Morphology-Tunable Fluorescent Nanoparticles: Synthesis, Photophysical Properties and Two-Photon Cell Imaging

Article abstract of DOI:10.1002/cjoc.201500248

A novel water-soluble fluorescent amphiphile based on amino polyethylene glycol (PEG-NH₂) substituted oligo(p-phenyleneethynylene) (OPE) was designed and synthesized successfully. Taking anion OPE amphiphile as a comparison, the photophysical f

Full text of DOI:10.1002/cjoc.201500248

FULL PAPER
DOI: 10.1002/cjoc.201500248
Morphology-Tunable Fluorescent Nanoparticles: Synthesis,
Photophysical Properties and Two-Photon Cell Imaging
Xiaoming Hu,†,a Chao Yin,†,a Wenbo Hu,a Zhen Yang,a Jie Li,a Xiang Li,a Xiaomei Lu,a,b
Hui Zhao,a Yufu Tang,a Quli Fan,*,a and Wei Huang*,a,b
a Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials, Jiangsu
National Synergetic Innovation Center for Advanced Materials, Nanjing University of Posts & Telecommunications,
Nanjing, Jiangsu 210023, China
b Key Laboratory of Flexible Electronics & Institute of Advanced Materials, Jiangsu National Synergetic Innovation
Center for Advanced Materials, Nanjing Tech University, Nanjing, Jiangsu 211816, China
A novel water-soluble fluorescent amphiphile based on amino polyethylene glycol (PEG-NH2) substituted oligo-
(p-phenyleneethynylene) (OPE) was designed and synthesized successfully. Taking anion OPE amphiphile as a
comparison, the photophysical features were investigated through ultraviolet absorption (UV) and photolumines-
cence (PL) analyses. Due to the hydrophobic-hydrophilic property of the OPE conjugated molecule, self-assembled
nanoparticles with the size ranging from 19.6 to 93.5 nm along with the change of morphology from “grain” to
“strawberry” were conveniently prepared via adjusting concentrations of OPE aqueous solution. Interestingly, after
aging for a period of time, homogeneous hollow nanospheres were spontaneously constructed with a diameter of
about 200 nm. Cytotoxicity test and cellular uptake behavior of the nanoparticles were further investigated to evalu-
ate their potential biomedical applications. Subsequently, the promising applications of two-photon cell imaging
were explored using human pancreatic cancer cells (PANC-1 cells), which indicated that the nanoparticles were
mainly located within the cell cytoplasm.
Keywords water-soluble fluorescent nanoparticles, oligo(p-phenyleneethynylene) amphiphile, hollow nano-
spheres, two-photon cell imaging
Introduction
acid))[13] or amphiphilic molecules (such as phosphol-
ipids and DSPE-PEG)[14] through noncovalent bond
self-assembly. The second approach is transferring oil-
soluble conjugated polymers into water dispersed con-
jugated polymer dots (CPdots) which show relatively
steady fuorescence and ultrahigh brightness through a
reprecipitation method.[15] As for the third approach,
amphiphilic conjugated polymers are directly dispersed
in a selective solvent to form various well-defined su-
pramolecular architectures, such as micelles and vesi-
cles,[16] which provide wide application in chemistry,
biology, and materials science.[17] Compared with the
first two approaches, this is an exactly fast and facile
method to fabricate water-soluble conjugated polymer
nanoparticles. For example, homogeneous nanoparticles
were obtained through self-assembly using amphiphilic
hyperbranched conjugated polyelectrolytes possessing
hydrophobic PF internal cores and hydrophilic PEG
external shells and their live cell imaging was further
investigated by Liu and co-workers.[18,19]
Recent years have witnessed the rapid development
of water-soluble fluorescent π-conjugated oligomers/
polymers and their various bioapplications, including
highly efficient chemosensors or biosensors,[1-4] optical
imaging in vitro and in vivo,[5] gene delivery,[6] drug
screening, anticancer therapy[5] and so on. A new useful
characteristic of water-soluble conjugated oligomers or
polymers is their potential ability to form nanoparticles
(NPs), which present great potential in biological imag-
ing and drug delivery[7] applications for their superior
features, such as facile chemical synthesis,[8] excellent
photostability,[9] high brightness,[10] tunable photolumi-
nescent properties,[11] and versatile surface modifica-
tion.[12] For this, three approaches are usually adopted to
prepare conjugated polymer nanoparticles. In the first
approach, water-soluble fluorescent nanoparticles were
constructed from hydrophobic conjugated polymers/
oligomers and water-soluble polymers (such as poly-
(ethylene glycol), poly(galactose) and poly(acrylic
However, applications in biological system of these
*
E-mail: iamqlfan@njupt.edu.cn, wei-huang@njtech.edu.cn; Tel.: 0086-025-85866396; Fax: 0086-025-85866533; † Xiaoming Hu and Chao Yin are the
co-first authors.
