Highly efficient Far Red/Near-Infrared fluorophores with aggregation-induced emission for bioimaging

Article abstract of DOI:10.1016/j.dyepig.2017.04.004

Organic fluorescent probes play an important role in modern biomedical research, such as biological sensing and imaging. However, the development of organic fluorophores with efficient aggregate state emissions expanded to the red to near-infrared region

Full text of DOI:10.1016/j.dyepig.2017.04.004

Dyes and Pigments 142 (2017) 491e498
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Highly efficient Far Red/Near-Infrared fluorophores with
aggregation-induced emission for bioimaging
,
**
Fengli Zhang a, Yizeng Di a, Yue Li b, Qingkai Qi a, Jingyu Qian a, Xueqi Fu b, Bin Xu a
,
,
*
Wenjing Tian a
a State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, 130012, PR China
b College of Life Sciences, Jilin University, Changchun, 130012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Organic fluorescent probes play an important role in modern biomedical research, such as biological
sensing and imaging. However, the development of organic fluorophores with efficient aggregate state
emissions expanded to the red to near-infrared region is still challenging. Here, we present a series of
highly efficient Far Red/Near-Infrared (FR/NIR) fluorescent compounds with aggregation-induced emis-
sion (AIE) properties by attaching electron donor and accepter to tetraphenylethene (TPE) moieties
Received 16 January 2017
Received in revised form
1 April 2017
Accepted 2 April 2017
Available online 8 April 2017
through
a simple synthesis method. These compounds exhibited the pronounced fluorescence
enhancement in aggregate state, the red to near infrared emission, and can be facilely fabricated into
uniform compounds-loaded Pluronic F127 NPs. The emission maximum of the NPs fabricated by the self-
assembly method is in the range of 550e850 nm and the highest fluorescent quantum yield can get to
15.2%. The biological imaging of NPs of compound 1 and 2 for A549 lung cancer cell indicates that these
compounds are effective fluorescent probes for cancer cells with high specificity, high photostability and
good fluorescence contrast.
Keywords:
Aggregation-induced emission
Tetraphenylethene derivatives
Biocompatible dye
Bioimaging
Far Red/Near-Infrared
Fluorescent compounds
© 2017 Elsevier Ltd. All rights reserved.
1. Introduction
or upon interaction with bioanalyses, which is the so-called ag-
gregation-caused quenching (ACQ) effect [7e10]. In 2001, Tang
Fluorescent probes have attracted extensive attentions due to
their imaging of biological species and monitoring ability of the life
process [1,2]. Among various fluorescent probes, inorganic semi-
conductor quantum dots (QDs) have been widely used because of
their unique optical and electronic properties, i.e. size-tunable light
emission, superior signal brightness, resistance to photo bleaching,
and broad absorption spectra for simultaneous excitation of mul-
tiple fluorescence colors [3]. Whereas, QDs induce notorious cyto-
toxicity owing to the release of heavy metal ions, especially in
anoxidative environment, which limited their applications in some
fields of biological imaging [4,5]. In comparison with QDs, organic
dye-loaded fluorescent nanoparticles (NPs) have exhibited superior
cytocompatibility and fluorescence stability [6]. However, con-
ventional organic dye may suffers weakened or annihilated fluo-
rescence in nanoparticles matrices due to the planar aromatic
et al. reported a series of silole derivatives which were non-
emissive in dilute solutions but exhibited strong luminescence
when the molecules were aggregated in concentrated solutions or
casted into solid films due to the restriction of intramolecular
rotation [11e13]. That is aggregation-induced emission (AIE).
Recently, many AIE fluorophores with highly twisted structures
have been synthesized and their various applications have been
explored, especially in optoelectronic and biological areas [12e18].
TPE is one of the typical AIEgens. Most of the TPE derivatives give
blue or green emission, limited their applications for bioimaging
[12,19e33]. Meanwhile, far FR/NIR fluorophores play a crucial role
in fluorescent bioimaging because of their low tissue absorption
and weak tissue auto fluorescence in FR/NIR region, thus can
minimize the background interference and improve the image
sensitivity [34e39]. Tang et al. reported a series of AIE fluorophores
by conjugating a common AIE molecule with ACQ red fluorophores,
emitted fluorescence in the FR/NIR region and assembled into
nanoparticles for vitro bioimaging of cancer cells [10,19,33,40,41].
Herein, we designed and synthesized FR/NIR AIE fluorophores
by selecting proper combination of electron donor (D) and acceptor
structure which can lead to non-radiative process via pep stacking
* Corresponding author.
** Corresponding author.
E-mail addresses: xubin@jlu.edu.cn (B. Xu), wjtian@jlu.edu.cn (W. Tian).
http://dx.doi.org/10.1016/j.dyepig.2017.04.004
0143-7208/© 2017 Elsevier Ltd. All rights reserved.

