Facile synthesis of AIEgens with wide color tunability for cellular imaging and therapy

Article abstract of DOI:10.1039/c8sc05805a

Luminogens with aggregation-induced emission (AIE) characteristics are nowadays undergoing explosive development in the fields of imaging, process visualization, diagnosis and therapy. However, exploration of an AIE luminogen (AIEgen) system allowing for extremely wide color tunability remains challenging. In this contribution, the facile synthesis of triphenylamine (TPA)-thiophene building block-based AIEgens having tunable maximum emission wavelengths covering violet, blue, green, yellow, orange, red, deep red and NIR regions is reported. The obtained AIEgens can be utilized as extraordinary fluorescent probes for lipid droplet (LD)-specific cell imaging and cell fusion assessment, showing excellent image contrast to the cell background and high photostability, as well as satisfactory visualization outcomes. Interestingly, quantitative evaluation of the phototherapy effect demonstrates that one of these presented AIEgens, namely TTNIR, performs well as a photosensitizer for photodynamic ablation of cancer cells upon white light irradiation. This study thus provides useful insights into rational design of fluorescence systems for widely tuning emission colors with high brightness, and remarkably extends the applications of AIEgens.

Full text of DOI:10.1039/c8sc05805a

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ARTICLE
Facile synthesis of AIEgens with wide color tunability for cellular
imaging and therapy
Received 00th January 20xx,
Accepted 00th January 20xx
Wenhan Xu,b,‡ Michelle M. S. Lee,b,‡ Zhihan Zhang,b Herman H. Y. Sung,b Ian D. Williams,b Ryan T.
K. Kwok,b Jacky W. Y. Lam,b Dong Wang*a and Ben Zhong Tang*b
DOI: 10.1039/x0xx00000x
Luminogens with aggregation-induced emission (AIE) characteristics are nowadays undergoing an explosive development
in the fields of imaging, process visualization, diagnosis and therapy. However, exploration of an AIE luminogen (AIEgen)
system allowing for extremely wide color tunability remains challenging. In this contribution, the facile synthesis of
triphenylamine (TPA)-thiophene building block-based AIEgens having tunable maximum emission wavelengths covering
violet, blue, green, yellow, orange, red, deep red and NIR regions is reported. The obtained AIEgens can be utilized as
extraordinary fluorescent probes for lipid droplets (LDs)-specific cell imaging and cell fusion assessment, showing excellent
image contrast to the cell background, high photostability, as well as satisfying visualization outcome. Interestingly,
quantitative evaluation of phototherapy effect demonstrates that one of these presented AIEgens, namely TTNIR, well
performs as photosensitizer for photodynamic ablation of cancer cells upon white light irradiation. This study thus
provides useful insights into rational design of fluorescence system for widely tuning emission colors with high brightness,
and remarkably extends the applications of AIEgens.
