Aggregation Induced Emission Mediated Controlled Release by Using a Built-In Functionalized Nanocluster with Theranostic Features

Article abstract of DOI:10.1021/acs.jmedchem.5b01622

We report biological evaluation of a novel nanoparticle delivery system based on 1,1,2-triphenyl-2-(p-hydroxyphenyl)-ethene (TPE-OH, compound 1), which has tunable aggregation-induced emission (AIE) characteristics. Compound 1 exhibited no emission in DMS

Full text of DOI:10.1021/acs.jmedchem.5b01622

Article
pubs.acs.org/jmc
Aggregation Induced Emission Mediated Controlled Release by
Using a Built-In Functionalized Nanocluster with Theranostic
Features
‡
∥
‡
,†,‡,§
Zhan Zhou, Cheng Cheng Zhang, Yuhui Zheng, and Qianming Wang*
†Key Laboratory of Theoretical Chemistry of Environment, Ministry of Education, School of Chemistry & Environment, South China
Normal University, Guangzhou 510006, China
‡
School of Chemistry & Environment, South China Normal University, Guangzhou 510006, China
§
Guangzhou Key Laboratory of Materials for Energy Conversion and Storage, Guangzhou 510006, P. R. China
∥
Departments of Physiology and Developmental Biology, University of Texas, Southwestern Medical Center, Dallas, Texas
7
5390-9133, United States
*
S Supporting Information
ABSTRACT: We report biological evaluation of a novel
nanoparticle delivery system based on 1,1,2-triphenyl-2-(p-
hydroxyphenyl)-ethene (TPE-OH, compound 1), which has
tunable aggregation-induced emission (AIE) characteristics.
Compound 1 exhibited no emission in DMSO. In aqueous
media, compound 1 aggregated, and luminescence was
observed. The novel membrane−cytoplasm−nucleus sequen-
tial delivery strategy could induce apoptosis in four different
kinds of cancer cells (including three adherent cell lines and
one suspension cell line). The nanoparticles remained in the
cytoplasm with intense blue emissions, whereas doxorubicin
was observed in the nucleus with striking red luminescence. The nanoassembly was internalized in cells through an energy-
dependent process. Three sorts of chemical inhibitors were used to clarify the endocytosis mechanism based on the AIE type
prodrug. Furthermore, we have developed the first AIE theranostic system where drug targeting and release have been applied in
an animal model.
INTRODUCTION
quenching phenomenon would be a main disadvantage for the
design of biosensors used in on-site detection. To circumvent
these issues, Tang and his team members invented compounds
■
The majority of human cancers are solid tumors. Therapeutic
efficacy relies on delivery of the anticancer drugs to tumor cells.
Chemotherapeutic agents must be transported across vascular
walls into blood and, finally, must enter target cancer cells.
Often penetration, accumulation, and retention of therapeutic
regents in tumor cells are very limited. In order to circumvent
the obstacles, novel nanoplatforms with high loading capacities
and delivery capabilities have been reported.
the small-molecule drugs have been loaded onto the nanoscale
delivery systems and are ready for releasing at particular tumor
sites within physiological environments based on stimulus-
responsive features, such as temperature, magnetic or electric
field, and enzymatic and ultrasound triggers.
Among the above external activations, several studies have
18−20
that emit light upon aggregate formation,
and numerous
1
,2
aggregation-induced emission (AIE) species have now been
2
1,22
synthesized.
Therefore, systems capable of tumor targeting
23,24
3
,4
based on AIE species have been prepared.
Hence, as
described in Figure 1, we developed a self-emissive nanoscale
prodrug in terms of the novel tetraphenylethylene bearing
hydroxyl group (abbreviated as TPE-OH, compound 1). The
anticancer agent doxorubicin (DOX) was formulated with 1 in
stable nanoassemblies (abbreviated as TD NPs).
This new theranostic method has provided an appealing
chance by which the therapeutic process will be connected with
the self-indicating diagnostic imaging. The potential strategy
includes the detection and treatment of the disease at the same
time. It is highly expected to fabricate the apoptosis related
biochemical theranostic system that could finely monitor the
specific response in the situation, but the biological behavior of
AIE and DOX nanoparticles, especially concerning their cellular
5
−10
In this way,
1
1−15
offered deeper insights into drug delivery in a controllable
manner derived from fluorescent signals.
16,17
However, overlap
in emissions between the luminescent dyes and the drug
molecules and quenching due to the biological environment
prohibits the real-time monitoring of the therapeutic agents
when this strategy is used. In addition, the low molecular
weight molecules will automatically accumulate onto the
surfaces of biological structures and the typical concentration
Received: October 16, 2015
©
XXXX American Chemical Society
A
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Figure 1. Representation of functionalized TD NPs used for LL2 tumor therapy in a mice model.
uptake mechanism, is not well understood. Exploring the
principles of how nanodomains could pass through cell
membranes would clarify the complicated pathways and
decrease the risk of toxic effects. Here we used three different
chemical inhibitors (chlorpromazine, cytochalasin D, and
methyl-β-cyclodextran) to investigate the particular forms of
endocytosis, and the basic results contribute to identify cellular
entry modes. The elucidation of the exact transport channels
based on AIE including nano pro-drug has never been reported.