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F. Zhang et al. / Dyes and Pigments 142 (2017) 491e498
(A) to create FR/NIR fluorophores. By introducing the dimethyl-
amine and cyano moieties into TPE, four novel FR/NIR fluorescent
compounds 1, 2, 3 and 4 with distinct AIE characteristics have been
synthesized. The highest quantum yield of the four compounds in
solid state can reach to 40%. These compounds were easily fabri-
cated into uniform compound-loaded NPs. Remarkably, compound
1-loaded Pluronic F127 NPs showed the long emission wavelength
at the peak of 650 nm and high fluorescence quantum yield of
15.2%. The biological imaging of NPs of compound 1 and 2 for A549
lung cancer cell indicates that these compounds are effective
fluorescent probes for cancer cells.
2.4. Preparation of fluorogen-loaded F-127 NPs
The fluorogen-loaded F-127 NPs were prepared by using the
self-assembly method as described previously for conjugated
polymers [38]. In THF solution (3 mL) containing F-127 (200 mg),
TPE derivatives (from 0.25 to 2 mg) was poured into water (10 mL).
And then the mixture was evaporated to completely remove the
organic agent (THF) by nitrogen purge. The NP suspension was
further purified with a 0.2 mm syringe filter to obtain fluorogen-
loaded F-127 NPs.
2.5. Cell culture
2. Experimental section
The A-549 cell line was maintained in DMEM (Gibco) supple-
mented with 10% FBS (Gibco), and 100 U/mL of penicillin under a
humidified atmosphere containing 95% air and 5% CO2 at 37 ꢁC. The
cells were precultured until confluence was reached prior to ex-
periments. The cells were harvested by briefly rinsing with culture
media followed by incubation with trypsin-EDTA solution (0.25%
w/v trypsin, 0.53 mM EDTA) for cell imaging.
2.1. Materials
Tetrahydrofuran (THF) was distilled from sodium benzopheno-
neketyl under nitrogen immediately prior to use. And all the other
chemicals were purchased from Aladdin and used as received
without further purification.
2.2. Instruments
2.6. Cell viability and cytotoxicity using an MTT assay
1H NMR spectra were recorded on a 500 MHz Bruker Avance,
using DMSO as solvent. 13C NMR spectra were recorded on a
125 MHz Bruker Avancespectrometer, using CDCl3 as a solvent and
We determined the cytotoxic effects of F-127 dots on A549 cells
and the cell viability of A549 using an MTT assay. A549 cells need to
be incubated with F-127 dots in the dark for 24 h before interacting
TMS as an internal standard (
d
¼ 0.00 ppm). LC-HRMS was obtained
with MTT assay. After incubation, a sample of 100 mL of MTT (5 mg/
by Agilent 1290-microTOF Q II. Element analyses were performed
on a FlashEA1112 spectrometer. GCMS were recorded on a Thermo
Fisher ITQ1100. UV/Vis spectra were measured on a Shimadzu UV-
2550 spectrophotometer. Fluorescence spectra were recorded by
Shimadzu RF-5301 PC spectrometer and Maya2000Pro optical fiber
spectrophotometer. Solid photoluminescence (PL) efficiencies were
measured by using an integrating sphere (C-701, Labsphere Inc.),
with 365 and 470 nm Ocean Optics LLS-LED as the excitation
source, and the light was introduced into the integrating sphere
through optical fiber. The confocal laser scanning microscopy im-
ages were obtained on an Olympus FV1000 confocal laser scanning
microscope.
ml) in phosphate buffer solution (PBS) was added to each well of a
96-well plate and cultured for 4 h at 37 ꢁC. After removing the
medium containing MTT,150 mL of warm dimethyl sulfoxide (DMSO)
was added to each well to dissolve the formazan. The samples were
shaken for 10 min at room temperature in the dark. The absorbance
was read at 490 nm using a microplate reader. Percentage cell
viability and cytotoxicity were calculated from the following for-
mulas: Percentage cell viability ¼ (average Abs. value of experi-
mental group e average Abs. value of blank group)/(average Abs.
value of control group e average Abs. value of blank group) ꢂ 100%;
percentage cell cytotoxicity ¼ [1 e (average Abs value of experi-
mental group e average Abs value of blank group)/(average Abs
value of control group e average Abs value of blank group)] ꢂ 100%;
percentage cell cytotoxicity ¼ (1 e Percentage cell viability) ꢂ 100%.
2.3. Synthesis
Under N2 atmosphere, a three-necked flask equipped with a
magnetic stirrer was charged with zinc powder (4.2 g, 65 mmol)
and 40 mL THF. The mixture was cooled to ꢀ5e0 ꢁC, and TiCl4
(3.6 mL, 32.4 mmol) was slowly added by a syringe with the tem-
perature kept under 10 ꢁC. The suspending mixture was warmed to
room temperature and stirred for 0.5 h, then heated at reflux for
2.5 h. The mixture was again cooled to ꢀ5e0 ꢁC, charged with
pyridine (0.24 mL, 3 mmol) and stirred for 10 min. The solution of
two carbonyl compounds (in 6:6 mmol to 6:7.2 mmol mole ratios,
in 15 mLTHF) was added slowly. After addition, the reaction
mixture was heated at reflux until the carbonyl compounds were
consumed (monitored by TLC). The reaction was quenched with
10% Na2CO3 aqueous solution and taken up with CH2Cl2. The
organic layer was collected and concentrated. The crude material
was purified by flash chromatography to give the desired products.
The 1-(4-bromophenyl)-1,2,2-triphenylethylene was solved in
THF, the added n-BuLi at ꢀ78 ꢁC for 2 h, followed by addition of
dimethylformamide, the crude material was kept at room tem-
perature for 2 h. The mixture was quenched by water and purified
by flash chromatography, gave the expected aldehyde.
2.7. Cell imaging
A million A549 cells in 100 mL labeling buffer (PBS containing
1% BSA and 2 mM EDTA) were incubated with F-127 dots for 4 h.
After washing two times, cell suspension was dropwise to a
coverslip, then covered with a glass slide and imaged immediately
under confocal laser scanning microscope.
2.8. Determination of quantum yields
By using rhodamine B as the reference (fluorescence quantum
yield equal to 0.69 in dilute ethanol solution with excitation
wavelength of 365 nm), the fluorescence quantum yield of the
near-infrared emission dots were measured according to the
following equation.
ꢀ
ꢁꢀ ꢁꢀ
ꢁ
Ar
As
Is
Ir
nr2
ns2
hs
¼
hr
where hr is the quantum yield of reference, Ar is the absorbance of
reference at the excitation wavelength, As is the absorbance of
sample at the excitation wavelength, Ir is the area under the
emission peak on a wavelength scale of reference, Is is the area
Knoevenagel reaction was taking place between the aldehyde
and the cyano derivative under alkaline conditions. The mixture
was purified by flash chromatography, gave the target molecular.