www.rsc.org/
which is notoriously known as aggregation-caused quenching
(ACQ);3 2) the difficulty of widely tuning emission colors by
simple modification of molecular structures; and 3)
Introduction
The exploration of fluorescent materials and technologies has
opened new avenues to scientific advancement, societal
development and public health,1 which is exemplified by the
Nobel Prize successively awarding to fluorescence-related
researches. As one of the most important branches of
fluorescent materials, fluorescent bio-materials that offer
researchers a powerful platform for analytical sensing and
optical imaging have been proven to be extremely useful for
biological visualizations, clinical diagnosis and diseases
treatment by virtue of their non-invasion, in situ workability,
excellent accuracy, superb sensitivity and simple operation.2
Although many types of fluorophores have been
commercialized for biological applications, current situation is
still far from ideal, mainly due to some limitations: 1) inherent
fluorescence quenching upon aggregates formation due to
intermolecular π-π stacking and other nonradiative pathways,
complicated and laborious syntheses of fluorophores.4
As an anti-ACQ phenomenon, aggregation-induced
emission (AIE) was coined in 2001 by Tang’s group,5 which
refers to a unique phenomenon that a novel class of
fluorophores are non-emissive or weakly emissive in the
molecularly dissolved state but they emit intensively in
aggregated states owing to the restriction of the
intramolecular motions (RIM).6 Remarkably, the AIE principle
has triggered state-of-the-art developments in an array of
biological fields, ranging from bioimaging, biosensing, stimuli-
responsive systems, therapeutics to theranostics, mainly
resulting from various impressive advantages of AIE
luminogens (AIEgens), such as high photobleaching threshold,
high signal-to-noise ratio for imaging, excellent tolerance for
any concentrations, large Stokes shift, turn-on feature for
detecting analytes, and efficient photosensitizing ability.7
Although numerous AIEgens have been constructed on the
basis
of
different
structural
motifs
including
a. Center for AIE Research, College of Materials Science and Engineering, Shenzhen
University, Shenzhen 518060, China. E-mail: wangd@szu.edu.cn
b. Hong Kong Branch of Chinese National Engineering Research Center for Tissue
Restoration and Reconstruction, Department of Chemistry, 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
tetraphenylethene,8 hexaphenylsilole,9 tetraphenylpyrazine10
and distyrylanthracene,11 to the best of our knowledge, there
have been no single AIE system which allows arbitrarily tuning
emissions ranging from each color of visible light to near-
infrared (NIR) region. Considering the great significance of
tunable fluorescent system in the applications of multi-target
sensing, optoelectronic devices and full-color bio-imaging,12
the development of an AIE system exhibiting wide color
tunability is highly desired and remains a challenging task.
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
‡ W.X. and M.M.S.L. contributed equally to this work.
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Comparing with inorganic complexes and quantum dots, with intermediate product 1, which was isolated through Suzuki-
View Article Online
organic fluorophores are advantageous for bio-imaging, Miyaura
coupling
reaction
between
DOI: 10.1039/C8SC0580(54A-
diagnosis and therapy, benefiting from their good bio- (diphenylamino)phenyl)boronic acid and 2,5-dibromothiophene.
compatibility, tunable molecular structures and chemical
All compounds are comprised of sufficient moieties that can
compositions, and scalable synthesis.13 Evidently, the freely rotate in single-molecule state leading to energy
exploration of an organic fluorophore system with both AIE consumption of the excited state through non-radiative pathways,
attribute and emission color tunability across
a wide thus ensuring these compounds to be weekly emissive in solution.
wavelength range would captivate much interest. Herein, we Aiming to further study and deciphering of their optical properties
report for the first time the design and synthesis of a series of in aggregation state, single crystals of TTG, TTY and TTDR were
AIEgens having widely tunable emissions covering violet, blue, grown in DCM-MeOH mixtures by slow solvent evaporation. As
green, yellow, orange, red, deep red and NIR regions (Fig. 1). illustrated in Fig. 2, S1 and S2, twisted conformation of TPA
Each AIEgen comprising of triphenylamine (TPA)-thiophene segment extends the intermolecular distance (> 3.2 Å) between two
building block is facilely obtained through one- or two-step parallel planes, remarkably reducing or avoiding the intermolecular
reaction, and the emission colors are tuned by simple π-π interactions, and essentially preventing emission quenching in
alteration of HOMO-LUMO energy level by the introduction of its aggregation state. Moreover, the molecular conformation can be
electron donor (D)-acceptor (A) substituents.14 Moreover, strongly rigidified by abundant intermolecular interactions (such as
these AIEgens can be successfully utilized as extraordinary lipid C-H···O, C-H···C, S···C) resulting in the restriction of molecular
droplets (LDs)-specific bioprobes in cell imaging, determination motions, which is beneficial for enhancing the solid state emission
of cell fusion, and photodynamic cancer cell ablation.
efficiency. On the basis of above-mentioned XRD results, it is
believed that these synthesized compounds are potentially AIE-
active.
