Multifunctional polymer nanoparticles: Ultra bright near-infrared fluorescence and strong magnetization and their biological applications

Article abstract of DOI:10.1039/c6ra07520g

Multifunctional polymer nanoparticles with great promise for biomedical applications have attracted many attentions. Using an aggregation-enhanced emission (AEE) molecule DPPBPA and magnetic Fe₃O₄ as the core, and biocompatible polymer Pluronic F-127 as the encapsulation matrix, multifunctional polymer NPs Fe₃O₄/DPPBPA@F-127 were fabricated by self-assembly procedures. These NPs have bright near-infrared fluorescence λ_ex at 654 nm with a high fluorescence quantum yield of 18.3%, and strong magnetism, and superparamagnetism. With good monodispersity and biocompatibility, the NPs not only can show effective MRI ability, but also can stain in cytoplasm with a strong near-infrared fluorescent signal, as well as little toxicity to living cells, which show a very good prospect in the field of biological applications.

Full text of DOI:10.1039/c6ra07520g

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Multifuctional Polymer Nanoparitcles: Ultra Bright Near-infrared
Fluorescence and Strong Magnetization and Their Biological
Applications
Received 00th January 20xx,
Accepted 00th January 20xx
Lulin Yan, †a Yan Zhang , †a Guang Ji,a Lian Ma,a Jinlong Chen,a Bin Xu,*a Wenjing Tiana
DOI: 10.1039/x0xx00000x
www.rsc.org/
Multifunctional polymer nanoparticles with great promise for biomedical applications have attracted many attentions.
Using an aggregation-enhanced emission (AEE) molecule DPPBPA and magnetic Fe3O4 as the core, and biocompatible
polymer Pluronic F-127 as the encapsulation matrix, the multifuctional polymer NPs Fe3O4/DPPBPA@F-127 were
fabricated by self-assembly procedures. These NPs have bright near-infrared fluorescence λex at 654 nm with high
fluorescence quantum yield of 18.3%, and strong magnetism, superparamagnetism. With good monodispersity and
biocompatibility, the NPs not only can show effective MRI ability, but also can stain in cytoplasm with a strong near-
infrared fluorescent signal, as well as little toxicity to living cells, which show a very good prospect in the field of biological
applications.
required14. Recently, opposite to the ACQ effect, a novel class
of organic fluorophores15-19 with aggregation-induced emission
(AIE) characteristic has been developed. These fluorophores
Introduction
Multifunctional nanoparticles (NPs) with unique magnetic,
optical, catalytic, and electrical properties have hold great
promise for biomedical applications such as drug delivery
carriers 1, 2, diagnostic analysis3, magnetic resonance imaging4,
bioseperation5, and fluorescent labeling6, and attracted many
attentions in the past decades.7-11 The combination of
magnetic and fluorescent capabilities enables the design of
almost have no emission in their dilute solutions, but have
high fluorescence quantum yields in the aggregate state when
the intramolecular rotations are restricted15. These AIE
fluorophores opened a new gate to fabricate high brightness
NPs by encapsulating large amount of dyes.