The majority of current nanoscaled drug delivery systems
have been concentrated on the cellular levels and the relations
between independent tumor cells, and chemotherapeutic agents
were emphasized. But the performance of bioassays and therapy
in an animal model seems to be a real challenge, since complex
structures, such as solid tumors, are not a group of individual
cells and they reside in complex microenvironments. The
vascular density and low diffusion rate will be the main drug
transport barriers when the nanoparticles penetrate through the
solid tissue to reach the target cancer cells. With the aim of
studying the effects of drug release in tumor-affected tissues, the
tumor-bearing mice were treated with the promising
fluorescent nanoassembly to assess cancer targeting and organ
distribution. To the best of our knowledge, this is the first
example of drug targeting and release through an AIE based
nanoplatform in vivo.
Figure 2. Photoluminescence spectra of TPE-OH (50 μM) in
DMSO/water mixtures with different concentrations of water.
excitation at 330 nm, an immediate change was observed, and
the emission band located at 465 nm has decreased, since the
drug loading ratio was set as 10% (from blue line to red line in
Figure S2). It is estimated that the adjacent species between
TPE-OH NPs and DOX represented the existence of the
Forster resonance energy transfer (FRET) channel. But the
emission quenching from TPE-OH nanoparticles was also
accompanied by the reduction (from green line to purple line
in Figure S2) in the optical features from DOX (Ex = 477 nm),
indicating that TD NPs could largely increase local DOX
concentration and caused the aggregation induced quenching of
light emissions (peak at 595 nm). To get insight into the FRET
process, the emission of TPE-OH nanoparticles and the
absorption of DOX were evaluated (Figure S3). The good
spectral overlap between the donor and acceptor again
confirmed the effectiveness of FRET.
RESULTS AND DISCUSSION
■
Formulation and photophysical characterization of
an AIE based drug delivery system. The structure of
compound 1 is shown in Figure S1; the compound was
25
synthesized in one step, as previously described. Benzophe-
none was cross-coupled with 4-hydroxyl-phenylbenzophenone
in dried THF under nitrogen atmosphere using Zn powder as
the catalyst to give the target 1,1,2-triphenyl-2-(p-hydrox-
yphenyl)-ethene (compound 1). Before performing the cell
imaging and in vivo therapy, we tried to study whether the non-
benigh solvent (here is water) was able to induce the molecular
aggregations in TPE-OH (denoted as TPE-OH NPs). The
changes in the emission features were expressed by using the
steady-state fluorescence spectra. In DMSO, compound 1
exhibited almost no emission (Figure 2), likely due to the free
An efficient method that provides the concurrent release of
the optical signal changes and therapeutic agent would be
28,29
highly preferable to curing cancers.
It has been discussed
before that the improved drug release rate under lower pH
29,30
values would be desirable in tumor therapy.
Here the
capability of tunable drug delivery of AIE based carriers has
been demonstrated by the activation of the prodrug via the pH-
dependent process. As presented in Figure S4, no discernible
changes in the fluorescence intensities were observed between
neutral and acidic conditions, suggesting that TPE-OH
nanoassembly was maintained almost stable during the release
stage. In the case of DOX (Figure S5), however, a substantial
increase in the red emission has been detected in pH 5.0
solution. These attractive observations provide the possibility
that the majority of drug molecules might reach the tumor
targets due to the lower pH environment and the therapy
efficiency would be enhanced.
26
rotation of the aromatic rings. As the percentage of water was
increased to 99%, a 50-fold enhancement in the blue emission
band located at 465 nm was observed (Figure 2). The turn-on
fluorescent signal was completely consistent with the
observation (from invisible to bright blue) of the solutions
under UV light irradiation (inset of Figure 2). To determine
whether 1 was able to encapsulate an anticancer drug, we used
doxorubicin (DOX) as a model. This positively charged
molecule could be encapsulated into the nanocarriers due to
Morphological studies, stabilities, and structural
information. The microstructures of the aggregated species
27
electrostatic attractions and π−π stacking interactions. Under
B
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were explored by TEM. The individual TPE-OH nanoparticles
were irregularly shaped and had an average diameter of around
stretching vibrations. In the case of TD NPs, the peaks of the
C−H stretching vibration derived from DOX and the typical
bands from TPE-OH were clearly identified and the results
proved that TD NPs were formed. Moreover, the CO
8
0 nm, proving that small fluorophore molecules would
accumulate into nanoclusters in the presence of water (Figure
S6). After the drug molecules were loaded (DOX molar ratio =
−
1
absorption band of DOX shifted from 1730 to 1695 cm ,
indicating that there might exist hydrogen bond interactions
between DOX and TPE-OH. In order to figure out the local
chemical composition of the assembled TD NPs, energy
dispersive X-ray spectroscopy (EDX) was used (Figure S13).
