Highly Emissive AIEgens with Multiple Functions: Facile Synthesis, Chromism, Specific Lipid Droplet Imaging, Apoptosis Monitoring, and in Vivo Imaging

Article abstract of DOI:10.1021/acs.chemmater.8b03495

The development of new luminescent materials has allowed us to gain new knowledge and opened a new opportunity for scientific achievement and social development. In this work, luminogens, namely, (4-pyridinyl)-phenyldiphenylamine (TPAP) and 3-diphenylamin

Full text of DOI:10.1021/acs.chemmater.8b03495

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Article
Highly Emissive AIEgens with Multi-Functions: Facile Synthesis, Chromism,
Specific Lipid Droplet Imaging, Apoptosis Monitoring and In Vivo Imaging
Dongfeng Dang, Haixiang Liu, Jianguo Wang, Ming Chen, Yong Liu, Herman H.-
Y. Sung, Ian D. Williams, Ryan T. K. Kwok, Jacky W. Y. Lam, and Ben Zhong Tang
Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b03495 • Publication Date (Web): 02 Oct 2018
Downloaded from http://pubs.acs.org on October 2, 2018
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Chemistry of Materials
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Highly Emissive AIEgens with Multi-Functions: Facile Synthesis,
Chromism, Specific Lipid Droplet Imaging, Apoptosis Monitoring
and In Vivo Imaging
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Dongfeng Dang,a,b,‡ Haixiang Liu,a,‡ Jianguo Wang,a,c Ming Chen,a,c Yong Liu,a,c Herman H.-Y.
Sung,a Ian D. Williams,a Ryan T. K. Kwok,a Jacky W. Y. Lam,a Ben Zhong Tang a,c,d,*
a Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and
Reconstruction, Division of Life Science, Institute for Advanced Study, Institute of Molecular Functional Materials, and Depart-
ment of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon,
Hong Kong, China.
b School of Science, MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed Matter, Xi’an Jiaotong
University, Xi’an 710049, China.
c HKUST-Shenzhen Research Institute, No.9 Yuexing 1st RD, South Area, Hi-tech Park, Nanshan, Shenzhen 518057, China.
d NSCF Center for luminescence from molecular aggregates, SCUT-HKUST Joint Research Laboratory, State Key Laboratory of
Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China.
ABSTRACT: The development of new luminescent materials has allowed us to gain new knowledge and opened a new
opportunity to scientific achievement and social development. In this work, luminogens, namely (4-pyridinyl)-
phenyldiphenylamine (TPAP) and 3-diphenylamino-6-(2-pyridinyl)phenyldiphenylboron (TPAP-BB) were synthesized in
satisfactory yields through convenient synthetic routes and their properties were systematically investigated. These lu-
minogens exhibited a moderate fluorescence quantum yield in THF (14.6% for TPAP and 49.0% for TPAP-BB), whereas,
their values in thin films were much higher (69.4% and 88.4%), demonstrating a phenomenon of aggregation-induced
emission (AIE). Moreover, TPAP-BB not only exhibited piezo-chromism: its emission color could be tuned repeatedly by
grinding-heating cycle due to the transformation between crystalline and amorphous phases, but also showed on-off light
emission process by repeatedly fuming with acid and ammonia vapor. Furthermore, TPAP-BB exhibited impressive high
photo-stability and low toxicity to living cells. It could specifically stain lipid droplets (LDs) in live HeLa cells with higher
signal-to-noise ratio than Nile Red, a commercial LDs agent. This dye could also be applied for real-time monitoring of
cell apoptosis and in vivo imaging in Medaka fish. Such results were expected to create enthusiasm to generate new AIE
luminogens for further technological applications.
INTRODUCTION
various imaging techniques in recent years, including
Raman microscopy and transmission electron microsco-
py.16,17 However, although these techniques have been
widely known and well developed, their abilities in real-
time detection and in situ monitoring are still low. More-
over, the high cost and time-consuming measurement of
these techniques also limited their further applications in
biomedical studies. Hence, new approaches for real-time
detection of LDs and monitoring their dynamic biological
processes, such as apoptosis, are yet to be developed.