N
S
N
S
TTV
TTB
O
Photophysical property
The UV-vis absorption spectra of TTV, TTB, TTG, TTY, TTO, TTR,
TTDR and TTNIR were measured in acetonitrile (ACN). As shown in
Fig. 3A and Table S1, the solution of building block TTV displays
maximum absorption band at 348 nm, and the maximum
absorption peaks of these modified compounds are located ranging
from 383 nm to 512 nm. The gradually red-shifted absorption
wavelengths can be attributed to the orderly enhanced D-A effect
from TTV to TTNIR. To investigate their AIE features, ACN/H2O
mixture with different H2O fractions was utilized as solvent system.
It was observed that compounds TTB, TTG, TTY, TTO, TTR, TTDR and
TTNIR exhibit typical AIE features (Fig. 3C). Taking TTY as an
example, there is almost no fluorescence emission when H2O
fraction is below 60%. Afterwards, the PL intensity increases
dramatically along with raising fraction of water because of
activation of RIM by molecular aggregation and reaches to its
maximum at 90% water fraction, which is 185-fold higher than that
in ACN solution (Fig. 3B). Although the fluorescence intensity of TTV
is inversely proportional to water fraction, its quantum yield in solid
state (27.5%) is higher than that in solution state (18.6%), definitely
demonstrating an aggregation-induced emission enhancement
(AIEE) attribute. The gradually decreased fluorescence intensity of
TTV along with the increased water fraction could be attributed to
its twisted intramolecular charge transfer (TICT) feature,15 which
was determined by both the red-shifted emission wavelength and
the declined emission efficiency accompanying the raised solvent
polarity (Fig. S3). As one of the nonradiative pathways for the
excited state to relax and deactivate, TICT effect is competitive with
AIE properties in determining the PL intensity and efficiency using
ACN/H2O solution system. In the case of TTV, the AIE feature is
strongly depressed by TICT effect in the nanoaggregation state. As
illustrated in Fig. 3D, 3E and Table S1, these TPA-thiophene building
block-based AIEgens emit efficiently in both nanoaggregation and
N
NC
S
N
S
CN
O
S
O
TTNIR
TTG
O
N
N
S
S
CN
O
NC
O
TTY
TTDR
O
O
N
N
O
S
S
S
S
O
S
O
O
TTO
TTR
Fig. 1 TPA-thiophene-based AIEgens with widely tunable emissions.
Results and discussion
Synthesis and single crystal analysis
As depicted in Scheme 1, the desired compounds were facilely
prepared through one or two steps. TTV was synthesized through
Suzuki-Miyaura coupling reaction of 4-bromo-N,N-diphenylaniline
with thiophen-2-ylboronic acid in the presence of palladium catalyst
using mixed THF/H2O as solvent at 75 ℃. The same synthetic
procedure was successfully conducted by employing substituted 4-
bromo-N,N-diphenylaniline and modified thiophen-2-ylboronic acid
as starting materials, producing compounds TTG, TTY, TTO and TTR.
The reactions between TTG/TTO and malononitrile proceeded
smoothly, giving the corresponding products TTDR and TTNIR with
moderate yields. In addition, TTB was obtained by Suzuki-Miyaura
coupling reaction of (4-(1,2,2-triphenylvinyl)phenyl)boronic acid
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DOI: 10.1039/C8SC05805A
HO
S
Condition A
S
+
N
Br
B
N
HO
TTV
B(OH)2
Br
S
Condition A
OH
S
S
Br
Br
N
+
N
B
N
Condition A
OH
TTB
1
R
R
R
TTG, R = H, m = 1
NC
CN
TTY, R = OMe, m = 1
TTO, R = OMe, m = 2
TTR, R = OMe, m = 3
TTDR, R = H, m = 1
TTNIR, R = OMe, m = 2
HO
Condition A
Condition B
S
S
CHO
m
+
S
N
Br
N
N
B
CHO
m
m
HO
R
TTG, TTY, TTO, TTR
R
TTDR, TTNIR
R
Scheme 1. Synthetic routes to AIEgens with tunable emission colors. Condition A: Pd(PPh3)4, K2CO3, THF, H2O, 75 ℃, 12 h. Condition B:
EtOH, malononitrile, 78 ℃, 72 h.