The EDX analysis provided a relative composition of TD NPs,
and the corresponding elements C, N, O, and Cl were given in
1
0%), well-resolved nanospheres with monodispersed features
were achieved and have a uniform diameter of 10 nm (Figure
). The dimensions of these tiny particles are in the desired size
3
Table S1. The NMR spectrum of TD-NPs in DMSO-d was
6
provided in Figure S14.
Cell imaging features and behavior. Although the AIE
effect will allow the uses of concentrated solutions with
luminescence or their suspensions in aqueous environment for
potential sensing purposes, the cases of successful live cell stain
35,36
were very rare due to their poor solubilities.
To evaluate the
feasibility of TPE-OH NPs as a novel optical marker for living
cells imaging, mouse lewis lung carcinoma cells (LL2 cell) were
incubated with AIE molecules (TPE-OH NPs). It is clear that
the carcinoma cells displayed visible blue fluorescence, owing to
the intense emission from TPE-OH NPs, suggesting that
quantities of aggregated particles have been internalized into
the cells (Figure 4). In contrast, no fluorescence could be
Figure 3. TEM image of nanoparticles of TPE-OH (50 μM) and 10%
Dox formed in 99% of water.
range for tumor therapy and will be small enough for cell
31−33
membrane penetration.
This dramatic change in morphol-
ogy during the DOX incorporation process substantiated that
the electrostatic forces contributed to the formation of the
conjugate between donor and acceptor. This could be seen as
an indication of effective delivery of DOX in TPE-OH
nanoparticles. In order to explore the electrostatic forces
between TPE-OH and DOX, we have investigated the zeta
potentials of TPE-OH in the absence or presence of DOX.
Results showed that the surface charge density decreased from
−
22.1 mV to −11.6 mV after DOX was incorporated,
indicating that the electrostatic interaction existed between
TPE-OH and DOX (Figure S7). With a higher concentration
of DOX (molar ratio 15%), structures were nonuniform
(Figure S8). The closer proximity between AIE aggregates and
DOX (compared with independent TPE-OH nanoparticles)
has been evaluated by the photoluminescence studies. It has
been found that the blue emission was severely suppressed in
the DOX concentration experiments and the peak wavelength
slightly blue-shifted (Figure S9). According to the previous
literature, the interactions with drug molecules may benefit for
dispersing large aggregates in aqueous solution and facilitate the
Figure 4. Confocal microscopy images of LL2 cells cultured with TPE-
OH NPs (50 μM), free DOX (5 μM), TD NPs (50 μM), and
costained with Lysotracker Green after a 4-h incubation.
34
growth of small particles. As for the stability of TD NPs, we
measured the over quantum yields (Φ) through an integrating
sphere method under room temperature. It has been found that
the Φ value slightly changed even after 8 days (Figure S10).
The results indicated that TD NPs could keep the assembled
structures relatively stable. In addition, we also studied the
thermostability of TD NPs at different temperatures. As given
in Figure S11, the blue luminescence of TD NPs was
maintained to be stable. At high temperature (60 °C), the
reduction in peak emission intensity was around 10%. The FT-
IR spectra of DOX, TPE-OH, and TD NPs were given in
Figure S12. In the infrared curve of TPE-OH, the peaks at 3055
and 3015 cm− were ascribed to the unsaturated C−H
detected in the control sample. The intracellular and intra-
nuclear distributions of free DOX in LL2 cells were also
observed through confocal laser scanning microscopy images
(Figure 4). After incubation with TD NPs for 4 h, strong blue
luminescence throughout the cell cytoplasm demonstrated that
the substantial cellular uptakes of the regular nanospheres were
possible. Simultaneously, remarkable increases of bright red
emissions were found in the nucleoplasm of the cells treated
with TD NPs relative to those treated with TPE-OH NPs
(Figure 4). The cellular and nuclear uptakes of the new drug
delivery system have been realized in a consecutive cell
membrane-to-nuclear targeting manner. In this way, the
anticancer drug was released from the nanocarrier due to the
intracellular mild acidic conditions and translocated into the
cell nucleus. The utilization of Lysotracker Green for the
staining of lysosomes firmly proved that the location of TPE-
1
−1
stretching vibration. The intense band at 3525 cm was
caused by the O−H stretching vibration. In the curve of DOX,
−1
the peaks at 2981, 2932, and 2843 cm were attributed to the
saturated C−H stretching vibration. The peaks located at 3332
and 1730 cm− were assigned to the O−H and CO
1
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Figure 5. Representative distributions of THP-1 cells costained with Annexin V-APC/7-AAD. Cells were treated with (A) 0.5 μM DOX, (B) 5 μM
DOX, (C) 5 μM TD NPs (0.5 μM DOX), or (D) 50 μM TD NPs (5 μM DOX) for 24 h prior to analysis. Viable cells are negative for both APC and
7
7
-AAD (Q4 region), early apoptotic cells are APC positive and 7-AAD negative (Q3 region), late apoptotic or dead cells are positive, both APC and
-AAD (Q2 region), while the damaged cells are APC negative and 7-AAD positive (located in the region of Q1).
