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Nano Res
1
Electrically pulsatile responsive drug delivery platform
for treatment of Alzheimer’s disease
Li Wu1,2, Jiasi Wang1,2, Nan Gao1, Jinsong Ren1, Andong Zhao1,2, and Xiaogang Qu 1 ()
Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-015-0750-X
http://www.thenanoresearch.com on March 4, 2015
© Tsinghua University Press 2015
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Nano Research
DOI 10.1007/s12274-015-0750-X
TABLE OF CONTENTS (TOC)
Electrically Pulsatile Responsive Drug Delivery
Platform for Treatment of Alzheimer’s Disease
Li Wu1,2, Jiasi Wang1,2, Nan Gao1, Jinsong Ren1, Andong
Zhao1,2, and Xiaogang Qu 1*
1Laboratory of Chemical Biology and Division of
Biological Inorganic Chemistry, State Key Laboratory of
Rare Earth Resource Utilization, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences,
Changchun, Jilin 130022, China
2University of Chinese Academy of Sciences, Chinese
Academy of Sciences, Beijing 100039, China
A novel bifunctional platform by integrating
nonpharmacological and pharmacological cues in one system
for AD treatment has been developed. This
electrically-responsive platform can realize on-demand
controlled drug delivery. Intriguingly, electrochemical
stimulation can treat peripheral nerve injury (PNI) to stimulate
neurite outgrowth. The smart system can effectively inhibit Aβ
aggregate formation, decrease cellular ROS, protect cells from
Aβ-related toxicity and enhance neurite outgrowth.
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2 Nano Res.
Electrically Pulsatile Responsive Drug Delivery
Platform for Treatment of Alzheimer’s Disease
Li Wu1,2
, Jiasi Wang1,2
, Nan Gao1, Jinsong Ren
1, Andong Zhao
1,2, and Xiaogang Qu
1 ()
Received: day month year
Revised: day month year
Accepted: day month year
(automatically inserted by
the publisher)
© Tsinghua University Press
and Springer-Verlag Berlin
Heidelberg 2014
KEYWORDS
drug delivery, graphene,
mesoporous silica,
Alzheimer’s disease,
amyloid β-peptides
ABSTRACT
Metal ions are involved in Aβ aggregate deposition and neurotoxicity via
various processes, including acceleration of Aβ aggregation, disruption of
normal metal homeostasis and formation of reactive oxygen species (ROS).
Although metal chelation is a promising therapeutic strategy for Alzheimer’s
disease (AD), a significant challenge faces the wide use of chelation therapy. It
is hard to differentiate toxic metals associated with Aβ plaques from those
required with normal metal homeostasis. Furthermore, the multifactorial nature
of AD and the current lack of an accepted unitary theory to account for AD
neurodegeneration also restrict AD treatment by taking single therapeutic
strategy. Herein, a novel bifunctional platform by integrating
nonpharmacological and pharmacological cues in one system for AD treatment
has been presented. This electrically responsive drug release platform, based on
conducting polymer polypyrrole (PPy) incorporated with
graphene-mesoporous silica nanohybrids (GSNs) nanoreserviors, could realize
on-demand controlled drug delivery with spatial and temporal control.
Intriguingly, electrochemical stimulation can treat peripheral nerve injury (PNI)
to stimulate neurite outgrowth. The smart system can effectively inhibit Aβ
aggregate formation, decrease cellular ROS, protect cells from Aβ-related
toxicity and enhance neurite outgrowth. Therefore, our work presented here
would promote design of noninvasive remote-controlled multifunctional
systems for AD treatment.
Nano Research
DOI (automatically inserted by the publisher)
Research Article
1 Instructions
Controlled release drug delivery is of critical
importance because the dosage of a particular drug is
often limited to a narrow range, and restricted to a
particular target tissue [1-4]. Delivering drug
molecules to a specialized body compartment can
reduce a possible systemic drug effect, and preserve
the effect of the medications that might otherwise be
destroyed by the body. Thus, controlled release can
significantly improve treatment efficiency. Among
many different types of controlled release, pulsatile
drug delivery systems (PDDS) have attracted much
attention as they can deliver drug molecules at the
right place at the right time and in controlled amount,
thus providing spatial and temporal delivery and
increasing patient compliance [5-7]. PDDS can be
generally classified into: 1) time controlled systems
wherein the drug release is controlled primarily by
the delivery system; 2) stimuli induced PDDS in
which release is controlled by the stimuli, such as pH
[8] or enzymes [9] in drug delivery system; and 3)
externally regulated system where release is
programmed by external stimuli like magnetism [10,
11], ultrasound [12], electrical effect [13, 14] and
irradiation [15]. Among these actively controlled
stimuli, the electrical signal would be the best source
because it is portable and does not need large or
special equipment to trigger it. The signal can also be
easily and on demand controllable, and long cycles
are possible. Furthermore, when a sensor or
microchip system is combined, the feedback and
remote control outside the body is possible. To date,
electrically controlled release has found many
applications, and it is particularly attractive for
implantable devices such as neural electrode arrays
[16-20]. Additionally, according to the previous work,
electrical stimulation is also an efficient therapeutic
treatment for peripheral nerve injury to stimulate
neurite and axon extension or nerve regeneration in
vitro and or in vivo [17, 20-24]. Inspired by the above
discussion, an electrically triggered drug release
platform could be designed by integrating
nonpharmacological and pharmacological cues in
one system for nervous diseases treatment, such as
Alzheimer’s disease (AD).
