Chemically exfoliated WS nanosheets can efficiently ... · 300 ml of deionized H 2 O was added to...

Nano Res

1

Chemically exfoliated WS2 nanosheets can efficiently

inhibit amyloid β-peptide aggregation and use for

photothermal treatment of Alzheimer’s disease

Meng Li, Andong Zhao, Kai Dong, Wen Li, Jinsong Ren and Xiaogang Qu ()

Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-015-0821-z

http://www.thenanoresearch.com on May 25, 2015

© Tsinghua University Press 2015

Just Accepted

This is a “Just Accepted” manuscript, which has been examined by the peer-review process and has been

accepted for publication. A “Just Accepted” manuscript is published online shortly after its acceptance,

which is prior to technical editing and formatting and author proofing. Tsinghua University Press (TUP)

provides “Just Accepted” as an optional and free service which allows authors to make their results available

to the research community as soon as possible after acceptance. After a manuscript has been technically

edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP

article. Please note that technical editing may introduce minor changes to the manuscript text and/or

graphics which may affect the content, and all legal disclaimers that apply to the journal pertain. In no event

shall TUP be held responsible for errors or consequences arising from the use of any information contained

in these “Just Accepted” manuscripts. To cite this manuscript please use its Digital Object Identifier (DOI®),

which is identical for all formats of publication.

Nano Research

DOI 10.1007/s12274-015-0821-z

TABLE OF CONTENTS (TOC)

Chemically Exfoliated WS2 nanosheets Can Efficiently

Inhibit Amyloid β-Peptide Aggregation and Use for

Photothermal Treatment of Alzheimer’s Disease

Meng Li, Andong Zhao, Kai Dong, Wen Li, Jinsong Ren

and Xiaogang Qu*

Laboratory of Chemical Biology and State Key Laboratory

of Rare Earth Resource Utilization, Changchun Institute of

Applied Chemistry, University of Chinese Academy of

Sciences, Chinese Academy of Sciences, Changchun, Jilin

130022, China.

Herein, chemically exfoliated 2D WS2 nanosheets were

employed to strongly inhibit Aβ aggregation and dissociate Aβ

fibrils upon NIR irradiation by using the unique high NIR

absorption property of WS2. The low cellular toxic WS2

nanosheets possessed the potential ability to cross blood-brain

barrier (BBB) to overcome the drawback of previously reported

small-molecular-weight Aβ inhibitors.




Meng Li, Andong Zhao, Kai Dong, Wen Li, Jinsong Ren and Xiaogang Qu ()

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

Alzheimer disease;

WS2 nanosheets;

Aβ inhibitors;

photothermal treatment;

Aβ aggregation;

NIR

ABSTRACT

Polymerization of amyloid-β peptide (Aβ) into amyloid fibrils is a critical step

in the pathogenesis of Alzheimer’s Disease (AD). Inhibition of Aβ aggregation

and destabilization of preformed Aβ fibrils have shown promising against AD

and been used in clinic trials. Herein, we demonstrate for the first time the

application of WS2 nanosheets to not only effectively inhibit Aβ aggregation,

but also dissociate the preformed Aβ aggregates upon NIR irradiation. Further

studies indicated that the biocompatible WS2 nanosheets possess the potential

ability to cross blood-brain barrier (BBB) to overcome the drawback of the most

previously reported Aβ inhibitors. Through van der Waals and electrostatic

interactions between Aβ40 and WS2, Aβ40 monomers can selectively adsorb on

the surface of the nanosheet to inhibit the aggregation process of Aβ40.

Intriguingly, by using unique high NIR absorption property of WS2, amyloid

aggregates can be dissolved upon NIR irradiation. These results will promote

biological applications of WS2 and provide new insights into design of new

multifunctional nanomaterials for treatment of AD.

1 Introduction

As the most prevalent age-related

neurodegenerative disease, Alzheimer’s disease

(AD) has been characterized by progressive brain

atrophy/neuronal death which can result in

cognitive and memory impairment [1]. The

accumulation of extracellular amyloid β-peptide

(Aβ) plaques has been demonstrated to be a

pathological hallmark of AD [2-7]. Although the

mechanism of Aβ neurotoxicity is not fully

understood, recent advances have demonstrated

that polymerization of Aβ into amyloid fibrils is a

critical step in the pathogenesis [2-7]. Therefore,

diverse therapeutic strategies that inhibit Aβ

aggregation and dissociate the preformed Aβ fibrils

are being pursued.

For this purpose, a range of β-sheet breaker

Nano Research

DOI (automatically inserted by the publisher)

Research Article

Address correspondence to [email protected]

| www.editorialmanager.com/nare/default.asp

2 Nano Res.

peptides [3-10] and organic molecules [11-15] have

been designed and synthesized to inhibit Aβ

aggregation and toxicity. However, the weak

targeting [16] and poor permeability through the

blood-brain barrier (BBB) and/or toxic side effects

[17,18] lead to only moderate inhibition efficiencies

and weak disaggregation abilities of a majority of

these traditional inhibitors. To overcome these

limitations, nowadays nanomaterials as novel

therapeutic agents have been designed to inhibit Aβ

aggregation and disaggregate Aβ fibrils [19,20].