Monitoring and localizing organelles, such as nucleus,1,2
mitochondria,3 lysosomes,4 and cytoplasm membrane5 in
eukaryotic cells, have attracted tremendous attentions in
the past few decades for their critical roles in cellular
functions and health care. However, lipid droplets (LDs)
are ignored and have been regarded just as lipid-rich or-
ganelles in cells for a long time. Interestingly, recent stud-
ies show that LDs are closely related to many physiologi-
cal processes.6-8 For instance, cellular stress was found to
trigger apoptosis to induce extensive formation of LDs in
cells.9,10 Meanwhile, it should be mentioned that the ac-
cumulation of abnormal LDs can also lead to various dis-
eases, such as Alzheimer’s disease.11,12 Therefore, similar to
other well-studied organelles, it is important to localize
and track LDs in situ and in real time not only for the
understanding of their biological functions but also for
the early diagnosis of related diseases.13-15 To achieve this
goal, tremendous works have been devoted to developing
Currently, fluorescence imaging technique using
highly emissive visualizing agents is emerging as one of
the most powerful approaches in biomedical study for its
advantages of fast response, high sensitivity, easy and
noninvasive manner.18-27 Among these visualizing agents,
organic luminescent materials (OLMs) possess the unique
features of good chemical stability and excellent biocom-
patibility. The structural tailorability of OLMs also ena-
bles researchers to tune their properties easily to meet
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different needs.28-38 However, it is worth noting that alt-
tron-rich building blocks (D) and electron-deficient units
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hough many OLMs have been developed and commer-
cialized for fluorescence imaging, their drawbacks, such
as inferior emission features in aggregate state,39 high
background noise, poor photo-stability, small Stokes
shifts, and complicated synthetic process, have prevented
further realization of their potential for applications.
(A) were facilely synthesized through a simple synthetic
route (Figure 1). Owing to their D-A architectures and
molecular rotors introduced in molecular backbones,
both TPAP and TPAP-BB exhibited intramolecular charge
transfer (ICT) and AIE characteristics (Figure 1). Interest-
ingly, in contrast to the moderate photoluminescence
quantum yields (PLQYs) of TPAP in THF solution (14.6%)
and in thin film (49.0%), TPAP-BB exhibited more effi-
cient light emission with higher PLQYs (69.4% in THF
and 88.4% in thin film, respectively). In addition to its
superior fluorescent performance, TPAP-BB also showed
piezo- and acido-chromisms: its emission color and pro-
cess could be turned by grinding-heating and acid-base
vapor fuming cycles. Moreover, low toxicity to living cells
and also good photo-stability were also observed for
TPAP-BB to serve as visualizing agent for LDs specific
imaging with much higher signal-to-noise ratio than the
commercial LDs dyes of Nile Red. Finally, TPAP-BB was
successfully applied in the real-time monitoring of apop-
tosis in HeLa cells induced by hydrogen peroxide (H2O2)
and in vivo imaging of Medaka fish. Such highly emissive,
multi-functional AIEgens with low cytotoxicity is thus
expected to found wide applications, not only in biologi-
cal area but also in other fields such as optics and sensors.
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EXPERIMENTAL SECTION
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Instruments. H NMR and C NMR spectra were meas-
ured on a Bruker ARX 400 NMR using CDCl3 as solvent.
Mass spectra were obtained on a GCT premier CAB048
mass spectrometer operating in MALDI-TOF mode. UV-
Vis spectra were measured on a Milton Ray Spectronic
3000 array spectrophotometer. The PL spectra were col-
lected on a PerkinElmer LS 55 spectrophotometer. The
theoretical study was carried out on 6-31G** basis set in
Gaussian09 using the density functional theory (DFT)
approximated by B3LYP. Data collection from single crys-
tals was conducted using a Bruker Smart APEXII CCD
diffractometer equipped with graphite-monochromated
Cu Kα radiation (λ = 1.54178 Å). The absolute quantum
yield was measured using an Edinburgh Instrument
FLS980 Integrating sphere. The fluorescence lifetime was
measured using a Hamamatsu Compact Fluorescence
Lifetime Spectrometer C11367. Fluorescent images were
acquired using Olympus BX 41 fluorescence microscope.
Laser confocal scanning microscope images were collect-
ed using a Zeiss laser scanning confocal microscope
(LSM7 DUO) and analyzed using ZEN 2009 software (Carl
Zeiss).
Figure 1. Schematic diagram for molecular structures and
emission mechanism of TPAP and TPAP-BB (a, color code:
gray= C; yellow= N; and purple= B); Synthetic route to
TPAP and TPAP-BB (b).
In 2001, Tang et al. discovered an unusual photo-
physical phenomenon of ‘‘aggregation-induced emission’’
(AIE) in some propeller-like molecules and found that
these AIE luminogens (AIEgens) were generally non-
emissive in solution but emit intensively in the aggregate
state.40-42 Compared with commercially available OLMs,
AIEgens usually exhibit larger Stokes shifts to minimize
emission self-quenching to impart high sensitivity.43,44
Moreover, AIEgens also exhibit high photo-bleaching re-
sistance and photo-stability, making them suitable for
long-term process tracking and real-time monitoring.
