Redox-responsive Fluorescent Nanoparticles Based on Diselenide-containing AIEgens for Cell Imaging and Selective Cancer Therapy

Article abstract of DOI:10.1002/asia.201801527

A fluorescent, diselenide-containing 9,10-distyrylanthracene (DSA) derivative (SeDSA) with aggregation-induced emission (AIE) characteristic was successfully synthesized and SeDSA nanoparticles (NPs) were prepared through a nanoprecipitation method. SeDSA

Full text of DOI:10.1002/asia.201801527

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Redox Responsive Fluorescent nanoparticles based on
Diselenide-containing AIEgens for Cell imaging and Selective
Cancer Therapy
Authors: Wenkun Han, Song Zhang, Jingyu Qian, Jianxu Zhang,
Xuhang Wang, Zhigang Xie, Bin Xu, Yanqiu Han, and
Wenjing Tian
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To be cited as: Chem. Asian J. 10.1002/asia.201801527
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Chemistry - An Asian Journal
10.1002/asia.201801527
Redox Responsive Fluorescent nanoparticles based on
Diselenide-containing AIEgens for Cell imaging and Selective
Cancer Therapy
[
a]
[a]
[a]
[b]
[a]
[b]
Wenkun Han, Song Zhang, Jingyu Qian, Jianxu Zhang, Xuanhang Wang, Zhigang Xie, Bin
[
a]
[c]
[a]
Xu,* Yanqiu Han, and Wenjing Tian*
Abstract:
A
fluorescent molecule diselenide-containing 9, 10-
interests of researchers because it can provide a direct visual
method for cancer diagnosis and better understanding of
distyrylanthracene (DSA) derivative (SeDSA) with aggregation
induced emission (AIE) characteristic was successfully synthesized
and SeDSA nanoparticles (NPs) were prepared through
nanoprecipitation method. SeDSA could coassemble with the
antitumor prodrug, diselenide-containing paclitaxel (SePTX) which
could be obtained by precipitation, to form SeDSA-SePTX Co-NPs
[
2]
biological processes, metabolism and pharmacokinetics. Over
the past few decades, a variety of fluorescent agents have been
proverbially explored and applied for bioimaging owing to their
excellent merits, and organic fluorescent dyes with tunable
absorption and emission wavelengths are one of the significant
[
3]
(Co-NPs). Molecular dynamics (MD) simulations reveal that the
agents used in bioimaging. However, traditional dyes often
suffer from inherent aggregation-caused quenching (ACQ) effect
leading to no or weak fluorescence emission in solid state or in
aqueous media. More importantly, intrinsic drawbacks such as
poor stability, small stokes shift and poor biocompatibility have
driving forces for the self-assembly behaviors of SeDSA NPs and
SePTX NPs are π-π interactions and hydrophobic interactions,
respectively. While the driving forces for Co-NPs include
hydrophobic interactions between SeDSA and SePTX, π-π
interactions between SeDSA molecules and hydrophobic
interactions between SePTX molecules. Meanwhile, Se-Se bonds
play a crucial role in balancing the intramolecular forces. These
diselenide-containing nanoparticles (SeDSA NPs, SePTX NPs and
Co-NPs) exhibit high stability in physiological conditions and
excellent reduction-sensitivity under redox agent glutathione (GSH)
because of the selenium-sulfur exchange reaction between
diselenide and GSH. Both SeDSA NPs and Co-NPs show strong
orange fluorescence emissions on the account of the AIE feature of
SeDSA and they were easily internalized by HeLa and HepG2 cells.
Distinctively, the Co-NPs combine the advantage of SeDSA and
SePTX for cell imaging and antineoplastic activity and exhibit
selectivity of cytotoxicities between neoplasia cells and normal cells.
It highlights developing diselenide-containing AIEgens as a unique
approach to prepare uniform and stable fluorescent nanoparticles for
the application in cell imaging and tumor treatment.
[
4]
also impeded their further development for bioimaging. Since
the uncommon photophysical phenomenon aggregation induced
emission (AIE) by Tang and coworkers, considerable AIE
fluorogens (AIEgens) have been developed by worldwide
[
5]
research groups.
And many researchers proposed different
methods to increase the water solubility of AIEgens for their
applications in biology. For example, doping into the amphiphilic
polymers or silica matrix to generate water-dispersible AIE
[
6]
nanoparticles;
amphiphilic polymers to make AIEgens hydrophilic; modified
functionalized with hydrophilic peptides or
[
7]
[
8]
by polar groups or ions such as quaternary ammonium salt,
pyridinium salt and sulphonate to enhance their water solubility.
For the treatment of cancer ， chemotherapy using small
molecular anticancer drugs is one of the most extensively used
modalities and has achieved improved therapeutic effects in
[
9]
clinic. Unfortunately, most anticancer drugs are poorly water-
soluble resulting in poor bioavailability and undesired side
[
10]
effects.
To enhance the solubility and better therapeutic
efficacy of hydrophobic drugs, the dimer-induced self-assembly
of pure drugs has been explored by a crowd of researchers
because most hydrophobic drugs could be assembled as
Introduction
Cancer is one of the most serious diseases which severely
challenges the survival of people and results in several million
nanocarrier-based
drug
delivery
systems
based
on
[11]
supramolecular interactions,
such as π-π interactions,
[
1]
deaths every year. Fluorescence-based optical imaging has
successfully been applied in clinical medicine and aroused
hydrophobic interactions and electrostatic interactions. The
inserted linkers of dimers like disulfide bond or diselenide bond
have been broadly investigated as reduction responsive groups
because they could be cleaved under the high concentration of
[
12]
[
[
[
a]
b]
c]
W. Han, S. Zhang, J. Qian, X. Wang, Prof. B. Xu, Prof. W. Tian
State Key Laboratory of Supramolecular Structure and Materials
Jilin University
Changchun, Jilin 130012 (China)
E-mail: xubin@jlu.edu.cn, wjtian@jlu.edu.cn
J. Zhang, Prof. Z. Xie
State Key Laboratory of Polymer Physics and Chemistry
Changchun Institute of Applied Chemistry
Chinese Academy of Sciences
Changchun, Jilin 130022 (China)
Y. Han
redox agents (like GSH).