Due to the superparamagnetism, nontoxicity, and
biodegradability, magnetic iron oxide NPs have become
famous magnetic materials.20 However, as we know, when the
fluorescent material cones in contact with iron oxide, most of
the fluorescence is quenched.21, 22 For the AIE characters, the
NPs fabricated by combining AIE dyes and iron oxide may
overcome this problem. In order to get both high fluorescence
quantum yields and strong magnetism in the aggregate state,
Tang and co-workers succeeded in synthesizing silica NPs with
both efficient fluorescence and strong magnetization by using
AIE dyes and iron oxide firstly.12 And then, some other AIE
fluorescent magnetic NPs have been developed.23-26 However,
most of these dyes are blue or green emitters15, 27-29, whose
fluorescence is still quenched to some extent by the magnetic
materials, expecially iron oxide, and the intensity of
magnetism is always weak, which are not ideal for imaging
applications. Because of the low photodamage, deep tissue
penetration and minimal auto-fluorescence from biological
substrates, the fluorophores with intense emission in the far
red/near-infrared (FR/NIR) (650–900 nm) region have
attracted much interest30-32. Up to now, the investigations
about AIE dyes applied in near-infrared emission bioimaging
are just emerged, 32-36 and the reports on NPs with both bright
new
multifunctional
NPs
probes
in
multimodal
optical/magnetic resonance imaging.9-11 Because of the optical
transparence and biological compatibility, polymer have
become favorable encapsulation matrixes. Furthermore, the
encapsulation can protect dye molecules from the external
perturbation and prevent the magnetic nanoclusters from
agglomerating into large chunks.12
To prepare fluorescent magnetic NPs for imaging, the
luminescent QDs and organic dyes were both chosen in the
past studies. However, QDs are less chemically stable,
potentially toxic, and show fluorescence intermittence13, and
most commercially organic dyes typically exhibit rapid
photobleaching and a low fluorescence quantum yield in the
NPs due to the notorious aggregation-caused quenching (ACQ)
effect. Therefore, the luminescent materials with high photo-
stability, low optical background and bright light emission are
a.State Key Laboratory of Supramolecular Structure and Materials, Jilin University,
Changchun, 130012, P. R. China. E-mail: xubin@jlu.eu.cn
† These authors contributed equally to this work and should be considered co-first
authors.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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FR/NIR fluorescence and strong magnetism are quite limited. green line). A transmission electron microscopy (TEM) image
26
showed a mean size of OA-Fe3O4 particles of 10 nm in
Here, we present a simple strategy to fabricate multifunctional diameter (Figure 2A), which indicates that these particles are
polymer NPs Fe3O4/DPPBPA@F-127, by using oleic acid coated monodisperse. The magnetic NPs were dispersed in THF at 10
magnetic Fe3O4 (OA-Fe3O4) and an AEE molecule (2Z, 2'Z)-3, 3'- mg/mL for further study.
(2,
5-di
(piperidin-1-yl)-1,
4-phenylene)
bis
(2-
phenylacrylonitrile) (DPPBPA) as the core, and biocompatible
polymer Pluronic F-127 as the encapsulation matrix. During the
nanoparticle formation, OA-Fe3O4 and DPPBPA tended to
entangled with the hydrophobic domains of Pluronic F-127 to
afford the hydrophobic interiors, while the hydrophilic
domains of Pluronic F-127 extended into the aqueous phase,
which makes the NPs stable in the aqueous suspension. With
very little fluorescence quenching and magnetic intensity
reducing, these NPs possess bright near-infrared fluorescence
(λex = 654 nm, Фf = 18.3%), strong magnetism (Ms = 35.66
emu/g) and superparamagnetism (Mr = 0.3186 emu/g, Hci =
6.377 G), which show that the goals to prepare
Scheme 1. Schematic illustration of chemical formation of Fe3O4/DPPBPA@F127
nanostructureed
materials
with
high
near-infrared
fluorescence efficency and magnetic susceptibility have been
achieved. And these NPs are expected to facilitate their
biological applicataions.
Results and Discussion
Synthesis of DPPBPA and oleic acid coated Fe3O4 (OA- Fe3O4)
The synthetic route to DPPBPA is shown in Scheme S1
(2Z,2'Z)-3,3'-(2,5-dibromo- 1,4-phenylene)bis(2-
phenylacrylonitrile) ( was synthesized by coupling 2,5-
.
2
)
Dibromobenzene -1,4-dicarbaldehyde (1) and benzyl cyanide
through Knoevenagel reaction37. Under typical Suzuki reaction
conditions, the target molecule was derived from connect
38
piperidine to the 2,5-positions of compound
2
. All
compounds were characterized by standard spectroscopic
methods, from which satisfactory analysis data corresponding
to their molecular structures were obtained. The molecular
structure and purity were confirmed by 1HNMR and MS (Figure
S1 and S2 in the Supporting Information), which all gave
satisfactory spectroscopic data. The final product gives a [M +
H]+ peak at m/z 498 (calcd: 498.66) in the GC-MS spectra for
DPPBPA, confirming the formation of the expected adducts.
After the synthesis of AEE dye DPPBPA, we then worked on the
preparation of magnetic particles. Among various magnetic
materials, the Fe3O4 particles become a good candidate, since
its biocompatibility and wide biological applications such as
magnetic resonance imaging, enzyme and protein
immobilization. The Fe3O4 particles were prepared by a
coprecipitation method with oleic acid as stabilizer39. Ferric
chloride hexahydrate and ferrous chloride tetrahydrate was
dissolved in deionized (DI) water with the radio of 2:1.