Thus, AIEgens are potential materials for bio-medical
applications,45,46 such as lipid droplets imaging. Recently,
several easily prepared AIEgens were developed and re-
13,14,19
13
ported
with either photoactivatable characteristics
or two-photon and near infra-red (NIR) absorption fea-
14
tures for precise spatial and temporal imaging. For in-
X-ray crystallography. Crystal data for TPAP: C23H18N2,
MW= 322.39, monoclinic, P21/n, T/K= 100.01(10), a/Å=
13.98506(19), b/Å= 10.65635(14), c/Å= 11.37257(17), α/°= 90,
β/°= 99.9799(14), γ/°= 90, V/Å3 = 1669.20(4), Z= 4,
ρcalcg/cm3 = 1.283, μ/mm-1= 0.581, F(000)= 680.0, 4806
measured reflections, 2935 independent reflections (Rint=
0.0118, Rsigma= 0.0174), GOF on F2=1.005, R1= 0.0352, wR2=
0.0804 (all data). These data can be obtained free of
charge by The Cambridge Crystallographic Data Centre
(CCDC: 1842802). Crystal data for TPAP-BB: C35H27BN2,
MW= 486.39, monoclinic, P21/n, T/K= 220.00(10), a/Å=
stance, Jiang and Tang et al. prepared TPA-BI to achieve
the precise two-photon imaging of LDs with large Stokes
shift and also large two-photon absorption cross-section.14
However, it should be mentioned that although signifi-
cant progress has been achieved in this field currently,
AIEgens with super brightness for the investigation of real
time monitoring for lipid droplets and their related pro-
cess, such as apoptosis monitoring, is seriously limited.
Therefore, based on this consideration, in this work, new
AIEgens abbreviated as TPAP and TPAP-BB with elec-
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14.85425(18), b/Å= 10.61484(10), c/Å= 16.83686(18), α/°= 90,
cillin and 100 mg/mL streptomycin) at 37 °C and 5% CO2
humidity. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
β/°= 94.4742(10), γ/°= 90, V/Å3= 2646.67(5), Z= 4,
ρcalcg/cm3 = 1.221, μ/mm-1= 0.537, F(000)= 1024.0, 8124
measured reflections, 4619 independent reflections (Rint=
0.0138, Rsigma= 0.0188), GOF on F2= 1.002, R1= 0.0389, wR2=
0.0920 (all data). These data can be obtained free of
charge by The Cambridge Crystallographic Data Centre
(CCDC: 1842801).
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tetrazolium bromide (MTT) assay was used to evaluate
the cytotoxicity of TPAP-BB. Cells were seeded in 96-well
plates (Costar, IL, USA) at a density of 60,000 cells per
well. After an overnight incubation, the medium was re-
placed with 100 µL of fresh medium supplemented with
different concentrations of TPAP-BB. After 24 h incuba-
tion, 10 µL of MTT (5 mg/mL in PBS) was added. After 4 h
incubation, 100 µL of DMSO was then added. After 15 min,
the absorbance at 595 nm was measured using a plate
reader (Perkin-Elmer Victor3). Each experiment (i.e., each
TPAP-BB concentration) was conducted in quintuplicate.
Cell Imaging. Cells grown in a 35 mm petri dish with
glass cover at 37 °C were treated with 5 μM of TPAP-BB (1
µL of TPAP-BB stock solution in DMSO was added to 2
mL of cell culture medium and the final concentration of
DMSO was < 0.1 vol%) for a certain period. The cells were
washed three times with PBS prior to imaging. The imag-
ing was conducted at an excitation wavelength of 405 nm
using a laser scanning confocal microscope with 0.2%
laser power. Conducted in parallel, the cells were treated
with 500 nM of Nile Red (a commercial dye) and the im-
aging was carried out at an excitation wavelength of 560
nm.
Synthesis of TPAP. To a solution of 4-(diphenylamino)
phenylboronicacid (compound 1, 2.0 g, 6.9 mmol) and 2-
bromopyridine (compound 2, 1.1 g, 6.9 mmol) in a mixture
of toluene (80 mL) and ethanol (10 mL) were added
tetrakis(triphenylphosphine)palladium [Pd(PPh3)4] (150
mg) and potassium carbonate (K2CO3, 2M, 34 mL) under
nitrogen atmosphere. After reflux for 20 h, the mixture
was cooled and then extracted with dichloromethane
(DCM) three times (3×50 mL). The obtained organic solu-
tion was washed with water (3×100 mL) and after solvent
removal, the crude product was purified by column
chromatography to afford the pure TPAP as a white solid
(1.85 g, 83%). 1H NMR (400 MHz, CDCl3), δH = 8.68 (d, J =
4.8 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.76-7.68 (m, 2H),
7.36-7.24 (m, 4H), 7.20-7.16 (m, 7H), 7.10-7.06 (t, J = 7.8
Hz, 2H).
