Facile preparation of water dispersible red fluorescent organic nanoparticles and their cell imaging applications

Article abstract of DOI:10.1016/j.tet.2014.04.010

Red fluorescent organic nanoparticles (FONs) based on a cyano-substituted diarylethylene and tetraphenylethene derivative conjugated molecule (R-TPE) were facilely prepared via surfactant modification with lecithin for the first time. The obtained R-TPE-LEC FONs were characterized by a series of techniques including fluorescence and UV spectroscopy, Fourier transform infrared spectroscopy, and transmission electron microscopy. Biocompatibility evaluation and cell uptake behavior of R-TPE-LEC FONs were further investigated to explore their potential biomedical application. We demonstrated that such red FONs exhibit anti-aggregation-caused quenching property, broad excitation wavelength, high water dispersibility, uniform morphology (40-60 nm), and excellent biocompatibility, making them promising for cell imaging application.

Full text of DOI:10.1016/j.tet.2014.04.010

Tetrahedron 70 (2014) 3553e3559
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Facile preparation of water dispersible red fluorescent organic
nanoparticles and their cell imaging applications
,
y
y
*
*
Xiqi Zhang , Xiaoyong Zhang , Bin Yang, Yaling Zhang, Yen Wei
Department of Chemistry and the Tsinghua Center for Frontier Polymer Research, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Red fluorescent organic nanoparticles (FONs) based on a cyano-substituted diarylethylene and tetra-
phenylethene derivative conjugated molecule (R-TPE) were facilely prepared via surfactant modification
with lecithin for the first time. The obtained R-TPE-LEC FONs were characterized by a series of tech-
niques including fluorescence and UV spectroscopy, Fourier transform infrared spectroscopy, and
transmission electron microscopy. Biocompatibility evaluation and cell uptake behavior of R-TPE-LEC
FONs were further investigated to explore their potential biomedical application. We demonstrated that
such red FONs exhibit anti-aggregation-caused quenching property, broad excitation wavelength, high
water dispersibility, uniform morphology (40e60 nm), and excellent biocompatibility, making them
promising for cell imaging application.
Received 29 January 2014
Received in revised form 29 March 2014
Accepted 3 April 2014
Available online 12 April 2014
Keywords:
Red fluorescent organic nanoparticles
Tetraphenylethene
Lecithin
Surfactant modification
Cell imaging
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
including fluorescent conjugated polymers, polydopamine nano-
particles, and aggregation-induced-emission (AIE) or aggregation
Optical bioimaging is one of the rapid growing areas in biological
and biomedical research.1 Fluorescence cellular probes with red/
near-infrared (R/NIR, >600 nm) emission are highly desirable for
biological applications due to their low optical absorption and
autofluorescence of biological media.2e5 Up to date, various fluo-
rescent materials including organic dyes,6 fluorescent proteins,7
fluorescent inorganic nanoparticles (NPs)8e13 have been used as
R/NIR probes. However, many of these fluorescent materials often
suffer from obvious disadvantages, such as water insolubility, pho-
tobleaching, and toxicity, which severely limited their practical
bioimaging applications. For example, most of organic dyes are in-
trinsic hydrophobic and instable in biological media.14 Usage of
fluorescent proteins is often limited due to the high cost, low molar
absorptivity, and low photobleaching thresholds.15 While inorganic
NPs or quantum dots are non-biodegradable and often toxic to living
organisms.16e19 Compared with the current used fluorescent biop-
robes, fluorescent organic nanoparticles (FONs) have recently re-
ceived more and more attentions due to flexible synthetic
approaches of these small organic molecules and their bio-
degradation potential.20e25 Because of these virtues, various FONs
induced emission enhancement (AIEE) materials based FONs have
been reported in recent years.26e37 Among them, the AIE or AIEE
based FONs have attracted great research interest because they
could overcome the notorious aggregation-caused quenching (ACQ)
effect of most organic dyes. Different AIE or AIEE units, such
as siloles,38 diphenylacrylonitrile,39e43 tetraphenylethene,44e48
triphenylethene,49e54 distyrylanthracene derivatives55e59 conju-
gated molecules have been examined for chemosensors and bio-
imaging applications.
However, direct using of AIE or AIEE based FONs for bioimaging
has been proven problematic due to the strong hydrophobicity of
most of AIE or AIEE units. The introduction of charges into AIE (or
AIEE) materials could improve their solubility in aqueous media,
but the electric charges of the highly concentrated ionized dyes
may affect intracellular physiology and sometimes even kill
the cells.52 Therefore, facile preparation of novel FONs, which
exhibited enhanced water dispersibility, excellent biocompatibility,
and photoluminescent properties simultaneously is of great
significance.