According to literatures, there is a
more reductive environment in tumors with several times higher
[13]
GSH concentration than that in normal tumor tissue,
which
have attracted researchers to fabricate reduction responsive
drug delivery systems based on disulfide bond or diselenide
[
14]
bond.
Synchronously, the reduction responsive drug delivery
systems could show ability for selective release of prodrugs
between normal and tumor cells because of the higher
concentration of redox agents in tumor cells.
Department of Neurology
No.2 Hospital Jilin University
Changchun 130041, (China)
Compared with disulfide bond, the strength of diselenide bond is
much weaker leading to more reductive sensitivity of diselenide
bond linked molecules than disulfide-containing compounds
Supporting information for this article is given via a link at the end of
the document.
[15]
theoretically. According to the unique sensitivity of diselenide
bond towards the intracellular reductive environment, numbers
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Chemistry - An Asian Journal
10.1002/asia.201801527
of reductive sensitively diselenide-containing compounds have
been tactfully utilized for drug delivery and cancer therapy. For
instance, it has been reported that a serial of fluorescent dye
dimers using S-S or Se-Se bond connect BODIPY dyes could
self-assemble into nanoparticles for cell imaging and reduction
temperature and then the mixture solution was dialyzed to
remove the residual THF for 24h to form desired SeDSA NPs.
SePTX could also self-assemble into SePTX NPs to form stable
nanodrugs in water via nanoprecipitation method without any
other auxiliary approaches. After carefully dialyzing to remove
the remaining organic reagents, the acquired SePTX NPs were
obtained. By means of the reprecipitation method, hydrophobic
SeDSA and SePTX were dissolved in THF with sonication to
gain a homogeneous mixture solution. After the solution with
SeDSA and SePTX dropped into water with stirring, the obtained
mixtures were dialyzed in water for 24h to get rid of the needless
organic reagent so as to obtain the required SeDSA-SePTX NPs
(Co-NPs). The concentration of the SeDSA was measured using
a standard calibration curve technique by calculating the main
absorbance of SeDSA at 426 nm through UV-Vis absorption
responsive drug delivery systems; and
a
sequence of
diselenide-containing polymers, including main chain and side
chain block copolymers, dendrimers and hyperbranched
polymers, could self-assemble and disassemble under various
responsive stimuli for controlled drug delivery and enzyme
[
16]
mimics.
Here, we introduced diselenide bond into 9, 10-
distyrylanthracenederivative, a typical AIEgen to successfully
synthesize SeDSA which could self-assemble into nanoparticles
with enhanced fluorescence in aqueous suspension and co-
assemble with diselenide-containing paclitaxel (SePTX), the
antitumor prodrug, to form Co-NPs with dual functions of
fluorescence cell imaging and antitumor activity.
spectra in THF/H
2
O = 4:1 (Supporting Information, Figure S2).
The final concentration of SeDSA in SeDSA NPs was 12.6 μg
-1
mL . The concentration of SePTX NPs was determined through
high-performance liquid chromatography (HPLC, Shimadzu,
CBM-20A) with an UV-vis detector using methanol: acetonitrile:
Results and Discussion
water = 45: 45: 10 (v/v/v) as mobile phase. Similarly,
concentrations of SePTX and SeDSA in Co-NPs were
determined by HPLC and UV-vis absorption spectra. The final
concentrations of SeDSA and SePTX in Co-NPs were 21 and 38
The molecular structure and synthetic route of SeDSA and
SePTX were shown in Scheme S1. The required disodium
-1
μg mL , respectively.
diselenide (Na
powder using NaBH
of 3 was injected into the Na
2 2
Se ) were synthesized through reduction of Se
[
17]
4
.
Then the tetrahydrofuran (THF) solution
Se solution under Ar atmosphere.
2
2
2
After the mixture solution was heated at 70 °C for 24h, the
obtained SeDSA was measured and characterized by proton
1
nuclear magnetic resonance ( H NMR) and further confirmed by
matrix-assisted laser desorption/ionization time-of-light mass
spectrometry (MALDI-TOF), for which prominent signal at
m/z=1208.081 (m/z calcd: 1208.424) can be clearly observed.
The diselenide-containing SePTX was obtained via the
condensation reaction，and the compound were characterized
1
by H NMR and MALDI-TOF mass spectrometry (Supporting
Information, Figure S1).
Figure 1. (a) Normalized UV-vis absorption and fluorescence spectra of
SeDSA in THF and SeDSA NPs in water, respectively. (b) AIE property of
SeDSA in THF-water mixtures with different water fractions. Inset in (b) are
2
photos of SeDSA in THF: H O = 4:1 (v/v) solution (right) and SeDSA NPs in
water (left) under natural light and UV 365 nm light.
We measured the UV-vis absorption and fluorescence spectra of
SeDSA in THF and SeDSA NPs in water, respectively. As
shown in Figure 1a, the maximum absorption of SeDSA in THF
was at 426 nm and the main absorbance peak of SeDSA NPs in
water was at 436 nm, which has 10 nm red-shift relative to that
of SeDSA. The maximum fluorescence wavelength was
centered at 548 nm for SeDSA in THF, but enhanced
fluorescence was observed at 567 nm for SeDSA NPs due to
Scheme 1. The synthetic route of SeDSA.