B3LYP/6-31+G(d) level (Fig. 4). It was observed that from TTV to
TTNIR, the calculated HOMO-LUMO energy gaps are generally
decreased, and the results are in good accordance with
experimental data of emission maximums. The orderly declined
energy gaps are realized through ingenious modification of TPA-
thiophene building block with diverse electron-donating (thienyl or
methoxyl groups), electron-accepting (aldehyde or cyano groups)
units or π-bridge. Except for TTB, the HOMOs of the rest AIEgens
are delocalized at the TPA moiety, whereas their LUMOs are
distributed on the other side of the structures, demonstrating
typical D-A structural features. It has been demonstrated that the
separation of HOMO and LUMO distribution is essential to
effectively reduce singlet-triplet energy gap, which facilitates the
generation of reactive oxygen species (ROS),16 further endowing
these AIEgens with prominent potential for photodynamic therapy
(PDT) applications.17 On the contrary, TTB possesses evenly
distributed HOMO and LUMO, resulting from its both imperceptible
D-A effect and long π-conjugation bridges.
Fig. 2 A) Single crystal structure of TTG. B) Side view of the crystal
structure of TTG. C) Various inter- and intramolecular interactions in
crystals of TTG.
Bio-imaging, visualization of cell fusion and photodynamic therapy
In the preliminary bioimaging experiment, cell imaging study was
solid states exhibiting relatively high quantum yields ranging from conducted by using HeLa cells as a cell model. Cells were incubated
3.11% to 40.79%. Each maximum emission wavelength accurately with 1 μM of TTNIR for 20 min, as illustrated in Fig. 5B, the bright
peaks in violet (402 nm), blue (482 nm), green (531 nm), yellow fluorescence within cells can be observed showing excellent image
(580 nm), orange (612 nm), red (649 nm), deep red (667 nm) and contrast to the cell background. Co-localization study further
NIR (724 nm) regions, respectively, suggesting the extremely wide proceeded upon incubating HeLa cells with TTNIR and
emission color tunability, which is ascribed to both of their varied - BODIPY493/503 Green. The latter dye is a commercially available
conjugation and D-A effect. Additionally, the fluorescence decay bioprobe for the LDs, which are ubiquitous lipid-rich spherical
curves in the solid state show that their lifetimes range from 0.64 to organelles and actively involved in various biofunctions, such as
3.69 ns (Fig. 3F and Table S1).
signal transduction, lipid metabolism, and protein degradation. The
perfect overlap between TTNIR and BODIPY493/503 Green in cell
imaging output indicates the excellent LDs-specific targeting
capability of TTNIR (Fig. 5B, 5C, 5D). Photostability is a key criteria
Theoretical calculations
To better understand the optical properties of these AIEgens, for evaluating the overall stability of photosensitive substances.