Graphene oxide (GO), the water-soluble

derivative of graphene, is a two-dimensional (2D)

single atomic layer of carbon atoms arranged in a

honeycomb lattice, possessing unique properties,

such as atomic layered structures, large surface area

and easy functionalization [21-25]. It has been

proposed that GO can adsorb Aβ monomers via

π-π interactions and hydrophobic interactions to

prevent Aβ aggregation [26,27]. Recently, extensive

attention has been focused on the other 2D

nanomaterials, including the transition metal

dichalcogenides (TMDs) (e.g., WS2, etc.) due to their

2D layer structure analogous to graphene [28-35].

Being an ultrathin direct bandgap semiconductor,

WS2 with a layered structure has been used in the

area of nanoelectronics, optoelectronics, and

electrocatalysis [36-38]. Further biological

applications of WS2 nanosheets remain challenging.

Herein, for the first time, WS2 nanosheets were

found to strongly inhibit Aβ fibrillation and

dissociate Aβ fibrils.

As an alternative to the aforementioned materials,

chemically exfoliated WS2 is a mixed phase,

two-dimensional amphiphile that is easy to

synthesize in large batches and is directly

dispersible in water. Compared with GO, the

magnitude of its ζ potential (-23 mV) provides great

colloidal stability in aqueous media. The

physisorption of aromatic (e.g., pyridine, purine,

etc.) and conjugated compounds on the basal plane

of TMDs nanosheet has been reported using either

theoretical calculations or experimental studies

[39-41]. Therefore, we expect that WS2 can adsorb

Aβ monomer via the van der Waals force between

aromatic amino acids and the basal plane of WS2.

The sulfur existed in the nanosheet [42] can perturb

the formation of hydrogen bonding in Aβ

aggregation, which makes a further contribution to

the inhibition effect of WS2. Besides, the favorable

electrostatic interactions between cationic cluster

HHQK of Aβ [43] and negatively charged WS2 [44]

would enhance their binding. In considering these

special properties, WS2 can be a promising

candidate for inhibition of Aβ fibrillation.

On the other hand, owing to the high optical

absorption in the NIR region, a new direction for

TMDs nanosheet is in the field of biomedicine.

Recently, Chou and co-workers [45] and Liu’s

group [46,47] have explored biomedical

applications of MoS2 as near-infrared photothermal

agents for selective photoablation of cancer cells. As

we and others reported previously, local heat

generation (hyperthermia) can act as an effective

means to dissolve the amyloid aggregates of Aβ

[48-50]. The preformed Aβ fibrils can be dissociated

by utilizing TMD nanosheets strong NIR optical

absorption ability to generate local heat upon

low-power NIR laser irradiation. Therefore, WS2

nanosheet can not only effectively inhibit Aβ

aggregation, but also dissociate the preformed Aβ

aggregates upon NIR irradiation (Figure 1).

Photothermal therapy treatment possesses several

remarkable advantages, including their

non-invasive property, and unique site- and

time-specificity. Together with the ability of

nanomaterials to cross the BBB [51,52], WS2 can be a

promising candidate for treatment of AD. To the

best of our knowledge, there is no report of using

WS2-based material for treatment of AD.

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

3 Nano Res.

Figure 1. Schematic representation of WS2 nanosheets with high NIR absorbance used for AD treatment.

2 Experimental

2.1 ceWS2 Synthesis

First, 100 mg of WS2 powder was reacted with 3

ml of n-butyllithium (1.6 M in hexane) under a

nitrogen environment. After 2 day, the mixture was

filtered over Whatman #41 filter and washed 3

times with 100 ml of hexane. To achieve exfoliation,

300 ml of deionized H2O was added to the semi-dry

mixture and was sonicated for 1 hour. The solutions

were then centrifuged and washed with deionized

H2O for 3 times to remove the lithium cations and

the unexfoliated materials. It was then collected and

dialyzed against deionized H2O for 5 days.

2.2 Peptide Preparation

Aβ40 (lot no. U10012) was obtained from

American Peptide and prepared as previously

described. Firstly, the Aβ40 peptide power was

dissolved in 1,1,1,3,3,3-hexafluoropropan-2-ol

(HFIP) with a concentration of 1 mgmL-1. The

dissolved solution was kept in a sealed vial and

shaken at 4 °C for 4 h for further dissolution. After

that, the solution was stored at -20 °C as a stock

solution. Before use, the solvent HFIP was removed

by evaporation under a gentle stream of nitrogen

and the peptide was dissolved in water. Aβ40

self-aggregation was accomplished by incubating

the water solution in aggregation buffer (10 mM

Tris in 150 mM NaCl, pH 7.3) at 37 °C for 7 days.

2.3 ThT Fluorescence Measurements

The kinetics of Aβ40 aggregation was monitored

by ThT fluorescence assay. ThT is an

amyloid-specific dye that exhibits enhanced

fluorescence intensity upon binding to Aβ40 fibrils,

instead of Aβ40 monomers. When added to

samples containing β-sheet-rich aggregates, it

exhibits an excitation maximum at 444 nm and

enhanced emission at 482 nm. The reaction can be

completed within 1 min in an aqueous environment.

