Bright and photostable organic fluorescent dots with aggregation-induced emission characteristics for noninvasive long-term cell imaging

Article abstract of DOI:10.1002/adfm.201302114

Efficient long-term cell tracing in a noninvasive and real-time manner is of great importance to understand genesis, development, invasion, and metastasis of cancerous cells. Cell penetrating organic dots with aggregation- induced emission (AIE) characteristics are successfully developed as long-term cell trackers. The AIE dots enjoy the advantages of high emission efficiency, large Stokes shift, good biocompatibility, and high photostability, which ensure their good performance in long-term non-invasive in vitro cell tracing. Moreover, it is the first report that AIE dots exhibit certain permeability to cellular nucleus, making them attractive potential candidates for nucleus imaging. The AIE dots display superior performance compared to their counterparts of inorganic quantum dots, opening a new avenue in the development of fluorescent probes for monitoring biological processes. Encapsulation of orange-red fluorescent luminogens with aggregation-induced emission (AIE) characteristics in biocompatible matrix yields AIE dots with strong emission, large Stokes shift, good biocompatibility, and high photostability. Application of the AIE dots for in vitro cell tracing and nucleus imaging has been demonstrated using MCF-7 breast cancer cells as an example.

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Bright and Photostable Organic Fluorescent Dots with
Aggregation-Induced Emission Characteristics for
Noninvasive Long-Term Cell Imaging
Wei Qin, Kai Li, Guangxue Feng, Min Li, Zhiyong Yang, Bin Liu,* and Ben Zhong Tang*
intracellular
environments.[2]
Direct
E f fi cient long-term cell tracing in a noninvasive and real-time manner is of
great importance to understand genesis, development, invasion, and metas-
tasis of cancerous cells. Cell penetrating organic dots with aggregation-
induced emission (AIE) characteristics are successfully developed as long-
term cell trackers. The AIE dots enjoy the advantages of high emission
efficiency, large Stokes shift, good biocompatibility, and high photostability,
which ensure their good performance in long-term non-invasive in vitro cell
tracing. Moreover, it is the first report that AIE dots exhibit certain perme-
ability to cellular nucleus, making them attractive potential candidates for
nucleus imaging. The AIE dots display superior performance compared to
their counterparts of inorganic quantum dots, opening a new avenue in the
development of fluorescent probes for monitoring biological processes.
visualization of genesis, development,
invasion, and metastasis of cancerous
cells are ideal for medical diagnostics
and therapeutics of cancerization at the
early stage. Green fluorescent protein
(GFP) and its family have been adopted
for genetic cell tagging to achieve long-
term cell tracing.[3] Unfortunately, some
inherent drawbacks of GFP are unavoid-
able, such as the susceptibility to enzyme
degradation, small Stokes shifts, and poor
photostability.[4] Moreover, transfection of
fluorescent protein is a tedious and time-
consuming process, which often yields
low labeling efficiency.[5] Recent studies
also reveal that fluorescent protein trans-
fection can interfere with normal cell
functions, which hampers practical applications of the GFP-
1. Introduction
based probes.[6]
Fluorescence imaging as a highly sensitive and non-invasive
technology,[1] offers researchers a very useful tool to locate
and monitor biological targets within complex and dynamic
Besides GFP, inorganic semiconductor quantum dots (QDs)
are widely used as fluorescent probes for direct cell labeling,[7]
which have shown bright emission and high photobleaching
threshold under imaging conditions.[8] Unfortunately, the com-
monly used QDs (e.g., CdSe and CdTe) contain heavy metal ele-
ments, which are highly cytotoxic in oxidative environments.[9]
In addition, QDs are liable to aggregate in cells[10] and show
unstable fluorescence signals.[8,11] These detrimental effects
hinder their future application in clinical settings.
Organic fluorophore-loaded nanoparticles (NPs) have
recently emerged as a new generation of nanoprobes for bioim-
aging.[12] For practical biomedical applications, highly emissive
NPs are desirable to obtain high contrast imaging. Ideally, the
most straightforward strategy to enhance fluorescence signals
is to increase the number of encapsulated dye molecules in
each NP. However, there are several inherent limitations that
hamper full potential of conventional organic dye molecules as
sensitive fluorescence probes. First, many conventional organic
fluorophores have hydrophobic planar structures which show
strong intermolecular π–π interactions to favor dye aggrega-
tion, leading to quenched fluorescence at high concentrations
or in aggregates.[13] Second, the majority of commercial organic
dyes suffers from severe “self-absorption” resulting from the
small Stokes shift of less than 25 nm.[14] The poor separation
between the absorption and emission spectra also hampers the
use of optical filters to effectively block excitation light from
reaching the fluorescence detector. Moreover, low resistance to
photobleaching is another disadvantage of most organic dyes[15]
W. Qin, Dr. M. Li, Dr. Z. Yang, Prof. B. Z. Tang
Department of Chemistry
Institute for Advanced Study
Institute of Molecular Functional Materials
and Division of Biomedical Engineering
The Hong Kong University of Science & Technology (HKUST)
Clear Water Bay, Kowloon, Hong Kong, China
E-mail: tangbenz@ust.hk
Dr. K. Li, Prof. B. Liu
Institute of Materials Research and Engineering
3, Research Link, Singapore, 117602
E-mail: cheliub@nus.edu.sg
G. Feng, Prof. B. Liu
Department of Chemical and Biomolecular Engineering
National University of Singapore
4 Engineering Drive 4, Singapore, 117576
W. Qin, Dr. M. Li, Dr. Z. Yang, Prof. B. Z. Tang
HKUST-Shenzhen Research Institute
No. 9 Yuexing 1st RD, South Area, Hi-tech Park
Nanshan, Shenzhen, 518057, China
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 (SCUT)
Guangzhou, 51640, China
DOI: 10.1002/adfm.201302114
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due to photon-induced chemical damage,
which obviously compromises image quality.
