electrically pulsatile responsive drug delivery platform ... · table of contents (toc)...

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.

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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 [email protected]

mailto:[email protected]


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


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


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


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


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


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


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.


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.)


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


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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electrically pulsatile responsive drug delivery platform ... · table of contents (toc)...

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