[
19]
the AIE feature of SeDSA.
As shown in Figure 1b, SeDSA
NPs emitted strong orange fluorescence, while weak
fluorescence was observed for SeDSA in THF. The fluorescence
spectra of SeDSA in water and THF mixtures at different water
fractions (fw) were carefully measured and the results are
exhibited in Figure 1b. SeDSA is faintly fluorescence at 548 nm
in its benign solvent (THF) because the molecularly dissolved
One simple and effective method to prepare small organic
[
18]
nanoparticles is reprecipitation means in aqueous solution.
The characterized SeDSA was dissolved in THF with drastic
ultrasound, and then the uniform THF solution with SeDSA was
added into deionized water tardily with vigorous stirring at room
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Chemistry - An Asian Journal
10.1002/asia.201801527
state can consume the energy through free molecular motion,
PDI was 0.134 with good size distribution. The SePTX NPs and
Co-NPs also had excellent storage stability in water with no
significant changes in the size for nearly half a month (Figure
3a-c, Figure S7a).
[
20]
which favors nonradiative decay.
However, with increasing
water (poor solvent) fractions to 50%, SeDSA begins remarkable
enhanced fluorescence emission, showing a characteristic of
AIE. At 99% of fw, the fluorescence intensity at 572 nm is about
39-fold stronger than that in pure THF due to the formation of
aggregates which restrict the intramolecular motion to activate
[
21]
the AIE process.
However, the fluorescence emission of
SeDSA with different water fractions causes about 20 nm
redshift, which is attributed to the intramolecular charge transfer
(ICT) between nitrogen atom and DSA.
Figure 3. (a) DLS results of Co-NPs, Inside: Size, Z-Average and PDI of Co-
NPs; (b) TEM image of Co-NPs, Scale bar: 1 μm; (c) Changes of the co-NPs
and PDI as a function of time measured by DLS; (d) Size changes of co-NPs
over time determined by DLS in the presence of different concentrations of
GSH.
We further explored the self-assembling behaviors of SeDSA by
[
12]
Figure 2. (a) TEM image of SeDSA NPs, scale bar: 200 nm.(b) DLS results of
SeDSA NPs. (c) Changes of the SeDSA NPs and PDI as a function of time
measured by DLS.(d) Size changes of SeDSA NPs over time determined by
DLS in the presence of different concentrations of GSH.
molecular dynamics (MD) simulations
. SeDSA was initially
arranged approximately vertical with an unmanipulated
assembly process (Supporting Information, Figure S5A). After
10 ps in water, the final MD simulation position is shown in
The morphology and size distribution of SeDSA NPs, SePTX
NPs and Co-NPs were characterized by transmission electronic
microscopy (TEM) and dynamic light scattering (DLS) without
further treatment. As shown in Figure 2, TEM measurement
indicated that SeDSA NPs have uniform spherical structure and
the hydrodynamic diameter with the average size of 157 nm was
evaluated by DLS measurement, which was slightly larger than
the value recorded by TEM. The initial polydispersity index (PDI)
and zeta potential of SeDSA NPs are 0.133 and -30.8 mv. The
TEM image and DLS measurements showed SeDSA could self-
assemble into isolated nanoparticles in aqueous solution via
nanoprecipitation method. The unchanged PDI and average
sizes of SeDSA NPs after storing for 14 days indicated the
SeDSA NPs have good stability in aqueous system which was
also verified by TEM measurement (Supporting Information,
Figure S3b). In addition, the slight changes of PDI and the size
of SeDSA NPs after being incubated in phosphate buffer saline
Figure S9A. The obtained data indicated that four SeDSA
quickly flocked together to develop a cluster of tetramers and the
conformation of SeDSA was slightly changed. As shown in
Figure S5A, the two DSA derivatives of SeDSA are curved
inside the cluster and π-π interactions are found between DSA
derivatives which are the main driving forces for the self-
assembly behaviors of the SeDSA. Similarly, there are no direct
interactions between Se-Se bonds. The dihedral angle (77.5°)
between SeDSA planes in the self-assemblied tetramers of
SeDSA simulated by molecular dynamics (MD) (Supporting
Information, Figure S5) and negative charge near Se-Se bond
simulated by self-consistent reaction field (SCRF) method with
the polarizable continuum model (PCM)( Supporting Information,
Figure S10) indicated that Se-Se bond plays an indispensable
role in balancing the intermolecular forces that control the self-
[
12].
assembly behaviors of SeDSA.
The inserted Se-Se bond also
plays an indispensable role in balancing the intermolecular
forces that control the self-assembly behaviors of SeDSA.
However, when Se-Se bonds are broken, the segments of
SeDSA NPs may form more energetically favorable
conformations. Four SePTX rapidly aggregated into a cluster of
tetramers inside which the PTX derivatives are curved and there
are nonbonded hydrophobic interactions between SePTX. So
the driving forces for the self-assembly of SePTX are
nonbonded hydrophobic interactions. We further explored the
co-assembling behaviours between SeDSA and SePTX by
(PBS, pH 7.4) containing 10% fetal bovine serum (FBS) at 37℃
demonstrated the favorable stability of SeDSA NPs under
physiological conditions（Supporting Information, Figure S3a）.
The SePTX NPs showed spherical morphology with the average
size of 185.8 nm (Supporting Information, Figure S4a-c). TEM
images showed that Co-NPs exhibited isolated spherical
morphology with the average diameter of 190 nm, while the
average diameter of hydrated Co-NPs was 242.5 nm and the
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Chemistry - An Asian Journal
10.1002/asia.201801527
molecular dynamics (MD) simulation method. As shown in
Figure S5C, SeDSA and SePTX could come together to form a
cluster of tetramers and there are strong π-π interactions
between SeDSA molecules and hydrophobic interactions
between SePTX molecules. Considering the unique structure of
the tetramer of Co-NPs, the driving forces for the co-assembly of
Co-NPs are nonbonded hydrophobic interactions between
SePTX and SeDSA.
those in the reduced monomer, which means the fluorescence
of SeDSA may be quenched by the strong π-π interactions.