The combined drawbacks make many con-
ventional dyes not ideal for fabrication of
nanoparticle based probes.
Recently, we have successfully developed
a novel class of organic luminogens with
an extraordinary aggregation-induced emis-
sion (AIE) feature.[16] These luminogens, for
example, tetraphenylethene (TPE), are non-
emissive in dilute solutions but are induced
to luminesce intensely when aggregated
through a mechanism of restriction of intra-
molecular rotation (RIR).[17] Previous studies
revealed that AIE luminogens are ideal can-
didates for the fabrication of fluorescent NPs
due to their efficient emission in aggregate
state.[18] In addition, surface functionalization
of the NPs[19] with cell penetrating peptides,
such as Tat,[20] yielded surface functionalized
nanoparticles that can be internalized easily
by cells and thus further improve the sensi-
tivity of fluorescence imaging.[21]
Figure 1. Chemical structures and molecular geometries of a) TTB and b) TNB; the molec-
ular geometries were optimized by the DFT calculations using B3LYP/6-31G(d) basis set in
Gaussian 03 program; hydrogen atoms were omitted for clarity.
To further fine-tune the emission wave-
length and improve the fluorescence quantum yield of the
nanoparticles, we report the synthesis of two orange-red
emissive AIE-active compounds, TPE-TPA-BTD (TTB) and
TPE-NPA-BTD (TNB), and the fabrication of their AIE dots
for in vitro long-term cell tracing applications. Through
conjugation with cysteine on C-terminus of modified Tat
peptide(YGRKKRRQRRRC), the Tat functionalized AIE dots
show high cellular internalization efficiency. The performances
of the Tat-AIE dots in the in vitro studies were compared with
those of commercially available QDs of Qtracker 585 under
similar experimental conditions. It was found that the Tat-AIE
dots could efficiently trace MCF-7 breast cancer cells as long as
5 days in vitro, which outperform their counterparts of com-
mercial Qtracker 585 (1–2 days). Moreover, the first demonstra-
tion of AIE dots for intranuclear localization in live cells will
provide a promising platform for intranuclear fluorescence
imaging and future drug delivery. The excellent performance
of the AIE dots sheds light on tracking and monitoring cells for
cancer research and other cell-based therapies.
Detailed data are reported in the Experimental Section and
the mass spectra of TTB and TNB are shown in Figures S1,S2
(Supporting Information). Both TTB and TNB possess good
solubility in common organic solvents, such as tetrahydro-
furan (THF), toluene, dichloromethane and chloroform, but are
insoluble in water.
2.2. Optical Properties of AIE Luminogens
The compounds of TTB and TNB are comprised of arylamines
as the electron donor (D), benzothiadiazole core as the electron
acceptor (A) and TPE as iconic AIE unit. The D–A system is
designed for obtaining fluorophores with long wavelength
absorption and emission. The molecular fusion of the D–A
system and TPE groups is expected to generate new AIE-active
fluorogens with extended electronic conjugation. The absorp-
tion spectra of TTB and TNB in THF are peaked at 471 and
470 nm, respectively, indicative of very similar conjugation
length of the two molecules (Table 1). Density functional theory
calculations of the TTB and TNB were carried out using a suite
of Gaussian 03 program in order to understand their proper-
ties at the molecular level. The nonlocal density functional of
B3LYP with 6-31G(d) basis sets was used for the calculation.
The optimized molecular geometries are shown in Figure 1,
which adopt propeller-like conformations. The torsion angles
between the central benzothiadiazole plane and their adjacent
phenyl rings are ≈35° for both molecules (Tables S1,S2, Sup-
porting Information). In the TPE part of TTB or TNB, the dihe-
dral angles between any phenyl ring and the central double
bond are ≈50°. The dihedral angles between any two phenyl
rings of the arylamines in TTB are from ≈66° to ≈71° (Table S1,
Supporting Information). However, the two dihedral angles of
aromatic rings of arylamines in TNB are ≈82° (Table S2, Sup-
porting Information). The nearly perpendicular conformations
2. Results and Discussion
2.1. Synthesis and Characterization of TTB and TNB
The target molecules shown in Figure 1 were synthesized
according to Scheme 1. The key intermediate 4,7-bis(4-
bromophenyl)-2,1,3-benzothiadiazole (5) was prepared from
o-phenylenediamine (1) according to the previous report.[22]
The reaction between intermediate 5 and aryl amines in the
presence of palladium catalyst under basic conditions led to
the products with >70% yields. The purified products were
characterized by standard spectroscopic techniques, and the
data indicate the right chemical structures with high purity.
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Scheme 1. Synthetic routes to TTB and TNB.
of TNB should disfavor close molecular packing in the solid
state, leading to brighter fluorescence than that of TTB in con-
densed phase. In terms of the electronic structures, the lowest
unoccupied molecular orbitals (LUMOs) of both molecules
are dominated by the orbitals from the benzothiadiazole core.
While the electron clouds of the highest occupied molecular
orbitals (HOMOs) are mainly located on both the arylamine
unit and benzothiadiazole core (Figure S3, Supporting Infor-
mation). The difference in electron cloud distribution shows
intrinsic intramolecular charge transfer (ICT) property, which
is beneficial to red-shifted emission spectra.
emits orange-red light with an emission maximum at 616 nm.