Fluorescence measurements were carried out using

a JASCO FP6500 spectrofluorometer. The

fluorescence signal was recorded between 460 and

650 nm; 10 nm slits were used for both emission

and excitation measurements. In inhibition

experiment, the samples of Aβ40 peptide (50 μM)

with or without various concentrations of WS2 were

incubated at 37 °C for 7 days. At different times,

aliquots of each sample were taken for fluorescence

measurements. The concentration of Aβ40 used for

measurements was kept at 1 μM, and the ThT

concentration was 10 μM. In disaggregation

experiment, preformed Aβ40 fibrils were treated

with 40 μg/mL WS2 nanosheets and irradiated for

5min. Then aliquots of the Aβ40 solution were

taken for fluorescence measurements. The peptide

concentration used for measurements was 5 μM,

and the ThT concentration was 10 μM.

2.4 Circular dichroism (CD) measurements

The samples were measured in 10 mM Tris buffer

(pH 7.3) after incubation at 37 for 7 days. CD

spectra were collected at 37 with a JASCO J-810

spectropolarimeter using a 1 mm path length quartz

cell. The parameters were controlled as 0.1 nm

intervals, 4 seconds response, and each sample was

an average of three scans in a speed of 5 nmmin-1

over the wavelength range from 200 nm to 250 nm.

2.5 Attenuated Total Reflection Fourier-Transform

Infrared (ATR-FTIR) Spectroscopy


4 Nano Res.

For ATR-FTIR assay, peptides dissolved in Tris

buffer (Tris in 150 mM NaCl, pH 7.3) at a

concentration of 50 μM, treated with or without

WS2 nanosheets after 7 days of incubation, were

lyophilized and resuspended at a concentration of

50 μM in D2O. ATR-FTIR spectra were recorded

using a Nicolet 6700 infrared spectrometer. Spectra

of 128 scans were taken with a spectral resolution of

2 cm-1. The ATR out-of compartment accessory

consists of a liquid jacketed Piketech ATR

flow-through cell with a trapezoidal Ge-crystal

(Piketech, Madison, WI, 80 × 10 × 4 mm³, angle of

incidence: 45°). The ATR flow-cell was tempered to

25 °C. Spectra were processed using GRAMS

software (Thermo Electron). In this study, the final

concentration of Aβ40 and WS2 nanosheets were 50

μM and 40 μg/mL.

2.6 Atomic Force Microscopy

For the atomic force microscopy (AFM)

measurements, samples were diluted with

deionized H2O to yield a final concentration of 1

μM. Then the sample (20 μL) was applied onto

freshly cleaved muscovite mica and allowed to dry.

Data were acquired in the tapping mode on a

Nanoscope V multimode atomic force microscope

(Veeco Instruments, USA).

3 Results and discussion

3.1 Synthesis and Characterization of the WS2

Nanosheets

The WS2 nanosheet was synthesized according to

the Morrison method [53-55] which broke the weak

interlayer forces in bulk WS2 through Li ion

insertion and ultrasonication. As shown in X-ray

photoelectron spectroscopy (XPS), the peaks at

163.4 and 162.2 eV corresponded to the S2p1/2 and

S2p3/2 orbital of divalent sulfide ions (Figure 2B).

Besides, the W peaks shown in Figure 2A located at

38.9, 35.5, and 33.4 eV were assigned to W5p3/2,

W4f5/2, and W4f7/2, respectively. The energy

positions of these peaks indicated a W valence of +4,

indicating the formation of pure WS2 phase. In

addition, atomic force microscopy (AFM) (Figure

2C) demonstrated that the WS2 separated well with

an average topographic height of ～1.7±0.2 nm and

mean longest diagonal of 200 nm (n=50 sheets). The

relative small size made them possible to penetrate

the BBB.

Figure 2. XPS spectra of ceWS2 (A) W4f, (B) S2p. (C) AFM images and height profiles of the corresponding AFM topographies of typical nanosheets of WS2.

3.2 Effect of the WS2 Nanosheets on the Kinetics

of Aβ40 Fibrillation Process

To examine the influence of WS2 nanosheets on

the fibril formation of Aβ40 (Figure 3A), incubation

solutions of the Aβ40 with and without WS2 were

prepared. The kinetics of fibrillation was monitored

by a commonly used thioflavin T (ThT) assay. ThT,

an extrinsic fluorescent dye, is able to bind to

amyloid fibrils; upon binding, its fluorescence

intensity increases [56,57]. When fresh Aβ40 alone

incubated at 37 , ThT fluorescence as a function of

incubation time showed a sigmoidal shape (Figure

3B). This result was consistent with the

nucleation-dependent polymerization model.

However, in the presence of WS2, ThT fluorescence

did not increase, which indicated that Aβ40


5 Nano Res.

amyloid formation was suppressed. We also

conducted a control experiment to clarify that the

fluorescence of ThT was not influenced by the

addition of WS2 (Figure S1). Interestingly, after

binding with Aβ40 monomers, the ζ potential of

WS2 was changed (Figure S2). To investigate

whether WS2 nanosheets inhibit Aβ40 aggregation

by adsorbing monomers on the surface, tyrosine

intrinsic fluorescence and AFM assays were applied

to analyze the effect. AFM images (Figure S3)

clearly showed that Aβ40 monomers could be

adsorbed on the surfaces of WS2 nanosheets.