Therefore, the relatively low space occupancy and strong π-π
interactions may cause the lower fluorescence quantum yield of
SeDSA NPs than that of agglomerate. Also, we detected the
reduction-sensitive behaviors of SePTX NPs in the presence of
different concentrations of GSH in vitro. As Figure S4 shown, the
particle sizes of SePTX NPs increased gradually under GSH,
showing the nice reduction property of SePTX NPs (Supporting
Information, Figure S4d). Meanwhile, the reduction properties of
Co-NPs with 10 mM and 1 mM GSH were detected by DLS
through tracking the changes of the diameters and PDI. As
shown in Figure 3d, the diameter of Co-NPs increased from
242.5 nm to 1236 nm and PDI from 0.134 to 0.865 after being
treated with 10 mM GSH for 7 h. And the size of Co-NPs could
increase from 145.6 nm to 308.6 nm with 1mM GSH for 7 h. The
continuous increase of size and PDI of Co-NPs with GSH
demonstrated that Co-NPs are also reduction-sensitive.
Redox Sensitivity
The diselenide-containing compounds could react with redox
agent glutathione (GSH) in vitro and vivo because of selenium-
[
22]
sulfur exchange reaction between diselenide bond and GSH.
So we explored the reduction-sensitivity of prepared SeDSA
NPs in vitro using different concentrations of GSH to mimic the
intracellular GSH level. SeDSA NPs were coped with 1mM and
10 mM GSH for different time at 37 °C and the control group
was treated without redox agent GSH, then we monitored the
size changes of SeDSA NPs by DLS for 7h. According to DLS
results in Figure 2d, there were ignorable changes of the
average size and PDI for SeDSA NPs after SeDSA NPs were
incubated without reductive agents for 7h at 37 °C. Evident
increase of the size (from 159 nm to 7986 nm), PDI ( from 0.133
to 1.00 ) and zeta potential (from -37.4 mv to 8.85 mv) for
SeDSA NPs were observed when SeDSA NPs were incubated
with 10 mM GSH at 37 °C for 7 h, indicating SeDSA NPs were
reduction-sensitive under GSH. Moreover, large agglomerates of
SeDSA exhibited bright orange fluorescent emission in Figure
S6d because the disruption of diselenide bond under high
concentration of GSH can make hydrophobic fragments of
SeDSA aggregate into large agglomerates. Similarly, the
reduction-sensitive behaviors of SeDSA NPs with 1 mM GSH
were also detected by DLS. As displayed in Figure 2d, the
average size of SeDSA NPs had an increase from 157 nm to
The cell uptake of SeDSA NPs and Co-NPs
We confirmed the biocompatibility of SeDSA NPs in vitro
through measuring the cytotoxicity of SeDSA NPs toward
HepG2 cells quantified by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyl tetrazolium bromide (MTT) viability assay. As shown in
Figure S8, after incubation at 37 °C with different concentrations
of SeDSA NPs for 48 h, there were still 80% of cells survived
-1
with the concentration up to 7.2 μg mL , confirming the low
cytotoxicity and good biocompatibility of SeDSA NPs in vivo.
Flow cytometry was utilized to examine the quantitatively cellular
uptake of the SeDSA NPs and Co-NPs. As shown in Figure S9a,
it could be noticed that all internalization of the SeDSA NPs with
increasing incubation time from 0.5 to 4 h, evidenced the time-
dependent endocytosis.
2
022 nm after SeDSA NPs were treated with 1 mM GSH at
7 °C for 7 h. Furthermore, SeDSA NPs in serum containing
3
solution with 10 mM GSH still possess efficient reductive
behaviors. Figure S7b showed the size of SeDSA NPs
increased from 220 nm to 1718 nm and obvious naked-visible
suspension could be found under day light on the account of
disassembly of SeDSA NPs with reduction agents, which
demonstrated the efficient reductive behaviors of SeDSA NPs
under physiological conditions. In order to explain why the
PLQYs of SeDSA NPs and agglomerates were different, we first
calculated the space occupancy of SeDSA and reduced
monomer which are components of SeDSA NPs and
agglomerates by using MD simulation. As shown in Fig. S11, the
space occupancy of SeDSA was 65.44 ± 0.5 %, while that of
reduced monomer was 66.24 ± 0.4%. It means that SeDSA
could aggregate into a little bit tighter structure in nanoparticles
comparing with the reduced monomer in agglomerates, which
should result in the enhanced emission of SeDSA nanoparticles
due to the AIE characters of DSA derivative. Then we compared
the self-assembly behaviors of SeDSA and reduced monomer
(
Supporting Information, Fig. S12) and found the π-π
interactions between DSA parts in SeDSA are stronger than
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Chemistry - An Asian Journal
10.1002/asia.201801527
Figure 4. (a）CLSM images of (a) HeLa (b) HepG2 and (c) Skov 3 cells
treated with SeDSA NPs for 2 h. For each panel, the images from left to right
show cell nuclei stained by Hoechst (blue), SeDSA NPs fluorescence in cells
(orange) and overlays of both images, scale bar : 20 μm.
Figure 5. The representative CLSM images of HepG2 cells treated with Co-
NPs for (a) 0.5 and (b) 2 h. For each panel, the images from left to right show
cell nuclei stained by Hoechst (blue), SeDSA NPs fluorescence in cells
(orange) and overlays of both images, scale bar: 20 μm.