With gradual addition of water into THF (fw ≤ 40%), the emis-
sion of TTB is weakened and is bathochromically shifted from
616 to 624 nm, possibly due to the increase in solvent polarity
and hence the transformation to the twisted intramolecular
charge transfer (TICT) state. The light emission is invigorated
from fw ≈50% and is intensified with a further increase in fw,
indicating that TTB molecules form nanoaggregates due to
the poor solubility. In the aqueous mixture with fw of 90%, the
emission of TTB is hypsochromically shifted to 609 nm. These
data confirm that TTB is a luminogen with both TICT and AIE
features. TNB shows a similar behavior (Figure 2c,d). In pure
THF solution, the orange-red emission appears at 613 nm.
Upon addition of water (fw ≤ 40%), the emission is weakened
and red-shifted. Whereas in the aqueous mixtures with “large”
The photoluminescence (PL) spectra of TTB and TNB were
studied in THF/water mixtures with different water fractions
(fw), which is able to fine-tune the solvent polarity and the extent
of solute aggregation (Figure 2). The pure THF solution of TTB
Table 1. Optical Properties of TTB and TNB.
λ
em (nm)c)
aggr
Eg (eV)b)
2.26(2.45)
2.27(2.48)
soln
616
613
d)
λ
ab (nm)a)
471
film (ΦF,f)
TTB
609
617 (48.8%)
617 (63.0%)
TNB
470
604
a)Absorption maximum (λab) in THF; b)HOMO−LUMO band gap (Eg) calculated from the onset of the absorption spectrum, HOMO is the highest occupied molecular
orbital, LUMO is the lowest unoccupied molecular orbital. The values in the parentheses are derived from theoretical DFT calculations; c)Emission maximum (λem) in THF
solutions (soln, 10 μM), THF/water mixtures (aggr; 1:9 v/v; 10 μM), and solid thin films spin-coated from THF solution. d)
Φ
F,f is the fluorescence quantum efficiency in thin
film state measured by a calibrated integrating sphere.
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core consisting of AIE luminogens and the
hydrophilic PEG-Mal chains extended in
aqueous phase to afford surface maleimide
groups. After THF removal, the water sus-
pensions of TTB and TNB dots were further
mixed with Tat peptide, HIV-1 Tat (47-57),
which could facilitate dots to enter live cells.
The obtained Tat-TTB and Tat-TNB dots have
excellent colloidal stability in water as no
obvious precipitation from the stock suspen-
sions was observed after being stored at 4 °C
for months.
The sizes of Tat-TTB and Tat-TNB dots
were investigated by laser light scattering
(LLS), suggesting that the volume average
hydrodynamic diameters are ≈36 and 38 nm,
respectively (Figure 3a,c). The morphology
of Tat-AIE dots was further investigated
using high-resolution transmission electron
microscopy (HR-TEM), indicating that they
are in spherical shape and the dark dots are
due to the high electron density of AIE lumi-
nogens (Figure 3a,c). As shown in Figure
3b,d, Tat-TTB and Tat-TNB dots have the
same absorption peaks at 480 nm. The emis-
sion maxima of Tat-TTB and Tat-TNB dots
appear at 619 and 617 nm, respectively, with
intense emission tails extending to 850 nm.
It is noteworthy that the Stokes shifts of
the AIE dots are larger than 130 nm, which
greatly minimize the self-absorption effect
universally observed in conventional organic
dye molecules. The quantum yields of Tat-
TTB and Tat-TNB dots in water are 55% and
Figure 2. PL spectra of a) TTB and c) TNB in THF/water mixtures with different water fractions
(fw). Plot of peak intensity versus water fraction in the THF-water mixture of b) TTB and d) TNB.
Concentration: 10 μM; excitation wavelength: 470 nm.
amounts of water (fw > 40%), the emission is intensified and
blue-shifted to 604 nm with fw of 90%. The quantum yields (ΦF)
of TTB and TNB in solid state reach 48.8 and 63.0%, respec-
tively (Table 1), which make both molecules good candidates for
fabrication of AIE dots for bioimaging applications.
67%, respectively, measured using 4-(dicyanomethylene)-2-me-
thyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM) in methanol as
a standard (ΦF = 43%). The high quantum yields of AIE dots
are beneficial to high signal-to-noise ratios in cell imaging.
2.4. In Vitro Cell Imaging
2.3. Preparation and Characterization of Tat-AIE Dots
The application of as-prepared Tat-AIE dots in cell imaging was
studied by confocal laser scanning microscopy. In these experi-
ments, MCF-7 breast cancer cells were incubated with 2 nM
To facilitate further conjugation with biomolecules, the AIE dot
with maleimide groups on the surface were fabricated through a
modified nanoprecipitation method reported
by our group (Scheme 2).[23] A mixture of
1,2-distearoyl-sn-glycero-3-phosphoethanola-
mine-N--[methoxy(polyethylene glycol)-2000]
(DSPE-PEG2000)and1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[maleimide(poly-
ethylene
glycol)-2000]
(DSPE-PEG2000-
Mal) was employed as the encapsulation
matrix. In brief, a THF solution of TTB
or TNB (1 mg), DSPE-PEG2000 (1 mg) and
DSPE-PEG2000-Mal (1 mg) were mixed with
water under continuous sonication. During
the process, the hydrophobic DSPE seg-
ments were embedded in the hydrophobic Scheme 2. Schematic illustration of the preparation of Tat-functionalized AIE dots.
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imaging, TTB dots and TNB dots without
functionalization with Tat were also incu-
bated with MCF-7 cancer cells under the
same experimental conditions. As shown in
Figure S4 in the Supporting Information,
the TTB and TNB dots are found randomly
distributed in cell cytoplasm and no fluores-
cence from the dots can be detected in the
nucleus region. Moreover, the fluorescence
intensity from cell cytoplasm (Figure S4,
Supporting Information) is much lower as
compared to that in Figure 4. These results
not only verify that Tat peptides can greatly
enhance live cell internalization efficiency
but also further confirm the vital role of
Tat peptide functionalized on the surface
of AIE dots in facilitating the intranuclear
translocation.[25] Our findings are consistent
with previously reported inorganic nano-
particles studies, which showed that gold[26]
and silica[27] nanoparticles with diameters
of <50 nm could penetrate cell nuclei after
conjugation with Tat peptides. The first
demonstration of AIE dots for intranuclear
localization in live cells will provide a prom-
ising platform for intranuclear fluorescence
imaging and future drug delivery.