OH NPs was nearly overlapped with lysosomes (Figure 4) and
DOX could be efficiently delivered into nuclei of cancer cells
real-time monitoring of luminescence changes in situ. Actually,
to our knowledge, here is the first example of drug release
visualization through tunable aggregation induced emission
based on living suspension cells.
(Figure 4). The intracellular controlled drug delivery process
lies in the fact that the guest molecules would release
depending on the interactions between TPE-OH NPs with
negative surface charge and cationic DOX. It has been well
accepted that dysregulated pH is a common indication of
cancers, and lower pH environments in tumor cells were
detected. New physiological pH sensitive vehicles hold promise
for the therapeutic purpose of realizing high specificity. Here
the decrease of pH would result in the dissociation of TD NPs,
and the molecular electrostatic interactions were broken.
Therefore, the released DOX from the AIE type carrier was
internalized by tumor cells at lower pH, and improved cancer
therapy efficiency could be achieved. To further explore the
potential of using TD NPs as a simultaneous drug carrier and
imaging sensor, two typical adherent cell lines (human breast
adenocarcinoma, MCF-7, and human cervical cancer cells,
HeLa) were incubated with TPE-OH NPs, free DOX and TD
NPs, respectively (Figures S15 and 16). It can be seen that
intense blue fluorescence signals are collected from the
cytoplasm of cells and red emissions are highly accumulated
in the nuclei in terms of the cell membrane to nuclear
sequential strategy. In a subsequent study, the in vitro imaging
behavior of a suspension cell line (human leukemic monocytic
cells, THP-1) has been investigated (Figure S17). The results
clearly demonstrated that TD NPs could not only be applied
for targeted drug delivery, but also provide the possibility for
In vitro cytotoxicity analysis. The cytotoxicity of TPE-
OH, free DOX, and TD NPs has been studied in the above-
mentioned four cells by MTS assay, as given in Figures S18.
After 24 h of incubation with TPE-OH, cell viabilities were all
well above 90% of their original values, suggesting that TPE-
OH particles have excellent biocompatibility in live cells. In
general, the structural stability of TPE-OH might avoid
disruption of live cells. Moreover, the presence of multiple
aromatic moieties in TPE-OH may contribute to reduce the
undesired toxicity due to the hydrophobic interactions with cell
membranes. Additionally, the tightly packed structures would
bind to lipid membranes with affinity and transmigrate into
cells. However, when these cancer cells were treated with free
DOX and TD NPs, a clear tendency of reduced cell viability
was observed after 24 h. At each DOX dose concentration, the
viability of cells incubated with TD NPs was slightly lower than
that of free DOX molecules. This would be attributed to the
improved DOX delivery efficiency within cancer cells by AIE
edifices.
The DOX-triggered apoptosis process based on different
drug-carrier forms has been explored by the flow cytometry.
THP-1 cells were incubated with free DOX and TD NPs at
various concentrations (Figure 5). Loading of pure DOX and
TD NPs has led to a dramatic increase in the early and later
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Figure 6. Fluorescence detected by flow cytometry in cells cultured with free DOX (1 μM) and TD NPs (10 μM) for 4 h.
apoptosis rates of the cells. In particular, the later apoptosis
percentage reached 68.9%, which was higher than its counter-
part (free DOX) and the result was in agreement with the
previous experiments of the tumor cell viability. This enhanced
therapeutic outcome demonstrated that the current AIE based
delivery system was more efficient than individual DOX
molecules. We further used flow cytometry to investigate the
intracellular DOX concentrations. The collected curves
confirmed that, although the concentrations of DOX and TD
NPs in the culture medium remained the same, more DOX
could be detected in the aforementioned four cancer cells
incubated with TD NPs in contrast to the cells treated with
pure DOX (Figure 6). The results showed that the transport of
drug molecules across the cell membrane was more efficient
when the drug was encapsulated in the nanocarrier.