Potassium oleate and ammonium hydroxide was added into
the solution at 80 oC. The OA-Fe3O4 magnetic NPs were
centrifuged by supercentrifuge10. The oleic acid coated iron
oxide retained the characteristic X-ray diffraction pattern of
Fe3O4 at 2θ of 30.2, 35.5, 43.2, 53.3, 57.1, and 62.8 as listed in
ASTM XRD standard card (19-0629) (as shown in Figure S3,
Figure 1. Photographs of water solutions of Fe3O4/DPPBPA@F127 taken under A–C)
normal room lighting and B–D) UV illumination in the absence (A and B) and presence
(C and D) of external magnetic field from a bar magnet.
Nanoparticle preparation and Characterization
As shown in Scheme 1, the multifunctional polymer NPs
Fe3O4/DPPBPA@F-127 were fabricated by a self-assembly
procedure, where magnetic OA-Fe3O4 and DPPBPA served as
the core, and biocompatible polymer Pluronic F-127 served as
the encapsulation matrix40. Pluronic F127 is a copolymer
consisting poly (ethylene oxide)–poly (propylene oxide)–poly
(ethylene oxide) blocks, PEO100-PPO65–PEO100. The exterior
PEO corona provides an antifouling character to prevent
aggregation, protein adsorption, and recognition by the
reticulo-endothelial system (RES) and the hydrophobic PPO
core can be adapted to encapsulate the fluorophores. The self-
assembling characteristics of F127 have been extensively
explored in the form of micelles. OA-Fe3O4, DPPBPA and
Pluronic F-127 were dispersed in THF by a vigorous bath
sonicator. The solution mixture was quickly added into DI
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water in a vigorous bath sonicator. The THF was removed by Photophysical Properties
nitrogen stripping. The solution was concentrated by
The images in Figure
1
show that the suspension of
continuous nitrogen on a 90 ºC hotplate followed by filtration
Fe3O4/DPPBPA@F-127 in water exhibit good dispersion in the
solutions and can be attracted by a bar magnet. The intense
near-infrared light was observed in the suspension solution of
Fe3O4/DPPBPA@F-127 with and without magnet upon the UV
illumination12. It indicates that both DPPBPA and OA-Fe3O4
were suceessfully encapsulated into the NPs. The fluorescence
through a 0.2 micron filter41. During the formation procedure
of the NPs, the OA-Fe3O4 and DPPBPA tended to entangle with
the hydrophobic domains of Pluronic F-127 and afford the
hydrophobic interiors, while the hydrophilic domains of
Pluronic F-127 extend into the aqueous phase of the NPs.
quantum yield
(Фf) of Fe3O4/DPPBPA@F-127 NPs is
surprisingly as high as 18.3%. Compared with our previous
report, the fluorescence quantum yield of DPPBPA@F-127 NPs
without Fe3O4 encapsulated is 20% (Table S2).42 This means
that there is alomost no quenching of fluorescence when the
Fe3O4 co-encapsulated together with DPPBPA.
Figure 2. TEM image of OA-Fe3O4(A) and Fe3O4/DPPBPA@F127(B).
In order to optimize the size and morphology of the NPs, many
parameters should be taken into account. As the previous
studies, the sizes of the polymer NPs are affected by the
amount of polymer added. Under the condition shown in
Table S1, the NPs are formed with four different polymer
amount (mass of polymer: 6 mg, 8 mg, 10 mg, 12 mg). The
number average hydrodynamic diameters of the nanoparticle
are around 90-105 nm, and the NPs possess the uniform size
and monodisperse confirmed by dynamic light scattering (DLS)
, which show a narrow peak in the size distribution diagram, as
shown in Figure S4. Notably, when the addition amount of
polymer increase to 10 mg, the encapsulated NPs show the
narrowest peak and maximum yield. Therefore, we choose the
addition amount of polymer at 10 mg to further fabricate the
multifunctional polymer NPs. Zeta potential analyses reveal
that the Fe3O4/DPPBPA@F-127 NPs possess appreciable
surface charges and hence good colloidal stability, which
shows a negative charge of around −12.5 mV due to the
protonation of oxygen atom in the Pluronic F-127. These
negative charges on the surface will stabilize the NPs in
Figure 3. Normalized PL spectra of DPPBPA and NPs.