2.5. Photostability and Cytotoxicity of AIE
Dots
Figure 3. Particle size distributions studied by laser light scattering and HR-TEM images of
a) Tat-TTB and c) Tat-TNB dots. Absorption and emission spectra of b) Tat-TTB and d) Tat-TNB
dots suspended in water; λex = 488 nm.
Figure 5a shows the fluorescence change of
Tat-AIE dots and Qtracker 585 dots in cells
Tat-TTB or Tat-TNB suspensions for 6 h. The calculation of dot
concentration is described in the Supporting Information. Hoe-
chst 34580 was then used to stain the cell nuclei for 30 min
before live cell imaging studies. The cellular uptake and locali-
zation of Tat-AIE dots in live MCF-7 cancer
cells are shown in Figure 4. The images were
taken upon excitation at 405 nm (1 mW) while
440−480 nm and 600−800 nm bandpass fil-
ters were adopted for simultaneous collection
of fluorescence from Hoechst 34580 and Tat-
AIE dots, respectively. It is obvious that abun-
dant Tat-TTB dots (Figure 4a) and Tat-TNB
dots (Figure 4e) localize in the cytoplasm and
the perinuclear region. To a certain extent,
some orange-red emission is also observed
from the nuclei region (Figure 4c,g), indi-
cating that some AIE dots might locate in
the cell nuclei. 3D sectional images of MCF-7
cancer cells (Figure 4d,h) further reveal that
the AIE dots are indeed located in the nuclei,
possibly because some of them traverse the
upon continuous laser irradiation at 488 nm (2 mW) for 20 min.
After irradiation, the Tat-AIE dots possess ≈94% of their initial
fluorescence intensity, which is comparable to that of Qtracker
585 dots. Noteworthy is that the laser power employed is twice
Figure 4. Confocal images of live MCF-7 breast cancer cells after incubation with 2 nM a) Tat-
TTB and e) Tat-TNB dots for 6 h at 37 °C. b,f) The live cell nuclei were stained by Hoechst 34580
for 30 min. c,g) The corresponding overlay images. 3D sectional confocal images of MCF-7
breast cancer cells after incubation with 2 nM d) Tat-TTB and h) Tat-TNB dots for 6 h at 37 °C.
nuclear pore complex (NPC) to the nucleus
by passive diffusion.[24]
To further investigate the importance of
surface-conjugated Tat peptide in cellular The images (a–c,e–g) share the same scale bar.
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Figure 5. a) Photostability of Tat-TTB and Tat-TNB dots under continuous scanning at 488 nm (2 mW). Insets show confocal images of the Tat-TTB dot-
stained cells before (0 min, left) and after the laser irradiation for 20 min (right). I0 is the initial PL intensity, while I is that of the corresponding sample
after a designated time interval. b) Cell viability of MCF-7 cells after incubation with 1, 2, and 4 nM Tat-TTB, Tat-TNB dots for 24 h, 48 h, respectively.
as high as that used in in vitro fluorescence imaging studies (1
mW). Unlike traditional organic dyes which suffer from severe
photobleaching after several minutes of laser irradiation,[28] the
AIE dots exhibit excellent photostability, which will greatly ben-
efit their in vitro long-term cell tracing studies. The cytotoxicity
of the AIE dots was evaluated by metabolic viability of MCF-7
breast cancer cells after incubation with the different concen-
tration of dots. Figure 5b shows the cell viability after incu-
bation with Tat-TTB and Tat-TNB dots at dot concentrations
of 1, 2, and 4 nM for 24 and 48 h, respectively. Cell viabilities
remain above 95% after being treated with 1 and 2 nM Tat-AIE
dotswithin the tested periods of time. It still remains above
90% after being treated with 4 nM Tat-AIE dots even for 48 h,
indicating low cytotoxicity of the AIE dots in the test, which is
essential for long-term tracing applications.
Figure 6a shows that the labeling rate of Tat-TTB dots towards
MCF-7 cells is 99.8% on the 1st day and is above 93% on the
4th day. It remains above 86% on the 5th day as compared to
the untreated cells, which is still higher than that of Qtracker
585-treated cells on the 1st day (84%). The labeling rate for Tat-
TTB is 24.5% after continuous culture till the 9th day. Similarly,
the labeling rate of Tat-TNB towards MCF-7 cells is 99.9% on
the first day. It remains 94.9% on the 4th day and also above
86.0% at 5th day as compared to the untreated cells (Figure 6b).
The labeling rate is 28.8% after continuous culture till 8th day.
The gradually decreased fluorescence intensity in each single
cell can be attributed to the continuous cell division while the
internalized Tat-AIE dots are inherited by daughter cells. On
the contrary, the initial labeling rate of Qtracker 585 is around
84.0% on the 1st day, and only 26.9% of Qtracker 585 -treated
cells are labeled at the 5th day (Figure 6c). The results clearly
indicate the superior cell tracing ability of Tat-AIE dots over
Qtracker 585. These results were further confirmed by confocal
images (Figure 7). Only very weak fluorescence is detectable in
Qtracker 585-labeled cells while the Tat-AIE dot-labeled cells
show high fluorescence signal on the 3rd day. The fluorescence
of Qtracker 585 in MCF-7 cells fades out on the 5th day, while
the signal of Tat-AIE dots can still be collected on the 7th day.