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Synthesis of compound 3. In a two-neck flask, com-
pound TPAP (1.0 g, 3.1 mmol) and N,N-diisopropyl-
ethylamine (20 mg) were dissolved in DCM and then bo-
ron tribromide solution in DCM (1.0 M, 10 mL) was added
Medaka Fish Imaging. Live Medaka fish was soaked in
buffer solution containing 5×10-5 M of TPAP-BB for 30 min
at room temperature. Prior to fluorescence imaging, the
fish were washed three times with the buffer solution.
The imaging was conducted using a fluorescence micro-
scope (Olympus BX41).
o
dropwise at 0-5 C. Afterwards, the mixture was stirred
overnight at room temperature. Then, saturated K2CO3
solution was added to quench the reaction and the result-
ing mixture was extracted with chloroform (3×80 mL).
The collected organic layer was washed with water three
times (3×100 mL) and then dried over anhydrous magne-
sium sulfate (MgSO4). Finally, the solvent was removed
under reduced pressure to get compound 3 as an orange
solid (1.12 g, 74%). 1H NMR (400 MHz, CDCl3), δH = 8.81 (d,
J = 5.9 Hz, 1H), 8.06-8.02 (t, J = 7.8 Hz, 1H), 7.73 (d, J = 8.2
Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 2.1 Hz, 1H),
7.44-7.30 (m, 5H), 7.24-7.10 (m, 6H), 6.98-6.96 (m, 1H).
Synthesis of TPAP-BB. To a solution of compound 3 (0.5
g, 1.0 mmol) in toluene (30 mL) was added diphenylzinc
(0.45 g, 2.0 mmol) under nitrogen atmosphere. Then the
RESULTS AND DISCUSSION
Synthesis and Optical Properties. The synthetic route
to TPAP and TPAP-BB was outlined in Figure 1. TPAP was
synthesized in a high yield by Suzuki coupling of 4-
(diphenylamino)phenylboronic
acid
(1)
and
2-
bromopyridine (2) catalyzed by Pd(PPh3)4. Treatment of
TPAP with boron tribromide followed by the reaction
with diphenylzinc generated TPAP-BB in a moderate yield
of 67%. It is worth noting that the synthetic approach for
TPAP and TPAP-BB is simple and facile, which facilitates
large scale synthesis for commercialization purpose. The
chemical structures of TPAP and TPAP-BB were then
o
mixture was stirred at 70 C for 12 h. After that, 20 mL
1
characterized and confirmed by H NMR, 13C NMR, and
water was added, and the obtained mixture was extracted
with ethyl acetate (3×80 mL). The combined organic layer
was washed with brine (3×100 mL) and after solvent re-
moval, the crude product was purified by column chro-
matography to get the pure TPAP-BB as a yellow solid
(0.33 g, 67%). 1H NMR (400 MHz, CDCl3), δH = 8.44 (d, J =
5.3 Hz, 1H), 7.98-7.95 (t, J = 7.5 Hz, 1H), 7.88 (d, J = 7.9 Hz,
1H), 7.70 (d, J = 8.3 Hz, 1H), 7.49 (s, 1H), 7.30-7.17 (m, 19H),
MALDI-TOF-MS spectroscopies with satisfactory results
(see Figures S1-S5, supporting information).
The UV-Vis spectra of TPAP and TPAP-BB in solu-
tion and thin film were shown (see Figure S6, supporting
information). In THF, TPAP absorbed at 343 nm due to
ICT between the electron-rich TPA unit and the electron-
deficient pyridine ring.47,48 TPAP-BB displayed a redder
absorption than TPAP-BB caused by the enhanced ICT
effect for the vacant pz orbital of boron atom in back-
bone.49-52 The absorption of both TPAP and TPAP-BB
then red-shifted in the thin film state, suggestive of some
sort of intermolecular interactions in the solid state.
When the THF solutions of TPAP and TPAP-BB were
photo-excited, strong photoluminescence at 415 nm and
485 nm was then observed, giving Stocks’ Shift of 72 nm
13
7.09-7.06 (t, J = 7.0 Hz, 2H), 6.96 (d, J = 7.6 Hz, 1H). C
NMR (100 MHz, CDCl3), δC = 158.14, 150.60, 147.47, 143.98,
140.21, 133.12, 129.71, 129.24, 127.32, 125.59, 125.30, 123.73,
123.47, 122.59, 120.35, 120.22, 117.44. MALDI-MS calculated
for C35H27BN2: [M]+ 486.23; found 486.2288.