Received March 27, 2015; accepted May 27, 2015; published online July 22, 2015.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201500248 or from the author.
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Morphology-Tunable Fluorescent Nanoparticles
robust nanomaterials are usually hampered by the losing
tunability of their morphology and size due to polymers’
amorphous character and large molecular weight.[20]
Thus, lots of efforts have been made to solve these
problems such as changing solvent environments,[11]
adjusting molecular structures and shapes,[21-23] as well
as the control of relative proportion of hydrophilic and
hydrophobic parts.[24] Yet, all the methods abovemen-
tioned are not very facile and controllable in practice,
which still makes it complex to tune the morphology
and size of nanostructures precisely. Therefore, the de-
velopment of novel water-soluble conjugated polymer
nanoparticles with morphological tunability, good bio-
compatibility, and excellent photoelectric features for
bioimaging and biosensing is highly desired.
determined on a Shimadzu GC-MS-QP 2010 Plus mass
spectrometer. Matrix assistant laser desorption/ioni-
zation time-of-flight mass spectrometry (MALDI-TOF-
MASS) was measured on Bruker autoflex under the
reflector mode for data acquisition. Gel permeation
chromatography (GPC) results were obtained with Shi-
madzu LC-VP system with polystyrenes as the standard
and high purity of THF as the eluent. The UV-Vis ab-
sorption and PL emission spectra were recorded on
Shimadzu UV-3600 and RF-5301PC spectrometers, re-
spectively. FT-IR spectra were recorded with a Shimad-
zu IR Affinity-1 spectrometer by dispersing samples in
KBr. Morphological characterization of nanoparticles
was conducted using fluorescence microscopy (Olym-
pus IX71) and low resolution transmission electron mi-
croscope (TEM) (JEOL JEM-2100 transmission elec-
tron microscope operating at an acceleration voltage of
100.0 kV). Dynamic light scattering (DLS) and Zeta
potential results were obtained with Brookhaven system.
Two-photon fluorescence microscopy (2PFM) images in
PANC-1 cells were collected on a modified Olympus
Fluoview (FV1000) microscope system coupled to a
Coherent MIRA Ti: sapphire laser, which was used for
two-photon absorption measurements.
Here, a novel amphiphilic oligomer consisting of
oligo(p-phenyleneethynylene) (OPE) and amino poly-
ethylene glycol (PEG-NH2) was designed and synthe-
sized, and the photophysical properties, controllable
self-assembly behaviors and two-photon cell imaging
were further investigated. The employment of OPE and
PEG stems from high quantum yields and photostability
of OPE chromophore,[25] and prominent biocompatibil-
ity of PEG chains. Due to the hydrophobic-hydrophilic
property of the OPE conjugated molecule, it can spon-
taneously aggregate to form nano-clusters ranging from
19.6 to 93.5 nm along with the morphological conver-
sion from “grain” to “strawberry” just by controlling
concentrations of OPE aqueous solution (from 0.0642 to
0.2168 mg/mL). More interestingly, after the solution
was aged for one week at room temperature, homoge-
neous hollow nanospheres with a diameter of about 200
nm were obtained easily. All these provided a facile and
convenient access to tuning the morphology and size of
water-soluble fluorescent nanoparticles. The morphol-
ogy and size of nanoparticles were characterized by
transmission electron microscopy (TEM) and dynamic
Cytotoxicity test
The cytotoxicity of the amphiphilic oligomer was
evaluated by determining the viability of human pan-
creatic cancer cells (PANC-1) after incubation with
DMEM containing our amphiphilic material at a variety
of concentrations from 0.0001 to 0.5 mg/mL. Cell vi-
ability testing was carried out via the reduction of the
MTT reagent. PANC-1 cells were seeded at a density of
104 cells per well for 24 h before the medium was re-
placed with one containing amphiphilic oligomer at dif-
ferent concentrations. Then the cells were incubated at
37 ℃ with 5% CO2 for 24 h. After the designated time
intervals, the wells were washed twice with PBS buffer
and freshly prepared MTT (10 μL, 5 mg/mL in PBS)
solution in a culture medium was added into each well.
The MTT medium solution was carefully removed after
3 h incubation in the incubator. After that, DMSO (150
μL) was then added into each well and the wells were
gently shaken for 10－20 min at room temperature to
dissolve all the precipitate formed. The optical absorb-
ance was then measured at 490 nm on a microplate
reader (Tecan GENios). Cell viability was expressed by
the ratio of the absorbance of the cells incubated with
the amphiphilic oligomer solution. Each result is an av-
erage of data from six wells and the standard deviation
was also calculated. 100% cell viability was determined
using untreated cells.