F. Zhang et al. / Dyes and Pigments 142 (2017) 491e498
493
under the emission peak on a wavelength scale of sample, nr is the
refractive index of the reference solvent, ns is the refractive index of
the sample solvent. Absorption spectrum and emission spectrum
measured by the Maya 2000 pro fiber spectrometer (Ocean Optics).
Solid state PL efficiencies were measured by using an integrating
sphere (C-701, Labsphere Inc.) with a 365 nm Ocean Optics LLS-LED
as the excitation source, and the laser was introduced into the
sphere through the optical fiber.
3. Result and discussion
3.1. Synthesis and characterizations
Compound 1, 2, 3 and 4 were synthesized by a series of chemical
reactions according to Scheme 1. The detailed synthetic procedure
and characterizations are described in the Experimental Section
and Supporting Information.
3.2. Optical properties
Fig. 1. UV-vis absorption and normalized photoluminescence spectra of 10 mM of each
compound 1, 2, 3 and 4 in THF/water (v/v ¼ 1/9).
The UV-vis absorption (solid lines) and normalized PL spectra
(dashed lines) of compound 1, 2, 3 and 4 are shown in Fig. 1. Each
compound has a high absorption peak at around 330 nm, which is
shown in Fig. 2, the molecular orbital density of the Highest
Occupied Molecular Orbital (HOMO) is mainly located in the central
tetraphenylethene and dimethylamine moieties. The Lowest Un-
occupied Molecular Orbital (LUMO) level is primarily localized in
cyan group, which suggests a strong intramolecular charge transfer
between dimethylamine and cyano moieties within the com-
pounds, leading to the FR/NIR emission.
The four compounds exhibited remarkable AIE properties. For
example, compound 1 was almost non-emissive in THF. As shown
in Fig. 3, in dilute THF solution, compound 1 exhibited weak fluo-
rescence. When fw (volume fraction of water) varied from 0 to 50%,
the PL spectra of the solutions changed little. When fw was 60% or
higher, PL intensity and fluorescence quantum yield both increased
evidently. When the water fraction was increased to 90%, the PL
attributed to pep* transition, and a weak absorption ranging from
440 to 490 nm, which represents the intramolecular charge
transfer (ICT) transition between dimethylamine and cyano moi-
eties. The emission peak of the compounds ranged from red to the
NIR region with large Stokes shifts, which facilitates to improve the
signal/background ratios of fluorescent images by avoiding imaging
interference between the emission and excitation. Compound 1
exhibited high solid state FR/NIR emission with excellent quantum
yields of 0.40 measured by an integrating sphere (C-701, Labsphere
Inc.).
In order to better understand the ICT transitions of these fluo-
rophores, we performed density functional theory (DFT) calcula-
tions for compound 1e4 by using Gaussian 09/B3LYP/6-31G(d). As
Scheme 1. The synthetic route of TPE derivatives.