Cellular entry mechanism. The rational design of
luminescent sensors that can pass through cell membranes
and enter a specific organelle needs a full understanding of the
entry mechanism. Recent work has revealed that the nano-
particles are internalized into cells through two major uptake
channels: passive diffusion and endocytosis. The latter energy-
dependent case would be hindered at low temperature or under
ATP-depleted conditions. As given in Figure S19−23, the
studies performed at 4 °C or after ATP depletion presented
very weak emissions in contrast to the normal conditions,
indicating that the endocytosis pathway is the preferred process
in the cellular internationalization. Since endocytosis takes
many forms, the encapsulation of different inhibitors can
disclose molecular components required for defining modes of
cellular entry. The first chemical inhibitor (chlorpromazine) is a
cationic amphipathic drug that induces the aggregation of
adaptor proteins and clathrin and leads to depletion, thus
blocking clathrin-mediated endocytosis. The results showed it
causes an inhibition rate of around 60.6% on HeLa cells,
suggesting that clathrin-mediated endocytosis was involved in
the possible uptake ways. The second compound that could
consistently inhibit macropinocytosis is cytochalasin D, which
depolymerizes F-actin to suppress the process. An inhibition
rate of 44.3% was achieved, demonstrating that this particular
class of endocytosis (macropinocytosis) could be easily
identified. Finally, we also included methyl-β-cyclodextran,
which removes cholesterol from the plasma membrane and
inhibits lipid raft mediated endocytotic pathways. The treat-
ment of HeLa cells with methyl-β-cyclodextran did inhibit
cellular uptake by approximately 49.2%. In addition, the
systematic studies based on a number of inhibitors in three
other cell lines showed slightly different yet generally similar
results for the inhibition tests (Figures S20−S23). Con-
sequently, it is estimated that clathrin-mediated endocytosis,
macropinocytosis, and lipid raft-mediated endocytosis were all
involved in the internalization mechanism. The selection from a
screen of inhibitors for a targeted biochemical activity based on
an aggregated-induced-emission (AIE) optical probe will
contribute to a deeper understanding of the physiological
functions.
In vivo anticancer efficiency. To determine whether TD
NPs were effective within tumor-affected tissues, a tumor
implantation mouse model was tested to assess the targeting
capability. The experiments were performed by using a C57BL/
6 mice model created subcutaneous inoculation with LL2
cancer cells. Then the extent of tumor growth was monitored.
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In the parallel experiments, a single dose (at day 7) or two
doses (at day 7 and day 14) of free DOX (5 mg/kg) and TD
NPs (5 mg/kg) were intravenously injected into mice and
administered on 18 days in each group. In the mice treated with
5
mg/kg of free DOX, a slight reduction in tumor volume was
observed (Figure 7). However, the inhibitory effects derived
Figure 8. Representative H&E stained images of slices of indicated
organs, including lung, heart, liver, kidney, and spleen, collected from
mice treated with PBS, DOX (10 mg/kg), and TD (10 mg/kg) after
1
8 days.
organ slices, indicating that no appreciable toxic side effects
have been found. Based on our results, doxorubicin formulated
in TPE-OH nanoparticles induced apoptosis in cancer cell lines
and had antitumor effects in vivo. It could effectively deliver
DOX into tumor cells through visualization changes and show
antiproliferative activity in animal models.
Figure 7. Quantitative analysis of the effects of various treatments on
tumor size (top), and photos of mice after various treatments
(
bottom).
EXPERIMENTAL SECTION
Chemicals and materials. Benzophenone, 4-methoxybenzophe-
none, phosphorus tribromide, titanium(IV) chloride, doxorubicin
■
from TD NPs were demonstrated on a significant reduction in
tumor volume compared with control and free DOX. This
confirmed that the AIE-loaded new prodrug really works in
terms of design purpose in vivo as well as in vitro. Since the
treatment of DOX would induce negative responses or the
decrease of white blood cell inside the human body, the total
toxicity of free DOX and TD NPs has been studied. Mice
treated with 10 mg/kg of TD NPs achieved almost equal body
weight to that of control on day 9. At the planned end point
hydrochloride (DOX), NaN , 2-deoxy-D-glucose, chlorpromazine,
3
cytochalasin D, and methyl-β-cyclodextran were purchased from
Sigma-Aldrich. Fetal bovine serum (FBS), antibiotics penicillin (PS),
Dulbecco’s modified medium (DMEM), and RPMI1640 medium were
obtained from Life Technologies. The CellTiter 96 AQueous One
Solution Reagent (MTS kit) was obtained from Promega. Lysotracker
Green and the Annexin V-APC/7-AAD kit were purchased from BD
Biosciences (USA). All the other chemicals were analytical reagents
and used without further purification.