Figure 6. Confocal microscopy images of HepG2 cells, (a) without any
pretreatment, and (b) pretreated with GSH (10 mM) for 15 min (c) pretreated
-1
with NEM (1 mM) for 15 min, then treated with SeDSA NPs (0.88 μg mL ) for
h. For each panel, the images from left to right show cell nuclei stained by
Hoechst (blue), SeDSA NPs fluorescence in cells (orange), and overlays of
both images. Scale bar: 20 μm.
2
We used confocal laser scanning microscopy (CLSM) to
investigate the cellular uptake of SeDSA NPs towards HepG2,
HeLa and Skov 3. As shown in Figure 4, strong intracellular
orange fluorescence was observed for HepG2 and HeLa cells
after incubating with SeDSA NPs for 2 h, but there was weak
fluorescence in Skov 3 cells evaluated by CLSM. It manifested
that SeDSA NPs could be well internalized by HepG2 and HeLa
cells. We further explored the reduction-response of SeDSA
NPs in vitro by CLSM. HepG2 cells were first preprocessed with
We carried out the quantitatively cellular uptake of the Co-NPs
using flow cytometry. The flow cytometry analysis (shown in
Figure S9B) indicated that a significant increase in orange
fluorescence intensity was detected after the HeLa cells were
treated with Co-NPs for different time, identifying the time-
dependent endocytosis for Co-NPs. To investigate the cellular
uptake and further explore the potential of using of Co-NPs for
bimolecular imaging in live cells, HepG2 cells were incubated
with the same concentrations of Co-NPs for 0.5 h and 2 h in cell
incubator. After staining the cell nuclei by Hochest 33258, the
cells were evaluated by CLSM. As shown in Figure 5, orange
fluorescence was observed for the cells, indicating that Co-NPs
could be endocytosed by HepG2 cells. In addition, the orange
fluorescence intensity in HepG2 cells obviously increased in the
wake of the increasing incubation time.
1
mM N-ethylmaleimide (NEM) or 10 mM GSH for 15 min which
[
23]
could reduce or increase the concentrations of GSH in cells,
and then HepG2 cells were incubated with SeDSA NPs (0.87 μg
-1
mL ) for 2 h in cell incubator at 37 °C. The control group only
incubated with the same potion of SeDSA NPs without
pretreatment. As shown in Figure 6, compared with the control
group, HepG2 cells pretreated with GSH gave strong orange
fluorescence emission and the group pretreated with NEM which
could effectively quench biological thiol groups in the cytoplasm,
gave weak orange fluorescence emission. The results indicated
that the intracellular reducing agents like GSH could make a fast
breaking of diselenide bond in SeDSA NPs.
The cellular toxicity of Co-NPs toward the tumor cells (HeLa
cells and HepG2 cells) and the normal cells (293T cells) was
studied using MTT assays. Figure 7 showed that after 48 h
incubation with different concentrations of free PTX and Co-NPs,
Co-NPs have shown lower cytotoxicity than free PTX under the
same concentration of PTX, which might ascribe the prompt
diffusion of PTX into cytoplasm other than the endocytosis of
Co-NPs. Surprisingly, we also found obvious cell cytoxicities
against tumor cells compared with normal cells. More than 70%
of HepG2 cells and 50% of HeLa cells were killed at the
-1
concentration of 5 μg mL of PTX. In contrast, no more than 15%
of 293T cells were killed, as shown in Figure 7. The IC50 values
-1
toward HeLa cells and HepG2 cells were 3.96 5 μg mL and
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Chemistry - An Asian Journal
10.1002/asia.201801527
-
1
4
.45 5 μg mL , which are lower than that of the normal cells (>5
and the results also revealed Co-NPs have an effect on
selectivity treatment of tumor cells. So the redox responsive
fluorescent nanoparticles based on diselenide-containing
AIEgens show the potential applications in cell imaging and
reduction-sensitive drug delivering.
-1
μg mL ) (Supporting Information, Table S1). These results
manifested Co-NPs could distinguish the normal cells and tumor
cells and kill the tumor cells selectively, due to the selenium-
induced reactive oxygen species (ROS) in cancer cells causing
[
12, 24]
the apoptosis.
Experimental Section
Material and instruments
Selenium powder, 1, 6-dibromohexane, 4-Hydroxybenzaldehyde and N-
ethylmaleimide (NEM) were all purchased from Shanghai Energy
Chemical. T-BuOK was purchased J&K Scientific Ltd, Glutathione (GSH)
was purchased from Dingguo Biotech (Beijing, China), Sodium
borohydride was purchased from Shanghai Macklin Biochemical Co., Ltd.
Tetrahydrofuran (THF) and dichloromethane (DCM) were dried by
distillation using sodium as drying agent and benzophenone as indicator.
All non-aqueous reactions were carried out under argon atmosphere in
oven-dried glassware. Milli-Q water (18.2 MΩ) was used to prepare the
buffer solutions from 1L PBS stock buffer. Unless otherwise indicated, all
reagents were purchased from commercial available and used as
1
received without further purification. H NMR spectra were measured in
CDCl
3
at room temperature by an AV-500 NMR spectrometer from
Bruker. The average size, PDI and the zeta potential of the nanoparticles
were recorded by Malvern Zeta-sizer Nano for dynamic light scattering
Figure 7. In vitro cytotoxicities of different concentrations of PTX and Co-NPs
toward HepG2 (a) HeLa cells, (b) HepG2 cells, and (c) 239T cells incubated
for 48 h. (d) Comparison of cytotoxicities in vitro toward tumor cells (HeLa and
HepG2 cells) and normal cells (293T cells) after being incubated with Co-NPs
for 48 h. Statistical significance analysis was assessed by SPSS via one-way
ANOVA test; **P ≤ 0.01 was considered statistically highly significant; ***P ≤
(DLS). The DLS measurement was recorded at 25 °C and the scattering
angle was fixed at 90°. Transmission electron microscopy (TEM) was
conducted by a JEM-2100F electron microscope. Matrix assisted laser
desorption/ionization time-off light (MALDI-TOF) mass spectra (MS) were
measured on a Bruker/Auto Reflex III mass spectrometer equipped
0.001 was considered statistically highly significant.