Cell Culture and Cytotoxicity Study. HeLa cells were
cultured in minimal essential medium (MEM) supple-
mented with 10% FBS and antibiotics (100 units/mL peni-
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Figure 2. PL spectra of TPAP (a) and TPAP-BB (c) in THF solution with different water fractions (fw); Plots of relative PL
intensity (I/I0) and emission wavelength versus fw in THF/Water mixture for TPAP (b) and TPAP-BB (d); PL spectra of
TPAP-BB in hexane, toluene, THF and DMSO (e); Transient decay spectra of TPAP and TPAP-BB in THF and thin film (f).
Table 1. Photo-physical properties of TPAP and TPAP-BB in solution and thin film states.[a]
UV (sol/film)
PL (sol/film)
Compounds
λabs
(nm)
λem
(nm)
PLQYs
(%)
τ
kr
knr
kr/knr
(ns)
(×108 s-1) (×108 s-1)
TPAP
TPAP-BB
343/355
392/415
415/425 14.6/49.0 2.39/1.55 0.61/3.16 3.57/3.29 0.17/0.96
485/490 69.4/88.4 5.42/5.80 1.28/1.52 0.56/0.20 2.28/7.60
[a] Abbreviation: sol= THF solution, film= solid thin film, λabs= absorption maximum, λem= emission maximum, PLQYs=
photoluminescence quantum yields, τ= fluorescence lifetime, kr= radiative decay constant, knr= non-radiative decay con-
stant.
and 93 nm respectively (Figure 2a and 2c). To further
study whether these molecules are AIE-active, their PL in
THF/Water mixture was studied. The relative PL intensity
(I/I0) and wavelength versus different water fraction (fw)
in THF/Water mixture for TPAP and TPAP-BB were also
plotted and given in Figure 2b and 2d. As observed, for
both compound TPAP and TPAP-BB, emissive features
can be observed in their solution state, but when water
was added gradually, the fluorescence intensity was de-
creased caused by the twisted intramolecular charge
transfer (TICT) effect, which is a common phenomenon
for the D-A structured molecules, such as TPAP and
TPAP-BB. However, it should be mentioned that the
change of PL intensity when much more water was added
to form the nano-aggregates is generally the key criterion
to estimate whether they are AIE active or not. For TPAP,
although the emission in THF/Water solution (fw= 90%)
is much weaker than that in THF, slight enhancement
was still observed when fw is larger than 70%, indicating
that TPAP should be AIE active, but with an inferior AIE
performance. This poor AIE features for TPAP finally led
to the much lower fluorescence intensity in its aggrega-
tion state than that in solution state. On the other hand,
in contrast to TPAP, TPAP-BB exhibited much twisted
molecular structures to further prevent the emission
quenched π-π stacking, therefore, the PL features for
TPAP-BB was strengthened significantly during the ag-
gregates formation (fw> 70%), finally leading to the large
PL enhancement in aggregated states (fw= 90%), which is
actually much higher than that in THF solution, indicat-
ing that TPAP-BB exhibited a much more promising and
much better AIE performance than TPAP. Moreover, the
corresponding emission wavelength also varied with the
fw values due to the TICT effect.53,54 This was further
proved by the PL spectra of TPAP-BB measured in sol-
vents with different polarities, where a large emission red-
shift from 445 nm to 529 nm was observed when the sol-
vent was changed from nonpolar hexane to polar dime-
thylsulfoxide (DMSO) (Figure 2e).
To further investigate and understand the emission
of TPAP and TPAP-BB, their PLQYs and transient decay
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on the electron-deficient building block.57 However, alt-
spectra in both solution and aggregate state were meas-
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ured (Figure 2f). The data was summarized in Table 1.
The PLQYs of TPAP were moderate being 14.6% and 49.0%
in solution and thin film, respectively. Much higher
PLQYs, however, (69.4% in solution and 88.4% in thin
film, respectively) were observed in TPAP-BB, due to its
higher molecular conjugation and better AIE performance.