To get indepth understanding of emission behavior of the
Fe3O4/DPPBPA@F-127 NPs, we check the effect of both
DPPBPA and OA-Fe3O4 loading amounts (Table
1). With
enlarging the loading amount of DPPBPA from 0.5 mg to 1 mg,
the fluorescence quantum yield can increase. On the contrary,
the fluorescence quantum yield shows obvious decrease when
adding more OA-Fe3O4 particles. Although the combination of
luminescent dye and magnetic particles often quench the
fluorescence, the high quantum yield of these NPs can be
achieved, even that a low dye loading was used for the
nanoparticle fabrication and relative high loading of OA-Fe3O4
in the polymer NPs. It is well known that the luminescent dyes
with AIE or AEE active have the enhancement emission in their
aggregated states, which originate from the restriction of
intramolecular rotations (RIR). The properties of enhancement
emission is mainly affected by the aggregate state and quite
different from the traditional luminescence process, such as
fluorescence resonance energy transfer (FRET) 43or
photoinduced electron transfer (PET)44,
45
, which often
aqueous medium by electrostatic repulsion
(Figure S5).
accompanied by the electronic process in the excited state of
luminescent dye. Therefore, we infer that the extraordinary
emission properties of these NPs are probably caused by less
interaction between the excited state of the DPPBPA and
magnetic Fe3O4.
According to the TEM, the average diameter of the NPs is
about 65 nm. This size is smaller than that of NPs obtained by
DLS analysis due to the shrinkage of the outer hydrophilic
chains of Fe3O4/DPPBPA@F-127, which often occur under the
high vacuum in the TEM chamber.
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Table 1. The fluorescent quantum yield(Фf) and spinlattice relaxation time T1 with different content of DPPBPA and Fe3O4
DPPBPA
0.5mg
1mg
OA-Fe3O4
1mg
F127
THF
5mL
5mL
5mL
5mL
H2O
Фf
1/T1(S-1
)
1
2
3
4
10mg
10mg
10mg
10mg
15mL
15mL
15mL
15mL
16.4%
18.3%
7%
1mg
2mg
0.24
0.30
0.5mg
1mg
2mg
14%
nearly no change in the spectrum, after put on shelves for
several weeks without protection from light and air. The
leaching issue is also researched. There was little change in the
spectrum after continuous ultrasound in a long time (Figure
S7), which suggest that only few dyes leak out from the
nanoparticles.
Magnetism
Owing to nanoscopic magnetic Fe3O4 contained into
Fe3O4/DPPBPA@F-127 NPs, they are expected to be
magnetically susceptible. Figure
4 shows the plots of
magnetization versus applied magnetic field at 300 K for OA-
Fe3O4 and Fe3O4/DPPBPA@F-127. With the increasing of the
magnetic field strength (Maximum filed=18000G), the
magnetization of Fe3O4/DPPBPA@F127 swiftly increases and
ultimately reaches a saturation magnetization (Ms) of 35.66
emu/g. There is no hysteresis and both remanence
(Mr=0.3186emu/g) and coercivity (Hci=6.377G) are nearly zero,
which indicates that the NPs are superparamagnetic46. It
should be noted that the saturation magnetization often
reduce when the iron oxide was cladded in some matrix.
Although the Ms value of Fe3O4/DPPBPA@F127 is a little bit
lower than that of OA- Fe3O4 (Ms= 55.629 emu/g), the
magnetization of Fe3O4/DPPBPA@F127 is already superior to
those NPs reported previously.
Figure 4. Plots of magnetization versus applied magnetic field at 300 K for OA-
Fe3O4 and Fe3O4/DPPBPA@F127.
Figure 5. MRI results of A) water and B) Fe3O4/DPPBPA@F127
DPPBPA is a typical AEE active luminogen, which show weak
near-infrared emission peaked at 674 nm in its THF solution,
and strong emission located at 654 nm in the solid state with
high quantum yield (Фf = 78%). The emission spectra of
Fe3O4/DPPBPA@F-127 and DPPBPA@F-127 NPs are almost
same and with the peak located at 654 nm under the same
measurement conditions, which are similar with the emission
spectrum in solid state (Figure 3). The fluorescence quantum
yield of Fe3O4/DPPBPA@F-127 shows slight decrease
compared with that of DPPBPA@F-127. In addition, time
resolved fluorescence spectra of Fe3O4/DPPBPA@F-127 and
DPPBPA@F-127 NPs shown in Figure S6 reveals the similar
fluorescence lifetime τFL of both systems, 8.25 ns for
Fe3O4/DPPBPA@F-127 and 8.63 ns for DPPBPA@F-127,
respectively. Since Φf equals the product of τFL and radiative
deactivation rate (kr) , kr and non-radiative deactivation rate (
knr ) can be approximately estimated as listed in Table S2. It
shows obviouly that the radiative deactivation process of
exicted state have not be affected when the magnetic OA-
Fe3O4 particles added, which is in agreement with the
observation of high solid state fluorescence quantum yield.