Tyrosine fluorescence signals of Aβ40 peptides

were acquired after titration with different

concentrations of WS2 nanosheets. Figure S4A

revealed a marked quenching of the tyrosine

fluorescence signal when Aβ40 was mixed with the

WS2 nanosheets, which meant that the large surface

area of WS2 sheets can adsorb and bind Aβ40

monomers to quench the tyrosine fluorescence

signal. The amount of Aβ40 monomers bound to

WS2 nanosheets was determined via calculation of

the difference of absorbance at 276 nm between the

Aβ40 solution before and after incubation with WS2,

which was defined as 436.96 μmol/g WS2 (Figure

S4B). A significant body of data has indicated

trapping the monomers and/or blocking the

growing oligomer ends on the surface of

nanomaterials could disturb the monomer-critical

nuclei equilibrium, thereby decreasing their

solution concentration and interfering with their

elongation to form fibrils [19,58,59]. In order to

check whether TMDs nanosheets with similar

structures could inhibit Aβ40 self-assembly and

demonstrate the superiority of WS2 over GO, we

examined the inhibition effects of MoS2 and GO

nanosheets. MoS2 sheets with an average

topographic height of ～ 1.6±0.4 nm and mean

longest diagonal of 200 nm (Figure S5A) were

synthesized according to the same method as WS2.

While GO obtained from graphite powder via a

modified Hummers’ method [25] possessed an

average topographic height of ～1.5±0.3 nm and

mean longest diagonal of 200 nm (Figure S5B).

Figure S5C clearly indicated that MoS2 exhibited

comparable inhibition effect on Aβ40 aggregation

with WS2. Furthermore, both of them can decrease

ThT fluorescence in a dose-dependent manner

(Figure S5D). Interestingly, when compared the

inhibition effects of WS2 on Aβ40 aggregation with

GO, WS2 showed a stronger inhibition effect than

GO under the same experimental conditions from

the results of ThT fluorescence assay (Figure S5D)

and CD spectra (Figure S5E). The enhanced

inhibition effect of WS2 could be attributed to the

large excess sulfur in the nanosheets [42] by

forming hydrogen bonding between peptides and

WS2 nanosheets.

Inhibition effect on Aβ40 aggregation was further

studied by AFM (Figure S6A and S6B). AFM has

been widely used to study the morphology of Aβ40

amyloid fibrils. The sample of Aβ40 alone formed

typical unbranched Aβ40 amyloid fibrils longer

than 1 μm. In the presence of the WS2, there was

almost no obvious aggregates or fibrils observed.

As shown in Figure S6, Aβ40 monomers can

selectively attach on the surface of WS2 to inhibit

the aggregation process.

Aβ oligomers, protofibrils and fibrils all share the

common β-sheet structure which drives Aβ

aggregation and toxicity. Circular dichroism studies

clearly indicated that WS2 could inhibit structural

transition from native Aβ40 random coil to β-sheet

conformation in solution (Figure 3C). As a control

experiment, the presence of WS2 nanosheets in the

Aβ40 monomer solution could not change the Aβ40

random coil structures (Figure S6C). For further to

study the structures of Aβ40 on the surface of WS2

nanosheets, we applied the surface-sensitive

attenuated total reflection Fourier-transform

infrared (ATR-FTIR) spectroscopy technique [60-62]

to be able to monitor the amyloid formation in the

presence of WS2. As shown in Figure 3D, after Aβ40

alone incubated for 7 days, formation of


6 Nano Res.

β-sheet-rich amyloid fibrils was characterized by

their specific amide I subband which appeared at

approximately 1623 cm-1. When WS2 nanosheets

were included in the buffer, no such aggregation

subband was observed. Rather, broad IR bands at

about 1640-1645 cm-1 appeared, which were

characteristic of a large contribution of disordered

conformations arising from the presence of

monomeric and small oligomeric Aβ40 particles in

solution.

Figure 3. (A) Inhibition of Aβ40 aggregation by WS2 nanosheets. (A) Schematic representation of WS2 nanosheets used for inhibiting Aβ40 aggregation. (B) Fibrillation kinetics of Aβ40 as monitored by the development of thioflavin T binding in the absence or presence of WS2 nanosheets. The concentration of Aβ40 was 50 μM. The data points shown are the mean values ± SEM from three independent experiments. (C) CD spectra of Aβ40 in the absence or presence of WS2 nanosheets. The samples were measured in 10 mM Tris buffer (pH 7.3) after incubation at 37 for 7 days. (D) ATR-FTIR spectra of Aβ40 in the absence or presence of WS2 nanosheets. In this study, the final concentrations of Aβ40 and WS2 nanosheets were 50 μM and 40 μg/mL, respectively.

3.3 The Hyperthermic Effects of WS2 on

Dissociating the Aβ Fibrils

Having established the inhibition effect of the

WS2 nanosheets, we next investigated the ability of

the novel nanosheet to dissociate the Aβ40

aggregates upon NIR irradiation. Similar to GO,

WS2 exhibited a high optical absorption (Figure S7).