Accordingly, the emission lifetime (τ) of TPAP-BB was
also larger than TPAP. Their radiative rates (kr) and non-
radiative decay rates (knr) in solution and thin film were
then calculated according to the equations: kr = PLQYs/τ
and knr = 1/τ-kr.55,56 As shown in Table 1, the large kr and
small knr values of TPAP-BB lead to high PLQYs in solu-
tion. The much smaller knr value in thin film indicated
that the non-radiative decay channel was efficiently
blocked to endow the TPAP-BB film with intense light
emission. The kr/knr ratio was also calculated and com-
pared. As expected, the higher kr/knr values of TPAP-BB
than TPAP resulted in its much higher PLQYs values in
both solution and thin film. On the other hand, it’s
known that the fluorescence lifetime for OLMs is inverse-
ly proportional to kr and knr values. Therefore, the much
smaller kr value for TPAP in solution resulted a much
longer lifetime than that in solid state.
hough the orbitals of TPAP were somewhat overlapped,
those of TPAP-BB were better separated, indicative of
more efficient ICT effect and redder emission in TPAP-
BB. Moreover, the significant dense electron cloud in the
boron atom of LUMO for TPAP-BB further demonstrated
the stronger ICT effect in TPAP-BB than in TPAP.58
To further confirm the molecular structures and un-
derstand their optical properties, the single crystals of
TPAP and TPAP-BB were grown and analyzed crystallo-
graphically. As illustrated in Figure 4, the obtained crys-
tal structures were similar to their DFT-optimized ones
with twisted molecular backbones and also large dihedral
angles. The distance between neighboring planes of TPAP
and TPAP-BB were calculated to be 6.646 Å and 7.112 Å,
respectively, which were long enough to prevent the oc-
currence of determined π-π stacking. On the other hand,
abundant interactions, such as C-H…π, were observed in
the crystal lattice of TPAP-BB to restrain its molecular
rotations.59 All these factors made the molecules highly
emissive in the aggregate state.
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Figure 4. Structures (a and d), twisting angles of aromatic
units (b and e), intermolecular stacking distances (c and f)
and intermolecular interactions (g) in crystal lattices of
TPAP (a, b, and c) and TPAP-BB (d, e, and f).
Figure 3. The optimized geometry, HOMO and LUMO of
TPAP and TPAP-BB calculated at B3LYP/6-3/G** level
using DFT.
Piezo- and Acido-chromisms. As observed, TPAP-BB
exhibited much superior fluorescent properties in con-
trast to TPAP, therefore, we choose this newly developed
compound to further investigate its piezo-chromic and
acido-chromic performances. Upon grinding the pristine
sample of TPAP-BB, its emission red-shifted significantly
(Figure 5a). Analysis by powder X-ray diffraction showed
that the pristine sample exhibited many sharp diffraction
peaks, which disappeared upon grinding (Figure 5b). This
suggested that the piezo-chromism of TPAP-BB was
caused by the conformational change from crystalline to
amorphous state.60 Interestingly, the ground sample re-
crystallized upon heating to recover the crystal-state
Theoretical Calculations and Crystal Structure Anal-
ysis. The ground-state geometries of TPAP and TPAP-BB
were optimized using DFT at B3LYP/6-31G** level. As
shown in Figure 3, both TPAP and TPAP-BB exhibited
twisted molecular structures with large dihedral angles.
This prevented the π-π stacking between neighboring
molecules in the aggregate state to avoid emission
quenching. The highest occupied molecular orbitals
(HOMO) and the lowest unoccupied molecular orbitals
(LUMO) of TPAP and TPAP-BB were also presented in
Figure 3. The HOMO of both TPAP and TPAP-BB was
contributed mainly by their electron-donating unit, while
the orbitals of their LUMO were located predominately
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emission. On the other hand, since organoboron fluoro- scanning using a confocal laser scanning microscope
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phores have been found that can undergo protonation
easily to quench their emission, a filter paper loaded with
TPAP-BB was employed to study its acido-chromic prop-
erty. The dye-doped filter paper exhibited a strong emis-
sion at 475 nm but became non-luminescent upon fuming
with vapor of hydrochloric acid (Figure 5c). The emission
was then subsequently “turned-on” when the filter paper
was exposed with ammonia vapor for 30 s. Such “on/off”
cycle could be repeated for more than 5 times without
considerable intensity decay (Figure 5d), indicating that
TPAP-BB may be potentially used as a solid-state “on/off”
luminescent switch.61,62
(CLSM). While the signal of Nile Red was attenuated to
about 60% after 90 scans, that of TPAP-BB was virtually
unchanged, indicating that TPAP-BB possessed much
higher photo-bleaching resistance and superior photo-
stability than Nile Red (Figure 6b). TPAP-BB was then
successfully utilized in the imaging of HeLa cells using 3D
CLSM (insert in Figure 6b).
After confirming the cytotoxicity and photo-stability of
TPAP-BB, detailed cell-imaging experiments were then
conducted. As displayed in Figure 6c, bright fluorescence
was observed mainly in the lipid droplets of HeLa cells.