AD is a chronic progressive, brain disorder resulting
in a loss of memory, reasoning, language skills, and
the ability to care for one’s self [25, 26]. Although the
molecular mechanisms of AD pathogenesis are not
clearly understood owing to its complexity, recent
advances have demonstrated that interactions
between amyloid β peptide (Aβ) and transition metal
ions are associated with the pathophysiology of AD
[27-29]. There is accumulating evidence that metal
ions are involved in Aβ aggregate deposition and
neurotoxicity via various processes, including
modification of aggregation pathway and formation
of reactive oxygen species (ROS) [30-34]. The
emergence of redox-active metals as key players in
AD pathogenesis strongly argues that
amyloid-specific metal-chelating agents and
antioxidants be investigated as possible
disease-modifying agents for treating this horrible
disease [35-37]. Although metal chelation may be a
promising therapeutic strategy for AD, a significant
problem faces the wide use of chelation therapy.
Most of the chelators possess limited ability to
differentiate toxic metals associated with Aβ plaques
from those associated with normal metal homeostasis
[38]. A complementary approach that overcomes this
limitation is the use of prochelator as an agent that
does not interact with metal ions until activated to its
chelator form under specific conditions after they
have entered the target organ [39-43]. In addition, the
development of bifunctional or multifunctional
molecules via a rational structure-based
incorporation approach by integrating an Aβ
interacting framework with a metal chelation moiety
into a single molecule also provides alternative
avenues to pharmacotherapy of AD [44, 45].
Although pharmacotherapy appears to slow aspects
of AD symptom progression, the current limits on
the effectiveness of drugs and the requirement for a
range of options highlight the need of new concept
for the treatment of AD, such as introducing
controlled-drug release system or the
nonpharmacological therapeutic intervention in AD.
In previous reports, we demonstrated that controlled
drug release system have lent a strong impetus to
chelation therapy in AD by overcoming the limitation
mentioned above [46-48]. However, since the nature
of AD is multifactorial and currently there is a lack of
Address correspondence to xqu@ciac.ac.cn
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4 Nano Res.
an accepted unitary theory to account for AD
neurodegeneration, the therapeutic effect by taking
the single therapeutic strategy is restricted. To the
best of our knowledge, no attempt has been made to
address the effect of nonpharmacological and
pharmacological cues integrated in one system on
treatment of AD.
Scheme 1. Electrically controlled pulsatile drug delivery system
for AD therapy. A) Schematic of the drug loading process of
GSNs nanoreserviors and the fabrication process of GSNs/PPy
nanocomposite on the surface of ITO electrode. Drug solution is
filled into the pore channels of GSNs through sonication. Pyrrole
is added to the suspension containing GSNs and
electropolymerization is carried out; B) CQ is released from
GSNs nanoreserviors to surroundings in response to electrical
stimulation; C) CQ can chelate Cu2+ to disassemble Aβ plaques.
Herein, we introduce graphene-mesoporous silica
nanohybrids (GSNs) as the dopant confined in
conducting polymer film for electrically controlled
drug release in AD therapy. Conducting polymers
(e.g., polypyrrole (PPy)) offer the possibility of
control-lable drug administration through electrical
stimulation [14, 49, 50]. However, the application of
conductive polymer in delivery system has been
restricted due to some intrinsic technical barriers. For
instance, the dopant may disturb the biocompatibility,
impedance and charge injection density of the
electrode, the drug loading capacity of a
conventional conducting polymer film is limited, and
the amount of drug release per stimulation is neither
steady nor sustainable. Especially for hydrophobic
drug, the direct doping in polymer backbone as
counter ions through electropolymerization is not
possible. Incorporation of nanomaterials as drug
carriers into the conducting polymer matrix can
enhance drug delivery performance because of their
unique structures and tunable properties [49]. As
shown in Scheme 1, the 2D sandwich like GSNs were
firstly prepared via soft template-assisted reducing
process as nanoreserviors for drug delivery. The drug
loaded GSNs were then encapsulated in PPy films
through electropolymerization. On-demand drug
release from PPy/GSNs film can be realized through
electrical stimulation. As the nanoreserviors, GSNs
provided a higher drug loading capacity for both
hydrophobic and hydrophilic drugs, and also
possessed a sustainable release profile than that of a
conventional PPy film. The system presented here
introduced a new concept to realize the
spatially/temporally controlled drug release and
electrical stimulation for AD therapy.
2 Experimental
2.1 Materials
Graphite was purchased from Sinopharm Chemical
Reagent (Shanghai, China). Tetraethylorthosilicate
(TEOS) and (3-aminopropyl) trimethoxysilane
(APTES) were obtained from Sigma-Aldrich and
used as received. Pyrrole (98%) was purchased from
Sigma-Aldrich, vacuum distilled and stored frozen.
N–cetyltrimethylammonium bromide (CTAB) was
obtained from Alfa Aesar. Hydrazine (85%) was
purchased from Beijing Chemicals Inc. (Beijing,
China). All other reagents were all of analytical
reagent grade and used as received. All aqueous
solutions were prepared with nanopure water (18.2
MΩ cm, Milli-Q, Millipore).