To verify the potential of using WS2 in

photothermal therapy, the WS2 solution was

exposed to an 808 nm NIR laser with water as the

control. In marked contrast to the water sample, the

WS2 solution showed a rapid increase of

temperature when exposed to the laser within a

short time. Furthermore, the solution of WS2

exhibited a concentration-dependent (from 10 to 40

μgmL-1) and radiant energy-dependent (from 0.5 to

2 W cm-2) photothermal heating effect (Figure S8).

Motivated by the strong NIR optical absorption

property of WS2, we then carried out a

photothermal study on Aβ40 aggregation (Figure

4A). Aβ40 fibrils in aggregation buffer (10 mM Tris,

150 mM NaCl, pH 7.3) were incubated with WS2 for

20 min at 37 to maximize the targeting effect of

WS2 and Aβ40 fibrils. The presence of Aβ40 fibrils

enhanced the fluorescence of ThT. However, after

we exposed the solution to a laser with a power

density of 1 Wcm-2 to irradiate the WS2-Aβ40

aggregates, the fluorescence signal of ThT

decreased gradually following the increase of

irradiation time, indicating that the amount of Aβ40

aggregates was diminished by the local heat

generated by WS2 upon NIR irradiation (Figure S9).

In marked contrast, for the irradiation of Aβ40

fibrils alone or the WS2 with Aβ40 aggregates

without laser irradiation, the fluorescent signals

remained almost unchanged (Figure 4B),

demonstrating that neither the NIR laser irradiation

alone nor WS2 by itself can affect the Aβ40 structure

in a short time under our experiment condition. To

demonstrate the ability of WS2 for dissociating

Aβ40 aggregates upon NIR irradiation more clearly,

CD spectroscopy was employed to measure the

secondary structure change (Figure 4C). According

to the computer fit results, after photothermal

treatment, the content of random-coil had an

increase of 22.1% (from 21.2% to 43.3%), while the

β-sheet decreased from 54.8% to 22.4%. The

decreased content of β-sheet conformation

supported that Aβ fibrils were dissociated after

photothermal treatment.


7 Nano Res.

The same results were also observed by using

AFM. When incubated with WS2, the morphology

of the Aβ40 fibrils remained almost unchanged

illustrating that WS2 could hardly alter the assembly

of Aβ40 (Figure 4D). However, after irradiation

with NIR laser, numerous small, relatively

amorphous aggregates were observed in the WS2

treated Aβ40 samples, demonstrating the excellent

efficacy of WS2 to disaggregate the preformed Aβ40

aggregates upon NIR laser irradiation (Figure 4E).

In contrast, without WS2, exposure to the laser alone

could not change the morphology of the amyloid

fibrils (Figure S10). These results further supported

the above results and indicated that our design can

effectively dissociate Aβ40 fibrils.

Figure 4. The influence of the photothermal effect of WS2 nanosheets on Aβ40 disaggregation. (A) Schematic representation of WS2 nanosheets used for dissociating Aβ40 aggregation. (B) The influence of the photothermal effect of WS2 nanosheets on Aβ40 aggregation monitored by fluorescence spectroscopy. The signals were collected after irradiation for 5 min. The concentrations of Aβ40 and WS2 nanosheets were 50 μM and 40 μg/mL, respectively. (C) CD spectra of Aβ40 fibrils in the presence of WS2 nanosheets treated with or without NIR irradiation. The final concentrations of Aβ40 and WS2 nanosheets were 50 μM and 40 μg/mL, respectively. (D) The morphology of Aβ40 fibrils in the presence of WS2 nanosheets. (E) The morphology of Aβ40 fibrils in the presence of WS2 nanosheets under laser irradiation.

It is well known that Aβ accumulated in

cerebrospinal fluid (CSF) is a diagnostic and

therapeutic target for AD [50]. Therefore, it is

important to perform the experiment in CSF in

order to determine whether WS2 can work well in a

biological matrix which contains various other

proteins. As shown in Figure 5, WS2 could not only

effectively inhibit Aβ40 aggregation (Figure 5A) but

also dissociate the preformed Aβ40 aggregates

upon NIR irradiation (Figure 5B) even in CSF.

To show whether other proteins can also be

adsorbed by WS2, we took bovine serum albumin

(BSA) as the example of proteins. As shown in Fig.

S11, BSA could associate with WS2 with an

efficiency of 0.63 g/g WS2 to form a protein corona

on the surface of WS2. Although the interactions

between WS2 and Aβ were not specific, the protein

coated WS2 (WS2-BSA) nanosheets still possessed

the ability to inhibit Aβ aggregation via binding

and adsorbing Aβ monomers, which were clearly

demonstrated by the fluorescence titration, ThT

assay and AFM study (Figure S12). It has been

widely reported that formation of the protein

corona can largely define the biological identity of

the nanomaterials [26]. However, our study clearly

demonstrated that the ability of WS2 to inhibit Aβ

aggregation was not affected by adsorbing other

proteins on the surface.

Figure 5. The influence of WS2 on Aβ aggregation monitored by ThT assay in mice CSF. (A) The dose-dependent inhibition effects on Aβ40 aggregation of WS2. The concentration of Aβ40 was 50 μM. (B) The influence of the photothermal effect of WS2 nanosheets on Aβ40 fibrils monitored by fluorescence spectroscopy. The concentrations of Aβ40 fibrils and ThT were 5 μM and 10 μM, respectively.