(
Bremen, Germany). UV-vis absorption spectra were conducted by a
Shimadzu UV-2550 spectrophotometer. Fluorescence spectroscopy was
taken with Shimadzu RF-5301 PC spectrometer. Confocal laser
a
Conclusions
scanning microscopy (CLSM) images were performed using an Olympus
FV1000 confocal laser scanning microscope. Flow cytometry was
obtained on Guava easyCyte 6-2L Base System (Merck Millipore, USA).
In summary, we successfully synthesized a novel fluorescent
molecule SeDSA containing diselenide bond and AIEgens.
SeDSA could self-assemble into uniform nanoparticles with
orange fluorescence through usual nanoprecipitation method.
Simultaneously, SeDSA and SePTX which is the reported
diselenide-containing antitumor prodrug could coassemble into
Co-NPs. The self-assembly driving forces of SeDSA NPs,
SePTX NPs and Co-NPs explored by molecular dynamics (MD)
simulations are π-π interactions, hydrophobic interactions, and
hydrophobic interactions between SeDSA and SePTX, π-π
interactions between SeDSA molecules and hydrophobic
interactions between SePTX molecules, respectively. And there
is indispensable contribution of the diselenide bond balancing
the intermolecular forces. All the obtained nanoparticles linked
by diselenide bond exhibited favorable stability in aqueous
solution and physiological environment, which showed excellent
redox sensitivity under the reductive environment with GSH.
Because of the AIE property of SeDSA and the redox
responsive property of diselenide bond, Co-NPs could be used
for cell imaging and exerting the antitumor activity. The
cytotoxicity experiments demonstrate that SeDSA NPs are
biocompatible with low cytotoxicity and Co-NPs have lower
cytotoxicity than free PTX under the same concentration of PTX,
Synthesis of the compound 2
4-Hydroxybenzaldehyde (2.44 g, 0.02 mol), K
2 3
CO (5.52 g, 0.1 mol) were
dissolved in 40 mL of CH CN, and then 1, 6-dibromohexane (16.6ml,
3
0.10 mol) was injected into the solution. The mixture solution was
refluxed for 12 h. After filtration, the solvent was removed with a rotary
evaporator and the residue was poured into 50 mL of water and
extracted with CH
MgSO and then the solvent was removed, the crude was further purified
by flash column chromatography (dichloromethane/petroleum ether, 1:1
2 2
Cl . The organic phase was collected, dried with
4
1
v/v) to afford 2 as a white solid. H NMR (500 MHz, CDCl
3
) δ 9.90 (s, 1H),
.85 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 8.6 Hz, 2H), 4.07 (t, J = 6.4 Hz, 2H),
.45 (t, J = 6.7 Hz, 2H), 1.89 (dp, J = 26.6, 6.6 Hz, 4H), 1.66-1.45 (m, 4H).
7
3
Synthesis of the compound 3
Compound 1 (236.8 mg, 0.5 mmol) and compound 2 (154.5 mg, 0.6
2
mmol) were dissolved in 20 ml dry THF under N with stirring, and t-
BuOK (67.3 mg, 0.6 mmol) in 10 mL dry THF was added into the solvent
under ice water bath. After the mixture stirred at room temperature for 24
h, the reaction mixture was quenched with 5 mL water, and then the
solvent was removed with a rotary evaporator and the residue was
2 2 2 2
poured into 50 mL of CH Cl and 50 mL brine, extracted with CH Cl (50
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Chemistry - An Asian Journal
10.1002/asia.201801527
ml) three times. The organic phase was dried over MgSO
4
, filtered and
[4]
[5]
R. Deans, J. Kim, M. R. Machacek, T. M. Swager, J. Am. Chem. Soc.,
2000, 122, 8565-8566.
the filtrate was concentrated and purified by chromatography
dichloromethane/petroleum ether, 2: 1 v/v) to give compound 3 as an
(
a) J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu, H. S. Kwok,
X. Zhan, Y. Liu, D. Zhu, B. Z. Tang, Chem. Commun. 2001, 37, 1740-
orange solid.
1741; b) R. T. K. Kwok, C. W. T. Leung, J. W. Y. Lam, B. Z. Tang,
Chem. Soc. Rev. 2015, 44, 4228-4238.
Synthesis of SeDSA
[
6]
a) Z. Wang, L. Yan, L. Zhang, Y. Chen, H. Li, J. Zhang, Y. Zhang, X. Li,
B. Xu, X. Fu, Z. Sun, W. Tian, Polym. Chem. 2014, 5, 7013-7020; b) X.
Zhang, X. Zhang, B. Yang, L. Liu, J. Hui, M. Liu, Y. Chen, Y. Wei, RSC
Adv. 2014, 4, 10060-10066; c) D. Li, J. Yu, Small 2016, 47, 6478-6494;
d) W. Wu, D. Mao, F. Hu, S. Xu, C. Chen, C. -J. Zhang, X. Cheng, Y.
Yuan, D. Ding, D. Kong, B. Liu, Adv. Mater. 2017, 29, 1700548; e) X.
Zhang, X. Zhang, S. Wang, M. Liu, Y. Zhang, L. Tao, Y. Wei, ACS Appl.
Mater. Interfaces. 2013, 5, 1943-1947.
