-
probe, the photostabilities of the presented AIEgens were
assessed by continuous excitation and sequential scanning with
confocal microscope. As depicted in Figure 5, for both TTMN
and MeTTMN, their fluorescence intensities remain almost
constant after 40 scans within 15 min irradiation, while the
fluorescence intensities of TPMN and MeOTTMN keep at 84%
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Figure 4. Colocalization imaging of HeLa cells stained with BODIPY493/503 Green and AIEgens. A,E,I,M) Bright-field and B,C,F,G,J,K,N,O) confocal
images of HeLa cells stained with B) TPMN, F) TTMN, J) MeTTMN, N) MeOTTMN, and C,G,K,O) BODIPY493/503 Green. D,H,I,P) Merged images
of panels (B) and (C), (F) and (G), (J) and (K), as well as (N) and (O). λex: 488 nm (1% laser power). Concentrations: TPMN (200 × 10−9 m), TTMN
(200 × 10−9 m), MeTTMN (200 × 10−9 m), MeOTTMN (2 × 10−6 m), and BODIPY493/503 Green (100 × 10−9 m). Scale bar = 20 µm.
Figure 5. Confocal images of HeLa cells A–D) before (0 min, upper panel) and E–H) after the laser irradiation for 15 min (lower panel) stained with
A,E) TPMN, B,F) TTMN, C,G) MeTTMN, and D,H) MeOTTMN. I) Loss in fluorescence of HeLa cells stained with AIEgens and BODIPY493/503 Green
with the number of scans of laser irradiation. Concentration: 200 × 10−9 m (TPMN, TTMN, and MeTTMN), 2 × 10−6 m (MeOTTMN), and 100 × 10−9
m
(BODIPY493/503 Green); λex: 488 nm; scanning rate: 22.4 s per frame; laser power of confocal fluorescence microscope: 0.3 µW; scale bar = 20 µm.
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Figure 6. ROS generation upon white light irradiation and PDT study of both MeTTMN and MeOTTMN. A) Relative change in fluorescent intensity
(I/I0 − 1) at 534 nm of H2DCF-DA, TPMN, TTMN, MeTTMN, MeOTTMN, and mixtures of each AIEgen and H2DCF-DA in PBS upon white light
irradiation for different time. Concentrations: 10 × 10−6 m (AIEgens) and 5 × 10−6 m (H2DCF-DA). Cell viability of HeLa cells stained with different
concentrations of B) MeTTMN or C) MeOTTMN in the absence or presence of white light irradiation.
and 89%, respectively of its initial values in this process. By con-
trast, BODIPY493/503 Green suffers an obvious drop of fluo-
rescence intensity to 35% of its initial intensity under the same
conditions. Evidently, the photostabilities of these AIEgens are
greatly superior to that of commercial BODIPY493/503 Green.
Apart from the application of LD-specific cell imaging, the
presented AIEgens are also proven as powerful photosensi-
tizers in ROS generation. In this experimental study, H2DCF-
DA that emits fluorescence at around 534 nm triggered by
ROS was employed as an ROS indicator. Considering the
strong absorption of these presented AIEgens in the visible
light region, white light was utilized as excitation light source.
As illustrated in Figure 6A, in the presence of TPMN, TTMN,
MeTTMN, and MeOTTMN, the emission of H2DCF-DA was
gradually and rapidly intensified with the increase of irradiation
time. After 90 s exposure to white light, the emission intensi-
ties of H2DCF-DA were 17, 13, 25, and 19 times higher than
the original emission intensities without light irradiation,
respectively. Such change, however, was not observed in AIE-
gens or H2DCF-DA alone under the same conditions. These
results demonstrate the high ROS generation efficiencies of
these AIEgens. In particular, both MeTTMN and MeOTTMN
could serve as better sensitizers for ROS generation than the
other two AIEgens. In this context, MeTTMN and MeOTTMN
were chosen as photosensitizers to investigate the therapeutic
effect of these AIEgens through PDT, which was quantitatively
evaluated on HeLa cells by standard MTT assay. Both of the
two AIEgens exhibit very low cytotoxicity in dark condition,
regardless of the AIEgen concentration used for cell staining
(Figure S10, Supporting Information and Figure 6). Low cyto-
toxicity in dark condition is one of the essential features of
photosensitizers for PDT application. With white light irradia-
tion, a dose-dependent toxicity is observed for both of them. In
the case of MeTTMN, the HeLa cell viability decreased grad-
ually to around 50% with concentration of 1 × 10−6 m, while
increasing the concentration to 2.5 × 10−6 m eventually lead to
almost complete cell apoptosis (Figure 6B). When MeOTTMN
was examined, cell viability remained the same with those con-
centrations lower than 2.5 × 10−6 m; however, a sharp decline
of cell viability was found with the concentration of 5 × 10−6
m
(Figure 6C). These results clearly reveal that both of the two
AIEgens are remarkably effective for killing cancer cells by
PDT pathway. In addition, MeTTMN shows higher efficiency
than MeOTTMN that perfectly match with the outcomes of
ROS generation. Meanwhile, at the imaging concentrations of
200 × 10−9 m (MeTTMN) and 2 × 10−6 m (MeOTTMN), these
two AIEgens do not cause obvious damage of cell viability even
under light irradiation, making them ideal for dual applications
of cell imaging and therapy through controlling AIEgen con-
centrations. It seems reasonable to infer that due to the fluidity
and various biofunctions of LDs, the ROSs that are generated
by the AIEgens with specific-targeting feather toward LDs can
efficiently initiate to cell apoptosis.