2.2 Apparatus and Characterization
Transmission electron microscopic (TEM) images
were recorded using a FEI TECNAI G2 20
high-resolution transmission electron microscope
operating at 200 KV. SEM images were obtained
with a Hitachi S-4800 FE-SEM. AFM measurements
were performed using a Nanoscope V multimode
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5 Nano Res.
atomic force microscope (Veeco Instruments, USA).
FT-IR characterization was carried out on a BRUKE
Vertex 70 FT-IR spectrometer. The samples were
thoroughly ground with exhaustively dried KBr.
X-ray photoelectron spectroscopy (XPS) spectra
were obtained with an ESCALAB Thermal 250
instrument and monochromatic Mg-Ka (E=1253.6
eV) was used for photoexcitation. N2
adsorption-desorption isotherms were recorded on
a Micromeritics ASAP 2020M automated sorption
analyzer. The samples were degassed at 150 ºC for 5
h. The specific surface areas were calculated from
the adsorption data in the low pressure range using
the BET model and pore size was determined
following the BJH method. UV-vis spectroscopy
was carried out with a JASCO V-550 UV/vis
spectrometer. Cellular imaging was visualized
using an Olympus BX−51 optical system microscope
(Tokyo, Japan). Pictures were taken with an
Olympus digital camera. Electrochemical
measurements were performed with a CHI 660B
Electrochemistry Workstation (CHI, USA). A
three-electrode setup was used with a common
Ag/AgCl reference electrode, an ITO (Indium Tin
Oxide) working electrode and a Pt wire auxiliary
electrode placed in the central buffer solution.
2.3 Synthesis and Chemical Modification of the
GSNs Surface
Graphene oxide (GO) was synthesized from
graphite by modified Hummers method [51]. The
procedure of synthesizing MSGNs was followed by
the literature with some modification [52]. Briefly,
5.8 mL the as synthesized GO (3.8 mg/mL) aqueous
solution was added into 44.2 mL water containing
0.5 g CTAB and 20 mg NaOH, and then
ultrasonically treated for 1 h. After magnetic
stirring for 2 h at 40°C, tetraethylor-thosilicate
(TEOS, 400 µL dissolved in 1.6 mL ethanol) was
slowly added to the above mixture. After reaction
for 12 h, 80 µL of hydrazine was additionally
introduced into the above mixture, and then heated
at 70 °C for 5 h. The obtained product was
centrifuged and washed with warm ethanol for
three times. The product was then mixed with 200
µL APTES in 50 mL ethanol and stirred for 12 h at
80 °C under reflux before centrifugation. Finally, the
product was dispersed in 50 mL acetone stirred at
40 °C for 24 h. The product was collected by
centrifugation and washed by warm ethanol for
three times. The product GSNs-NH2 was then
placed under high vacuum to remove the remaining
solvent in the mesopores. The GSNs-NH2 (50 mg)
was reacted with succinic anhydride (1.00 g) in
N,N-dimetylformamide solution (20 mL) under N2
gas for 8 h with continuous stirring. By doing so,
carboxyl groups were formed onto the GSNs
surface, obtaining GSNs-COOH. The preparation of
MSNs-NH2 and MSNs-COOH were according to
our previous report [53].
2.4 CQ Loading Experiments
The purified GSNs-COOH (100.0 mg) was added in
a solution of CQ (1 mM) in methanol solution under
sonication for 2h and then stirred for 24 h in dark,
following by centrifugation and washing gently
with PBS to remove physisorbed CQ from the
exterior surface of the material. The resulting
precipitate was isolated and dried using
freeze-drying.
2.5 Preparation of Drug-loaded PPy Films
ITO electrode surface was cleaned successively with
acetone, ethanol, and water under sonication for 30
min, respectively. Subsequently, the surface was
immersed in piranha solution for 5 seconds and
then washed with pure water. 0.4 M pyrrole was
added to the solution containing 1.0 mg/mL
CQ-loaded GSNs, and the cleaned ITO electrode
was immersed into the solution for
electropolymerization. The electropolymerization of
pyrrole was carried out at a constant current of
0.00025 A for 600s, and the PPy films incorporated
with drug-loaded nanoparticles were thus formed.
For comparison, PPy films doped with CQ-loaded
MSNs and conventional PPy films without GSNs
but with CQ were electropolymerized with similar
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6 Nano Res.
ways.
2.6 Electrochemically Controlled Drug Release
After electrodeposition, the as-prepared electrodes
were thoroughly washed with PBS solution (10 mM,
pH 7.4) for three times to remove the adsorbed
nanoparticles or drugs. The electrochemically
controlled release of drug from the PPy films was
carried out in an electrochemical cell with 1 mL 2%
(v/v) methanol/PBS (10 mM, pH 7.4). A square wave
electrical stimulation with 50% duty cycle was used
for drug release [49]. The applied potentials were
-2.0 V for 5 s followed by 0.0 V for 5 s (aggressive
stimulation for quick drug release), or -0.5 V for 5 s
followed by 0.5 V for 5s (mild stimulation for
sustainable drug release). The solution with the
released drug was sampled after specifical cycles of
square wave electrical stimulation in each
experiment description. The solution with released
drug was then measured by UV-vis spectrum with
the absorbance band of CQ at 325 nm. Drug
diffusion was tested with similar procedure, but
without actually applying the electrical potential.