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F. Zhang et al. / Dyes and Pigments 142 (2017) 491e498
Fig. 2. Chemical structures and HOMOeLUMO distributions of the compound 1, 2, 3 and 4 optimized structures of the HOMO and LUMO were calculated by TD-DFT (Gaussian 09/
B3LYP/6-31G(d)).
Fig. 3. (a) PL spectra of 1 in THF/water mixtures with different water fractions (fw). (b) Plot of peak intensity of 1 versus water fraction in the aqueous mixtures. [1] ¼ 10 ꢂ 10ꢀ6 M,
lex ¼ 365 nm.
intensity was about 550 times greater than that in pure THF. The
enhancements were ascribed to aggregation formation, which
were induced by the addition of water. In aggregates, the propeller
shape of compound 1 packed closely, leading to the restriction of
the rotations of phenyls in TPE units, which is the progress that
exhausts the excited energy and renders it non-radiative. The
emission maximum for the aggregates of compound 1 is at 650 nm,
while the PL spectrum ranges from 570 to 860 nm, covered a rather
large area in NIR region. Compounds 2e4 show similar properties
as compound 1 (see Figs. S1e3). The FR/NIR emission and desirable
fluorescence quantum yield suggest these molecules can be used as
potential fluorescent probes for bioimaging.
as the encapsulation matrix. A THF solution (3 mL) containing
compound 1 (0.25e2 mg) and F-127 (fixed at 200 mg) was injected
slowly into water (10 mL). Compound 1 was aggregated and
entangled within the hydrophobic domains of the F-127 chains,
accompanied its hybridization with the hydrophobic fluorogen.
And then the mixture was evaporated to completely remove the
organic agent (THF) by nitrogen purge. During compound 1-loaded
F-127 NPs formation, the hydrophobic units of F-127 molecule
tended to locate inside the NPs while the hydrophilic units local-
ized on the surface of the NPs in the aqueous environment.
Transmission electron microscopy (TEM) images of the compound
1 loaded F-127 NPs with 1 mg of compound 1 are shown in Fig. 4 as
an example. The images indicate that the AIE-active fluorogen-
loaded NPs had a spherical shape and a smooth surface with an
almost uniform size of around 30 nm. All the F-127 NPs loaded
variable fluorogen have a narrow particle size distribution around
35e40 nm (see Figs. S7e9).
3.3. Nanoparticle preparation and characterization
In order to apply these compounds as the biosensors in living
tissue cells, we prepared organic fluorescent AIE nanoparticles
(NPs) through a modified nanoprecipitation method by using F-127
Table 1 shows the fluorescence quantum yield of the compound