The fluorescence signal in Figure 7 is from the Tat-AIE dots
since cell autofluorescence is not detectable under the same
experimental conditions (Figure S5, Supporting Information).
2.6. In Vitro Cell Tracing
MCF-7 human breast cancer cells were chosen as a model to
demonstrate the ability of Tat-AIE dots for in vitro cell tracing
application. The MCF-7 cancer cells were first incubated with
2 nM Tat-AIE dots ovenight at 37 °C. The labeled cells were then
subcultured for designated time intervals and the fluorescence
profiles were recorded using flow cytometry by counting 10 000
events with 488 nm excitation and, 575/25 nm bandpass filter.
Figure 6. Flow cytometry histograms of MCF-7 breast cancer cells after overnight incubation with 2 nM a) Tat-TTB, b) Tat-TNB dots, and c) Qtracker
585 dots at 37 °C and then subcultured for designated time intervals (day). The untreated MCF-7 cells were used as the control (blank).
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phosphoethanolamine-N-[maleimide(polyethylene
glycol)-2000]
(DSPE-PEG2000-Mal)
was
a
commercial product of Avanti Polar Lipids, Inc.
Dulbecco’s Modified Eagle Medium (DMEM), and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) were purchased from Sigma-Aldrich.
Hoechst 34580, penicillin-streptomycin solution,
fetal bovine serum (FBS) and trypsin-EDTA solution
were purchased from Life Technologies, Singapore.
HIV-1 Tat (47-57) derived from transactivator of
transcription proteins was modified with cysteine
on C-terminus (YGRKKRRQRRRC), which was
a commercial product customized by GenicBio
(Shanghai, China). Milli-Q water was supplied by
Milli-Q Plus System (Millipore Corporation, Breford,
USA). MCF-7 breast cancer cells were provided by
American Type Culture Collection.
1
Characterization: H- and 13C-NMR spectra were
measured on a Bruker AV 300 or 400 spectrometer
in CDCl3 or CD2Cl2 using tetramethylsilane
(TMS; δ = 0) as internal reference. The UV-vis
spectra were measured on a Shimadzu UV-1700
spectrometer. The fluorescence spectra of dots
were recorded using a fluorometer (LS-55, Perkin
Elmer, USA). High resolution mass spectra
(HRMS) were recorded on a GCT premier CAB048
mass spectrometer operating in MALDI-TOF
mode. Elemental analysis was performed on a
ThermoFinnigan Flash EA1112. Fluorescence
Figure 7. CLSM images of MCF-7 on designated days after the overnight incubation of a) Tat-
TTB dots, b) Tat-TNB dots, and c) Qtracker 585 dots at a concentration of 2 nM. The confocal
images were taken under excitation at 488 nm (≈1 mW) with signal collected above 505 nm.
As Qtracker labeling kits are the most commonly used long-
term fluorescent tracing probes, the Tat-AIE dots can serve as
an ultrabright and ultrastable probe in long-term cell tracing
and imaging applications.
quantum efficiencies of the organic compounds in films were measured
using a calibrated integrating sphere. The average particle size and size
distribution were determined by laser light scattering with a particle size
analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of
90° at 24 °C. The morphology of dots was studied by high-resolution
transmission electron microscope (HR-TEM, JEM-2010F, JEOL, Japan).
Preparation of TTB and TNB Nanoaggregates: Stock solutions of
the compounds in THF with a concentration of 10−4M were prepared.
Aliquots of the stock solution were transferred to 10 mL volumetric
flasks. After appropriate amounts of THF were added, water was added
dropwise under vigorous stirring to furnish 10−5M solutions with different
water contents (0–90 vol%). The PL measurements of the resultant
solutions were then performed immediately.
3. Conclusions
In summary, we have synthesized two AIE luminogens (TTB
and TNB), fabricated the corresponding functionalized AIE
dots and explored their in vitro bioimaging applications. The
two AIE luminogens possess both TICT and AIE features. The
formulated Tat-TNB dots exhibit higher emission efficiency
compared to Tat-TTB dots, while the two AIE dots have similar
performance in imaging: both AIE dots show high emission
efficiency, large Stokes shift, good biocompatibility, and high
photobleaching resistance, exhibiting superior performance
in long-term cell tracing as compared to that for commercial
QD-based cell tracing probes. Further efforts will be made to
control the size and modify the surface of AIE dots to improve
their internalization efficiency for cellular nucleus imaging.
Preparation of TTB: A mixture of compound 5 (223 mg, 0.5 mmol), N-(4-
(1,2,2-triphenylvinyl)phenyl)benzenamine (10a) (634 mg, 1.5 mmol),
Cs2CO3 (1.14 g, 3.5 mmol), Pd(OAc)2 (11.2 mg, 0.05 mmol), P(t-Bu)3
(30.3 mg, 0.15 mmol) and toluene (40 mL) was heated at 40 °C for
2 h. And then the reaction mixture heated at 110 °C for 24 h. After the
mixture was cooled to room temperature, water (30 mL) and chloroform
(300 mL) were added. An organic layer was separated and washed
with brine, dried over anhydrous MgSO4, and evaporated to dryness
under reduced pressure. The crude product was purified by column
chromatography on silica gel using hexane/toluene as eluent, affording
1
orange red solid in 71% yield (401 mg). H NMR (400 MHz, CD2Cl2),
δ (TMS, ppm): 7.87 (d, J = 8.8 Hz, 4H), 7.74 (s, 2H), 7.27 (t, J = 8.0
Hz, 4H), 7.16–7.02 (m, 40H), 6.93–6.91 (m, 4H), 6.88–6.86 (m, 4H). 13
C
NMR (75 MHz, CDCl3), δ (TMS, ppm): 154.30, 147.88, 147.41, 145.74,
144.15, 143.87, 143.70, 140.86, 140.80, 138.79, 132.44, 132.29, 131.53,
131.50, 131.17, 129.97, 129.42, 127.82, 127.76, 127.56, 126.61, 126.56,
126.49, 125.20, 123.75, 123.42, 123.18. HRMS (MALDI-TOF, m/z):
[M+] calcd for C82H58N4S, 1130. 4382; found, 1130.4374. Anal. calcd for
C82H58N4S: C, 87.05; H, 5.17; N, 4.95; S, 2.83. found: C, 86.81; H, 5.13;
N, 4.92.