2.7 Aβ Preparation
Aβ 1-40 (lot no. U10012) was purchased from
American Peptide and prepared as previously
described [54]. Briefly, the powered Aβ peptide was
first dissolved in 1,1,1,3,3,3 hexafluoro-2-propanol
(HFIP) at the concentration of 1 mg/mL. The
solution was shaken at 4 °C for 2 hours in a sealed
vial for further dissolution and subsequently stored
at −20 °C as a stock solution. Before use, the solvent
HFIP was removed by evaporation under a gentle
stream of nitrogen and then the peptide was
dissolved in water. Cu2+ induced aggregation of Aβ
1–40 was accomplished by mixing an aliquot of the
peptides and CuCl2 at a molar ratio of 1:1 into 10
mM HEPES (150 mM NaCl, pH 6.6) at 37 °C for 24
h.
2.8 Native Polyacrylamide Gel Electrophoresis
Aβ 40 peptide (10 μM) was incubated at 37 °C for
24h under different conditions. Samples (10 mL)
were analysed by 12% native PAGE. Gels were run
in a Tris/glycine system and developed by the
silver-stain method.
2.9 Intracellular Determination of ROS
The generation of reactive oxygen radicals was
monitored using 2’,7’-dichlorofluorescein diacetate
(DCFH-DA), a nonfluorescent compound which
reacts with intracellular free radicals and generating
the fluorescent product dichloro-fluorescein (DCF).
The DCF fluorescence intensity correlates with the
amount of intracellular reactive oxygen radicals. To
perform the experiment, 20 mM DCFH-DA solution
was added to the PC-12 cells and the mixture was
incubated at 37 oC for 1h. The cells were then
washed twice with PBS solution and finally the
fluorescence intensity was monitored by flow
cytometric analysis and fluorescence
spectrofluorometer.
2.10 Cell Toxicity Assays
PC-12 cells (rat pheochromocytoma, American Type
Culture Collection) were cultured in IMDM (Gibco
BRL) medium supplemented with 5% FBS, 10%
horse serum in a 5% CO2 humidified environment
at 37 °C. For the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu
m bromide, Sigma-Aldrich) assay, cells were plated
at a density of 10,000 cells per well on 96-well plates
for 24 h, followed by introduction of Aβ (5 μ M),
CuCl2 (5 μ M), and CQ or CQ-loaded PPy,
PPy/GSNs and PPy/MSNs films with or without
aggressive or mild electrical stimulation for 20 h.
After 48 h, the cells were treated with 10 μL MTT (5
mg mL-1 in PBS) for 4 h at 37 oC and then were lysed
in DMSO for 10 min at room temperature in the
dark. Absorbance values of formazan were
determined at 570 nm with an automatic plate
reader.
2.11 In Vitro Electrical Stimulation of Cells
Differentiation of PC-12 cells into a neural
phenotype was induced by placing cells in
proliferation media (IMDM supplemented with
10% horse serum and 5% fetal bovine serum)
overnight on PPy films coated ITO electrode surface
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7 Nano Res.
with or without incorporation of nanoparticles, then
changing to differentiation media (IMDM
supplemented with 2% horse serum and 50 ng/mL
nerve growth factor). For the investigation of cell
differentiation in the presence of Aβ, Aβ-Cu2+
complex with or without chelator was added into
the differentiation media. Electrochemical
stimulation of PC-12 cells growing on substrates
was undertaken by using a steady potential of 100
mV for 2 h [21]. The integration of electrical
stimulation and drug delivery platform was carried
out by using 50% duty cycle of square wave: -0.5V
for 5 s followed by 0.5 V for 5 s, repeatly for 2h. For
electrochemical stimulation, the modified ITO
electrode served as the anode and a Pt wire placed
at the opposite end of the well as the cathode. The
reference electrode used in the system was a
common Ag/AgCl reference electrode. CHI 660B
Electrochemistry Workstation (CHI, USA) was used
as the source of constant voltage. Cells were
maintained in a CO2 incubator during the period of
electrical stimulation. After electrical stimulation,
the cells were incubated for an additional 48 h. A
seeding density of about 1×104 cells/cm2 was
maintained for all experiments. Cells were fixed
with 2% glutaraldehyde and 2% parafprmaldehyde
in 0.01 M phosphate buffered saline (PBS) for 30
min at 4 oC. Then calcein / propidium iodide (PI)
dye mix in PBS was added to the cells, followed by
10 min of incubation in dark. The cells were then
washed twice with PBS, and viewed with an
Olympus BX-51 optical system microscope (Tokyo,
Japan) with a blue filter. Pictures were taken with
an Olympus digital camera.
3 Results and discussion
As shown in Figure S1, upon hydrazine reduction
treatment and the soft-template removing, the
free-standing GSNs nanosheets were successfully
collected with mesoporous silica coated on the
surface of graphene sheets homogenously. The
characterizations of the as-synthesized nanomaterials
were given in supporting information (Figures S2-S4).