Synthesis of 1,1,2-triphenyl-2-(p-hydroxyphenyl)-ethene
(compound 1). Zinc powder (1.56 g, 24 mmol) and anhydrous
THF (40 mL) were added into a three-necked flask equipped with a
magnetic stirrer under nitrogen atmosphere. The mixture was cooled
(
day 18), the mouse that was administered TD NPs was slightly
heavier than the one treated with free DOX (Figure S24). This
observation leads us to say that this new drug delivery system
has less signs of toxicity compared with free drug. Generally,
mice treated with anticancer drugs would show a weight loss
due to serious side-effects in chemotherapy. Here the
encapsulation of the drugs (free DOX) in the AIE delivery
system would reduce systemic toxicity because of slower release
in body fluid. The sustainable release from the nanovehicle
could prevent the overexposure of DOX drugs to the normal
cells. In addition, the tolerance capability of mice and their
status might be different during the administration of drugs.
Therefore, a gradual weight gain was achieved in the presence
of TD NPs. To provide further evidence for the nanotoxicity of
in vivo therapy, major organs such as lung, heart, liver, kidney,
and spleen from the mice were collected. As shown in Figure 8,
hematoxylin and eosin (H&E) stained images exhibited that no
discernible signal of organ damages were identified from the
to 0 to −5 °C, and 1.3 mL of TiCl (12 mL) was slowly added. The
4
suspension was warmed to room temperature. After 30 min, the
mixture was heated with refluxing for 2.5 h. Then, the above solution
was again cooled to 0 to −5 °C, charged with pyridine (0.5 mL, 6
mmol), and stirred for 10 min. The solution of a mixture of
benzophenone (436 mg, 2.4 mmol) and 4-hydroxybezophenone (475
mg, 2.4 mmol) in 15 mL of THF was added slowly. Then, the reaction
mixture was heated with refluxing for 10 h. Excess of 10% aqueous
K
CO
solution was added to quench the reaction, and product was
2
3
taken up with CH Cl . The organic layer was collected, dried with
2
2
anhydrous NaSO , and concentrated. The crude product was purified
4
1
by column chromatography to obtain the target compound. H NMR
(
8
3
400 MHz, DMSO-d ): δ (ppm) 6.51 (d, J = 8.4 Hz, 2H), 6.74 (d, J =
6
.4 Hz, 2H), 6.92−7.15 (m, 15H); Mass spectrum (ESI-MS): m/z
+
71.2 [M + Na] .
Preparation of TPE-OH NPs (nanoparticles of compound 1)
and TD NPs. A solution of TPE-OH in DMSO was dropwise added
F
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5
into water under vigorous stirring, and TPE-OH NPs were obtained
after 5 min. Similarly, TD NPs were prepared first adding TPE-OH in
DMSO dropwise into water and then adding DOX.
10 cells/well) were seeded in 12-well plates and incubated for 24 h.
Chlorpromazine (10 μg/mL) and cytochalasin D (5 μM), or methyl-
β-cyclodextran (5 mg/mL) were added into the medium and cultured
at 37 °C for 45 min. TD NPs (50 μM) were then added and incubated
for another 2 h. Then, cells were washed with PBS three times,
centrifuged, and resuspended in PBS. The cellular uptake efficiency
was determined using a flow cytometer.
Photophysical studies. Fluorescence emission and excitation
spectra were measured on a Hitachi F-2500. Absolute quantum yields
were detected at room temperature through an integrating sphere
(
Hamamatsu C9920-02 system) method, and the sample was placed
inside the integrating sphere. The excitation light was entered into the
sphere by the optical fiber. When excitation wavelength (330 nm) was
selected, the quantum yield values were calculated automatically by the
software.
In vivo studies. The LL2 implantation experiment was performed
4
0
6
as we described. Briefly, 1 × 10 cells in 200 μL PBS were injected
subcutaneously into the female C57BL/6 mice (4−6 weeks old).
3
When the tumors reached a size of approximately 100 mm (at day 7),
Cell lines and cell culture. Four cancer cell lines, LL2 (mouse
lewis lung carcinoma cell), MCF-7 (human breast adenocarcinoma),
HeLa (human cervical cancer cell), and THP-1 (human leukemic
monocyte cell), were used in these studies. LL2, HeLa, and MCF-7
cells were cultured in Dulbecco’s modified medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics
the mice were divided into several groups for subsequent experiments.
Mice bearing LL2 tumors in different groups were intravenous
injected with free DOX (5 mg/kg, single dose at day 7), free DOX (10
mg/kg, two doses at day 7 and 14), and TD NPs (5 mg/kg, single
dose at day 7), TD NPs (10 mg/kg, two doses at day 7 and 14),
respectively. Tumor size was measured with a digital caliper every
three days, and tumor volume was calculated by (length of tumor) ×
penicillin (PS) and maintained at 37 °C with 5% CO . THP-1 cells
2
2
was incubated in RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS) and 1% antibiotics penicillin (PS) and maintained
at 37 °C with 5% CO2.