Visualization of the biological structures and processes on in
vivo level is of great importance.[19] However, imaging of cells
in vitro by bioprobes often does not represent the real circum-
stances in vivo, because of the separation from native environ-
ments.[20] Encouraged by the excellent cell imaging results by
using the presented AIEgens, further investigations were done
for in vivo imaging of living zebrafish embryos. Zebrafish is an
ideal vertebrate model for biological in vivo imaging, owing to
the exceptionally high optical clarity in embryonic and larval
stages. In this experiment, living zebrafish embryos were
stained by these AIEgens with the concentration of 5 × 10−6 m for
30 min. As depicted in Figure 7, AIEgens TPMN (Figure 7G),
TTMN (Figure 7H), and MeTTMN (Figure 7I) provided fluo-
rescent images with bright red emission showing the contour
profile of zebrafish. In contrast, in the case of MeOTTMN, an
unclear image of zebrafish was obtained, perhaps due to its long
emission wavelength located in NIR region. These preliminary
results of zebrafish imaging suggest the great potential of these
AIEgens for observing biological processes toward in vivo level.
3. Conclusion
In summary, we have developed a facile one-pot synthetic
approach to red/NIR AIEgens with emission wavelengths
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Figure 7. In vivo zebrafish imaging. A–E) Bright-field, F–J) fluorescence microscope images of living zebrafish embryos stained with B,G) TPMN, C,H)
TTMN, D,I) MeTTMN, E,J) MeOTTMN, as well as A,F) images of zebrafish embryo without staining. Concentration: 5 × 10−6 m; staining time: 30 min.
1
Characterization: H spectra were measured on Bruker ARX 400 NMR
spectrometers using CDCl3 as the deuterated solvent. High-resolution
mass spectra (HRMS) were recorded on a Finnegan MAT TSQ 7000 Mass
ranging from 648 to 719 nm in solid state. This AIEgen
system exhibits several impressive characteristics including
simple structures, small molecular weights, good solubility
in common organic solvents, large Stokes shifts, bright emis-
sions, and high processabilities. These AIEgens have been
proven to be considerably powerful for cell imaging, in which
they show excellent LDs-targeting specificity, much higher
photostability than commercial LDs-staining fluorophore, as
well as high brightness even with a low concentration down
to 200 × 10−9 m. These impressive properties make them as an
ideal alternative of commercial LD-specific dyes. The high pen-
etrability of these AIEgens enables the living zebrafish embryos
to be brightly stained through simple incubation procedure for
in vivo imaging. Moreover, with white light irradiation, their
high ROS generation efficiency enables the effective application
for photodynamic ablation of cancer cells, and the apoptosis of
cancer cell was significantly induced by using low concentra-
tion of AIEgens. To the best of our knowledge, this would be
the first report on using LD-specific AIEgens as photosensi-
tizers for PDT application.
Our findings in this study may perfectly solve the problem of
complicated synthetic procedures of red/NIR AIEgens and effi-
ciently provide an ideal model for design, synthesis, and biological
utilizations of red/NIR AIEgens. Furthermore, this study would
significantly promote new strategies in the construction, modifi-
cation, and functionalization of bioprobes sharing both AIE fea-
ture and long emission wavelengths on the basis of the presented
system, and consequently benefit the developments of AIE study
in the areas of fluorescent imaging and therapeutic applications.
Spectrometer System operating in a Matrix-Assisted Laser Desorption/
Ionization Time of Flight (MALDI-TOF) mode. UV absorption spectra
were taken on a Milton Ray Spectronic 3000 array spectrophotometer.