8 Nano Res.

3.4 The WS2 Nanosheets Rescue Aβ40-Induced

Cytotoxicity

Since WS2 nanosheets can inhibit Aβ40

aggregation, disaggregation of the amyloid

aggregates upon laser irradiation, the question

arose as to whether the nanosheets can be used to

block Aβ40-mediated cellular toxicity. To

demonstrate this question, we used PC12 cells to

perform

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu

m bromide (MTT) assay to probe cellular

metabolism. Figure 6 showed that Aβ40 fibrils (5

μM) led to a decrease of 44% in cellular reduction of

MTT (fAβ40 sample). However, treatment of the

cells with Aβ40 which was pre-incubated with 4

μg/mL WS2 for 7 days increased the survival of the

cells to about 80% within the experimental errors

(Aβ40-WS2 samples). On the other hand, even after

the Aβ40 fibrils preformed, treatment of the cells

with Aβ40 fibrils in the presence of WS2 under NIR

laser irradiation for 5 minutes could effectively

decrease the cytotoxicity of Aβ40 (fAβ40-WS2-NIR

sample). In order to decrease the side toxicity effect

of WS2 on PC12 cells upon irradiation, we used the

lower power density of laser (0.5 W/cm2) to carry

out this experiment. Furthermore, WS2 can prevent

Aβ40-induced cell death in a dose dependent

manner (Figure 6). Aβ40 fibrils treated with WS2 in

the absence of NIR laser irradiation (fAβ40-WS2

samples) or Aβ40 untreated with WS2 under NIR

laser irradiation (fAβ40 sample), however, did not

increase the cell viability. As depicted in Figure S13,

WS2 nanosheets themselves with or without NIR

irradiation did not affect the survival of PC12 cells

at low dose of less than 20 μg/mL and WS2

incubated with cells did not affect the output of the

MTT assay under the same conditions as Aβ40.

Furthermore, the effect of WS2 on MTT was ruled

out by the unchanged signal in presence of WS2

(Figure S14).

Figure 6. Effect of WS2 on the cell toxicity of Aβ40. Cell viability was determined using the MTT method and data points shown are the mean values ± standard error of the mean (SEM) from four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Control: Aβ40 untreated cells, [Aβ40] = 5 μM.

Figure 7. (A) Schematic illustration for WS2 decreasing Aβ40 aggregates-induced cytotoxicity. (B) Measurements of 5(6)-CF leakage from liposome induced by Aβ40 aggregates (10 μM) at 4 h, 12 h and 24 h in the absence or presence of incubation with 8 μg/mL WS2 nanosheets.


9 Nano Res.

In order to clarify the mechanism of WS2 reduced

the Aβ40-induced cytotoxicity, fluorescent leakage

assay was performed. Previous studies suggested

that nerve cell of AD patients were somehow killed

through Aβ-induced damage of the cell membrane

[63,64]. Herein, we took the liposome to mimic the

cell membrane [65]. As shown in Figure 7, with the

addition of Aβ40 aggregates, the leaked

5(6)-carboxyfluorescein (5(6)-CF) [66] from the

liposome was increased with increasing the

incubation time. In contrast, the leakage was

decreased significantly when Aβ40 was

co-incubated with WS2 in advance (Figure 6).

Furthermore, introduction of the Aβ40 aggregates

which was treated with NIR irradiation in the

presence of WS2 to the liposome also caused a

decrease of 5(6)-CF leakage (Figure S15A). We

conducted a control experiment to clarify that

leaked 5(6)-CF would not be quenched by the

added WS2 nanosheets (Figure S15B). These results

demonstrated that WS2 could effectively prevent

Aβ40-induced damage of the cell membrane.

3.5 The Ability of WS2 Nanosheets to Cross the

BBB

As suitable candidates for AD treatment, the

multifunctional nanosheets should cross BBB. To

determine whether WS2 can passively accumulate

in the brain of living animals, we used ICP-MS to

measure the amount of WS2 in the cerebrospinal

fluid after intraperitoneal injection for 6 hours. A

significant level of tungsten was found in the

cerebrospinal fluid (CSF) of the mouse that treated

with WS2 compared to the control group. The

efficiency of WS2 accumulation in the cerebrospinal

fluid was about 0.53 ± 0.05 % (Table 1), indicating

WS2 possessed the potential ability to cross BBB.

Similar with other nanomaterials, the accumulation

of the nanosheets in the CSF may occur via

endocytosis of the nanosheets by the brain capillary

endothelial cells followed by nanosheets

transcytosis through these cells [67,68]. Critically,

the higher X-ray attenuation and shortening the

longitudinal relaxation time by tungsten [69-72] can

endow WS2 nanosheets with excellent X-ray

computed tomography imaging capabilities. These

results further supported that WS2 could act as

promising therapeutic agents and contrast agents

for AD treatment and brain tumor diagnosis.

Table 1 The amount of WS2 accumulation in the cerebrospinal

fluid of the mouse treated with or without the nanosheets.

Samples treated with WS2 Control samples

ICP results

(ng/mL-1)

Amounts

(%)

ICP results

(ng/mL-1)

Amounts

(%)

1 26.16 0.47 1.987 0.036

2 31.31 0.57 2.348 0.043

3 29.65 0.54 1.398 0.025

4. Conclusions

In conclusion, WS2 nanosheet can effectively

inhibit Aβ40 aggregation. Through van der Waals

and electrostatic interactions between Aβ40 and

WS2, Aβ40 monomers can selectively adsorb on the

surface of the nanosheet. More importantly, with

the unique high NIR absorption property of WS2,

WS2 can also dissociate the preformed Aβ40 fibrils

upon NIR irradiation. Compared with traditional

small-molecular-weight Aβ inhibitors, WS2

nanosheets can cross the BBB which overcomes the

drawbacks of unstable small chemicals or peptides.