density functional theory (DFT) calculations were carried out at Continuous scanning method was then utilized to quantitatively
B3LYP/6-31+G(d) level with molecular geometries optimized at TD- study and compare the photostability of TTNIR and BODIPY493/503
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DOI: 10.1039/C8SC05805A
200
TTV
TTB
TTG
TTY
TTO
TTR
f
w (vol %)
A
B
C
95
90
80
70
60
50
40
30
20
10
0
100
25
TTV
TTB
TTG
TTY
TTO
TTR
TTDR
TTNIR
20
15
10
5
TTDR
TTNIR
0
450
500
550
600
650
700
0
20
40
60
80
100
300
400
500
600
700
Wavelength (nm)
Water fraction (vol%)
Wavelength (nm)
D
F
TTV
TTB
TTG
TTY
TTO
TTR
TTV
TTB
TTG
TTY
TTO
TTR
Aggregation
1000
100
10
TTDR
TTNIR
TTDR
TTNIR
TTV TTB TTG TTY TTO TTR TTDR TTNIR
Solid
350
450
550
650
750
850
950
0
10
20
30
40
50
Wavelength (nm)
Time decay (ns)
Fig. 3 A) Normalized absorption spectra of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR in ACN solution. B) PL spectra of TTY (1×10-5M) in
ACN/water mixtures with different water fraction (w); ex: 410 nm. C) The plot of the emission maximum and the relative emission
intensity (I/I0) versus the composition of the aqueous mixture of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR. D) Normalized PL spectra
of TTV (em: 417 nm), TTB (em: 489 nm), TTG (em: 539 nm), TTY (em: 583 nm), TTO (em: 603 nm), TTR (em: 659 nm), TTDR (em: 684 nm),
and TTNIR (em: 706 nm) in solid state. E) Fluorescence photographs of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR (from left to right) in
ACN/water mixtures with 95% water fractions (upper) and in solid state (below) taken under 365 nm UV irradiation. F) Fluorescence decay
curves of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR in solid state.
LDs-staining (Figure S13). It was observed that Nile Red suffered an
TTV
TTB
TTG
TTY
obvious signal loss with remained signal intensity around 60% after
15 minutes of laser irradiation, strongly suggesting that the
photostability of TTNIR is superior to that of commercially available
bioprobes. To further prove its applicability, this staining and
imaging strategy using TTNIR is exploited for other cell lines,
including NCM460, DLD1, SW480, SW620 and COS-7 (Fig. S4). In
each case after incubation with TTNIR for 20 min, it shows strong
and specific internalization into the LDs. Moreover, other AIEgens
including TTV, TTB, TTG, TTY, TTO, TTR and TTDR were also
investigated for cell imaging. It was observed that LDs can be clearly
visualized with excellent image contrast to the cell background
through respective incubation of cells with these presented AIEgens
(Fig. 6). Pearson’s correlation coefficients between AIEgens and
commercially available LDs-bioprobes were determined to be 90-
95%, solidly demonstrating the high specificity of these AIEgens for
staining LDs (Fig. S5-S12). Their excellent LDs-staining specificity
reasonably results from the lipophilic property, which brings about
efficient accumulation of them in the hydrophobic spherical LDs
due to the “like-like” interactions. Evidently, these AIEgens possess
various impressive features, such as high brightness, excellent
targeting specificities to LDs, extraordinary photostabilities and
widely tunable emission colors, making them remarkably important
in visualization of biological structures and processes.
LUMO
HOMO
3.211 eV
2.423 eV
2.486 eV
1.963 eV
TTO
TTR
TTDR
TTNIR
LUMO
HOMO
1.828 eV
1.764 eV
1.929 eV
1.342 eV
Fig. 4 Molecular orbital amplitude plots of HOMO and LUMO energy
levels of TTV, TTB, TTG, TTY, TTO, TTR, TTDR and TTNIR calculated at
B3LYP/6-31+G(d) level based on the geometries optimized at TD-
B3LYP/6-31+G(d) level.
Green. As shown in Fig. 5E-I, after 15 minutes of laser irradiation,
the fluorescence intensity of BODIPY493/503 Green encounters
obvious decline, whereas TTNIR shows negligible photobleaching.
Moreover, the photostability assessment was also conducted
towards Nile Red, which is another commercially available dye for
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120
100
80
60
40
20
0
Bright field
TTNIR
BODIPY Green
Merged Image
20 μm
0 min
15 min
0 min
15 min
TTNIR
BODIPY 493/503
5
10 15 20 25 30 35 40
Number of Scan
Fig. 5 Colocalization imaging of HeLa cells stained with BODIPY493/503 Green and TTNIR, and the investigation of photostability. A) Bright-
field and B) confocal images of HeLa cells stained with TTNIR and C) BODIPY493/503 Green. D) Merged images of panels (B) and (C). λex:
488 nm (1% laser power). Concentrations: TTNIR (1 μM), BODIPY493/503 Green (500 nM). Confocal images of HeLa cells E, G) before (0
min) and F, H) after the laser irradiation for 15 min stained with E,F) TTNIR, and G,H) BODIPY493/503 Green. I) Loss in fluorescence of HeLa
cells stained with TTNIR and BODIPY493/503 Green with the number of scans of laser irradiation. Scanning rate: 22.4 s per frame. Scale bar
= 20 µm.