To confirm TPAP-BB targets specifically to LDs, the HeLa
cells were co-stained with Nile Red (Figure 6d).64 High
Pearson’s correlation co-efficiency of up to 97% was final-
ly achieved (Figure 6e and 6f), which implied that TPAP-
BB was highly specific to LDs. To gain further insight into
such high specificity, the HeLa cells were respectively
stained with TPAP-BB and Nile Red, then observed under
different emission intensity (Figure 7). LDs-specific im-
ages were firstly obtained from both TPAP-BB and Nile
Red at low emission intensity (Figure 7a). When the emis-
sion intensity was gradually increased, fluorescence sig-
nals associated with Nile Red were then also observed in
the cytoplasm of HeLa cells, while those associated with
TPAP-BB were still confined in LDs (Figure 7b and 7c).
After that, the signal-to-noise ratio was calculated using
the average emission intensity in LDs and cytoplasm in
three different cases. As suggested (see Figure S8, sup-
porting information) and Figure 7d, TPAP-BB exhibited a
much higher signal-to-noise ratio than Nile Red. This
result revealed that TPAP-BB was potential and promising
fluorescence agent for bio-imaging of LDs in biomedical
study.
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Figure 5. The normalized PL spectra of pristine, ground,
and ground+ heating TPAP-BB powder (a, inset shows the
piezo-chromic behaviors of TPAP-BB); XRD diffracto-
grams of pristine, ground, and ground+ heating TPAP-BB
powder (b); PL spectra of pristine, HCl-fumed, and
HCl+NH3-fumed filter paper loaded with TPAP-BB (c);
Reversible switching the light emission of TPAP-BB by
repeatedly HCl-NH3 fuming cycle (d).
Lipid Droplets Imaging. Although TPAP-BB shows effi-
cient solid-state emission, to serve as visualizing agent for
bio-imaging, it should also exhibit low cytotoxicity.
Therefore, the cytotoxicity of TPAP-BB to living cells was
firstly evaluated using MTT assay.63 Owing to that Hela
cells have been demonstrated as one of the most widely
used model cells for bio-imaging and process monitoring.
Therefore, we chose this easily available cell here.14,59 As
shown in Figure 6a, the HeLa cells treated with TPAP-BB
at a high concentration of 50 μM displayed a viability of
90%, indicating that TPAP-BB exhibited low toxicity to
living cells. On the other hand, photo-stability is another
key factor for bio-imaging. As shown (see Figure S7, sup-
porting information), the emission of TPAP-BB in both
solution and aggregate state remained nearly constant
after an extended period of irradiation, suggestive of its
high photo-stability. Then, the photo-stability of TPAP-
BB in living cells was further studied and compared with
that of Nile Red, a commercial bio-probe, by continuous
Figure 6. Cell viability of HeLa cells treated with TPAP-
BB determined by MTT assay (a); Attenuation of fluores-
cence for TPAP-BB- and Nile Red-stained HeLa cells as a
function of number of scan (b, inset shows the 3D CLSM
image of TPAP-BB-stained HeLa cells); CLSM images of
HeLa cells incubated with 5 μM of TPAP-BB (c) and Nile
Red (d) in DMSO at 37 oC for 30 min; Dark-field image (e)
of merged (c) and (d); Bright-field image (f) of merged (c)
and (d).
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Figure 7. CLSM images of HeLa cells stained with TPAP-BB and Nile Red with different emission intensity of 1.0 (a), 2.5
(b), 3.0 (c) and their corresponding signal-to-noise ratios (d).
TPAP-BB was further used to monitor the dynamic
movement of LDs using a confocal microscope. The imag-
es captured at different time showed that TPAP-BB was
able to track the spatial distribution of LDs and their dy-
namic movement in living cells (see Figure S9, supporting
information).65 On the other hand, it is important to real-
ize the emission behaviors of co-stained bio-probes in
biological environment to minimize the possible cross-
talk. For TICT-featured molecules, such as TPA-BB, their
tunable emission could also provide information on the
polarity of the surrounding environment. Therefore, the
imaging of LDs using TPAP-BB was further investigated
using a confocal microscope operated in a lambda-mode.
The fluorescent images of TPAP-BB stained HeLa cells
were taken at different emission wavelengths (see Figure
S10, supporting information) and the intensity of each
image was plotted against the wavelength, from which an
emission spectrum similar to that measured in toluene
was obtained (see Figure S11, supporting information).
This further indicated the non-polar biological environ-
ment near the LDs in living cells.
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Figure 8. CLSM images of TPAP-BB- and MT-Red-stained
HeLa cells treated with 5 mM of H2O2 for 0 min (a), 20
min (b), 40 min (c) and 60 min (d).