Furthermore, the hyperthermic effects of WS2 have

been considered important and useful in clinical

treatment with reduced side effects. Our results

would accelerate biomedical applications of WS2

and provide new insights into design of new

multifunctional nanomaterials for treatment of AD.

Acknowledgements

This work was supported by 973 Project


10 Nano Res.

(2011CB936004, 2012CB720602), and NSFC (21210002,

91213302, 21431007, 91413111). References [1] Rauk, A. The chemistry of Alzheimer's disease. Chem.

Soc. Rev. 2009, 38, 2698-2715.

[2] Jakob-Roetne, R.; Jacobsen, H. Alzheimer's disease: from

pathology to therapeutic approaches. Angew. Chem. Int.

Ed. 2009, 48, 3030-3059.

[3] Scott, L. E.; Orvig, C. Medicinal inorganic chemistry

approaches to passivation and removal of aberrant metal

ions in disease. Chem. Rev. 2009, 109, 4885-4910.

[4] Gaggelli, E.; Kozlowski, H.; Valensin, D.; Valensin, G.

Copper homeostasis and neurodegenerative disorders

(Alzheimer's, prion, and Parkinson's diseases and

amyotrophic lateral sclerosis). Chem. Rev. 2006, 106,

1995-2044.

[5] Hamley, I. W. The amyloid beta peptide: a chemist's

perspective. Role in Alzheimer's and fibrillization. Chem.

Rev. 2012, 112, 5147-5192.

[6] Faller, P.; Hureau, C.; Berthoumieu, O. Role of metal ions in

the self-assembly of the Alzheimer's amyloid-β peptide.

Inorg. Chem. 2013, 52, 12193-12206.

[7] Taniguchi, A.; Sasaki, D.; Shiohara, A.; Iwatsubo,

T.; Tomita, T.; Sohma, Y.; Kanai, M. Attenuation of the

aggregation and neurotoxicity of amyloid-β peptides by

catalytic photooxygenation. Angew. Chem. Int. Ed. 2014,

53, 1382-1385.

[8] Tjernberg, L. O.; Näslund, J.; Lindqvist, F.; Johansson,

J.; Karlström, A. R.; Thyberg, J.; Terenius, L.; Nordstedt,

C. Arrest of beta-amyloid fibril formation by a

pentapeptide ligand. J. Biol. Chem. 1996, 271,

8545-8548.

[9] Takahashi, T.; Mihara, H. Peptide and protein mimetics

inhibiting amyloid beta-peptide aggregation. Acc. Chem.

Res. 2008, 41, 1309-1318.

[10] Frydman-Marom, A.; Rechter, M.; Shefler, I.; Bram,

Y.; Shalev D. E.; Gazit, E. Cognitive-performance

recovery of Alzheimer's disease model mice by

modulation of early soluble amyloidal assemblies. Angew.

Chem. Int. Ed. Engl. 2009, 48, 1981-1986.

[11] Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons,

M. R.; Ambegaokar, S. S.; Chen, P. P.; Kayed, R.; Glabe,

C. G.; Frautschy, S. A.; Cole, G. M. Curcumin inhibits

formation of amyloid beta oligomers and fibrils, binds

plaques, and reduces amyloid in vivo. J. Biol. Chem. 2005,

280, 5892-5901.

[12] Barnham, K. J.; Kenche, V. B.; Ciccotosto, G. D.; Smith, D.

P.; Tew, D. J.; Liu, X.; Perez, K.; Cranston, G.

A.; Johanssen, T. J.; Volitakis, I.; Bush, A. I.; Masters, C.

L.; White, A. R.; Smith, J. P.; Cherny, R. A.; Cappai; R.

Platinum-based inhibitors of amyloid-beta as therapeutic

agents for Alzheimer's disease. Proc. Natl. Acad. Sci.

U.S.A. 2008, 105, 6813-6818.

[13] Ehrnhoefer, D.E.; Bieschke, J.; Boeddrich, A.; Herbst,

M.; Masino, L.; Lurz, R.; Engemann, S.; Pastore,

A.; Wanker, E. E. EGCG redirects amyloidogenic

polypeptides into unstructured, off-pathway oligomers.

Nature Struct. Mol. Biol. 2008, 15, 558-566.

[14] Necula, M.; Kayed, R.; Milton, S.; Glabe, C. G. Small

molecule inhibitors of aggregation indicate that amyloid

beta oligomerization and fibrillization pathways are

independent and distinct. J. Biol. Chem. 2007, 282,

10311-10324.

[15] Nie, Q.; Du, X.G.; Geng, M. Y. Small molecule inhibitors

of amyloid β peptide aggregation as a potential

therapeutic strategy for Alzheimer's disease. Acta

Pharmacol. Sin. 2011, 32, 545-551.

[16] Ouberai, M.; Dumy, P.; Chierici, S.; Garcia, J. Synthesis

and biological evaluation of clicked curcumin and clicked

KLVFFA conjugates as inhibitors of beta-amyloid fibril

formation. Bioconjugate Chem. 2009, 20, 2123-2132.

[17] Liu, L.; Xu, K.; Wang, H.; Tan, P. K.; Fan,

W.; Venkatraman, S. S.; Li, L.; Yang, Y. Y. Self-assembled

cationic peptide nanoparticles as an efficient

antimicrobial agent. Nat. Nanotechno. 2009, 4, 457-463.