with different emission ranges is definitely reliable. Evidently, the
TTB
TTG
TTY
TTV
developed AIEgens having widely tunable emissions and high
emission efficiencies are potentially useful in the fundamental study
of cell fusion.
Intense fluorescence in near-infrared (NIR) region is highly
desirable for many clinical processes, due to the salient advantages
of deep tissue penetration, minimal photodamage to biological
structures, and high image contrast to the physiological
background.21 Moreover, NIR emission is generally realized by
intensifying D-A effect of the structure, resulting in the separation
of HOMO and LUMO distribution, as well as the decrease of singlet-
triplet energy gap, thus facilitating the generation efficiency of ROS.
Therefore, the AIEgen TTNIR with both bright NIR emission and
TTO
TTR
TTDR
TTNIR
20 m
Fig. 6 Confocal images of HeLa cells stained with A) TTV, B) TTB, C)
TTG, D) TTY, E) TTO, F) TTR, G) TTDR and H) TTNIR. Concentration:
AIEgen (1 μM).
As a common phenomenon in nature, cell fusion is highly
associated with many cellular processes, including fertilization,
development of placental, regeneration of skeletal muscle,
oncogenesis, aneuploidy, chromosomal instability and DNA
damage.18,19 In addition, recent study shows that cell fusion could
play a vital role in alternative therapies for restoring organ function
through repairing cellular dysfunction.19 Therefore, the
development of effective methods for visualizing cell fusion is of
great importance. Encouraged by the excellent cell imaging results
and homology of the presented AIEgens, a straightforward method
for visualization of cell fusion outcome was conducted by using the
combination of TTG and TTNIR as cell imaging agents, due to their
minimal overlap of the emission range. In this experiment, two sets
of cells were respectively stained with TTG and TTNIR, which were
then mingled and treated by polyethylene glycol (PEG) to induce
cell fusion.20 As illustrated in Fig. 7, after treatment by PEG, both
green and red fluorescence of lipid droplets were observed within
one single cell, suggesting that cell fusion between TTG- and TTNIR-
staining cells successfully proceeded. In addition, the cell fusion
outcome was also solidly verified through a commercially available
nuclei-staining agent Hoechst 33258. The appearance of two
stained nuclei within one single cell (Fig. 7D) indicated that the
visualization strategy of cell fusion outcome by using two AIEgens
Fig. 7 Cell fusion of COS-7 cells induced by 50% polyethylene glycol
(PEG), and visualized through fluorescence imaging of TTG and
TTNIR. A) Confocal image and B) bright-field of mixed cells
respectively stained with TTG and TTNIR. C) Merged images of
panels A) and B). D) Confocal image and E) bright-field of mixed
cells respectively stained with TTG, TTNIR and Hoechst 33258. F)
Merged images of panels D) and E). For TTG, λex: 405 nm (1% laser
power), λem: 425-540 nm. For TTNIR, λex: 560 nm (6.5% laser
power), λem: 600-740 nm. For Hoechst 33258, λex: 405 nm (3.5%
laser power) emission: 425-540 nm. Concentrations: TTG (500 nM),
TTNIR (2 μM), Hoechst 33258 (2.5 μM). Scale bar = 20 µm.