1
light scattering (DLS), respectively. Besides, H NMR
and Zeta potential confirmed the existence of amino
groups on particle surface, which can be available for
the subsequent modification. Furthermore, cytotoxicity
test and cellular uptake behavior were investigated to
confirm excellent biocompatibility of the OPE nanopar-
ticles. Based on these, two-photon excited live cell im-
aging was conducted with confocal laser scanning mi-
croscopy, which presented strong fluorescence from
cellular cytoplasm with considerably high contrast and
spatial resolution. Due to the unique hollow structure of
obtained nanospheres, they may become carrier of drugs
and the template for fabricating multifunctional nano-
probes for biological application.
Experimental
Live cell imaging
General methods
PANC-1 cells were first seeded into confocal micro-
scope dishes at 5×105/mL in complete DMEM medium
and cultured for 24 h at 37 ℃. The medium was then
replaced by DMEM medium with OPE nanoparticles
All NMR spectra were recorded on a Bruker Ultra
Shield Plus 400 MHz NMR at 22 ℃ and tetramethyl-
silane was used as internal reference. GC-MS was
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solution (0.2168 mg/mL). After incubation for 24 h at
37 ℃, the cells were rinsed three times with PBS buffer.
2PFM images in PANC-1 cells were collected on a
modified Olympus Fluoview (FV1000) microscope
system.
and diamino PEG. The product was then obtained as
loose-yellow powder by freeze drying for 2 d (0.11 g).
1H NMR (CDCl3) δ: 8.16, 7.49, 6.87 (the chemical shift
of benzene ring), 5.30 (NH2), 3.97 (Ph-O-CH2), 3.64
(PEG chain), 1.26 (alkyl chain). GPC analysis: Mw＝
7389, Mn＝6622, and polydispersity distribution index
(PDI) is 1.12. To synthesize anion OPE amphiphile (5),
0.15 g 3 was concentrated in a methyl amine solution
(25%－30 wt% water). The residual methyl amine was
then removed via rotary evaporation and the remaining
salt was obtained by freeze drying.
Synthesis
The OPE amphiphile (4) was synthesized by the
route shown in Scheme 1. First, 1 was prepared accord-
ing to the previous report[14] with a yield of 77.25% [1H
NMR (CDCl3) δ: 7.35 (d, J＝9.2 Hz, 2H), 6.78 (d, J＝
9.2 Hz, 2H), 3.91 (t, J＝6.8 Hz, 2H), 1.73－1.80 (m,
2H), 1.26 (s, 18H), 0.88 (t, J＝7.2 Hz, 3H). GC-MS
(m/z): 340 (M＋)]. Then, 2 was synthesized based on the
Pd-catalyzed Sonogashira reaction by “one-pot” ap-
proach: 4.68 g (13.73 mmol) of 1, 1.73 g (13.73 mmol)
of 1,4-diethynylbenzene, 3.75 g (13.73 mmol) of di-
methyl 5-bromoisophthalate, 0.795 g (0.687 mmol) of
Pd(PPh3)4 and 0.132 g (0.693 mmol) of CuI were
poured into a 250 mL round-bottom flask with magnetic
stirring bar and reflux condenser under dark condition.
Then 120 mL diisopropylamine was injected into the
round-bottom flask under nitrogen protection. And the
mixture was stirred at 85 ℃ for 24 h. The product was
then purified as a light yellow solid (2.16 g, yield
27.22%) through silica gel eluting with PE/DCM＝3/1
[1H NMR (CDCl3) δ: 8.63 (s, 1H), 8.37 (s, 2H), 7.45－
7.51 (m, 6H), 6.89 (d, J＝8.8 Hz, 2H), 3.95－3.99 (m,
8H), 1.76－1.82 (m, 2H), 1.26 (s, 18H), 0.88 (t, J＝7.2
Hz, 3H). GC-MS (m/z): 578 (M＋)]. Subsequently, all of
the products (2) obtained above were dissolved in 30
mL THF, and a hydrolysis reaction under 65 ℃ for 5 h
with KOH (3.0 g) and tetrabutyl ammonium bromide
(0.8 g) was conducted. After the residual THF was re-
moved by rotary evaporation, excess hydrochloric acid
aqueous solution was poured into the system until white
floccus precipitation emerged. Then 3 was obtained as a
yellow-green solid (1.76 g, yield 85.64%) after the liq-
uid was filtered away and the precipitation was dried in
a vacuum chamber at 40 ℃ for 12 h [1H NMR
(dimethyl sulfoxide-d6) δ: 8.44 (s, 1H), 8.25 (s, 2H),
7.47－7.68 (m, 6H), 6.95 (d, J＝8.8 Hz, 2H), 3.98 (t,
J＝6.8 Hz, 2H), 1.66－1.72 (m, 2H), 1.13－1.31 (m,
18H), 0.83 (t, J＝7.2 Hz, 3H); 13C NMR (dimethyl
sulfoxide-d6) δ: 166.3, 159.7, 136.1, 133.6, 132.5, 131.9,
130.5, 123.9, 115.4, 70.2, 68.1, 56.5, 31.8, 29.5, 29.2,
25.9, 22.6, 14.4. MALDI-TOF, m/z: calcd 550.3; found
550.3 (M＋ )]. Finally, the OPE amphiphile (4) was
synthesized according to the following route: 3 (20 mg),
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hy-
drochloride (EDC•HCl) (60 mg), N-hydroxysuccinimide
(NHS) (36 mg), and poly(ethylene glycol) bis(3-amino-
propyl) terminated (diamino PEG) (Mn≈1500) (0.3272
g) were dissolved in 20 mL of DMF. And the resulting
mixture was stirred at room temperature for 74 h.