Furthermore, the flourescence is so stable that there was
Figure 6. Confocal laser scanning microscopy images of MCF-7 cells after
incubation with Fe3O4/DPPBPA@F127 for 6h at 37 ℃. A: bright field image; B:
fluorescence image; C: overlay of A and B. AEE NPs Concentration: 50ppm. Scale
bar for all images =30μm.
Next we explore the magnetic resonance imaging (MRI) ability
of the NPs, due to the excellent magnetic characters of
Fe3O4/DPPBPA@F127. Normally, with the same imaging
parameters, the higher concentration of the contrast agent is
the larger change of water relaxation in tissue. The spinlattice
relaxation time T1 of Fe3O4/DPPBPA@F127 NPs with different
concentration of Fe3O4 loading in water was performed. From
Table 1, we can know that 1/T1 increases with increasing the
concentration of Fe3O4 from 1 mg to 2 mg. Figure 5 shows the
compartion of the T1 contrast MRI image of water without and
with the presence of Fe3O4/DPPBPA@F127 NPs carried out in
vitro MRI experiments. It can be clearly observed that the
different MRI contrast between map A and B, where the
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contrast for map A, which is made with pure water, is fairly that they are good superparamagnets with high
poor, whereas that for map B, which contain the magnetization, which show effective MRI ability. The
Fe3O4/DPPBPA@F127 NPs, is much better. The contrast with Fe3O4/DPPBPA@F-127 NPs show less cytotoxicity in aqueous
the presence of Fe3O4/DPPBPA@F127 is significantly solution, suggesting NPs are biocompatible nanocarriers. Due
enhanced, suggesting the effective MRI ability.
to its high brightness, low cytotoxicity and excellent stability,
the obtained NPs have been successfully utilized in in vivo
fluorescence imaging with high fluorescence contrast. We
anticipate that this strategy of multifunctional NPs will inspire
the development of a novel biological nanomaterials with high
efficient fluorescence and strong magnetic susceptibility.
Intracellular Imaging
The cytotoxicity of Fe3O4/DPPBPA@F-127 in the MCF-7 cells
was studied by using 3-(4, 5-dimetylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) assay. Figure S8 shows the
cell viability after incubation with the NPs suspension at
concentrations of 0, 5, 10, 20, 40, 60, 80, 100 ppm for 24 h,
respectively. It was observed that cell viabilities for all the
fluorogen concentrations within the tested periods of time
Experimental Section
Materials
were
more
than
95%,
indicating
that
the
Fe3O4/DPPBPA@F-127 NPs had low cytotoxicity or good
biocompatibility. The low cytotoxicity makes the NPs
promising for bioimaging applications and superior to QDs,
which are well-known for their concentration-dependent
cytotoxicity.
All reagents and starting materials are commercially available
and were used without further purification, unless otherwise
noted. 1, 4-dibromo-2, 5-dimethylbnzene was purchased from
Aladdin (Shanghai, China). Benzyl cyanide was purchased from
J&K Scientific Ltd (Beijing China). Pluronic F-127 and MTT were
purchased from Sigma–Aldrich (St. Louis, USA). Deionized
water (18.2 MΩ·cm resistivity) from a Milli-Q water system
was used throughout the experiments before being used as
solvents.
The in vitro cellular imaging of Fe3O4/DPPBPA@F-127 was
performed by using confocal laser scanning microscopy (CLSM)
with a 405 nm laser excitation and the fluorescent signals
were collected at 620nm – 680nm to monitor the cell uptake
behaviour to the NPs. Previous studies have showed that the
FSNPs could selectively stain the cytoplasmic regions of the
living cells. Phagocytosis may be the possible mechanism for
intracellular uptake of the NPs. The NPs were enclosed by the
cell membrane to form small vesicles, which were then
internalized by the cells. The NPs were further processed in
endosomes and lysosomes and are eventually released into
the cytoplasm. As shown in Figure 6, after incubating with 50
ppm of Fe3O4/DPPBPA@F-127 for 6 h at 37 °C in the culture
medium, an intense red fluorescence was observed in the
cellular cytoplasms, which suggested the intracellular uptake
of the NPs. These results manifested that the
Fe3O4/DPPBPA@F-127 NPs were effective FR/NIR fluorescent
bioprobes for cellular imaging with a high fluorescence
contrast.