F. Zhang et al. / Dyes and Pigments 142 (2017) 491e498
495
Fig. 4. (aeb) TEM images of 1@F-127 NPs in different magnifications. (c) Diameter statistics of NPs via the counting of the NPs in the TEM images. More than 100 NPs were counted.
Table 1
water were measured using Rhodamine B in ethanol as the stan-
dard. The fluorescence quantum yield initially increased and then
Fluorescence quantum yield of compound 1 in different weight loaded F-127 NPs.
decreased with increasing the amount of compound 1. It eventually
reached a value of 15.2% at a compound 1 loaded weight of 1.5 mg.
Therefore, in order to obtain the best fluorescence imaging result,
the NPs were prepared by using a precursor solution with the
proper mass ratio of 200: 1.5 (F-127: compound 1). The quantum
yield also reached to 6.4%, 5.7%, and 4.3% for compound 2e4,
respectively (see Figs. S4e6).
Excellent fluorescence stability in aqueous media is essential for
biological applications, especially in long-term studies. The fluo-
rescence intensity evolution of compound 1 and 2 loaded F-127 NPs
was investigated by monitoring their fluorescence changes upon
incubation in a phosphate buffer solution (PBS) under different
incubation time. As shown in Fig. 5a and its inset, no obvious
Compound 1 loading
weight (mg)
Pluronic F-127 loading
weight (mg)
Fluorescent quantum
yield (%)
0.25
0.50
0.75
1.00
1.25
1.5
200
200
200
200
200
200
200
200
4.52
5.64
5.84
7.09
10.8
15.2
14.7
14.6
1.75
2.00
1 loaded F-127 NPs prepared at different weight of compound 1.
The fluorescence quantum yield values of the 1-loaded F-127 NPs in
Fig. 5. PL intensity evolution of compound 1 loaded F-127 NPs (a) and compound 2 loaded F-127 NPs (b) upon continuous excitation at 405 nm (0.5 mW) following the incubation
time until 24 h. I0 is the initial fluorescence intensity and I is the fluorescence intensity of the sample after continuous exposure for designed time intervals.
Fig. 6. Metabolic viability of A549 cancer cells after incubation with compound 1 and 2 loaded F-127 NPs and compound 1 and compound 2 at various fluorogen concentration for
24 h.