In our recent report, we have demonstrated that hydrophobic
AIE unit (An18) could be facilely transferred into hydrophilic FONs
via mixing of An18 and a commercial surfactant pluronic F127.30
The An18 based FONs showed good water solubility and excellent
biocompatibility are promising for cell imaging applications.
However, one of the disadvantages of these AIE-based FONs for
* Corresponding authors. Tel.: þ86 10 6279 2604; e-mail addresses: sychyzhang@
126.com (X. Zhang), weiyen@tsinghua.edu.cn (Y. Wei).
y
These authors contributed equally to this work.
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.04.010

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X. Zhang et al. / Tetrahedron 70 (2014) 3553e3559
bioimaging applications is that their maximum emission wave-
length is not long enough (filled into 530e550 nm), which will
inevitably interfered by the body optical absorption, light scatter-
ing, and autofluorescence of biological media.
2. Results and discussion
2.1. Characterization of R-TPE-LEC FONs
Herein, we report a facile method for the preparation of red
FONs based on a cyano-substituted diarylethylene and tetraphe-
nylethene derivative (R-TPE) (derivatized from 4,8-bis[5-(2-
ethylhexyl)-2-thienyl]benzodithiophene core structure with bis
[2-(tetraphenylethene)-acrylonitrile] side groups, its synthetic
route was shown in Scheme 1) by surfactant modification of leci-
thin (Scheme 2). R-TPE was firstly dissolved in THF, mixed with the
aqueous solution of surfactant lecithin by sonication. With the re-
moval of THF, the red FONs R-TPE-LEC formed. The hydrophobic
surface of R-TPE was changed to hydrophilic by surrounding with
lecithin, thus the R-TPE-LEC FONs exhibit good water dispersibility
and anti-ACQ property. To exploit the potential biomedical appli-
cations of such red FONs, their biocompatibility as well as cell
imaging applications were further investigated.
2.1.1. Fluorescence spectra. The fluorogen R-TPE was prepared fol-
lowing the synthetic route shown in Scheme 2 (see Experimental
procedure). Its structure was characterized and confirmed by
standard spectroscopic methods. The fluorescent dye R-TPE is hy-
drophobic and emits strong crimson fluorescence in solid state.
When R-TPE is dissolved in DMSO, it has strong orange fluores-
cence in dispersed state. After modified by surfactant lecithin, R-
TPE could be dispersed well in water by the hydrophobic in-
teraction of lecithin, meanwhile, the fluorogen is still in aggregated
state in the R-TPE-LEC nanoparticles, and the R-TPE-LEC composite
emitted strong red fluorescence (612 nm for the maximum emis-
sion wavelength) in water with almost the same fluorescent in-
tensity like that in a pure DMSO solution (Fig. 1(A)), which show
obvious anti-ACQ property. Meanwhile, fluorescence quantum
Scheme 1. Synthetic route of R-TPE.
Scheme 2. Schematic showing transformation of R-TPE from hydrophobic to hydrophilic red R-TPE-LEC FONs with lecithin and their use as cell imaging probes.

X. Zhang et al. / Tetrahedron 70 (2014) 3553e3559
3555
Fig. 1. (A) Fluorescence emission spectra of R-TPE in DMSO and surfactant modificated nanoparticles (R-TPE-LEC) dispersed in water. Inset: fluorescent image of R-TPE in DMSO
and R-TPE-LEC in water taken at 365 nm UV light; (B) fluorescence excitation spectrum of R-TPE-LEC FONs in water.
yields (FF) of R-TPE in DMSO and R-TPE-LEC FONs in water were
estimated using Rhodamine 6G in ethanol as standard (FF¼95%),
the absorbance of the solutions was kept around 0.05 to avoid in-
ternal filter effect. We demonstrated that the FF of R-TPE in DMSO
and R-TPE-LEC FONs in water were about 10.3% and 8.7%, re-
spectively. When the nanoparticles formed, the fluorescent wave-
length of the fluorescence dye emerged a 46 nm red-shifted
emission than that of R-TPE in DMSO. The observed spectral shift
is attributed presumably to the torsion-locking planarization of the
distorted solution geometry of R-TPE by aggregation, which nar-
transmission electron microscopy (TEM) images further confirmed
the formation of the conjugated FONs. Some small organic spheres
with diameters ranging from 40 to 60 nm, which derived from the
assembly of R-TPE and lecithin were observed (Fig. 2(C)). The for-
mation of such nanostructures is likely due to the strong in-
teractions between the alkyl chain of R-TPE and the hydrophobic
segments of lecithin, meanwhile, the hydrophilic segments of lec-
ithin were covered on the spheres to render them water dispersible.