Thereafter, the resulting mixture was purified by dialy-
sis against water for 3 d with a dialysis bag (MWCO＝
3500 Da) to remove the excess EDC•HCl, NHS, DMF
Self-assembly of 4 in water
Appropriate amount of 4 was dissolved in deionized
water to prepare three bottles of solution with different
concentrations: 0.0642, 0.1297, and 0.2168 mg/mL. Af-
ter ultrasonic treatment for 10 min, the mixtures were
stirred vigorously for another 24 h. The light-cloudy
solution obtained was then filtered through 0.22 μm size
filter membrane for further investigation. Hollow nano-
spheres were prepared through aging the solution
(0.2168 mg/mL) for one week at room temperature.
Results and Discussion
Synthesis and structure characterization
The preparation process of OPE amphiphile (4) is
shown in Scheme 1. Particularly, the preparation of 2
was accomplished via Sonogashira reaction in the mix-
ture of diisopropylamine solution in the presence of
Pd(PPh3)4/CuI catalyst at 85 ℃ for 24 h. This “one-
pot” reaction to synthesize asymmetric OPE conjugated
molecular provided a rapid and convenient approach of
asymmetric synthesis with a relatively high yield. Actu-
ally, more efforts have been made to prepare 2 through
“step-by-step” reaction, however, a quite low yield
(about 10%) was obtained owing to the complex reac-
tion process. Subsequently, 3 was prepared through hy-
drolysis reaction and then acid treatment. After the acti-
vation of the carboxylic groups on 3 with EDC•HCl and
NHS, diamino PEG was linked to 3 via the amidation
reaction. It is worth noting that large excess of diamino
PEG (ndiamino PEG∶n3＝6∶1) was used to make it pos-
sible that only one amino group of diamino PEG can be
employed rather than formation of large crosslinking.
However, GPC analysis indicated the formation of
crosslinking with about three OPE backbone units. Ad-
ditionally, anion OPE amphiphile (5) was easily ob-
tained under alkali treatment. The correct structure of all
the products was confirmed by NMR, GC-MS,
MALDI-TOF and GPC analysis.
The existence of free amino groups could be deter-
mined by 1H NMR spectra (Figure 1). 4 exhibited a dis-
tinct peak at δ 5.3 when the solvent was CDCl3 (Figure
1c). Interestingly, this specific peak disappeared after
adding a drop of CH3OD into the solution (Figure 1b),
which can be attributed to the replacement of hydrogen
atoms corresponding to NH2 by deuterium atom of
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Morphology-Tunable Fluorescent Nanoparticles
Scheme 1 The preparation process of oligo(p-phenyleneethynylene) amphiphiles (4 and 5)
(a)
(b)
COOCH3
COOCH3
Br
1-Bromododecane
Br
OH
Br
O
K2CO3, Aceton
Pd(PPh3)4:CuI
(i-Pr)2NH
1
(a) THF
KOH/TBAB/H2O
COOCH3
COOCH3
O
(b) HCl, H2O
2
COOH
COOH
Diamino PEG 1500
EDC/NHS, DMF
O
3
H2N-GEP-HNOC
CONH-PEG-HNOC
CONH-PEG-
HNOC
CONH-PEG-NH2
O
O
O
4
H
N
+
+
H
H
H
-
COO
CH3
Methyl amine
O
H
H
N
3
-
COO
CH3
5
CH3OD for the activity of amino group. Furthermore,
when 4 was reconstituted into D2O, specific peaks of
OPE and alkyl chains disappeared at their proton spectra
while only the specific peaks of PEG were shown (Fig-
ure 1a). These results indicated that OPE and alkyl
chain were condensed in the core and PEG chains acted
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as the outstretched shell, which strongly confirmed the
formation of nanoparticles while the OPE amphiphile
was reconstituted in water.[26]
5 with the same concentration of 0.1 mg/mL in different
solvents were shown in Figure 3. In tetrahydrofuran
(THF) solution, 4 exhibited a strong absorption peak
occurring at 330 nm with a vibronic band shoulder at
351 nm and a tail absorption band at 388 nm (Figure 3d).