Preparation of10b: The compound was prepared from compound 9
and 1-Naphthylamine according to the reported methods.[11]1H NMR
(400 MHz, CDCl3), δ (TMS, ppm): 7.98–7.96 (m, 1H), 7.86–7.83 (m,
1H), 7.55–7.44 (m, 3H), 7.39–7.35 (m, 2H), 7.18–7.01 (m, 15H), 6.91
(d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H). 13C NMR (75 MHz, CDCl3),
δ (TMS, ppm):144.34, 144.20, 144.12, 142.89, 140.90, 140.00, 138.53,
4. Experimental Section
Materials: 4,7-Dibromo-2,1,3-benzothiadiazole 3,[29] 4,7-bis(4-
bromophenyl)-2,1,3-benzothiadiazole 5,[22] TPE derivative, 10,[11] were
prepared according to the literature methods. THF was distilled from
sodium benzophenone ketyl under dry nitrogen immediately prior to use.
All the chemicals and other regents were purchased from Aldrich and used
as received without further purification. All reactions and manipulations
were carried out under nitrogen gas with the use of standard inert
atmosphere.
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-
[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was a gift from
Lipoid GmbH (Ludwigshafen, Germany).1,2-distearoyl-sn-glycero-3-
©
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Adv. Funct. Mater. 2013,
DOI: 10.1002/adfm.201302114
2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
7

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136.07, 134.79, 132.58, 131.61, 131.52, 128.66, 127.85, 127.73, 127.72,
127.54, 126.48, 126.37, 126.31, 126.20, 126.06, 125.71, 122.89, 121.76,
116.59, 115.62. HRMS (MALDI-TOF, m/z): [M+] calcd for C36H27N,
473.2143; found 473.1956.
In Vitro Cell Tracing: MCF-7 breast cancer cells were cultured in 6-well
plates (Costar, IL, USA) to achieve 80% confluence. After medium
removal and washing with 1× PBS buffer, 2 nM Tat-AIE dots or Qtracker
585 in DMEM were then added to the wells. After overnight incubation
at 37 °C, the cells were washed twice with 1× PBS buffer and detached
by 1× tripsin and resuspended in culture medium. Upon dilution, the
cells were subcultured in 6-well plates for 1 to 10 day regeneration,
respectively. After designated time points, the cells were washed twice
with 1× PBS buffer and then trypsinized to suspend in 1× PBS buffer. The
fluorescence intensities of cells were then analyzed by flow cytometry
measurements using Cyan-LX (DakoCytomation) and the histogram of
each sample was obtained by counting 10 000 events (λex = 488 nm,
575/25 nm bandpass filter). For confocal image studies, the cells were
first labeled by 2 nM Tat-AIE dots or Qtracker 585. The labeled cells were
then washed twice with 1× PBS buffer and trypsinized to suspend in
culture medium. The cells were then diluted and subcultured in 6-well
plates containing cell culture coverslips for designated passages. Upon
reaching designated time points, the cells were washed twice with 1×
PBS buffer and then fixed by 75% ethanol for 20 min. The coverslips
were sealed with mounting medium and used for confocal imaging.
Preparation of TNB: The compound was prepared from compound
5 (223 mg, 0.5 mmol), N-(4-(1,2,2-triphenylvinyl)phenyl)naphthalen-1-
amine (10b) (710 mg, 1.5 mmol), Cs2CO3 (1.14 g, 3.5 mmol), Pd(OAc)2
(11.2 mg, 0.05 mmol), and P(t-Bu)3 (30.3 mg, 0.15 mmol), following
the same procedure described above, affording orange red solid in 78%
yield (480 mg). 1H NMR (300 MHz, CDCl3), δ (TMS, ppm): 7.92–7.87 (t,
4H), 7.77 (t, J = 8.4 Hz, 6H), 7.64 (s, 2H), 7.46 (t, J = 8.0 Hz, 4H), 7.41–
7.35 (m, 4H), 7.14–6.99 (m, 34H), 6.86 (s, 8H). 13C NMR (75 MHz,
CDCl3), δ (TMS, ppm):154.28, 148.48, 146.22, 144.13, 143.96, 143.76,
143.13, 140.81, 140.60, 137.83, 135.43, 132.32, 132.16, 131.50, 131.34,
130.27, 129.87, 128.55, 127.76, 127.73, 127.51, 127.38, 126.84, 126.53,
126.47, 126.41, 126.29, 124.44, 121.67, 121.11. HRMS (MALDI-TOF,
m/z): [M+] calcd for C90H62N4S, 1230.4695; found, 1230.5199). Anal.
calcd for C90H62N4S: C, 87.77; H, 5.07; N, 4.55; S, 2.60. found: C, 87.91;
H, 5.15; N, 4.62.
Synthesis of Tat Peptide-Functionalized AIE Dots: Two AIE
chromophores were used in synthesis of Tat peptide-functionalized AIE
dots. TTB (1 mg), DSPE-PEG2000 (1 mg) and DSPE-PEG2000-Mal (1 mg)
were dissolved in 1 mL of THF solution, followed by mixing with 9 mL of
water. The mixture was then sonicated for 60 seconds using a microtip
probe sonicator at 15 W output (XL2000, Misonix Incorporated, NY).