Instrumentations
Mass spectra were recorded on an Agilent 1100 LC-MS system.
1H NMR spectra were recorded on Bruker AVANVE 500 MHz
spectrometer or Varian 300 MHz with tetramethylsilane as the
internal standard. UV-vis absorption spectra were recorded
using a Shimadzu UV-3600 UV-vis spectrophotometer. Solid
state PL efficiencies were measured 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. Photoluminescence
spectra were collected on
a
Shimadzu RF-5301PC
spectrophotometer. DLS and Zeta potential measurement was
performed using a Malvern Zetasizer Nano ZS size analyser at
room temperature. Transmission electron microscopy (TEM)
images were obtained using
a transmission electron
microscope (TEM, JEM-2100F). The cellular imaging was
performed on an Olympus IX71 microscope with Mercury lamp
as the excitation source.
Conclusions
In summary, we succeeded in developping a simple strategy to
fabricate multifunctional polymer NPs Fe3O4/DPPBPA@F-127,
by using oleic acid coated magnetic Fe3O4 and an AEE molecule
DPPBPA as the core, and biocompatible polymer Pluronic F-
127 as the encapsulation matrix. The structure, morphology,
and property of the NPs are characterized and investigated.
The fluorescent magnetic NPs could be successfully dispersed
in aqueous solution and showed significantly near-infrared
emission (654nm) with high fluorescence quantum yields
(18.3%). The high quantum yield of the fluorescent magnetic
NPs are attributed to the restricted intramolecular rotation of
the DPPBPA molecules in the spatially confined hydrophobic
core environment of the NPs, which prevents direct
interactions of DPPBPA molecules. The NPs are magnetically
susceptible with zero remanence and coercivity, suggesting
Measurements of fluorescence quantum yields
： Fluorescence
quantum yields for the solutions and suspensions were
obtained by comparing to the fluorescence spectrum of
rhodamine in ethanol (absorbance value < 0.1, excitation
wavelength:365 nm, PL efficiencies Φr= 69%) with corrections
of refractive index differences using eqn47.

2 





A
Is ns
r








Φs = Φr
2


As Ir
nr



Where Φr and Φs are the fluorescence quantum yields of
standards and the samples, respectively. Ar and As are the
absorbances of the standards and the measured samples at
the excitation wavelength, respectively. Ir and Is are the
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integrated emission intensities of standards and the samples. for 15 h and then allowed to cool to room temperature. The
nr and ns are the refractive indices of the corresponding reaction mixture was passed through a short pad of neutral
solvents of the solutions, respectively.
alumina (activated level I). The filtrate was concentrated by
evaporation. Then the crude product was purified by silica gel
Measurements of the leaching issue
The leakage degrees of the dye in the matrix were chromatography (hexane/CH2Cl2=1:1) to give a red solid. Then,
investigated using fluorescence spectra. After continuous the pure product was obtained by sublimation in the yield of
1
vigorous sonicating, Fe3O4/DPPBPA@F-127 NPs were 15%. HNMR: δ (500 MHz, CDCl3) 8.06 (2 H, s), 7.93 (2 H, s),
H
separated by magnet from 1mL aqueous solution and then 7.78 (4 H, d, J 7.6), 7.51 (4 H, t, J 7.6), 7.43 (2 H, t, J 7.4), 3.06 –
dispersed in 1mL water by sonicator. After that, the 2.93 (8 H, m), 1.80 – 1.71 (8 H, m), 1.62 (4 H, s), GC-MS: C34 H34
fluorescence was measured.
N4, calcd 498.66; found 498.66.