Similar situation has occured to 4 in aqueous solution
(Figure 3d), which also exhibited a strong absorption
peak at 330 nm. However, an enhanced tail absorption
band at 388 nm was observed in comparison with that in
THF. Furthermore, the concentration dependent absorp-
tion spectra of 4 in water were detected in Figure S2,
which indicated a gradually increasing trend of the tail
absorption band with the concentration ranging from
0.05 to 0.25 mg/mL. Correspondingly, the photolumi-
nescence peak maximum of 4 in water appeared at 454
nm with the concentration of 0.1 mg/mL, which was
beyond 47 nm red shift from the emission in THF (Fig-
ure 3b). All these optical properties revealed the typical
J-type aggregation[28,14] of 4 in aqueous solution. Figure
3c demonstrated the absorption band of 5 in water and
THF, and the most striking feature is that the vibronic
band shoulder at 351 nm of 5 in THF is much stronger
than that of 4 (Figure 3d). Besides, a slight bathochro-
mic shift happened from 4 to 5 with about 10 nm in
emission spectra in THF (Figure 3a and Figure 3b), and
all of these indicated that the conjugated segments of 5
may have a strong tendency to form interchain aggre-
gates[29] for its weaker solubility than 4 in THF. It can be
hypothesized that carboxylic acid ions (COO−) en-
hanced the polarity and inorganic nature of 5, which can
not be dissolved very well in THF.
Figure 1 1H NMR of 4 in different solvents (D2O, CDCl3 and
CDCl3＋CH3OD).
The covalent linkage of diamino PEG to 3 was fur-
ther confirmed by FT-IR spectrometry (Figure 2). The
absorption peak at 1725 cm−1 in Figure 2b is attributed
to the carboxylic carbonyl groups of 3 (C＝O stretching)
which is absent in Figure 2c. Yet a new peak at ca. 1657
cm−1 has arised that can be assigned to the amide car-
bonyl groups. This provided reasonable evidence that
diamino PEG was successfully linked to 3 by amidation
reaction. Different from the previous literature report,[27]
the carboxyl groups of 3 have disappeared completely in
our product for the superfluous diamino PEG we used.
Besides, the FT-IR spectrum of 4 showed a strong band
at 1112 cm−1 attributed to the stretching vibration of
ether bond (C－O－C) of PEG chains, which further
confirmed the successful modification of diamino PEG
to 3.
Interestingly, almost the opposite results were ob-
tained in aqueous solution. In comparison with 4, the
absorption (Figure 3c) and emission (Figure 3a) peaks
of 5 presented obvious blue-shift. This possibly resulted
from the mutual repulsion among the negative charges
(COO−) leading to a more twisted main chain conforma-
tion, and hence a decreased effective conjugation
length.[30] The abovementioned results also supported
that adjusting relative proportion of hydrophilic-hydro-
phobic parts would play effective roles to control pho-
tophysical properties as well as the aggregation states of
amphiphilic conjugated molecules.
c 4
a Diamino PEG1500
b 3
2
1
a
b
Morphological and superficial properties
Like a host of amphiphilic molecules, 4 contains dis-
tinct hydrophobic and hydrophilic segments, which
would undergo self-assembly in aqueous solution to
form diversified structures. To intuitively investigate the
morphology of OPE nanoparticles, fluorescence mi-
croscopy and TEM measurements were carried out.
Fluorescence micrograph of 4 in water was shown in
Figure 4, which presented that the nanoparticles which
emitted vivid blue light were dispersed well in aqueous
solution, and Brownian motion of the nanoparticles can
also be seen clearly. To explore the representative re-
sults of self-assembly, three bottles of aqueous solution
with different concentrations (0.0642, 0.1297, and
1725
c
0
1657
-1
1112
3500 3000 2500 2000 1500 1000 500
Wavenumber/cm-1
Figure 2 FT-IR of 4, 3 and diamino PEG 1500.
Photophysical properties
The UV-vis and photoluminescence spectra of 4 and
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Morphology-Tunable Fluorescent Nanoparticles
454 nm
417 nm
407 nm
b
451 nm
a
1.0
0.5
0.0
1.0
0.5
0.0
4 in water
4 in THF
5 in water
5 in THF
300
400
500
Wavelength/nm
600
300
400
500
600
Wavelength/nm
330 nm
1.2
1.0
0.8
0.6
0.4
0.2
0.0
d
1.2
294 nm
5 in water
331 nm
351 nm
c
4 in water
4 in THF
5 in THF
1.0
0.8
0.6
0.4
0.2
0.0
351 nm
388 nm
300
400
Wavelength/nm
500
600
300
400
Wavelength/nm
500
600
Figure 3 UV-vis (c and d) and photoluminescence spectra (a and b) of 4 and 5 in different solvents (0.1 mg/mL). The excited wave-
length of emission spectra was fixed at 340 nm.
Figure 4 Representative fluorescence micrograph of the nano-
particles formed by self-assembly of 4 in water.