After filtration using a 0.2 μm syringe driven filter, the suspension was
stirred vigorously at room temperature overnight to yield TTB dots in
water (8 mL). The AIE dot suspension (2 mL) was further mixed with
the modified HIV1-Tat peptide (5 × 10−5 M). After reaction overnight at
room temperature, the solution was dialysed against MilliQ water for
2 days to eliminate the excess peptides. The Tat-TTB dots were collected
for further use. TNB was also used to fabricate Tat-TNB dots following
the same experimental procedures.
Cell Culture: MCF-7 breast cancer cells were cultured in DMEM
containing 10% fetal bovine serum and 1% penicillin streptomycin
at 37 °C in a humidified environment containing 5% CO2. Before
experiment, the cells were pre-cultured until confluence was reached.
In Vitro Cell Imaging: MCF-7 breast cancer cells were cultured in
8-well confocal chambers (Costar, IL, USA) to achieve 80% confluence.
The medium was then removed and cell monolayer was washed twice
with 1× PBS buffer. Tat-TTB dots or Tat-TNB dots in DMEM (2 nM) were
then added to the sample wells. After 6 h incubation at 37 °C, the cells
were washed twice with 1× PBS buffer and freshly prepared Hoechst
34580 solution in DMEM (1 μg mL−1) was added into the wells for
further 30 min incubation. The cells were then washed twice with 1× PBS
buffer. After addition of fresh DMEM, the cells were immediately imaged
by Leica TCS SP 5X. The laser at 405 nm (1 mW) was used to obtain
the one-photon excited fluorescence images with 440–480 nm and
600–800 nm bandpass filters for simultaneous collection of fluorescence
from Hoechst 34580 and Tat-AIE dots, respectively.
Cytotoxicity of AIE Dots and Tat-AIE Dots: The metabolic
viability of MCF-7 breast cancer cells were evaluated using
methylthiazolyldiphenyltetrazolium bromide (MTT) assays. MCF-7
breast cancer cells were seeded in 96-well plates (Costar, IL, USA) at an
intensity of 6 × 104 cells mL−1, respectively. After 24 h incubation, the
old medium was replaced by Tat-AIE dot suspension at concentrations
of 1, 2, and 4 nM, and the cells were then incubated for 24 h and 48 h,
respectively. The wells were then washed twice with 1× PBS buffer and
100 μL of freshly prepared MTT (0.5 mg mL−1) solution in culture
medium was added into each well. The MTT medium solution was
carefully removed after 3 h incubation. Filtered DMSO (100 μL) was
then added into each well and the plate was gently shaken for 10 min
to dissolve all the precipitates formed. The absorbance of MTT at
570 nm was monitored by the microplate reader (Genios Tecan). Cell
metabolic viability was expressed by the ratio of the absorbance of the
cells incubated with Tat-AIE dot suspension to that of the cells incubated
with culture medium only.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgment
W.Q. and K.L. contributed equally to this work. This work was partially
supported by the National Basic Research Program of China (973
Program, 2013CB834701), the RPC and SRFI Grants of HKUST
(RPC11SC09 and SRFI11SC03PG), the Research Grants Council of Hong
Kong (HKUST2/CRF/10 and N_HKUST620/11), and the University
Grants Committee of Hong Kong (AoE/P-03/08), and Singapore
Institute of Materials Research and Engineering (IMRE/13-8P1104;
IMRE/13-1P0909), and National Research Foundation (R279-000-390-
281). The authors thank the support of the Guangdong innovative
Research Team Program (201101C0105067115). The authors also thank
Professor Kam Sing Wong in Department of Physics, HKUST for the
measurement of fluorescence quantum yields of solid powder samples.
Received: June 21, 2013
Revised: July 12, 2013
Published online:
[1] a) B. N. G. Giepmans, S. R. Adams, M. H. Ellisman, R. Y. Tsien, Sci-
ence 2006, 312, 217; b) T. Terai, T. Nagano, Curr. Opin. Chem. Biol.
2008, 12, 515.
[2] a) D. W. Domaille, E. L. Que, C. J. Chang, Nat. Chem. Biol. 2008,
4, 168; b) R. Y. Tsien, Angew. Chem. Int. Edit. 2009, 48, 5612;
c) M. Y. Hu, L. Li, H. Wu, Y. Su, P. Y. Yang, M. Uttamchandani,
Q. H. Xu, S. Q. Yao, J. Am. Chem. Soc. 2011, 133, 12009.
[3] a) S. Wang, T. Hazelrigg, Nature 1994, 369, 400; b) C. J. Daly,
J. C. McGrath, Pharmacol. Ther. 2003, 100, 101.
[4] a) F. Q. Chen, D. Gerion, Nano Lett. 2004, 4, 1827; b) J. K. Jaiswal,
E. R. Goldman, H. Mattoussi, S. M. Simon, Nat. Methods 2004, 1,
73; c) L. S. Shah, P. A. Clark, E. K. Moioli, M. A. Stroscio, J. J. Mao,
Nano Lett. 2007, 7, 3071.
[5] Y. G. Meng, J. Liang, W. L. Wong, V. Chisholm, Gene 2000, 242, 201.
[6] E. Dellambra, G. Pellegrini, L. Guerra, G. Ferrari, G. Zambruno,
F. Mavilio, M. De Luca, Hum. Gene Ther. 2000, 11, 2283.
[7] a) X. H. Gao, Y. Y. Cui, R. M. Levenson, L. W. K. Chung, S. M. Nie,
Nat. Biotechnol. 2004, 22, 969; b) X. Michalet, F. F. Pinaud,
©
8
wileyonlinelibrary.com
2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Funct. Mater. 2013,
DOI: 10.1002/adfm.201302114

www.afm-journal.de
www.MaterialsViews.com
L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan,
A. M. Wu, S. S. Gambhir, S. Weiss, Science 2005, 307, 538.
[8] A. M. Smith, H. W. Duan, A. M. Mohs, S. M. Nie, Adv. Drug Delivery
Rev. 2008, 60, 1226.