Steady-state fluorescence spectra were recorded on a Perkin Elmer LS
55 spectrometer. Fluorescence images were collected on Olympus BX 41
fluorescence microscope. Laser confocal scanning microscope images
were collected on Zeiss laser scanning confocal microscope (LSM7
DUO) and analyzed using ZEN 2009 software (Carl Zeiss).
Cell Culture: HeLa cells were cultured in the minimal essential
medium (MEM) containing 10% FBS and antibiotics (100 units mL−1
penicillin and 100 mg mL−1 streptomycin) in a 5% CO2 humidity
incubator at 37 °C.
Cytotoxicity Study: MTT assays were used to evaluate the cytotoxicity
of the presented AIEgens. Cells were seeded in 96-well plates (Costar, IL,
USA) at a density of 6000–8000 cells per well. After overnight culturing,
medium in each well were replaced by 100 µL fresh medium containing
different concentrations of the presented AIEgens. The volume fraction
of dimethyl sulfoxide (DMSO) was below 0.2%. 24 h later, 10 µL MTT
solution (5 mg mL−1 in PBS) was added into each well. After 4 h of
incubation, 100 µL SDS-HCl aqueous solution (10% SDS and 0.01
m
HCl) was added to each well. After incubation for 4 h, the absorption
of each well at 595 nm was recorded via plate reader (Perkin-Elmer
Victor3). Each trial was performed with six wells parallel.
Cytotoxicity of MeTTMN and MeOTTMN to Cancer Cells under Light
Irradiation: HeLa cells were seeded in 96-well plates (Costar, IL, USA) at
a density of 6000–8000 cells per well. After overnight culturing, medium
in each well was replaced by 100 µL fresh medium containing different
concentrations of MeTTMN or MeOTTMN. The volume fraction of
DMSO is below 0.2%. After incubation for 25 min, plates containing
HeLa cells were exposed to white light (around 10 mW) for 30 min, and
another array of plates with cells was kept in dark as control. Then, the
plates were conducted the same treatment as the biocompatibility test.
Cell Imaging: Cells were grown in a 35 mm petri dish with a coverslip
at 37 °C. The live cells were incubated with certain dye at certain
concentration for certain time (by adding 2 µL of a stock solution in
DMSO solution to 2 mL of cell culture medium, DMSO < 0.1 vol%).
After incubation with AIEgens, the cells were washed with PBS for three
times. The AIEgen-labelled cells were mounted and imaged using a laser
scanning confocal microscope (LSM7 DUO) at 488 nm with 1% laser
power (the scanning rate was 22.4 s per frame). The emission filter was
600–744 nm.
4. Experimental Section
Materials: Dulbecco’s Modified Essential Medium and RPMI-1640
were purchased from Gibco (Life Technologies). Ultrapure water was
supplied by Milli-Q Plus System (Millipore Corporation, United States).
Phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin,
streptomycin, and BODIPY 493/503 green were purchased from Thermo
Fisher Scientific. H2DCF-DA was purchased from Sigma-Aldrich and
used as received. 4-Bromo-N,N-diphenylaniline 4-bromo-N,N-di-p-
tolylaniline, 4-bromo-N,N-bis(4-methoxyphenyl)aniline, (4-formylphenyl)
boronic acid, (5-formylfuran-2-yl)boronic acid, and (5-formylthiophene-
2-yl)boronic acid were purchased from J&K or Meryer. All the chemicals
used as received without further purification.
Confocal Colocalization: For costaining with lipid dye BODIPY493/503
Green, cells were first incubated with AIEgens and BODIPY493/503
Green (100 × 10−9 m) at 37 °C for 30 min. The medium was then
removed and the cells were rinsed with PBS for three times and then
imaged under confocal microscope. For AIEgens, the emission filter was
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600–740 nm; for BODIPY493/503 Green, the excitation was 488 nm and
the emission filter was 510–553 nm.
Grants Committee of Hong Kong (Grant No. AoE/P-03/08), the Research
Grants Council of Hong Kong (Grant Nos. 16301614, 16305015, and
N_HKUST604/14), Innovation and Technology Commission (Grant
No. ITC-CNERC14SC01), the National Science Foundation of China
(Grant Nos. 81372274, 81501591, and 8141101080), the Science and
Technology Planning Project of Guangdong Province (Grant Nos.