(width of tumor) /2. The body weight of mice without tumors was
also monitored. After 18 days, the major organs of those mice were
collected and fixed in 4% formalin for at least 3 days. The tissues were
stained with hematoxylin and eosin (H&E), and observed under a
BX50 bright field microscope (Olympus).
MTS assay. The viability of LL2, HeLa, MCF-7, and THP-1 cells
exposed to TPE-OH, free Dox, and TD was measured with MTS kits.
3
1
The cells (about 5 × 10 cells/well) within replicate 96-well plates
Measurements. H NMR spectra were recorded at 293 K using a
were precultured at standard culture conditions for 1 day. After
removal of the medium, cells were incubated with fresh medium
containing TPE-OH, free Dox, or TD (with various concentration) for
another 24 h. Twenty μL MTS solution was added to each well. After
incubation for 2 h, the absorbance at 490 nm using a Polarsar
microplate reader was recorded.
Varian 400 (400 MHz) with TMS as an internal standard; all chemical
shifts were reported in the standard notation of parts per million. LC-
MS was measured on a Thermo Finnigan LCQ Deca XP Max. FT-IR
spectra were measured using a Shimadzu Prestige-21. TEM was
measured using a JEOL JEM-2100HR transmission electron micro-
scope. Energy dispersive X-ray (EDX) analysis data were measured
based on a scanning electron microscope equipped with an energy-
dispersive spectrometer (S-4800, Hitachi, Japan). The surface charges
of TPE-OH and TD-NPs were measured through dynamic light
scattering (Malvern Zetasizer Nano ZS90).
Flow cytometry and apoptosis analysis. Flow cytometry and
37,38
apoptosis assays were performed as we described.
Briefly, the
above-mentioned four kinds of cells were cultured with free Dox (1
μM) and TD NPs (10 μM) for 2 h, respectively. Then, cells were
washed and resuspended in PBS, and measured by flow cytometry on a
BD Accuri C6 Flow Cytometer. To investigate apoptosis analysis,
untreated cells and cells treated with Dox (0.5 or 5 μM) and TD NPs
CONCLUSIONS
■
As our understanding of different drug delivery systems
increases, it becomes more important to embrace one particular
drug carrier as a new diagnostic tool for a specific mode of
therapy. Here the use of aggregation induced emission type
compounds appears to offer better choices than the conven-
tional fluorophores. Based on our findings, TPE-OH NPs
exhibited very weak fluorescence signals in organic solvents. But
the exposure to aqueous solution induces a 60-fold increase in
the emission intensity. The drug loaded nanoassembly (TD
NPs) could be internalized effectively within four sorts of
cancer cells (including three adherent cell lines and one
suspension cell line). The excellent biocompatibility showed
that this AIE based nanoprobe possessed the potential as a cell
imaging agent and antitumor drug carrier simultaneously. The
identification of energy-dependent uptake and the use of
inhibitors will be valuable as we start to figure out the
physiological functions and mechanisms of cellular entry
derived from a new theranostic system. More importantly, it
allows one to target the drug release and evaluate the
therapeutic efficiency in vivo, which is fundamental in
developing novel responsive materials in clinical cancer
treatment.
(
5 or 50 μM) were both stained with Annexin V-APC/7-AAD. THP-1
5
cells (5 × 10 cells/well) were dispersed within replicated 12-well
microtiter plates and incubated for 24 h at 37 °C with 5% CO . After
2
removal of the medium, cells were incubated with fresh medium (here
fresh medium refers to DMEM supplemented with 10% fetal bovine
serum and 1% antibiotics penicillin or RPMI 1640 medium
supplemented with 10% fetal bovine serum and 1% antibiotics
penicillin) containing Dox (0.5 or 5 μM) and TD NPs (5 or 50 μM)
for another 24 h. After washing with binding buffer twice, centrifuging,
and adding another 100 μL binding buffer, 3 μL of 7-AAD and
Annexin V-PE were added to each sample. Samples were incubated for
1
5 min and analyzed by flow cytometry.
Cell imaging. Cell imaging was performed as we described.
3
9
Briefly, the cells were plated in Lab-Tek chambered cover glass (8-well,
4
Thermo Scientific, USA) with a density of 10 cells/mL and grown to
70% confluency. The cells were washed with medium and incubated in
the fresh medium containing TPE-OH NPs (50 μM), DOX (5 μM),
or TD NPs (50 μM) for another 4 h. Cells were washed three times
with PBS and costained with Lysotracker Green (200 nM for 20 min),
then imaged on a Leica confocal laser scanning microscope (TCS SP2
AOBS).