Synthesis of 2,5-Dibromobenzene-1,4-dicarbaldehyde (1)
Synthesis of oleic acid coated Fe3O4
2,5-Dibromobenzene-1,4-dicarbaldehyde (1) was synthesized A solution of 0.01 mol ferric chloride hexahydrate and 0.005
according to the procedure shown in Scheme S1. All chemicals mol ferrous chloride tetrahydrate in 100 mL of de-ionized (DI)
were purchased commercially, and used without further water in a 3-neck round bottom flask was stirred by
purification. 4g of 1, 4-dibromo-2, 5-dimethylbnzene was mechanical stirrer at 80 ºC with nitrogen bubbling for 30 min.
dissolved in 20 mL of acetic acid and 40 mL of acetic anhydride 0.01 mol of potassium oleate was added, and the mixture was
at 0 °C. 14 mL of sulfuric acid was added dropwise into the stirred for another 30 min. 35 mL of 4% ammonium hydroxide
solution, which was stirred for another 10 min. CrO3 was was added to the mixture. The reaction system turned to black
grinded into powders, and then added to the mixture in immediately. The reaction continued at 80 ºC under nitrogen
portions. The resulting mixture was stirred vigorously for bubbling for 30 min. The black liquid was centrifuged at
another 5 h at the temperature under 10°C.38 The greenish 60000r/min for 20min at 20 ºC to separate unreacted oleic
slurry was poured into ice water and filtered. The solid was acid from magnetic NPs. The magnetic NPs were dispersed in
washed with water and microscale cold methanol. The white THF at 10 mg/mL for further application.
compound would be get. The diacetate was then hydrolyzed
Preparation of Fe3O4/DPPBPA@F-127
by refluxing with a mixture of 20mL of water, 20 mL of ethyl
alcohol, and 2 mL of sulfuric acid for 5 h. After the mixture
cooled, the pale yellow product was separated by filtration.
The crude product was purified by recrystallization from
chloroform. Yellow crystal will be get(1.83g, 40%). 1 H NMR δ
H (500 MHz, CDCl3) 10.38 (2 H, s), 8.19 (2 H, s).
In a typical preparation, the fluorescent DPPBPA was first
dissolved in tetrahydrofuran (THF) to make a 0.5 mg/mL stock
solution. OA-Fe3O4 (1mg, 2mg), DPPBPA (0.5mg, 1mg) and
Pluronic F-127 (6mg, 8mg, 10mg, 12 mg) with different
contentions were dispersed in 5mL THF by a vigorous bath
sonicator. The solution mixture was quickly added to DI water,
and the THF was removed by nitrogen stripping in a vigorous
bath sonicator. The solution was concentrated by continuous
nitrogen on a 90 ºC hotplate followed by filtration through a
0.2 micron filter. During nanoparticle formation, the OA-Fe3O4
and DPPBPA tended to entangled with the hydrophobic
domains of Pluronic F-127, to afford the hydrophobic interiors,
while the hydrophilic domains of Pluronic F-127 extend into
the aqueous phase of the NPs.
Synthesis of (2Z,2'Z)-3,3'-(2,5-dibromo-1,4-phenylene)bis(2-
phenylacrylonitrile) (2)
The mixture of benzylcyanide (201 mg, 1.5 mmol) and
compound 1 (659 mg, 3 mmol) in tert-butyl alcohol (10 mL)
was stirred at 46 °C for 30 min. Then, potassium tert-butoxide
(1
M
solution in tetrahydrofuran, 0.25 mL) and
Tetrabutylammonium hydroxide (TBAH, 1 M solution in
methanol, 0.25 mL) were slowly added, and stirred for 60
minutes37. The resulting precipitate was filtered and purified
by column chromatography using dichloromethane.
Cell Culture
Compound 2 (348 mg) was obtained in yield of 70% by
1
evaporated the solvent. H NMR: δ (500 MHz, CDCl3) 8.39 (2
MCF-7 breast cancer cells were provided by American Type
Culture Collection (ATCC). The cells were cultured in DMEM
medium containing 10% heat-inactivated FBS (Invitrogen), 100
U/ml penicillin, and 100 μg/ml streptomycin (Thermo
H
H, s), 7.79 (2 H, s), 7.77 (4 H, dd, J 7.9, 1.2), 7.56 – 7.43 (6 H,
m).