Apoptosis Monitoring. Cell apoptosis is generally close-
ly associated with mitochondrial dysfunction to result
extensive formation of LDs.66 Therefore, the developed
LDs-specific TPAP-BB was used to monitor the dynamic
process of cell apoptosis induced by reactive oxygen spe-
cies generated by hydrogen peroxide (H2O2).67 Commer-
cial MitoTracker-Red (MT-Red) was also employed to
track the mitochondrial changes during apoptosis. As
shown (see Figure S12a, supporting information), the LDs
and mitochondria of HeLa cells were respectively stained
with TPAP-BB and MT-Red. At low H2O2 concentration (1
mM), no apoptosis occurred as the morphology of mito-
chondria only changed slightly after 2 h (see Figure S12b,
supporting information). However, apoptosis was induced
at high H2O2 concentration (5 mM; Figure S12c, support-
ing information). Afterward, the apoptosis process was
real-time monitored using TPAP-BB and MT-Red (Figure
8).
Initially, the emission of LDs and mitochondria was
distinguishable (Figure 8a), and the morphology of mito-
chondria was gradually altered after 20 min (Figure 8b).
At longer time (40 min and 60 min), the mitochondria
were found to show the green color from the TPAP-BB
stained LDs (Figure 8c and 8d). As a control, TPAP-BB
and MT-Red were used to co-stain HeLa cells in the pres-
ence of light irradiation for 60 min. Interestingly, the
above phenomenon was not observed (see Figure S13,
supporting information), indicating that its cause was due
to apoptosis induced by H2O2. On the other hand, owing
to that apoptosis can largely decrease the membrane po-
tential of mitochondrion to allow entry of neutral TPAP-
BB,68 therefore, to prove that TPAP-BB observed in the
CLSM images is bound to lipid droplets, rather than
TPAP-BB’s entering into mitochondria, carbonyl cyanide
3-chlorophenyl-hydrazone was employed to decrease the
mitochondrial membrane potential.69 Although the mor-
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phology of mitochondria was altered, the green signals Supporting Information. The NMR spectra, High reso-
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associated TPAP-BB-stained lipid droplets were still ob-
served at the same positions (see Figure S14, supporting
information). This results further suggested that it’s the
formation of LDs during apoptosis played roles here, indi-
cating the great potential of TPAP-BB in apoptosis moni-
toring. Interestingly, the cell fragments denoted “apoptot-
ic bodies” were also successfully monitored during apop-
tosis process (see Figure S15, supporting information).
lution mass spectrum, UV-Vis spectra of TPAP and TPAP-
BB, Photo-stability of TPAP-BB in THF and THF/Water
mixture, CLSM images of TPAP-BB-stained HeLa cells at
different moments and emission wavelengths were listed
in Supporting Information. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
B.Z. Tang: tangbenz@ust.hk
Author Contributions
‡ D. Dang and H. Liu contributed equally.
Notes
9
In vivo imaging of Medaka fish. Based on the impres-
sive cell imaging results in HeLa cells, as a proof of con-
cept, the developed TPAP-BB was then applied to in vivo
imaging using Medaka fish for its high optical transpar-
ence. The images showed that TPAP-BB was able to stain
the living Medaka fish after 30 min incubation at a con-
centration of 5×10-5 M (Figure 9e-h). TPAP-BB was found
to probably localize in the fish’s excretory system. Like
the H2O2-treated HeLa cells, Medaka fish fed with H2O2-
containing food also exhibited much stronger emission of
TPAP-BB (Figure 9i-l), suggesting that some fish cells had
underwent apoptosis. These findings further demonstrate
our developed TPAP-BB could potentially be applied for
in vivo imaging, a technique used for monitoring of vari-
ous biological processes.
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The authors declare no conflict of interest.
ACKNOWLEDGMENT
This work was supported by the National Science Founda-
tion of China (21788102, 81372274, 81501591 and
8141101080), the Research Grant Council of Hong Kong
(N_HKUST604/14, C6009-17G, A-HKUST605116 and
C2014-15G), the Innovation and Technology Commission
(ITC-CNERC14SC01 and ITS/251/17), the Shenzhen Sci-
ence and Technology Program (JCYJ20160509170535223,
JCYJ20166428150429072, JCYJ20160229205601482 and JCY
20170818113602462), and the International Science &
Technology Cooperation Program of Guangzhou
(20170403069). D. Dang also thanks the financial supports
by National Natural Science Foundation of China
(51603165) and young talent fund of university association
for science and technology in shaanxi, China (20180601).
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