The size distribution of R-TPE-LEC FONs in water was also de-
termined using a zeta Plus particle size analyzer. Results showed
that the size distribution of R-TPE-LEC FONs in pure water was
170.7ꢀ3.8 nm with narrow polydispersity index (0.214). As com-
pared with the size distribution in pure water, the size of the FONs
characterized by TEM was significantly smaller likely due to
shrinkage of micelle during the drying process. Taken together, due
to excellent water solubility, uniform morphology, biodegradable
potential, and unique photoluminescent (PL) properties (including
anti-ACQ, broad excitation wavelength and long emission wave-
length), we expect that R-TPE-LEC FONs can be potentially used for
bioimaging applications.
rows the optical bandgap by lengthening the effective
p-electron
conjugation.56 The appearance of a longer-wavelength emission
also suggests that close stacking of the planarized R-TPE molecules
is possibly accompanied with enhancing fluorescence J-type
p-
aggregation, often occurring in diarylacrylonitrile derivatives.23,27
As shown in Fig. 1(B), the fluorescence excitation spectrum of R-
TPE-LEC FONs in water exhibited broad excitation wavelength from
350 nm to 580 nm. It is noteworthy that when the excitation
wavelength exceeds 540 nm, the fluorescence excitation intensity
of such red FONs even significantly continuous increase with al-
most 3-fold as the wavelength reaches 580 nm. This excellent
feature is greatly benefited for their potential cell imaging
applications.
2.1.3. FT-IR spectra. Furthermore, successful formation of R-TPE-
LEC FONs was confirmed by FT-IR spectroscopy. As shown in Fig. 3,
the peak located at 1746 cmꢁ1 can be ascribed to the C]O
stretching vibration band of ester bond in lecithin, and another
peak located at 1073 cmꢁ1 can be assigned to the bending vibration
band of CeO in lecithin. As compared with R-TPE, lecithin, and R-
TPE-LEC FONs, these two characteristic peaks located at 1746 cmꢁ1
and 1073 cmꢁ1 can be clearly observed in R-TPE-LEC FONs, dem-
onstrating the successful modification of lecithin with R-TPE.
2.1.2. UV spectrum and TEM. As shown in the inset of Fig. 2(A), after
surface modification with surfactant lecithin, the surfactant mod-
ificated nanoparticles R-TPE-LEC were readily dispersed in water,
suggesting the successful formation of hydrophilic conjugated
FONs. The UV absorption spectrum of R-TPE-LEC FONs in water
showed that the maximum absorption wavelength located at
434 nm (Fig. 2(A)). The absorption spectrum of the FONs was not
consistent with the excitation spectrum, and this phenomenon
often could be seen in the FONs system, which might be due to the
light scattering effect.60e62 The UV absorption spectrum of R-TPE
dissolved in DMSO was shown in Fig. 2(B) for comparison. The
2.2. Biocompatibility of R-TPE-LEC FONs
To test the potential biomedical applications of R-TPE-LEC FONs,
their biocompatibility with A549 cells were subsequently
Fig. 2. (A) UV absorption spectrum of R-TPE-LEC FONs dispersed in water. Inset: visible image of R-TPE-LEC FONs in water using the logo of ‘Tsinghua University’ as background; (B)
UV absorption spectrum of R-TPE dissolved in DMSO; (C) TEM image of R-TPE-LEC FONs dispersed in water, scale bar¼100 nm.

3556
X. Zhang et al. / Tetrahedron 70 (2014) 3553e3559
120 m
g mLꢁ1, further confirming the excellent biocompatibility of
R-TPE-LEC FONs.
2.3. Cell imaging applications of R-TPE-LEC FONs
The cell imaging applications of R-TPE-LEC FONs were further
explored. The confocal laser scanning microscope (CLSM) images of
cells incubated with 100 m
g mLꢁ1 of R-TPE-LEC FONs for 3 h were
shown in Fig. 5. Bright field images (Fig. 5(A)) indicated that cells
still kept their normal morphology, further evidencing the good
biocompatibility of R-TPE-LEC FONs. When cells were excited with
543 nm laser, the cell uptake of R-TPE-LEC FONs can be clearly
distinguished due to they were stained by R-TPE-LEC FONs. Fur-
thermore, many dark areas, which were surrounded by R-TPE-LEC
FONs areas could also be found from CLSM images. These dark areas
are likely the location of cell nucleus. In the previous report,22
amphiphilic block copolymers were employed to disperse hydro-
phobic organic dyes to form polymeric micelles, thus FONs were
then carried out for intracellular imaging. Phagocytosis was be-
lieved as the possible mechanism for intracellular uptake by the
cell, while the micelles could remain intact in the cells. In our work,
phagocytosis may also be the possible mechanism for intracellular
uptake by A549 cells, as compared with the size of FONs and nu-
cleus pore (9ꢂ15 nm), these FONs are considered could not directly
enter the cell nucleus. Therefore, we could expect that the R-TPE-
LEC FONs should be promising candidates for various biomedical
applications due to the combined advantages for bio-applications,
such as unique PL properties, good water solubility, excellent bio-
compatibility, and biodegradable potential.