During PPy/GSNs film polymerization, negatively
charged species are loaded into the polymer matrix
to balance positive charges formed on the backbone
of the growing polymer [50]. The GSNs are
negatively charged as a consequence of carboxylic
acid groups formed on their surface through the
conjugation of succinic anhydride, enabling them to
be incorporated into the PPy film as the dopant
agents. The presence of carboxylic groups on the
surface of as-synthesized carboxyl functionalized
GSNs (COOH-GSNs) was supported by the FT-IR
spectrum and zeta potential measurement (Figure S5).
The emerging band at around 1700 cm-1 in the
sample COOH-GSNs can be assigned to C=O
stretching of the carboxyl groups contained within
the attached succinic acid molecules. For
construction of drug delivery system, it is expected
that aqueous solution containing drug can flow into
the inner pores of GSNs, especially with the help of
sonication. To keep the loaded drug inside the GSNs,
an electropolymerized PPy film was used to seal the
pores. When electropolymerization was carried out
in the presence of pyrrole monomer and drug-filled
GSNs, some of the GSNs may be incorporated in the
deposited PPy film. The morphology of polymerized
PPy film containing GSNs on ITO electrode was
characterized by the scanning electron microscope
(SEM) (Figure 1). As illustrated, the GSNs were
distributed within the PPy film uniformly and the
PPy films were grown around the GSNs due to the
high conductivity of GSNs, sealing the opened pores
of GSNs and keeping the drug encapsulated in the
inner cavity of GSNs.
Figure 1. Typical (A) and magnified (B) SEM imges of
PPy/GSNs film.
Electrochemical impedance spectroscopy (EIS) is a
highly sensitive characterization technique, which
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8 Nano Res.
was often used for probing the interfacial properties
at the electrode surface [55]. Here we compared the
EIS spectra of ITO electrodes with different
conductive polymer-nanomaterials film coated
(Figure S6). The introduction of PPy had obviously
decreased the impedance of ITO electrode, which
could be ascribed to the increased effective surface
area of the electrode by the polymer film.
Additionally, further incorporation of GSNs into the
PPy film led to a decreased impedance at all
frequencies (0.1-100 KHz), which was possibly due to
the increase of effective surface area (shown in SEM
image in Figure 1) and the high electrical
conductivity of the incorporated nanocomposites.
Although mesoporous silica nanoparticles (MSNs)
owned highly ordered and unique pore structure as
GSNs (Figure S7), the poor conductivity of MSNs
crippled their performance in electrically controlled
drug release system.
Figure 2. (A) CQ releasing profiles of different controlled drug
release systems for 360 stimulation times. The electric
stimulation applied: 50% duty square wave potential stimulation.
For each stimulation time, the applied stimulus was -0.5 V for 5 s
followed by 0.5 V for 5 s, and the solution was sampled every 10
stimulation times; (B) CQ released from different PPy films
under electric stimulation for 20 h. The electric stimulation
applied: 50% duty cycle of square wave, -0.5 V for 5 s followed
by 0.5 V for 5 s.
According to previous work, Cu2+ can accelerate the
aggregation of Aβ [40, 44, 47]. To investigate the
feasibility of the designed drug delivery system on
reversing Cu2+-induced Aβ aggregation, clioquinol
(CQ) was then chosen as a guest molecule. The metal
chelator, CQ has shown promising results in animal
models and clinical trials, and new generation of
metal ligand-based therapeutics are currently under
development [56]. The aqueous solution containing
CQ can flow into the inner cavity of GSNs by
sonication. Once the PPy film was formed around the
GSNs through electropolymerization, the pores of
GSNs were closed, sealing the guest molecules inside
GSNs. The drug release experiments were carried out
in 2% (v/v) methanol/PBS (10 mM, pH 7.4) with
different electrical stimulus. For many practical drug
delivery systems, “zero-premature release” and
“stimuli-responsive controlled release” of the
pharmaceutical cargo are two very important
prerequisites that impact the therapeutic efficacy and
cytotoxicity of drug delivery. As shown in Figure S8,
compared to the release initiated with electrical
stimulation, the amount of CQ which diffused from
the PPy/GSNs film to the PBS solution was negligible.
Once the dedoping process during negative bias
occurred, the positively charged polymer backbone
was electrochemically reduced to a neutral state
causing the break of the polymer film. Upon water
ingress, drug is released from GSNs after rupturing
the surrounding polymer layer, due to pressure
build-up within the system. The actuation of the
pores of GSNs was successfully realized by changing
the electrochemical state and the dynamic
monitoring of drug release depended on the applied
electrical stimulations. The electrically controlled
drug release of PPy/GSNs with an aggressive
electrical stimulation (repeated stimulation of -2 V
for 5 s followed by 0 V for 5 s) and a milder electrical
stimulation (repeated stimulation of -0.5 V for 5 s
followed by 0.5 V for 5 s) for 12 h was tested (Figure
S8). The drug release profile is nearly linear within
the whole drug delivery process for milder
stimulation, while for aggressive stimulation, it is
curved and levels off (Figure S8). Thus release in the
milder model can be more sustainable, avoiding the
burst effect.