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F. Zhang et al. / Dyes and Pigments 142 (2017) 491e498
decrease of the fluorescence intensity was observed after excitation
at 405 nm (0.5 mW) for 24 h, which suggests the well stability of
the prepared NPs. Thus, the excellent optical properties of the NPs,
such as near-infrared emission, large Stokes shift, high fluorescent
quantum yield and good photostability make them suitable can-
didates for bioimaging.
Fig. 7. Confocal fluorescence images of F-127 NPs for A549 and NIH/3T3; (a)e(c) compound 1 loaded F-127 NPs for A549; (d)e(f) compound 1 loaded F-127 NPs for NIH/3T3. (g)e(i)
compound 2 loaded F-127 NPs for A549; (j)e(l) compound 2 loaded F-127 NPs for NIH/3T3; (a)e(l) were incubated for 3 h. Incubation concentration of NPs was 30 mg/mL. Excitation
light: 405 nm.

F. Zhang et al. / Dyes and Pigments 142 (2017) 491e498
497
3.4. Cytotoxicity
21674041) and Program for Chang Jiang Scholars and Innovative
Research Team in University (No. IRT101713018) and Program for
Changbaishan Scholars of Jilin Province.
We took compound 1 and 2 loaded F-127 NPs as examples to
investigate the toxicity. The cytotoxicity was evaluated with
A549 cells through MTT assay. First, cancer cells were seeded in 96-
well plates (104 cells per mL) and incubated for 24 h. Then, various
concentrations of fluorogen-loaded F-127 NPs were added into the
96-well plate, and the cells with the NPs were incubated at 37 ꢁC.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2017.04.004.
After 24 h, 100
m
L of MTT solution (5 mg mLꢀ1 in PBS) was added
L of DMSO were added
into each well and incubated for 4 h. 100
m
References
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The absorbance at 570 nm was measured with a microplate reader
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by the ratio of the absorbance of the cells incubated with com-
pound 1 and 2 loaded F-127 NPs suspension to that of the cells
incubated with culture medium only. Fig. 6 shows the cytotoxicity
results of the variable concentration of compound 1 and 2
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3.5. In vitro cellular imaging
Compound 1 and 2 loaded F-127 NPs are expected to be good
candidates for biological imaging due to their suitable size, good
stability and good luminescence performance. To evaluate the
performance of specific cellular imaging of these NPs, A549 lung
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NPs in cellular imaging was investigated by confocal laser scanning
microscopy (CLSM). A549 cancer cells and 3T3-L1 cells were incu-
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concentration of 30 mg/mL. The CLSM images of A549 cancer cells
after incubation with compound 1 and 2 loaded F-127 NPs are
shown in Fig. 7aec and gei, respectively. Strong fluorescence could
be found in the cytoplasm. Only weak fluorescence could be found
in NIH/3T3 cells when the same NPs were incubated (Fig. 7def and
jel). Therefore, compound 1 and 2 loaded F-127 NPs are more
effectively internalized in the cytoplasm of A549 cancer cells than
NIH/3T3 due to the faster endocytosis.
4. Conclusion
In this work, we successfully synthesized four species of FR/NIR
organic fluorescent molecules with AIE feature. These fluorophores
were assembled into FR/NIR organic fluorescent NPs with the flu-
orophores as the core and biocompatible F-127 as the encapsula-
tion matrix. It has been showed that the fluorogen-loaded F-127
NPs have small size, strong near-infrared emission and large Stokes
shift. These superior performances make the fluorogen-loaded F-
127 NPs a potential material in bioimaging. This study provides an
efficient strategy to design highly emissive water soluble organic
NPs for a wide range of biological detection. Moreover, the devel-
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This work was supported by (2013CB834701), the Natural Sci-
ence Foundation of China (No. 51373063, 51573068, 21221063,

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