Fig. 3. FT-IR spectra of R-TPE, lecithin, and R-TPE-LEC FONs.
examined. Fig. 4(A)e(C) shows the microscopy images of cells
when they incubated with different concentrations of R-TPE-LEC
FONs for 24 h, and no significant cell morphology changes were
observed. It can be seen that cells still attached very well to cell
plate even the concentration of R-TPE-LEC FONs was up to
120
m
g mLꢁ1. The optical microscopy images implied the good
biocompatibility of R-TPE-LEC FONs. Cell viability of R-TPE-LEC
FONs was further determined to quantitatively evaluate the bio-
compatibility of R-TPE-LEC FONs.63 As shown in Fig. 4(D), no ob-
vious cell viability decrease was found when cells were incubated
3. Conclusions
with 40
values were about 90% even the concentrations increased to
m
g mLꢁ1 of R-TPE-LEC FONs for 8 and 24 h. The cell viability
In summary, a novel strategy for fabrication of red FONs (R-TPE-
LEC) was developed via mixing of a hydrophobic cyano-substituted
Fig. 4. Biocompatibility evaluation of R-TPE-LEC FONs. (AeC) Optical microscopy images of A549 cells incubated with different concentrations of R-TPE-LEC FONs for 24 h, (A)
control cells, (B) 40
g mLꢁ1, (C) 120 g mLꢁ1, (D) cell viability of R-TPE-LEC FONs with A549 cells for 8 and 24 h, respectively. Cell viability was determined by the WST assay.
m
m

X. Zhang et al. / Tetrahedron 70 (2014) 3553e3559
3557
Fig. 5. CLSM images of A549 cells incubated with 100
images. Scale bar¼20 m.
m
g mLꢁ1 of R-TPE-LEC FONs for 3 h. (A) Bright field, (B) fluorescent image, which were excited with 543 nm laser, (C) merge
m
diarylethylene and tetraphenylethene derivative conjugated mol-
ecule (R-TPE) and a commercial surfactant lecithin. Red FONs with
diameter from 40 to 60 nm could be facilely obtained with uniform
morphology and were subsequently utilized for cell imaging ap-
plications. Our results demonstrated that such red FONs show re-
markable PL properties (anti-ACQ properties and broad excitation
wavelength), excellent water solubility and biocompatibility, which
are promising for bioimaging applications.
potassium carbonate aqueous solution (10 mL). The mixture was
stirred at room temperature for 0.5 h under Ar gas followed adding
Pd(PPh3)4 (0.010 g, 8.70ꢂ10ꢁ3 mmol) and then heated to 90 ꢃC for
24 h. After that the mixture was poured into water and extracted
three times with ethyl acetate. The organic layer was dried over
anhydrous sodium sulfate. After removing the solvent under re-
duced pressure, the residue was chromatographed on a silica gel
column with petroleum ether/CH2Cl2 (3:1 by volume) as eluent to
give intermediate 1 (1.30 g, 68% yield). 1H NMR (400 MHz, CDCl3)
d
(ppm): 3.65 (s, 2H, eCH2e), 6.98e7.06 (m, 10H, phenyl-H),
4. Experimental procedure
7.07e7.15 (m, 9H, phenyl-H); HRMS calcd for C28H21N [MþH]þ:
372.1752, found 372.1759. Elemental analysis calcd for C28H21N: C,
90.53; H, 5.70; N, 3.77. Found: C, 90.37; H, 5.81; N, 3.69.
4.1. Materials and characterization
A solution of 2 (0.10 g, 0.16 mmol) and 1 (0.16 g, 0.43 mmol) in
ethanol (10 mL) was stirred at room temperature. Then terabutyl
ammonium hydroxide solution (0.8 M, five drops) was added and
the mixture was heated to reflux for 2 h precipitating a dark red
solid. The reaction mixture was cooled to room temperature and
filtered, washed with ethanol for several times obtaining a dark red
Thiophene, 3-(bromomethyl)heptane, thiophene-2-carboxylic
acid, thionyl chloride, dimethylamine, n-butyllithium, stannic
chloride, 1-bromo-1,2,2-triphenylethene, 4-(cyanomethyl)phe-
nylboronic acid, toluene, Aliquat 336, potassium carbonate, tetra-
butylammonium hydroxide (0.8 M in methanol) purchased from
Alfa Aesar were used as received. All other agents and solvents
were purchased from commercial sources and used directly
without further purification. Ultra-pure water was used in the
experiments.