The drug release profile of different systems was
studied using a milder stimulus for up to 360
stimulation times and the results are shown in Figure
2. It is expected that the PPy film with incorporated
nanocontainer can load and release more drug than
the pure PPy film, as the hydrophobic property of
CQ weakened its doping ability in PPy film. The
nano drug container serves as a “nano-train”,
offering higher drug loading capacity and permitting
the direct dopant in PPy film by surface
functionalization. For PPy/MSNs and PPy/GSNs
Figure 3. Determination of the inhibition effects of compounds on the Cu2+-induced formation of Aβ aggregation by AFM: (A) Control
(Aβ), (B) Aβ-Cu2+ complex, (C) Aβ-Cu2+ complex with CQ, (D) Aβ-Cu2+ complex with CQ released from PPy/GSNs film by
immersion, (E) Aβ-Cu2+ complex with CQ released from PPy/GSNs film by electrical stimulation with -0.5 V for 5 s followed by 0.5 V
for 5 s, (F) Aβ-Cu2+ complex with CQ released from PPy/GSNs film by electrical stimulation with -2 V for 5 s followed by 0 V for 5 s.
[Aβ]=10 µM, [Cu2+]=10 µM, [CQ]=20 µM. Buffer: 10 mM HEPES, 150 mM NaCl, pH 6.6.
films, the actuation effect of PPy film upon
electrochemical stimulation may cause the expansion
and contraction of the polymer, temporally
controlling the cap on GSNs and accelerating the
drug release in a sustainable way (Figure 2A). The
total amount of drug released from PPy/GSNs film is
more than that from PPy/MSNs film and PPy film,
indicating the GSNs possess the superiority in
electrically controlled drug delivery system (Figure
2B).
To verify the feasibility of the PPy/GSNs system for
AD therapeutic applications, we investigate the
bioactivity of the released CQ. All the samples,
including Aβ-metal complex or Aβ-metal complex
treated with CQ, were incubated in a weak acidic
buffer (10 mM HEPES, 150 mM NaCl, pH 6.6) at 37oC
for 24h. Using atomic force microscope (AFM) assay,
we first studied the inhibition effect of the CQ
released from PPy/GSNs on the Cu2+-induced Aβ
aggregation. AFM has proven well suited to the
study of Aβ and other amyloidogenic proteins,
because it generates detailed three-dimensional
information at a nanometer scale [54, 57]. After 24 h
incubation at room temperature for Aβ alone, the
majority of Aβ remained as unassembled structures.
The height value for the individual peptide
structures as measured by AFM was about 1.0 nm
(Figure 3A), which agreed the expected size of a
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10 Nano Res.
single Aβ monomer [57]. When incubated in the
presence of Cu2+ for 24 h, Aβ assembled into
predominantly 4-7 nm amorphous oligomeric
structures (Figure 3B). While in the presence of CQ,
the metal-induced Aβ oligomer formation was
inhibited and the height value was approximately
1-1.5 nm (Figure 3C). As for milder stimulation, the
size of oligomeric structures was reduced,
demonstrating a certain amount of CQ was delivered.
The same result was obtained from the sample
treated with CQ released from aggressive electrical
stimulation (Figure 3E-F). However, the simple
immersion of PPy/GSNs film in buffer could not
activate the delivery platform and the majority of Aβ
remained 4-7 nm height assembled structures (Figure
3D). The above results were further supported by
TEM study (Figure S9). To further confirm the
inhibition effect of the controlled-release system on
the Cu2+-induced Aβ aggregation, native
polyacrylamide gel electrophoresis (PAGE) assay was
performed (Figure S10). The obtained results were
consistent with the release experiments that electrical
stimulus could enhance the drug release and realize
better inhibition efficacy.
It has been suggested that the formation of ROS by
Aβ–Cu2+ is another proposed mechanism of AD
pathogenesis, which can cause oxidative stress and
trigger a series of damages of cellular components
such as DNA, lipids, and proteins [58]. Accordingly,
we then investigated the effect of PPy/GSNs system
against ROS. DCFH-DA (dichlorofluorescindiacetate)
was used as a probe for intracellular ROS profiling,
which could diffuse into cells and become fluorescent
DCF (dichlorofluorescin) via oxidation by
intracellular ROS. The changes of intracellular ROS
were monitored by flow cytometry (Figure 4 A-E)
and the quantification was given in Figure 4 F.
Compared to Aβ-Cu2+ untreated control cells, the
relative fluorescence intensity of cells exposed to
Aβ-Cu2+ complex increased to 185% (Figure 4 B),
indicating the increased amount of generated ROS.
The level of ROS obviously decreased for cells
cultured with Aβ-Cu2+ complex in the presence of CQ
or CQ released from PPy/GSNs by electrical
stimulation (Figure 4 C-E). The successful inhibition
Figure 4. Cells were treated with 5 μM Aβ+Cu2+ in the absence
or presence of compounds for 48 h and ROS generation was
measured using DCF fluorescence. Flow cytometry analysis to
monitor the changes of intracellular ROS: (A) control, (B)
Aβ+Cu2+, (C) Aβ+Cu2+ with CQ, (D) Aβ+Cu2+ with CQ released
from PPy/GSNs film by electrical stimulation with -0.5 V for 5 s
followed by 0.5 V for 5 s, (E) Aβ+Cu2+ with CQ released from
PPy/GSNs film by electrical stimulation with -2 V for 5 s
followed by 0 V for 5 s. (F) Quantification of the changes of
intracellular ROS: 1) control, 2) Aβ+Cu2+, 3) Aβ+Cu2+ with CQ,
4) Aβ+Cu2+ with CQ released from PPy/GSNs film by electrical
stimulation with -0.5 V for 5 s followed by 0.5 V for 5 s, 5)
Aβ+Cu2+ with CQ released from PPy/GSNs film by electrical
stimulation with -2 V for 5 s followed by 0 V for 5 s. Control:
Aβ+Cu2+-untreated cells, [Aβ]=5 μM, [Cu2+]=5 μM, [CQ]=10
μM.
of Aβ aggregation and intracellular ROS formation
indicates that this system can be effective free-radical
scavenger. Methylthiazolyl tetrazolium (MTT)
experiments were further carried out to examine the
effects of this delivery system on Aβ-induced
cytotoxicity using rat pheochromocytoma PC-12 cells.