[9] a) J. A. Kloepfer, R. E. Mielke, M. S. Wong, K. H. Nealson, G. Stucky,
J. L. Nadeau, Appl. Environ. Microb. 2003, 69, 4205; b) A. M. Derfus,
W. C. W. Chan, S. N. Bhatia, Nano Lett. 2004, 4, 11; c) S. J. Cho,
D. Maysinger, M. Jain, B. Roder, S. Hackbarth, F. M. Winnik, Lang-
muir. 2007, 23, 1974.
[10] a) C. Kirchner, T. Liedl, S. Kudera, T. Pellegrino, A. M. Javier,
H. E. Gaub, S. Stolzle, N. Fertig, W. J. Parak, Nano Lett. 2005, 5,
331; b) J. F. Weng, J. C. Ren, Curr. Med. Chem. 2006, 13, 897.
[11] K. Li, W. Qin, D. Ding, N. Tomczak, J. Geng, R. Liu, J. Liu, X. Zhang,
H. Liu, B. Liu, B. Z. Tang, Sci. Rep. 2013, 3, 1150.
[16] Y. N. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Soc. Rev. 2011, 40, 5361.
[17] Y. N. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Commun. 2009, 4332.
[18] a) W. Z. Yuan, P. Lu, S. M. Chen, J. W. Y. Lam, Z. M. Wang,
Y. Liu, H. S. Kwok, Y. G. Ma, B. Z. Tang, Adv. Mater. 2010, 22, 2159;
b) Z. Zhao, S. Chen, X. Shen, F. Mahtab, Y. Yu, P. Lu, J. W. Y. Lam,
H. S. Kwok, B. Z. Tang, Chem. Commun. 2010, 46, 686.
[19] M. Li, J. W. Y. Lam, F. Mahtab, S. J. Chen, W. J. Zhang, Y. N. Hong,
J. Xiong, Q. C. Zheng, B. Z. Tang, J. Mater. Chem. B 2013, 1, 676.
[20] L. N. Patel, J. L. Zaro, W. C. Shen, Pharmaceut. Res. 2007, 24, 1977.
[21] V. P. Torchilin, Adv. Drug Delivery Rev. 2008, 60, 548.
[22] J. Liu, L. J. Bu, J. P. Dong, Q. G. Zhou, Y. H. Geng, D. G. Ma,
L. X. Wang, X. B. Jing, F. S. Wang, J. Mater. Chem. 2007, 17, 2832.
[23] K. Li, Y. Jiang, D. Ding, X. Zhang, Y. Liu, J. Hua, S.-S. Feng, B. Liu,
Chem. Commun. 2011, 47, 7323.
[12] a) A. Palma, L. A. Alvarez, D. Scholz, D. O. Frimannsson, M. Grossi,
S. J. Quinn, D. F. O’Shea, J. Am. Chem. Soc. 2011, 133, 19618;
b) A. Wagh, S. Y. Qian, B. Law, Bioconjugate. Chem. 2012, 23, 981.
[13] a) I. B. Berlman, Handbook of Fluorescence Spectra of Aromatic Mol-
ecules, 2nd ed., Academic Press, New York 1971; b) M. Shimizu,
T. Hiyama, Chem. Asian J. 2010, 5, 1516.
[24] C. W. Pouton, K. M. Wagstaff, D. M. Roth, G. W. Moseley, D. A. Jans,
Adv. Drug Delivery Rev. 2007, 59, 698.
[25] a) S. Nakielny, G. Dreyfuss, Cell 1999, 99, 677; b) F. Alber,
S. Dokudovskaya, L. M. Veenhoff, W. H. Zhang, J. Kipper, D. Devos,
A. Suprapto, O. Karni-Schmidt, R. Williams, B. T. Chait, A. Sali,
M. P. Rout, Nature 2007, 450, 695.
[14] a) C. A. Bertolino, G. Caputo, C. Barolo, G. Viscardi, S. Coluccia,
J. Fluoresc. 2006, 16, 221; b) W. Y. Lin, L. Yuan, Z. M. Cao,
Y. M. Feng, J. Z. Song, Angew. Chem. Int. Edit. 2010, 49, 375.
[15] a) L. L. Song, E. J. Hennink, I. T. Young, H. J. Tanke, Biophys. J. 1995,
68, 2588; b) C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel,
Anal. Chem. 1998, 70, 2651; c) U. Resch-Genger, M. Grabolle,
S. Cavaliere-Jaricot, R. Nitschke, T. Nann, Nat. Methods 2008, 5,
763.
[26] N. Pante, M. Kann, Mol. Biol. Cell 2002, 13, 425.
[27] L. M. Pan, Q. J. He, J. N. Liu, Y. Chen, M. Ma, L. L. Zhang, J. L. Shi,
J. Am. Chem. Soc. 2012, 134, 5722.
[28] a) H. Giloh, J. W. Sedat, Science 1982, 217, 1252; b) W. Qin, D. Ding,
J. Z. Liu, W. Z. Yuan, Y. Hu, B. Liu, B. Z. Tang, Adv. Funct. Mater.
2012, 22, 771.
[29] B. A. D. Neto, A. S. A. Lopes, G. Ebeling, R. S. Goncalves,
V. E. U. Costa, F. H. Quina, J. Dupont, Tetrahedron 2005, 61, 10975.
©
wileyonlinelibrary.com
Adv. Funct. Mater. 2013,
DOI: 10.1002/adfm.201302114
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