Figure 5 TEM images and hydrodynamic diameter distribution
of the nanoparticles in different morphologies (a, 0.0642 mg/mL;
b, 0.1297 mg/mL; c, 0.2168 mg/mL). Inset: photographs of the
nanoparticles aqueous solution (left) under ambient light and
(right) under a black lamp (λ＝365 nm).
0.2168 mg/mL) were employed. It is worth noting that
the data we chose were based on the photoluminescence
spectra of 4 with various concentrations (Figure S1),
which indicated that continuous increase of fluorescence
intensity of 4 in water has been observed with the con-
centration varying from 0.01 mg/mL to 0.1297 mg/mL,
yet a decrease with the concentration changing from
0.1297 mg/mL to 0.2845 mg/mL. Figure 5 showed the
TEM images and hydrodynamic diameter distribution of
the nanoparticles with different morphologies. In Figure
5a (c＝0.0642 mg/mL), the nanoparticles with a clear
grain-like structure were observed with a diameter of
(10±1.2) nm, which was a little smaller than DLS re-
sult (mean diameter: 19.6 nm). This is probably attrib-
uted to the stretch of PEG chains in aqueous solution
while shrink after dried on a copper grid coated with a
carbon film. With the concentration increased to 0.2168
mg/mL (Figure 5c), strawberry-like nanoparticles ap-
peared with a diameter of (93±2.2) nm which was in
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893

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accordance with the DLS result (mean diameter: 93.5
nm). Naturally, both the two kinds of aggregation mor-
phology can be observed in a middle-concentration,
such as 0.1297 mg/mL (Figure 5b). Interestingly,
through aging the solution (0.2168 mg/mL) for 3 d, ir-
regular nanostructure formed spontaneously (Figure 6a),
which further evolved into uniform hollow nanospheres
(Figure 6b) after one week. Based on these, we hy-
pothesized that monodisperse single nanoparticles
(similar with nanomicelles) can be formed by self-as-
sembly process at a low concentration of 0.0642 mg/mL
firstly, and subsequently, with the increase of amphi-
philic OPE concentration from 0.1297 to 0.2168 mg/mL,
large aggregates would be constructed from single
nanoparticles,[14] and their sizes sharply increased to ca.
93.5 nm (DLS result). However, these aggregates are
not stable enough, which would crimp (see arrows indi-
cated region in Figure 6a) to form more stable nanos-
tructures through aging the solution for 3 d. And finally,
hollow nanospheres were spontaneously constructed
(Figure 6b) after one week, which may result from high
thermodynamics and kinetics stability of these hollow
nanostructures. The possible self-assembly process of
the nanoparticles was shown in Scheme 2. Therefore,
we provided a facile access to tuning morphology and
size of nanostructures without complicated procedures,
such as changing solvent environments and adjusting
molecular structures. Compared with 4, no uniform
morphology was observed when 5 was dispersed in wa-
ter for its low water-solubility.
Scheme 2 Schematic of the possible formation process of the
hollow nanospheres
a
60
b
40
20
0
-20
-40
-60
3
4
5
6
7
8
9
10 11 12
pH
Figure 7 pH-dependent zeta potential curves of hollow nano-
spheres (a) and the irregular aggregates formed by 5 (b).
ited positive potentials when pH values were adjusted
below 9.18, resulting from the protonation of the amino
groups. Fortunately, the nearly neutral surface of nano-
spheres in physiological environments (pH≈7.4) would
lead to a longer blood circulation due to their weak
electrostatic interaction with proteins in comparison to
the charged nanoparticles.[27]
Cellular viability and imaging
To evaluate the potential biomedical applications of
OPE nanoparticles, their biocompatibility to PANC-1
cells was also assessed by an MTT assay. Figure 8
summarized the viability of PANC-1 cells cultured with
the OPE amphiphile (4) aqueous solution at different
concentrations ranging from 0.0001 mg/mL to 0.5
mg/mL for 24 h. It is clearly observed that all the cells
presented high viability (＞90%) after treating with the
OPE nanoparticles with different concentrations. The
influences of OPE nanoparticles to PANC-1 cells were
also examined by investigating the cellular morphology
at different time nodes (0 h and 24 h) using optical mi-
croscopy (Figure S3). As shown in Figure S3, cells still
adhered to the plate very well and no obvious cell mor-
phology change was observed after they were incubated
with OPE nanoparticles (0.5 mg/mL) for 24 h. All of
these proved the low cytotoxicity of OPE nanoparticles,
which provided large possibility for cellular imaging or
Figure 6 TEM images of the OPE nanoparticles aged for 3 d (a)
and one week (b) at room temperature (c＝0.2168 mg/mL).