As implied in Figure 5, for cells cultured with
Aβ-Cu2+ complex, the relative cell activities decreased
to 36%. The protection effect of CQ promised the
survival of the cells increased to about 78%. The
electrical stimulus of PPy/GSNs film alone illustrated
no cytotoxicity and it also showed no effect on the
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11 Nano Res.
toxity of Aβ-Cu2+ complex. PPy/GSNs film containing
CQ can prevent cell death in the condition of
electrical stimulation, indicating the decrease of
cytotoxicity was due to the release of CQ. The above
results demonstrated the feasibility of using
PPy/GSNs film as a noninvasive spatially/temporally
controlled drug delivery system for AD treatment.
Figure 5. Protection effects of compounds on Aβ-induced
cytotoxicity of PC-12 cells: (1) control, (2) Aβ, (3) Cu2+, (4)
Ppy/GSNs film without CQ electrically stimulated with -2 V for
5 s followed by 0 V for 5 s, (5) Aβ-Cu2+ complex, (6) Aβ-Cu2+
complex in the presence of PPy/GSNs film without CQ
electrically stimulated with -2 V for 5 s followed by 0 V for 5 s,
(7) Aβ-Cu2+ complex with CQ, (8) Aβ-Cu2+ complex with with
CQ released from PPy/GSNs film by immersion, (9) Aβ-Cu2+
complex with CQ released from PPy/GSNs film by electrical
stimulation with -0.5 V for 5 s followed by 0.5 V for 5 s, (10)
Aβ-Cu2+ complex with CQ released from PPy/GSNs film by
electrical stimulation with -2 V for 5 s followed by 0 V for 5 s.
Control: Aβ+Cu2+-untreated cells. [Aβ]=5 μM, [Cu2+]=5 μM,
[CQ]=10 μM. All assays were conducted under the same
conditions and data were normalized using the results from cells
cultured without Aβ-Cu2+ complex, which acted as a positive
control.
Importantly, in addition to their potential to combine
multiple therapeutic functions into a single platform,
the PPy/GSNs films enabled the integration of
nonpharmacological and pharmacological cues in
one system on the therapy of AD. The
amyloid-protein (Aβ) appears to play an essential
role in the pathogenesis of AD and the assemblies of
Aβ initiate a process leading to neuronal dysfunction
and cell death. For example, soluble Aβ oligomers
extracted from Alzheimer's disease brains potently
impair synapse structure and function [59].
Figure 6. SEM images of PPy film (A), PPy/MSNs film (B) and
PPy/GSNs film (C). Fluorescence microscopy images of
differentiated PC-12 cells without electrical stimulus on PPy
film (D), PPy/MSNs film (E) and PPy/GSNs film (F), with
electrical stimulus on PPy film (G), PPy/MSNs film (H) and
PPy/GSNs film (I).
Scheme 2. The schematic illustration of PC-12 cells incubated
with (A) Aβ-Cu2+ complex, (B) Aβ-Cu2+ complex in presence of
CQ, (C) electrical stimulus, (D) electrical stimulus and Aβ-Cu2+
complex in presence of CQ.
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12 Nano Res.
Electrochemical stimulation is an efficient therapeutic
treatment for peripheral nerve injury (PNI) to
stimulate neurite and axon extension or nerve
regeneration in vitro and/or in vivo. Here, combining
the bioactive surface with electrical stimulation, we
investigated the PC-12 interactions and cytotoxity
cultured on PPy/GSNs film in the presence of Aβ.
The high biocompatibility of PPy/GSNs film is one of
the most important parameters for achieving stable
communication with neurons. As illustrated in
Figure S11, PC-12 cells were capable of not only
adhering to the film but also spreading and
proliferating on the film. After incubation for 72 h,
the cells still had a good viability as evidenced by cell
morphology and fluorescent stain by calcein dye
molecules, indicating that PPy/GSNs is a suitable
material for in vitro nerve cell culture and for further
application of an electric stimulus to modulate the
cell behavior. Fluorescence micrographs of PC-12
cells cultured for 48 h in the presence of NGF on PPy,
PPy/MSNs and PPy/GSNs films with or without
electrical stimulation are shown in Figure 6. It is
apparent from the images that PC-12 cells can attach
and differentiate on PPy (D) and PPy/MSNs (E) films
and even better results are obtained on PPy/GSNs
films (F). Nanostructured materials have been
recently reported with enhanced cell-capturing
efficiency owing to its enhanced local topographic
interactions between the rough substrates (i.e.,
PPy/GSNs) and nanoscale cellular surface
components (e.g., microvilli and filopodia) and result
in vastly improved cell-capture affinity compared to
unstructured (i.e., flat PPy) substrates. The
nanoscaled surface created by selectively adapting
particle functionality, arrangement, and size, enables
control of surface chemistry as well as topography
(Figure 6 A-C). The application of an external
electrical stimulus through the substrate significantly
enhanced differentiation of PC-12 cells and neurites
extension (Figure 6 G-I), and cell spreading for the
stimulated cell population on PPy/GSNs was more
pronounced than that on PPy or PPy/MSNs films.