2014A030313033 and 2014A050503037), and the Shenzhen Science
and Technology Program (Grant Nos. JCYJ20130402103240486 and
JCYJ20160509170535223). B.Z.T. is also grateful for the support from
the Guangdong Innovative Research Team Program of China (Grant
No. 201101C0105067115). All the procedures used in this study
with Zebrafish were performed in accordance with the guidelines and
regulations set out by the Animal Ethics Committee of the Hong Kong
University of Science and Technology (HKUST).
Photostability: The dye-labeled HeLa cells were imaged by a confocal
microscope (Zeiss laser scanning confocal microscope LSM7 DUO)
using ZEN 2009 software (Carl Zeiss). Conditions: for AIEgens,
excitation wavelength: 488 nm; for BODIPY493/503 Green, excitation
wavelength: 488 nm (1% laser power).
Synthesis of Compound TPMN:[21] A dried Schlenk tube equipped with
a magnetic stirring bar was charged, under a nitrogen atmosphere, with
4-bromo-N,N-diphenylaniline (162 mg, 0.5 mmol), (4-formylphenyl)
boronic acid (112.5 mg, 0.75 mmol), K3PO4 (530 mg, 2.5 mmol),
Pd(OAc)2 (5.6 mg, 5 mol%), and EtOH (8 mL). The mixture was stirred
at 78 °C for 12 h, after cooling down to room temperature, the reaction
mixture was filtered. Then CNCH2CN (66 mg, 1 mmol) was added into
the obtained filtrate, the mixture was stirred at 78 °C for another
72 h. After cooling down to room temperature, the solvent was removed
under vacuum, then water (20 mL) was added into the mixture, which
was extracted with CH2Cl2 (5 mL × 3). The combined organic phase was
dried over Na2SO4 and filtered; the filtrate was removed under reduced
pressure in order to obtain the crude product, which was further purified
by silica gel chromatography (petroleum ether/CH2Cl2 as eluent) to yield
Conflict of Interest
The authors declare no conflict of interest.
1
TPMN with the yield of 42%. H NMR (400 MHz, CDCl3): 7.96 (d, J =
Keywords
8.4Hz, 2H), 7.73 (t, J = 6.6Hz, 3H), 7.52 (d, J = 8.8Hz, 2H), 7.28–7.32
(m, 4H), 7.07–7.16 (m, 8H). 13C NMR (100 MHz, CDCl3): 159.08,
148.97, 147.08, 146.80, 131.54, 131.49, 129.46, 129.12, 127.91, 127.05,
125.13, 123.78, 122.62, 114.12, 113.01, and 80.98. ESI HRMS: calcd. for
C28H19N3 [M]+: 397.1579, found: 397.1557.
aggregation-induced emission, facile synthesis, lipid droplet-specific
imaging, photodynamic therapy, red/NIR emission
Received: July 19, 2017
Revised: August 16, 2017
Published online:
Synthesis of Compound TTMN:[21] The synthetic process was similar
to TPMN except for the change of starting materials. 1H NMR (400
MHz, CDCl3): 7.75 (s, 1H), 7.68 (d, J = 4.0Hz, 1H), 7.53 (d, J = 8.8Hz,
2H), 7.29–7.33 (m, 5H), 7.10–7.16 (m, 6H), 7.04 (d, J = 8.8Hz, 2H). 13
C
NMR (100 MHz, CDCl3): 157.11, 150.24, 149.90, 146.59, 140.36, 133.06,
129.56, 127.54, 125.48, 124.81, 124.32, 123.25, 121.67, 114.54, 113.66,
and 74.99. ESI HRMS: calcd. for C26H17N3S [M]+: 403.1143, found:
403.1150.
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1
similar to TPMN except for the change of starting materials. H NMR
(400 MHz, CDCl3): 7.73 (s, 1H), 7.66 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H),
7.30 (s, 1H), 6.96–7.13 (m, 10H), 2.34 (s, 6H). 13C NMR (100 MHz,
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similar to TPMN except for the change of starting materials. 1H
NMR (400 MHz, CDCl3): 7.72 (s, 1H), 7.65 (d, J = 4 Hz, 1H), 7.47
(d, J = 8.8 Hz, 2H), 7.28 (d, J = 4 Hz, 1H), 7.09–7.11 (m, 4H), 6.86–6.89
(m, 6 H), and 3.81 (s, 6H). 13C NMR (100 MHz, CDCl3): 157.74, 156.83,
150.76, 150.13, 140.50, 139.40, 132.60, 127.51, 127.44, 123.04, 122.73,
118.75, 114.94, 114.72, 113.82, 74.27, and 55.48. ESI HRMS: calcd. for
C28H21N3O2S [M]+: 463.1354, found: 463.1381.
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