As we know, surface charge of nanoparticles plays a
vital role in biological system such as preventing rapid
nonspecific uptake in vivo[27] and binding biomole-
cules.[31] Therefore, the surface charge properties of the
hollow nanospheres (Figure 7a) and the irregular ag-
gregates formed by 5 (Figure 7b) were studied respec-
tively by measuring the zeta potentials as a function of
pH values. As shown in Figure 7, b exhibited negative
potentials in the pH range from 4.00 to 9.18, and the
zeta potential became more negative with the increase
of pH values, which can be attributed to the gradual
ionization of carboxylic groups on the surface of
nanoparticles. By contrast, hollow nanospheres exhib-
894
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© 2015 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Morphology-Tunable Fluorescent Nanoparticles
other biological applications.
nm. As seen in Figure 10(b), a strong fluorescence from
the cellular cytoplasm was observed and it was clear
that the 2PFM images provided considerably high con-
trast and spatial resolution. All of these demonstrated
that our material can be employed as an efficient fluo-
rescent probe for two-photon cell imaging, which pro-
vided opportunities for long-term imaging with reduced
photodamage in comparison with UV-excited imag-
ing.[32]
100
80
60
40
20
0
0.5
0.0001
0.001
0.1
0.01
-1
.
Concentration/(mg mL )
Figure 8 In vitro cell viability graph of 4 with different concen-
trations by MTT assay.
Figure 10 Two-photon images of PANC-1 cells treated with the
hollow nanospheres solution of 4 (0.2168 mg/mL). Two-photon
microscopy images were obtained with laser excitation at 720
nm.
Figure 9 showed the Z-scan measurement for 4 dis-
solved in methanol (0.1 mg/mL), which indicated a
two-photon absorption (2PA) feature. The 2PA cross-
section, δ, can be estimated according to the following
formula:
Conclusions
δ
hνβ/N
＝
In summary, a novel fluorescent water-soluble am-
phiphilic oligomer based on PEG and OPE was firstly
reported. In aqueous solution, this material can sponta-
neously self-assemble into nanoparticles with the size
ranging from 19.6 to 93.5 nm along with the morphol-
ogy change from “grain” to “strawberry” just through
adjusting concentration of the solution. After the solu-
tion was aged for one week, homogeneous hollow nano-
spheres were spontaneously constructed. These results
provided a facile approach to tuning morphology and
size of self-assembled nanostructures. The morphology
and size of nanoparticles were characterized by TEM,
fluorescence microscope and DLS, and their photo-
physical properties were also investigated. Good water-
solubility, low cytotoxicity and exactly high 2PA cross-
section made our material an excellent candidate as a
two-photon sensitizer and a new class of probe for bio-
imaging using 2PFM. Besides, this kind of nanospheres
may act as carriers of drug or inorganic material such as
magnetic nanoparticles (MNPs) and gold nanoparticles
(GNPs) to form multifunctional nano-probes. A more
comprehensive study of employing the nanospheres as
templates to fabricate multifunctional nanomaterials is
of great significance. More excitingly, abundant amino
groups on particle surface make it available for subse-
quent modifications, which would endow the OPE
nanoparticles more powerful functions in specific prob-
ing, multimodal imaging and multimodal therapy in
biomedical field.
N is the number of molecules per cm3 and hν is the
photon energy.
1.01
1.00
0.99
0.98
4
Methanol
0.97
0.96
-30
-20
-10
0
10
20
30
Z/mm
Figure 9 Z-scan signature for 4-methanol solution (0.1 mg/mL,
scatters) performed with 190 fs pulses at 720 nm. The solid lines
represent theoretical fittings with the parameters obtained in the
experiments.
Given that 4 exhibited exactly high 2PA cross-sec-
tion (δ was calculated to 1181 GM in methanol with
laser excitation at 720 nm), we set out to demonstrate
that our material could be used for intracellular imaging.
Two-photon excited live cell imaging using 4 was in-
vestigated with confocal laser scanning microscopy
coupled to a Coherent MIRA Ti: sapphire laser. After 24
h of incubation with the hollow nanospheres solution of
4 (0.2168 mg/mL), the cells were washed three times in
PBS buffer. The excitation wavelength was fixed at 720
Acknowledgement
This work was financially supported by the National
Basic Research Program of China (Nos. 2012CB933301
Chin. J. Chem. 2015, 33, 888—896
© 2015 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
895

Hu et al.
FULL PAPER
[14] Yin, C.; Song, W. L.; Jiang, R. C.; Lu, X. M.; Hu, W. B.; Shen, Q.
M.; Li, X.; Li, J.; Fan, Q. L.; Huang, W. Polym. Chem. 2014, 5,
5598.
and 2012CB723402), the Ministry of Education of Chi-
na (No. IRT1148), the National Natural Science Foun-
dation of China (Nos. 61378081, 21222404, 51173080
and 21104033) and the Innovation Program for Ordi-
nary Higher Education Graduate of Jiangsu Province of
China (No. KYLX_0796), Synergetic Innovation Center
for Organic Electronics and Information Displays and
the Natural Science Foundation of Jiangsu Province of
China (Nos. BZ2010043, NY211003, BM2012010).