Furthermore, there were no significant cytotoxic
effects in any of the electrochemically stimulated
groups, demonstrating the good biocompatibility of
electrical stimulation under our experimental
conditions.
Since PPy/GSNs film can be served as an effective
Figure 7. Fluorescence microscopy images of PC-12 cells
cultured for 48 h in the presence of NGF (A) control
(Aβ+Cu2+-untreated cells), (B) incubated with Aβ, (C) incubated
with Cu2+, (D) incubated with Aβ-Cu2+ complex, (E) incubated
with Aβ-Cu2+ complex in the presence of CQ, (F) incubated with
Aβ-Cu2+ complex and electrical stimulus, (G) incubated with
electrical stimulus, (H) incubated with Aβ-Cu2+ complex in the
presence of CQ and electrical stimulus. Viable cells were stained
green with calcein, dead cells were stained red with propidium
iodide (PI). (Scale bars: 50 μm.)
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13 Nano Res.
Figure 8. Fluorescence micaroscopy images of PC-12 cells
cultured for 48 h in the presence of NGF (A) incubated with
Aβ-Cu2+ complex, (B) incubated with Aβ-Cu2+ complex and CQ
released from CQ-loaded PPy/GSNs, (C) incubated with
electrical stimulus by using the same condition as mild
stimulated drug delivery, (D) incubated with Aβ-Cu2+ complex
and electrical stimulus by using the same condition as mild
stimulated drug delivery on CQ-loaded PPy/GSNs film.
platform for electrical stimulation of PC-12 cells, the
question remains as to whether the system can be
used as a nonpharmacological strategy for AD
therapy. To address this question, a series of
experiments were carried out to probe cellular
metabolism, as shown in Scheme 2. As depicted in
Figure 7 A and D, incubation of Aβ-Cu2+ complexes
for 48h induced an evident inhibition of neurite
extention and apoptosis of PC-12 cells, while Aβ and
Cu2+ alone had no cytotoxicity to cells (Figure 7B-C).
CQ protected cells from neurite atrophy and death
(Figure 7E) and electrical stimulus can also protect
cells from apoptotic death (Figure 7F-G). The
combination of nonpharmacological method by
introducing electrical stimulus and pharmacological
means by using CQ not only improved the survival
of cells, but also enhanced neurite outgrowth (Figure
7H). Image analysis was used to quantify the effect of
electrical stimulation on PC-12 differentiation. The
lengths of the outgrowth neurite under different
conditions were shown in Figure S12. It was obvious
that the median neurite length in the electrochemical
stimulation case was about twice that of the negative
controls without electrical stimulation. The same
results were obtained to the PC-12 cells treated by
Aβ-Cu2+ complex and CQ. We further explored the
effect of integrating electrical stimulus and drug
delivery in one system. As shown in Figure 8,
electrical stimulation of cells growing on CQ-loaded
PPy/GSNs film was undertaken using the same
condition as mild stimulated drug release. We first
validated that CQ collected from PPy/GSNs film
through mild stimulated drug release mode could
alleviate the cytotoxicity caused by Aβ-Cu2+
complexes (Figure 8A-B). The electrical stimulation
itself had no cytotoxicity to cells and the cells showed
enhanced neurite outgrowth (Figure 8C). The
electrical stimulation accompanied with drug
delivery had a positive effect on both cell activity and
neurite outgrowth (Figure 8D).
4 Conclusion
In summary, we have demonstrated a novel
electrically controlled AD drug release platform
based on polymer PPy incorporated with GSNs
nanoreserviors for AD treatment. As a nanocontainer,
GSNs may enable loading a variety of drugs or
biomolecules, not confined to the anionic species,
into the conductive polymer film. Owing to the
superior properties of GSNs, on-demand controlled
drug delivery with spatial and temporal control can
be realized, which provides more-effective way with
low toxicity. Additionally, electrochemical
stimulation is also an efficient therapeutic treatment
for peripheral nerve injury (PNI) to stimulate neurite
and axon extension or nerve regeneration in vitro
and/or in vivo. The two-in-one bifunctional platform
can effectively inhibit Aβ aggregate formation,
decrease cellular ROS, protect cells from Aβ-related
toxicity and enhance neurite outgrowth. To the best
of our knowledge, this is the first report that
nonpharmacological and pharmacological cues are
integrated in one system for AD treatment. In the
view of these advantages, the work presented here
may promote the design of noninvasive remote
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14 Nano Res.
controlled and multifunctional systems for AD
treatment.
Acknowledgements
This work was supported by 973 Project
(2011CB936004, 2012CB720602), and NSFC (21210002,
21431007, 91413111, 21402183).
Electronic Supplementary Material: Supplementary
material (Twelve supplementary figures) is available
in the online version of this article at
http://dx.doi.org/10.1007/s12274-***-****-*
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