solid R-TPE (0.17 g, yield 80%). 1H NMR (400 MHz, CDCl3)
d (ppm):
0.88e0.99 (m, 12H, eCH3), 1.30e1.48 (m, 16H, eCH2e), 1.63e1.75
(m, 2H, (methylene)3CeH), 2.86 (d, 4H, J¼6.8 Hz, aryleCH2e), 6.92
(d, 2H, J¼3.2 Hz, aryl-H), 6.97e7.20 (m, 34H, aryl-H), 7.34 (d, 2H,
J¼3.6 Hz, aryl-H), 7.39 (d, 4H, J¼8.4 Hz, aryl-H), 7.62 (s, 2H, aryl-H),
1H NMR and 13C NMR spectra were measured on a JEOL
400 MHz spectrometer, CDCl3 was used as solvent and tetrame-
thylsilane (TMS) as the internal standard. HRMS was obtained on
Shimadzu LCMS-IT-TOF high resolution mass spectrometry. Ele-
mental analysis was performed with an Elementar Vario EL ele-
mental analyzer. Fluorescence spectra were measured on a PE LS-
55 spectrometer with a slit width of 3 nm for both excitation and
emission. UV absorption spectra were recorded on an
UVeviseNIR PerkineElmer lambda750 spectrometer (Waltham,
MA, USA) using quartz cuvettes of 1 cm path length. Transmission
electron microscopy (TEM) images were recorded on a JEM-
1200EX microscope operated at 100 kV, the TEM specimens
were made by placing a drop of the nanoparticles suspension on
a carbon-coated copper grid. The size distribution of R-TPE-LEC
FONs in pure water was determined using a zeta Plus apparatus
(ZetaPlus, Brookhaven Instruments, Holtsville, NY). The FT-IR
8.02 (s, 2H, aryl-H); 13C NMR (100 MHz, CDCl3)
d (ppm): 146.83,
145.38, 143.47, 143.43, 143.29, 142.22, 140.26, 139.91, 139.68, 137.05,
135.77, 134.03, 132.20, 131.72, 131.46, 131.40, 131.38, 130.59, 128.01,
127.93, 127.79, 126.96, 126.82, 126.77, 125.94, 125.28, 124.94, 117.55,
110.96, 41.47, 34.36, 32.57, 28.99, 25.73, 23.14, 14.32, 10.97; HRMS
calcd for C92H80N2S4 [MþH]þ: 1341.5283, found 1341.5243. Ele-
mental analysis calcd for C92H80N2S4: C, 82.35; H, 6.01; N, 2.09; S,
9.56. Found: C, 82.59; H, 6.12; N, 2.01; S, 9.26.
4.3. Preparation of R-TPE-LEC FONs
The preparation of R-TPE-LEC FONs was carried out as follows.
Approximately 10 mg of synthesized dyes (R-TPE) was dissolved in
20 mL of THF and then added dropwise under sonication into the
solution of lecithin (50 mg) in 20 mL of H2O in a 100 mL vial. And
then the mixture was evaporated to completely remove the organic
agent (THF) on a rotary evaporator at 40 ꢃC. To remove the excess
lecithin, the R-TPE-LEC water dispersion was treated by repeated
centrifugal washing process for three times (centrifuged for 5 min
at a rotational speed of 10,000 r/min).
spectra were obtained in
a transmission mode on a Per-
kineElmer Spectrum 100 spectrometer (Waltham, MA, USA).
Typically, eight scans at a resolution of 1 cmꢁ1 were accumulated
to obtain one spectrum.
4.2. Synthesis of R-TPE
The intermediate 2 were prepared according to the literature
methods.31 The synthetic route of R-TPE is showed in Scheme 1.
1-Bromo-1,2,2-triphenylethene (1.74 g, 5.2 mmol) and 4-(cya-
nomethyl)phenylboronic acid (1.00 g, 6.2 mmol) were dissolved in
the mixture of toluene (40 mL), Aliquat 336 (10 drops), and 2 M
4.4. Cytotoxicity of R-TPE-LEC FONs
Cell morphology was observed to examine the effects of R-TPE-
LEC FONs to A549 cells. Briefly, cells were seeded in 6-well
microplates at a density of 1ꢂ105 cells mLꢁ1 in 2 mL of respective

3558
X. Zhang et al. / Tetrahedron 70 (2014) 3553e3559
9. Frangioni, J. V. Curr. Opin. Chem. Biol. 2003, 7, 626e634.
10. Chandra, S.; Das, P.; Bag, S.; Laha, D.; Pramanik, P. Nanoscale 2011, 3, 1533e1540.
11. Díez, I.; Ras, R. H. A. Nanoscale 2011, 3, 1963e1970.
12. Wu, X.; He, X.; Wang, K.; Xie, C.; Zhou, B.; Qing, Z. Nanoscale 2010, 2,
2244e2249.
media containing 10% FBS. After cell attachment, plates were
washed with PBS and the cells were treated with complete cell
culture medium, or different concentrations of fluoridated R-TPE-
LEC FONs prepared in 10% FBS containing media for 24 h. Then all
samples were washed with PBS three times to remove the unin-
ternalized FONs. The morphology of cells was observed using an
optical microscopy (Leica, Germany), the overall magnification was
ꢂ100.