Synthesis of (2Z, 2'Z)-3, 3'-(2, 5-di(piperidin-1-yl)-1, 4-phenylene)
bis(2-phenylacrylonitrile) (DPPBPA)
Scientific) and maintained in a humidified incubator at 37
℃
with 5% CO2. Before experiment, the cells were precultured
until confluence was reached. MCF-7 cells were seeded in a
96-well flat-bottomed microplate (10000 cells/well) and
A vial tube (25 mL) equipped with a magnetic stirring bar was
charged with Pd2(dba)3 (2.06mg, 22.5μmol) and Ruphos (
42mg, 90μmol). The vial tube was then capped with a rubber
septum, evacuated for 5 min and charged with nitrogen. The
evacuation –purge operation was repeated twice. Toluene (10
mL) was added to the vial at room temperature under a
nitrogen atmosphere and the resulting mixture was stirred at
room temperature for 5 min. To the solution were added
compound 2 (0.147 g, 0.3mmol), piperidine (0.297mL, 3mmol),
and K3PO4 (0.87g, 3mmol)48. The mixture was stirred at 100°C
cultured in 100 μL growth medium at 37
℃ and 5% CO2 for
24h. Cell culture medium in each well was then replaced by
100μL cell growth medium, containing Fe3O4/DPPBPA@F-127
with concentrations ranging from 0 to 100ppm. After
incubation for 20h, 20μL of MTT (5mg/mL in PBS solution) was
added to each well, and cells were incubated further for 4h at
37°C. The growth medium was removed gently, and 150 μL of
DMSO was then added to each well, sitting at room
6 | J. Name., 2012, 00, 1-3
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ARTICLE
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Reviews, 2011, 40, 5361-5388.
16 S. Zhang, J. Yan, A. Qin, J. Sun and B. Tang, Sci. China Chem.,
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temperature overnight to dissolve the formazan crystals
completely. The absorbance at the wavelength of 570 nm was
measured by Multiskan EX (Thermo Electron Corporation), and
each data point represents a mean +SD from triplicate wells.
Percentage cell viability = (average Abs value of experimental
group - average Abs value of blank group) / (average Abs value
of control group - average Abs value of blank group) ×100 %.
Percentage cell cytotoxicity = [1- (average Abs value of
experimental group - average Abs value of blank group) /
(average Abs value of control group - average Abs value of
blank group)] ×100 %.
18 J. He, B. Xu, F. Chen, H. Xia, K. Li, L. Ye and W. Tian, The
Journal of Physical Chemistry C, 2009, 113, 9892-9899.
19 X. Li, S. Zhu, B. Xu, K. Ma, J. Zhang, B. Yang and W. Tian,
Percentage cell cytotoxicity = (1- Percentage cell viability) ×100
%.
Nanoscale, 2013, 5, 7776-7779.
Cell imaging
20 B. Zhou, H. Shan, D. Li, Z.-B. Jiang, J.-S. Qian, K.-S. Zhu, M.-S.
Huang and X.-C. Meng, Magnetic Resonance Imaging, 2010,
28, 394-399.
21 C. G. Khoury and T. Vo-Dinh, The Journal of Physical
Chemistry C, 2008, 112, 18849-18859.
22 A. Narita, K. Naka and Y. Chujo, Colloids and Surfaces A:
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24 Y. Li, H. Yu, Y. Qian, J. Hu and S. Liu, Advanced Materials,
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To study the cellular uptake, MCF-7 cells were added to 6-well
plate, treated with various concentrations Fe3O4/DPPBPA@F-
127 (50ppm) for 12 h. The cells were washed with PBS before
observations on fluorescence microscopy. The cellular imaging
was performed on a Fluorescence microscope. Cellular uptake
of the NPs was investigated by using flow cytometer (FACS
Calibur, Becton and Dickinson Company) with excitation and
emission wavelengths of 460 nm and FLA-3. In total, 10,000
cells were analyzed per sample.
25 Y. Chen, M. Li, Y. Hong, J. W. Y. Lam, Q. Zheng and B. Z. Tang,
ACS Applied Materials & Interfaces, 2014,
26 K. Li, D. Ding, C. Prashant, W. Qin, C.-T. Yang, B. Z. Tang and B.
Liu, Advanced Healthcare Materials, 2013, , 1600-1605.
6, 10783-10791.
2
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Acknowledgements
This work was supported by (2013CB834701), the Natural
Science Foundation of China (No. 51373063, 51573068,
21221063) and Program for Chang Jiang Scholars and
Innovative Research Team in University (No. IRT101713018).
Wei, Polymer Chemistry, 2013, 4, 5060-5064.
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Chemistry of Materials, 2009, 21, 5819-5825.
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Magnetic fluorescent multifunctional polymer NPs Fe3O4/DPPBPA@F-127 and their application in
MRI and NIR imaging.