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Additionally, the high brightness and homoloVgieyw AortficletOhnelisnee
AIEgens endow them with excellenDtOI: p10e.1r0fo39rm/Ca8nSCc0e580f5oAr
visualizing cell fusion. To the best of our knowledge, this would
be the first report on using AIEgens as fluorescent probes for
assessing cell fusion. Notably, upon exposure to white light
irradiation, one of these presented AIEgens, namely TTNIR,
displays high ROS generation efficiency, enabling its effective
application for photodynamic ablation of cancer cells.
Our findings in this study provide an ideal fluorescence system
for widely tuning emission colors with high brightness at will. This
successful example would further facilitate the exploration of
organic fluorophores with AIE features for preclinical research and
clinical applications.
Fig. 8 ROS generation upon white light irradiation and PDT study of
TTNIR. A) Relative change in fluorescent intensity (I/I0) at 534 nm of
H2DCF-DA, TTNIR, and mixtures of TTNIR and H2DCF-DA in PBS
upon white light irradiation for different time. Concentrations: 10
μM (TTNIR) and 5 μM (H2DCF-DA). B) Cell viability of HeLa cells
stained with different concentrations of TTNIR in the absence or
presence of white light irradiation.
Experimental procedures
Materials and methods
Chemicals for synthesis were purchased from Sigma-Aldrich,
MERYER or J&K used without further purification. All solvents were
purified and dried following standard procedures. H spectra were
strong D-A effect is potentially efficient for PDT, which is an
extraordinary therapeutic modality, and has captivated much
interest for treating various malignant and non-malignant diseases
with minimal invasion and precise controllability. In the preliminary
test, the ROS generation efficiency of TTNIR was investigated using
H2DCF-DA as indicator, which can emit fluorescence at around 534
nm triggered by ROS. As shown in Fig. 8A, in the presence of TTNIR,
the emission of H2DCF-DA was rapidly intensified with the increase
of irradiation time using white light as irradiation source, reaching
36-fold enhancement in 6 min comparing with the original emission
intensity. On the contrary, the fluorescence intensities of AIEgens or
H2DCF-DA alone were very low and remained almost constant
under the same irradiation conditions. These results reveal the
good photo-sensitizing property for ROS generation. Quantitative
evaluation of phototherapy effect of TTNIR on HeLa cells was then
explored through standard 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. The dose-dependent
toxicity study exhibits that there is no obvious cytotoxicity observed
for the HeLa cells treated with TTNIR in dark, even with the TTNIR
concentration reaching as high as 20 μM (Fig. 8B). Upon white light
exposure, cell viability dropped gradually with raising the
concentration of TTNIR. Only 7% of cell viability remained with
utilizing 20 μM of TTNIR, demonstrating almost complete cell
apoptosis. Apparently, TTNIR hold high effectiveness for cancer cell
ablation by means of PDT.
1
measured on Bruker ARX 400 NMR spectrometers using CD2Cl2 or
CDCl3 as the deuterated solvent.
Mass spectrometric
measurements (HRMS) were performed on a Finnigan MAT TSQ
7000 Mass Spectrometer system operating in matrix-assisted laser
desorption/ ionization time of flight mass spectrometry (MALDI-
TOF) mode. UV-Vis spectra were measured on a Milton Ray
Spectronic 3000 array spectrophotometer. Steady-state
photoluminescence (PL) spectra were recorded on a PerkinElmer LS
55 spectrophotometer. Fluorescence images of AIEgens in solid
state and aggregation state were collected on Olympus BX 41
fluorescence microscope. The cellular fluorescence images were
taken using Zeiss laser scanning confocal microscope (LSM7 DUO)
and analyzed using ZEN 2009 software (Carl Zeiss).
Synthesis of compound TTV 7c,22
A mixture of bromide substituted triphenylamine moiety (1.2
mmol), thiophen-2-ylboronic acid moiety (1 mmol), THF (20 mL),
K2CO3 aqueous solution (2 M, 1.6 mL), and Pd(PPh3)4 (58 mg, 0.05
mmol) was degassed and charged with N2. The mixture was
refluxed overnight. The reaction was quenched by the addition of
water (30 mL) and extracted with CH2Cl2 (3 × 30 mL). The combined
organic layer was dried over anhydrous Na2SO4 and evaporated.