13. Zhang, X.; Wang, S.; Zhu, C.; Liu, M.; Ji, Y.; Feng, L.; Tao, L.; Wei, Y. J. Colloid
Interface Sci. 2013, 397, 39e44.
14. Resch-Genger, U.; Grabolle, M.; Cavaliere-Jaricot, S.; Nitschke, R.; Nann, T. Nat.
Methods 2008, 5, 763e775.
15. Cai, Z.; Ye, Z.; Yang, X.; Chang, Y.; Wang, H.; Liu, Y.; Cao, A. Nanoscale 2011, 3,
1974e1976.
The cell viability of R-TPE-LEC FONs on A549 cells was evaluated
by cell counting kit-8 (CCK-8) assay. Briefly, cells were seeded in 96-
16. Bhirde, A.; Xie, J.; Swierczewska, M.; Chen, X. Nanoscale 2011, 3, 142e153.
17. Smith, A. M.; Duan, H.; Mohs, A. M.; Nie, S. Adv. Drug Delivery Rev. 2008, 60,
1226e1240.
18. Wang, X.; Xu, S.; Xu, W. Nanoscale 2011, 3, 4670e4675.
19. Zhang, X.; Zhang, X.; Wang, S.; Liu, M.; Zhang, Y.; Tao, L.; Wei, Y. ACS Appl. Mater.
Interfaces 2013, 5, 1943e1947.
well microplates at a density of 5ꢂ104 cells mLꢁ1 in 160
mL of re-
spective media containing 10% FBS. After 24 h of cell attachment,
the cells were incubated with 10, 20, 40, 80,120
FONs for 8 and 24 h. Then nanoparticles were removed and cells
were washed with PBS three times.10 L of CCK-8 dye and 100 L of
m
g mLꢁ1 R-TPE-LEC
20. Li, K.; Pan, J.; Feng, S. S.; Wu, A. W.; Pu, K. Y.; Liu, Y.; Liu, B. Adv. Funct. Mater.
2009, 19, 3535e3542.
m
m
21. Lin, H. H.; Su, S. Y.; Chang, C. C. Org. Biomol. Chem. 2009, 7, 2036e2039.
22. Wu, W.-C.; Chen, C.-Y.; Tian, Y.; Jang, S.-H.; Hong, Y.; Liu, Y.; Hu, R.; Tang, B. Z.;
Lee, Y.-T.; Chen, C.-T.; Chen, W.-C.; Jen, A. K. Y. Adv. Funct. Mater. 2010, 20,
1413e1423.
23. An, B. K.; Gihm, S. H.; Chung, J. W.; Park, C. R.; Kwon, S. K.; Park, S. Y. J. Am.
Chem. Soc. 2009, 131, 3950e3957.
24. Kumar, M.; George, S. J. Nanoscale 2011, 3, 2130e2133.
25. Zhang, X.; Chi, Z.; Yang, Z.; Chen, M.; Xu, B.; Zhou, L.; Wang, C.; Zhang, Y.; Liu, S.;
Xu, J. Opt. Mater. 2009, 32, 94e98.
26. Thomas, S. W. I.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339e1386.
27. Pu, K. Y.; Li, K.; Shi, J.; Liu, B. Chem. Mater. 2009, 21, 3816e3822.
28. Zhang, X.; Wang, S.; Xu, L.; Ji, Y.; Feng, L.; Tao, L.; Li, S.; Wei, Y. Nanoscale 2012, 4,
5581e5584.
DMEM cell culture medium were added to each well and incubated
for 2 h at 37 ꢃC. Plates were then analyzed with a microplate reader
(VictorIII, PerkineElmer). Measurements of formazan dye absor-
bance were carried out at 450 nm, with the reference wavelength at
620 nm. The values were proportional to the number of live cells.
The percent reduction of CCK-8 dye was compared to controls (cells
not exposure to R-TPE-LEC FONs), which represented 100% CCK-8
reduction. Three replicate wells were used per microplate, and
the experiment was repeated three times. Cell survival was
expressed as absorbance relative to that of untreated controls. Re-
sults are presented as meanꢀstandard deviation (SD).
29. Liu, J.; Ding, D.; Geng, J.; Liu, B. Polym. Chem. 2012, 3, 1567e1575.
30. Zhang, X.; Zhang, X.; Wang, S.; Liu, M.; Tao, L.; Wei, Y. Nanoscale 2013, 5,
147e150.