Se powder (16 mg, 0.1 mmol) was dispersed in deionized water (2 mL)
2
under the sonication in Ar and sodium borohydride (7.6 mg, 0.2 mmol)
were also dissolved in deionized water (2 mL) during the sonication. The
sodium borohydride solvent was slowly injected into the Se dispersion
solution with stirring accompanying bubbling till the Se suspension liquid
became a colorless solution. After the reaction mixture was heated at
2 2
70 °C for 30 min, the dark wine solution indicated the Na Se was
[
7]
a) Y. Yuan, C.-J. Zhang, R. T. K. Kwok, S. Xu, R. Zhang, J. Wu, B. Z.
Tang, B. Liu, Adv. Funct. Mater. 2015, 25, 6586-6595; b) H. Li, X.
Zhang, X. Zhang, B. Yang, Y. Yang, Z. Huang, Y. Wei, RSC Adv. 2014,
obtained. Compound 3 (120 mg, 0.2 mmol) in 10 mL dry THF was
injected into the dark wine solution. The reaction was performed at 70 °C
for 14 h, and then the product was extracted with dichloromethane and
4
, 21588-21592; c) Y. Zhang, Y. Chen, X. Li, J. Zhang, J. Chen, B. Xu,
dried with anhydrous MgSO
4
. After evaporation, the crude product was
X. Fu, W. Tian, Polym. Chem. 2014, 5, 3824-3830; e) A. Han, H. Wang,
R. T. K. Kwok, S. Ji, J. Li, D. Kong, B. Z. Tang, B. Liu, Z. Yang, D. Ding,
Anal. Chem. 2016, 88, 3872-3878.
purified by a silica gel column (dichloromethane/petroleum ether, 2: 1,
1
v/v). H NMR (500 MHz, CDCl
3
) δ 8.37 (dd, J = 6.4, 3.7 Hz, 8H), 7.75 (d,
J = 16.4 Hz, 4H), 7.60 (t, J = 8.7 Hz, 8H), 7.48 – 7.41 (m, 8H), 7.26 (s,
[
8]
9]
a) Y. Yuan, S. Xu, X. Cheng, X. Cai, B. Liu, Angew. Chem. Int. Ed. 2016,
4
4
1
1
H), 7.00 – 6.96 (m, 4H), 6.85 (d, J = 16.4 Hz, 4H), 4.03 (t, J = 6.4 Hz,
5
5, 6457-6461; b) D. Wang, H. Su, R. T. K. Kwok, X. Hu, H. Zou, Q.
Luo, M. M. S. Lee, W. Xu, J. W. Y. Lam, B. Z. Tang, Chem. Sci. 2018, 9,
685-3693.
H), 3.07 (s, 12H), 2.97 (t, J = 7.4 Hz, 4H), 1.90 – 1.77 (m, 8H), 1.58–
+
.50 (m, 8H). MALDI-TOF: m/z: calcd. for (C76
H
76
N
2
O
2
Se
2
): [M] :
3
208.424; found: m/z 1208.081.
[
[
a) J. Zhang, S. Li, F. -F. An, J. Liu, S. Jin, J. -C. Zhang, P. C. Wang, X.
Zhang, C. -S. Lee， X. -J. Liang, Nanoscale, 2015, 7,13503-13510;
10] a) X. Wu, X. Sun, Z. Guo, J. Tang, Y. Shen, T. D. James, H. Tian, W.
Zhu, J. Am. Chem. Soc. 2014, 136, 3579; b) J. H. Atkins, L. J. Gershell,
Nat. Rev. Cancer, 2002, 2, 645.
Acknowledgements
[
11] a) D. He, W. Zhang, H. Deng, S. Huo, Y. -F. Wang, N Gong, L. Deng, X.
This work was supported by the Natural Science Foundation of
China (21835001, 51773080, 21674041, 51573068, 21221063),
Program for Changbaishan Scholars of Jilin Province, Jilin
Province project (20160101305JC), and the “Talents Cultivation
Program” of Jilin University.
-
J. Liang, A. Dong, Chem. Commun. 2016, 52, 14145-14148; b) Q.
Song, X. Wang, Y. Wang, Y. Liang, Y. Zhou, X. Song, B. He, H. Zhang,
W. Dai, X. Wang, Q. Zhang, Mol. Pharmaceutics 2016, 13, 190-201; c)
H. Kasai, T. Murakami, Y. Ikuta, Y. Koseki, K. Baba, H. Oikawa, H.
Nakanishi, M. Okada, M.Shoji, M. Ueda, H. Imahori, M. Hashida,
Angew. Chem. Int. Ed. 2012, 51, 10315-10318; D) Y. Ma, Q. Mou, X.
Zhu, D. Yan, Materials Today Chemistry 2017, 4, 26-39.
Conflict of interest
[
12] a) Y. Wang, D. Liu, Q. Zheng, Q. Zhao, H. Zhang, Y. Ma, J. K. Fallon, Q.
Fu, M. T. Haynes, G. Lin, R. Zhang, D. Wang, X. Yang, L. Zhao, Z. He,
F. Liu, Nano Lett. 2014, 14, 5577-5583; b) Q. Pei, X. Hua, S. Liu, Y. Li,
Z. Xie, X. Jing, Journal of Controlled Release 2017, 254, 23-33; c) W.
Zhang, W. Lin, Q. Pei, X. Hu, Z. Xie, X. Jing, Chem. Mater. 2016, 28,
The authors declare no conflict of interest.
Keywords: diselenide bond • AIEgens • self-assembly •
4440-4446.
bioimaging • drug delivery
[
[
13] Y. -C. Wang, Y. Li, T. -M. Sun, M. -H. Xiong, J. Wu, Y. -Y. Yang, J.