The residue was purified by column chromatography over silica gel
using petroleum ether to afford the desired product TTV with the
Conclusions
1
yield of 78%. H NMR (400 MHz, CD2Cl2): 7.60 (d, J = 6.8 Hz, 2H),
To sum up, we report the first series of AIEgens with widely
tunable emissions covering the whole visible region extending
to NIR area. These TPA-thiophene building block-based
AIEgens can be facilely prepared by extremely simple synthetic
protocols, and show high fluorescence quantum yields up to
40.79% in solid state benefiting from their intrinsic
aggregation-induced emission nature. They have been
successfully utilized for LDs-specific cell imaging, showing
excellent image contrast to the cell background and higher
photostability than the commercial LDs-staining fluorophore.
7.41 (d, J = 8 Hz, 2H), 7.37-7.33 (m, 4H), 7.13-7.06 (m, 9H). 13C NMR
(100 MHz, CDCl3): 147.49, 147.20, 144.26, 129.27, 128.54, 127.95,
126.71, 124.42, 123.98, 123.75, 123.02, 122.21. ESI HRMS: calcd. for
C22H17NS [M]+: 327.1082, found: 327.1066.
Cell imaging and confocal colocalization
6 | J. Name., 2012, 00, 1-3
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In 35 mm glass-bottomed dishes, the cells (NCM460, DLD1, The mixed culture was overlaid for 5 min at 37oVCiewwAirttihcle2OnmlinLe
o
SW480, and SW620) were seeded and cultured at 37 C. After PEG solution. Then the PEG solution wasDdOilIu: t1e0d.10w39it/hC8DSMC0E5M805inA
incubation with TTNIR (1 μM) for 20 min, the cells were gradient, after which the liquid was removed and replaced
washed with PBS for three times and subjected to imaging with DMEM. The obtained mixture was then subjected to
analysis using a laser scanning confocal microscope (Zeiss image analyses by confocal microscopy.
Laser Scanning Confocal Microscope; LSM7 DUO). The
excitation filter was 488 nm and the emission filter was 570-
Conflicts of interest
740 nm. For costaining assay, the AIEgens loaded COS-7 cells
were subjected for the incubation with BODIPY 493/503 Green
There are no conflicts to declare.
or Nile red for 20 min. Afterwards, the cells were washed by
PBS and then observed with CLSM. The cells were imaged
using proper excitation and emission filters for each dye. The
colocalization efficiency was analyzed with Olympus FV10-ASW
software, in which the calculated Pearson’s coefficient was
above 0.90.
Acknowledgements
This work was supported by the National Science Foundation
for Young Scientists of China (Grant No. 21801169), the
President Fund of Shenzhen University Foundation (848-
0000106), the National Natural Science Foundation of China
(21788102), the Research Grants Council of Hong Kong
(16301614, 16308016, 16305015, C6009-17G, C2014-15G,
N_HKUST604/14, and A-HKUST605/16), the Innovation and
Technology Commission (ITC-CNERC14SC01 and ITS/254117).
Photostability
For the photostability test, the cells were imaged using a
confocal microscope (Zeiss Laser Scanning Confocal
Microscope; LSM7 DUO) and analyzed using ZEN 2009
software (Carl Zeiss). Both TTNIR and BODIPY493/503 Green
were excited at 488 nm for one-photon imaging (1% laser
power). The scanning speed is 22.4 s per scan, and the
repeated image scans were taken for 40 times. The first scan
of both TTNIR and BODIPY493/503 Green was set to 100%,
followed by which the pixel intensity values were averaged
and plotted against the scan number. The resulting curve
represents the bleaching rate.
Notes and references
§ CCDC 1886931 and 1886933 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
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DOI: 10.1039/C8SC05805A
Facile synthesis and bio-applications of a series of AIEgens with widely tunable
emissions ranging from violet to near-infrared are reported.