Intracellular transport mechanism. First, the energy-dependent
uptake was performed with low temperature incubation and ATP
depletion treatments. The four cell lines (LL2, HeLa, MCF-7, and
THP-1) cultured at standard culture conditions were treated with TD
NPs (50 μM) and immediately maintained at 4 °C for 2 h. For the
ATP-depleted environments, cells were preincubated with 2-deoxy-D-
glucose (50 mM) and sodium azide (10 mM) for 30 min at 37 °C;
subsequently, cells were washed with PBS three times and then
incubated with TD NPs (50 μM). The intracellular fluorescence
intensity of TD NPs was detected using flow cytometry. Second, three
different inhibitors (chlorpromazine, cytochalasin D, methyl-β-cyclo-
dextran) were used to determine the modes of endocytosis. Cells (5 ×
ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01622.
Synthesis of TPE-OH; photoluminescence spectra of
TPE-OH NPs, TD NPs, and free DOX; overlap of the
G
DOI: 10.1021/acs.jmedchem.5b01622
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
absorption spectra of DOX and the emission spectra of
TPE-OH; fuorescence spectra of TD NPs when
dispersed in various pH buffers; TEM image of TPE-
OH nanoparticles; fluorescence changes of TPE-OH
when adding different molar ratios of DOX; confocal
microscopy images of MCF-7, HeLa, and THP-1; cell
viability of TPE-OH, free Dox, and TD NPs toward
various cells; study on the mechanism of cell uptake;
body weight changes of mice in the presence of different
treatments. (PDF)
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AUTHOR INFORMATION
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S.; Huang, Z. F.; Wei, Y. Fabrication of cross-linked fluorescent
polymer nanoparticles and their cell imaging applications. J. Mater.
Chem. C 2015, 3, 1854−1860.
(10) Qin, W.; Ding, D.; Liu, J. Z.; Yuan, W. Z.; Hu, Y.; Liu, B.; Tang,
B. Z. Biocompatible nanoparticles with aggregation-induced emission
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Corresponding Author
Tel.: 86-20-39310258. Fax: 86-20-39310187. E-mail address:
qmwang@scnu.edu.cn.
*
Notes
The authors declare no competing financial interest.
7
71−779.
ACKNOWLEDGMENTS
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Responsive fluorescent Bi O @PVA hybrid nanogels for temperature-
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(
Q.W. acknowledges the support from the National Natural
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Guangdong province (Yq2013053), the Guangzhou city
scientific research fund (2014J4100054), the Guangdong
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N a t u r a l S c i e n c e F o u n d a t i o n o f G u a n g d o n g
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ABBREVIATIONS USED
■
(
(
15) Marin, A.; Muniruzzaman, M.; Rapoport, N. Acoustic activation
AIE, aggregated-induced-emission; TPE-OH, 1,1,2-triphenyl-2-
p-hydroxyphenyl)-ethene; TPE-OH NPs, 1,1,2-triphenyl-2-(p-
of drug delivery from polymeric micelles: effect of pulsed ultrasound. J.
Controlled Release 2001, 71, 239−249.
(
S.; Kim, J. S. An activatable theranostic for targeted cancer therapy and
imaging. Angew. Chem. 2014, 126, 4558−4563.
(
C. T.; Neri, D. A small-molecule drug conjugate for the treatment of
carbonic anhydrase IX expressing tumors. Angew. Chem., Int. Ed. 2014,
5
(
Lam, J. W. Y.; Kwok, H. S.; Tang, B. Z. Aggregation-induced emission,
self-assembly, and electroluminescence of 4,4′-bis (1, 2, 2-triphenyl-
vinyl) biphenyl. Chem. Commun. 2010, 46, 686−688.
(19) Hong, Y. N.; Lam, J. W. Y.; Tang, B. Z. Aggregation-induced
emission. Chem. Soc. Rev. 2011, 40, 5361−5388.
(
hydroxyphenyl)-ethene nanoparticles; DOX, doxorubicin hy-
drochloride; TD NPs, 1,1,2-triphenyl-2-(p-hydroxyphenyl)-
ethene doxorubicin hydrochloride nanoparticles; LL2, mouse
lewis lung carcinoma cell; HeLa, human cervical cancer cell;
THP-1, human leukemic monocyte cell; MCF-7, human breast
adenocarcinoma; H&E, hematoxylin and eosin; FBS, fetal
bovine serum; PS, antibiotics penicillin; DMEM, Dulbecco’s
modified medium; ATP, adenosine triphosphate; APC,
allophycocyanin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; 7-
AAD, 7-aminoactinomycin D; TEM, transmission electron
microscope; NMR, nuclear magnetic resonance
16) Bhuniya, S.; Maiti, S.; Kim, E. J.; Lee, H.; Sessler, J. L.; Hong, K.
17) Krall, N.; Pretto, F.; Decurtins, W.; Bernardes, G. J. L.; Supuran,
3, 4231−4235.
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20) Wu, J. S.; Liu, W. M.; Ge, J. G.; Zhang, H. Y.; Wang, P. F. New
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