31. Zhang, X.; Zhang, X.; Yang, B.; Liu, M.; Liu, W.; Chen, Y.; Wei, Y. Polym. Chem.
2013, 4, 4317e4321.
32. Ibrahimova, V.; Ekiz, S.; Gezici, O.; Tuncel, D. Polym. Chem. 2012, 2, 2818e2824.
33. Zhang, X.; Zhang, X.; Yang, B.; Wang, S.; Liu, M.; Zhang, Y.; Tao, L.; Wei, Y. RSC
Adv. 2013, 3, 9633e9636.
34. Zhang, X.; Liu, M.; Yang, B.; Zhang, X.; Wei, Y. Colloids Surf., B 2013, 112, 81e86.
35. Zhang, X.; Liu, M.; Yang, B.; Zhang, X.; Chi, Z.; Liu, S.; Xu, J.; Wei, Y. Polym. Chem.
2013, 4, 5060e5064.
36. Chen, M.; Yin, M. Prog. Polym. Sci. 2014, 39, 365e395.
37. Zhang, X.; Zhang, X.; Yang, B.; Zhang, Y.; Wei, Y. ACS Appl. Mater. Interfaces 2014,
6, 3600e3606.
4.5. Confocal microscopic imaging of cells using R-TPE-LEC
FONs
_
€
A549 cells were cultured in Dulbecco’s modified eagle medium
(DMEM) supplemented with 10% heat-inactivated fetal bovine se-
rum (FBS), 2 mM glutamine,100 U mLꢁ1 penicillin, and 100 g mLꢁ1
m
of streptomycin. Cell culture was maintained at 37 ꢃC in a humidi-
fied condition of 95% air and 5% CO2 in culture medium. Culture
medium was changed every three days for maintaining the expo-
nential growth of the cells. On the day prior to treatment, cells were
seeded in a glass bottom dish with a density of 1ꢂ105 cells per dish.
On the day of treatment, the cells were incubated with R-TPE-LEC
38. Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.;
Liu, Y.; Zhu, D.; Tang, B. Z. Chem. Commun. 2001, 1740e1741.
39. An, B. K.; Kwon, S. K.; Jung, S. D.; Park, S. Y. J. Am. Chem. Soc. 2002, 124,
14410e14415.
40. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Liu, W.; Chen, Y.; Wei, Y. Polym.
Chem. 2014, 5, 689e693.
41. Zhang, X.; Zhang, X.; Yang, B.; Liu, M.; Liu, W.; Chen, Y.; Wei, Y. Polym. Chem.
2014, 5, 399e404.
42. Zhang, X.; Zhang, X.; Yang, B.; Liu, M.; Liu, W.; Chen, Y.; Wei, Y. Polym. Chem.
2014, 5, 356e360.
43. Zhang, X.; Ma, Z.; Liu, M.; Zhang, X.; Jia, X.; Wei, Y. Tetrahedron 2013, 69,
10552e10557.
FONs at a final concentration of 100 m
g mLꢁ1 for 3 h at 37 ꢃC. Af-
terward, the cells were washed three times with PBS to remove the
R-TPE-LEC FONs and then fixed with 4% paraformaldehyde for
10 min at room temperature. Cell images were taken with a Laser
Scanning Confocal Microscope (LCSM) Zesis 710 3-channel (Zesis,
Germany) with the excitation wavelengths of 543 nm.
44. Zhang, X.; Chi, Z.; Li, H.; Xu, B.; Li, X.; Zhou, W.; Liu, S.; Zhang, Y.; Xu, J. Chem.
dAsian J. 2011, 6, 808e811.
45. Zhang, X.; Chi, Z.; Li, H.; Xu, B.; Li, X.; Liu, S.; Zhang, Y.; Xu, J. J. Mater. Chem.
Acknowledgements
2011, 21, 1788e1796.
46. Zhang, X.; Chi, Z.; Zhang, Y.; Liu, S.; Xu, J. J. Mater. Chem.
3376e3390.
C 2013, 1,
This research was supported by the National Natural Science
Foundation of China (Nos. 21134004, 21201108), and the National
973 Project (No. 2011CB935700), China Postdoctoral Science
Foundation (2012M520243, 2013T60100).
47. Li, X.; Zhang, X.; Chi, Z.; Chao, X.; Zhou, X.; Zhang, Y.; Liu, S.; Xu, J. Anal. Methods
2012, 4, 3338e3343.
48. Zhou, X.; Li, H.; Chi, Z.; Zhang, X.; Zhang, J.; Xu, B.; Zhang, Y.; Liu, S.; Xu, J. New J.
Chem. 2012, 36, 685e693.