Wang, Macromol. Rapid Commun. 2010, 31, 1201-1206.
[
[
1]
2]
D. Cai; W. Gao, B. He, W. Dai, H. Zhang, X. Wang, J. Wang, X. Zhang,
Q. Zhang, Biomaterials 2014, 35, 2283-2294.
14] a) C. Sun, S. Ji, F. Li, H. Xu, ACS Appl. Mater. Interfaces 2017, 9,
12924-12929; b) X. Zheng, L. Wang, S. Liu, W. Zhang, F. Liu, Z. Xie,
a) E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych,
J. S. Bonifacino, M. W. Davidson, J. Lippincott Schwartz and H. F.
Hess, Science 2006, 313, 1642-1645; b) X. Feng, L. Liu, S. Wang D.
Zhu, Chem. Soc. Rev. 2010, 39, 2411-2419; c) G. Sun, M. Y. Berezin, J.
Fan, H. Lee, J. Ma, K. Zhang, K. L. Wooley, S. Achilefu, Nanoscale
Adv. Funct. Mater. 2018, 28, 1706507.
[
[
15] a) K. N. K. J., Chem. Educ. 1995, 72, 423-424; b) H. J. Reich, R. J.
Hondal, ACS Chem. Biol. 2016, 11, 821-841.
16] J. Xia, F. Li, S. Ji, H. Xu, ACS Appl. Mater. Interfaces 2017, 9, 21413-
21421; b) W. Zhou, L. Wang, F. Li, W. Zhang, W. Huang, F. Huo, H. Xu,
2010, 2, 548-558.
Adv. Funct. Mater. 2017, 27, 1605465; c) H. Xu, W. Cao, X. Zhang, Acc.
Chem. Res. 2013, 46, 1647-1658.
[
3]
a) V. Sokolova and M. Epple, Nanoscale 2011, 3, 1957-1962; b) K. Uno,
T. Sasaki, N. Sugimoto, H. Ito, T. Nishihara, S. Hagihara, T.
Higashiyama, N. Sasaki, Y. Sato, K. Itami, Chem. Asian J. 2017, 12,
[
[
[
17] D. L. Klayman, T. S. Griffin, J. Am. Chem. Soc. 1973, 95, 197-199.
18] L. Yan, Y. Zhang, B. Xu, W, Tian，Nanoscale, 2016, 8, 2471-2487.
233-238; c) S. Mulay, T. Yudhistira, M. Choi, Y. Kim, J. Kim, Y.-J. Jang,
19]
X. Zhang, X. Zhang, B. Yang, L. Liu, J. Hui, M. Liu, Y. Chen, Y. Wei,
S. Jon, D. -G. Churchill, Chem. Asian J. 2016, 11, 3598-3605; d) Y. Li,
W. Liu, H. Zhang, M. Wang, J. Wu, J. Ge, P. Wang, Chem. Asian J.
RSC Adv. 2014, 4, 10060-10066.
[
[
[
20] J. Zhang, S. Ma, H. Fang, B. Xu, H. Sun, I. Chan, W. Tian，Mater.
Chem. Front. 2017, 1, 1422-1429.
2
017, 12, 2098-2103; e) Y. Zhao, C. Shi, X. Yang, B. Shen, Y. Sun, Y.
Chen, X. Xu, H. Sun, K. Yu, B. Yang, Q. Lin, ACS Nano 2016, 10,
856-5863; f) W. Cheng, H. Cheng, S. Wan, X. Zhang, and M. Yin,
21] J. Mei, N. L. C. Leung, R. T. K. Kwok, J. W. Y. Lam, B. Z. Tang, Chem.
Rev. 2015, 115, 11718-11940.
5
Chem. Mater. 2017, 29, 4218−4226; g) S. Zhang, J. Li, J. Wei, M. Yin,
Science Bulletin 2018, 63, 101-107.
22] a) X. Han, X. Song, F. Yu, L. Chen, Chem. Sci. 2017, 8, 6991-7002; b)
X. Han, X. Song, F. Yu, L. Chen, Adv. Funct. Mater. 2017, 27, 1700769,
This article is protected by copyright. All rights reserved.

Chemistry - An Asian Journal
10.1002/asia.201801527
c) M. Gao, F. Yu, C. Lv, J. Chood, L. Chen, Chem. Soc. Rev. 2017, 46,
2237-2271.
[
[
23]
a) J. Ding, F. Shi, C. Xiao, L. Lin, L. Chen, C. He, X. Zhuang, X. Chen,
Polym. Chem. 2011, 2, 2857-2864; b) M. H. Lee, J. H. Han, P. -S.
Kwon, S. Bhuniya, J. Y.Kim, J. L. Sessler, C. Kang, J. S. Kim, J. Am.
Chem. Soc. 2012, 134, 1316-1322.
24] K. Han, Q. Lei, S. B. Wang, J. J. Hu, W. X. Qiu, J. Y. Zhu, W. N. Yin, X.
Luo, X. Z. Zhang, Adv. Funct. Mater. 2015, 25, 2961−2971；b) L. Zeng,
L. Li, T. Li, W. Cao, Y. Yi, W. Geng, Z. Sun, H. Xu, Chem. Asian J.
2014, 9, 2295-2302.
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FULL PAPER
Wenkun Han, Bin Xu*, Wenjing Tian*
Page No. – Page No.
Redox Responsive Fluorescent
nanoparticles based on Diselenide-
containing AIEgens for Cell imaging
and Selective Cancer Therapy
We reported AIEgens containing diselenide bond, which could co-assemble with
prodrug into nanoparticles. The self-assembly driving forces were explored by
molecular dynamics (MD) simulations. The NPs could be cracked in the cancer
intracellular environment that could be applied for bioimaging and cancer therapy.
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