[15] Wu, C.; Chiu, D. T. Angew. Chem., Int. Ed. 2013, 52, 3086.
[16] Ringsdorf, H.; Schlarb, B.; Venzmer, J. Angew. Chem., Int. Ed. 1988,
27, 113.
[17] Wang, C.; Wang, Z.; Zhang, X. Acc. Chem. Res. 2012, 45, 608.
[18] Pu, K. Y.; Li, K.; Shi, J. B.; Liu, B. Chem. Mater. 2009, 21, 3816.
[19] Li, K.; Pu, K. Y.; Cai, L. P.; Liu, B. Chem. Mater. 2011, 23, 2113.
[20] Zhang, G. W.; Lu, X. M.; Wang, Y. Y.; Huang, Y. Q.; Fan, Q. L.;
Huang, W. Polym. Int. 2011, 60, 45.
[21] Ryu, J.; Lee, M. J. Am. Chem. Soc. 2005, 127, 14170.
[22] Moon, K.; Kim, H.; Lee, E.; Lee, M. Angew. Chem., Int. Ed. 2007,
46, 6807.
References
[1] Thomas, S. W.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107,
1339.
[2] Fan, L. J.; Zhang, Y.; Murphy, C. B.; Angell, S. E.; Parker, M. F. L.;
Flynn, B. R.; Jones, W. E. Coord. Chem. Rev. 2009, 253, 410.
[3] Li, K.; Liu, B. Polym. Chem. 2010, 1, 252.
[4] Sun, P. F.; Fan, Q. L.; Liu, L. L.; Deng, W. X.; Lu, X. M.; Huang, W.
Acta Polym. Sin. 2014, 12, 1629.
[5] Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Chem. Rev. 2012, 112,
4687.
[6] Jiang, R. C.; Lu, X. M.; Yang, M. H.; Deng, W. X.; Fan, Q. L.;
Huang, W. Biomacromolecules 2013, 14, 3643.
[7] Liong, M.; Angelos, S.; Choi, E.; Patel, K.; Stoddart, J. F.; Zink, J. I.
J. Mater. Chem. 2009, 19, 6251.
[23] Moon, K.; Lee, E.; Lee, M. Chem. Commun. 2008, 3061.
[24] Photos, P. J.; Bacakova, L.; Discher, B.; Bates, F. S.; Discher, D. E. J.
Control. Release 2003, 90, 323.
[25] Clark, A. P. Z.; Shen, K. F.; Rubin, Y. F.; Tolbert, S. H. Nano Lett.
2005, 5, 1647.
[26] Kim, D. G.; Jeong, Y. I.; Choi, C.; Roh, S. H.; Kang, S. K.; Jang, M.
K.; Nah, J. W. Int. J. Pharm. 2006, 319, 130.
[27] Liu, D.; Wu, W.; Ling, J.; Wen, S.; Gu, N.; Zhang, X. Adv. Funct.
Mater. 2011, 21, 1498.
[28] Ajayaghosh, A.; Varghese, R.; Praveen, V. K.; Mahesh, S. Angew.
Chem., Int. Ed. 2006, 45, 3261.
[29] Tan, C.; Pinto, M. R.; Schanze, K. S. Chem. Commun. 2002, 446.
[30] Fan, Q. L.; Zhou, Y.; Lu, X. M.; Hou, X. Y.; Huang, W. Macromole-
cules 2005, 38, 2927.
[8] Wu, C.; Szymanski, C.; McNeill, J. Langmuir 2006, 22, 2956.
[9] Pecher, J.; Mecking, S. Chem. Rev. 2010, 110, 6260.
[10] Dey, S.; Sreenivasan, K. J. Mater. Chem. B 2015, 3, 824.
[11] Park, S. J.; Kang, S. G.; Fryd, M.; Saven, J. G.; Park, S. J. J. Am.
Chem. Soc. 2010, 132, 9931.
[31] Hong, R.; Fischer, N. O.; Emrick, T.; Rotello, V. M. Chem. Mater.
2005, 17, 4617.
[12] Yuan, Y.; Liu, J.; Liu, B. Angew. Chem. 2014, 126, 7291.
[13] Lu, X. M.; Fan, Q. L.; Zhang, G. W.; Pu, K. Y.; Huang, W. Sci. Chi-
na Chem. 2010, 53, 1122.
[32] Yong, K. T.; Qian, J.; Roy, I.; Lee, H. H.; Bergey, E. J.; Tramposch,
K. M.; He, S.; Swihart, M. T.; Maitra, A.; Prasad, P. N. Nano Lett.
2007, 7, 761.
(Zhao, X.)
896
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© 2015 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2015, 33, 888—896