49. Zhang, X.; Yang, Z.; Chi, Z.; Chen, M.; Xu, B.; Wang, C.; Liu, S.; Zhang, Y.; Xu, J. J.
Mater. Chem. 2009, 20, 292e298.
50. Zhang, X.; Chi, Z.; Xu, B.; Li, H.; Yang, Z.; Li, X.; Liu, S.; Zhang, Y.; Xu, J. Dyes Pigm.
References and notes
2011, 89, 56e62.
51. Zhang, X.; Chi, Z.; Xu, B.; Li, H.; Zhou, W.; Li, X.; Zhang, Y.; Liu, S.; Xu, J. J. Flu-
oresc. 2011, 21, 133e140.
52. Yu, Y.; Feng, C.; Hong, Y.; Liu, J.; Chen, S.; Ng, K. M.; Luo, K. Q.; Tang, B. Z. Adv.
Mater. 2011, 23, 3298e3302.
53. Chen, C.; Liao, J.-Y.; Chi, Z.; Xu, B.; Zhang, X.; Kuang, D.-B.; Zhang, Y.; Liu, S.; Xu,
J. RSC Adv. 2012, 2, 7788e7797.
54. Chen, C.; Liao, J.-Y.; Chi, Z.; Xu, B.; Zhang, X.; Kuang, D.-B.; Zhang, Y.; Liu, S.; Xu,
J. J. Mater. Chem. 2012, 22, 8994e9005.
55. Zhang, X.; Chi, Z.; Xu, B.; Chen, C.; Zhou, X.; Zhang, Y.; Liu, S.; Xu, J. J. Mater.
Chem. 2012, 22, 18505e18513.
1. Weissleder, R.; Pittet, M. J. Nature 2008, 452, 580e589.
2. Lee, S.; Cha, E. J.; Park, K.; Lee, S. Y.; Hong, J. K.; Sun, I. C.; Kim, S. Y.; Choi, K.;
Kwon, I. C.; Kim, K. Angew. Chem., Int. Ed. 2008, 120, 2846e2849.
3. Li, L.; Daou, T. J.; Texier, I.; Kim Chi, T. T.; Liem, N. Q.; Reiss, P. Chem. Mater. 2009,
21, 2422e2429.
4. Wang, F.; Banerjee, D.; Liu, Y.; Chen, X.; Liu, X. Analyst 2010, 135, 1839e1854.
5. Zhang, X.; Hui, J.; Yang, B.; Yang, Y.; Fan, D.; Liu, M.; Tao, L.; Wei, Y. Polym. Chem.
2013, 4, 4120e4125.
6. Lin, H. H.; Chan, Y. C.; Chen, J. W.; Chang, C. C. J. Mater. Chem. 2011, 21,
3170e3177.
7. Shu, X.; Royant, A.; Lin, M. Z.; Aguilera, T. A.; Lev-Ram, V.; Steinbach, P. A.; Tsien,
R. Y. Science 2009, 324, 804e807.
8. Michalet, X.; Pinaud, F.; Bentolila, L.; Tsay, J.; Doose, S.; Li, J.; Sundaresan, G.;
Wu, A.; Gambhir, S.; Weiss, S. Science 2005, 307, 538e544.
56. Zhang, X.; Chi, Z.; Zhang, J.; Li, H.; Xu, B.; Li, X.; Liu, S.; Zhang, Y.; Xu, J. J. Phys.
Chem. B 2011, 115, 7606e7611.
57. Zhang, X.; Chi, Z.; Zhou, X.; Liu, S.; Zhang, Y.; Xu, J. J. Phys. Chem. C 2012, 116,
23629e23638.

X. Zhang et al. / Tetrahedron 70 (2014) 3553e3559
3559
58. Chi, Z.; Zhang, X.; Xu, B.; Zhou, X.; Ma, C.; Zhang, Y.; Liu, S.; Xu, J. Chem. Soc. Rev.
2012, 41, 3878e3896.
61. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Chi, Z.; Liu, S.; Xu, J.; Wei, Y. Polym.
Chem. 2014, 5, 318e322.
59. Zhang, X.; Ma, Z.; Yang, Y.; Zhang, X.; Chi, Z.; Liu, S.; Xu, J.; Jia, X.; Wei, Y. Tet-
62. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Chi, Z.; Liu, S.; Xu, J.; Wei, Y. Polym.
rahedron 2014, 70, 924e929.
Chem. 2014, 5, 683e688.
60. Zhang, X.; Zhang, X.; Yang, B.; Hui, J.; Liu, M.; Chi, Z.; Liu, S.; Xu, J.; Wei, Y. J.
Mater. Chem. C 2014, 2, 816e820.
63. Zhang, X.; Hu, W.; Li, J.; Tao, L.; Wei, Y. Toxicol. Res. 2012, 1, 62e68.