[18] Pan, H.; Soman, N. R.; Schlesinger, P. H.; Lanza, G.

M.; Wickline, S. A. Cytolytic peptide nanoparticles

('NanoBees') for cancer therapy. Nanomed

Nanobiotechnol 2011, 3, 318-327.

[19] Cabaleiro-Lago, C.; Quinlan-Pluck F.; Lynch I.; Lindman


11 Nano Res.

S.; Minogue A. M.; Thulin E.; Walsh D. M.; Dawson, K.

A.; Linse, S. Inhibition of amyloid beta protein

fibrillation by polymeric nanoparticles. J. Am. Chem. Soc.

2008, 130, 15437-15443.

[20] Yoo S. I.; Yang, M.; Brender, J. R.; Subramanian, V.; Sun,

K.; Joo, N. E.; Jeong, S. H.; Ramamoorthy, A.; Kotov, N.

A. Inhibition of amyloid peptide fibrillation by inorganic

nanoparticles: functional similarities with proteins. Angew.

Chem. Int. Ed. 2011, 50, 5110-5115.

[21] Song, Y.; Qu, K.; Zhao, C.; Ren, J.; Qu, X. Graphene oxide:

intrinsic peroxidase catalytic activity and its application

to glucose detection. Adv. Mater. 2010, 22, 2206-2210.

[22] Dikin, D. A.; Stankovich, S.; Zimney, E. J.; Piner, R.

D.; Dommett, G. H.; Evmenenko, G.; Nguyen, S.

T.; Ruoff, R. S. Preparation and characterization of

graphene oxide paper. Nature 2007, 448, 457-460.

[23] Sun, X.; Liu, Z.; Welsher, K.; Robinson, J. T.; Goodwin,

A.; Zaric, S.; Dai, H. Nano-Graphene Oxide for Cellular

Imaging and Drug Delivery. Nano Res. 2008, 1, 203-212.

[24] Huang, X.; Yin, Z.; Wu, S.; Qi, X.; He, Q.; Zhang, Q.; Yan,

Q.; Boey, F.; Zhang, H. Graphene-based materials:

synthesis, characterization, properties, and applications.

Small 2011, 7, 1876-1902.

[25] Huang, X.; Qi, X.; Boey, F.; Zhang, H. Graphene-based

composites. Chem. Soc. Rev. 2012, 41, 666-686.

[26] Mahmoudi, M.; Akhavan, O.; Ghavami, M.; Rezaee,

F.; Ghiasi, S. M. Graphene oxide strongly inhibits

amyloid beta fibrillation. Nanoscale 2012, 4, 7322-7325.

[27] Yu, X.; Wang, Q.; Lin, Y.; Zhao, J.; Zhao, C.; Zheng, J.

Structure, orientation, and surface interaction of

Alzheimer amyloid-β peptides on the graphite. Langmuir

2012, 28, 6595-6605.

[28] Xiao, J; Choi D.; Cosimbescu, L.; Koech, P.; Liu,

J.; Lemmon, J. P. Exfoliated MoS2 Nanocomposite as an

Anode Material for Lithium Ion Batteries. Chem. Mater.

2010, 22, 4522-5424.

[29] Eda, G.; Fujita, T.; Yamaguchi, H.; Voiry, D.; Chen,

M.; Chhowalla, M. Coherent atomic and electronic

heterostructures of single-layer MoS2. ACS Nano 2012, 6,

7311-7317.

[30] Han, J. H.; Lee, S.; Cheon, J. Synthesis and structural

transformations of colloidal 2D layered metal

chalcogenide nanocrystals. Chem. Soc. Rev. 2013, 42,

2581-2591.

[31] Huang, X.; Zeng, Z.; Zhang, H. Metal dichalcogenide

nanosheets: preparation, properties and applications.

Chem. Soc. Rev. 2013, 42, 1934-1946.

[32] Zeng, Z.; Yin, Z.; Huang, X.; Li, H.; He, Q.; Lu, G.; Boey,

F.; Zhang, H. Single-layer semiconducting nanosheets:

high-yield preparation and device fabrication. Angew.

Chem. Int. Ed. 2011, 50, 11093-11097.

[33] Yin, Z.; Li, H.; Li, H.; Jiang, L.; Shi, Y.; Sun, Y.; Lu,

G.; Zhang, Q.;, Chen, X.; Zhang, H. Single-layer MoS2

phototransistors. ACS Nano 2012, 6, 74-80.

[34] Shaw, J. C.; Zhou, H.; Chen, Y.; Weiss, N. O.; Liu, Y.;

Huang, Y.; Duan, X. Chemical vapor deposition growth of

monolayer MoSe2 nanosheets. Nano Res. 2014, 7,

511-517.

[35] Kwon, H.; Choi, W.; Lee, D.; Lee, Y.; Kwon, J.; Yoo, B.;

Grigoropoulos, C. P.; Kim, S. Selective and localized

laser annealing effect for highperformance flexible

multilayer MoS2 thin-film transistors. Nano Res. 2014, 7,

1137-1145.

[36] Voiry, D.; Yamaguchi, H.; Li, J.; Silva, R.; Alves, D.

C.; Fujita, T.; Chen, M.; Asefa, T.; Shenoy, V. B.; Eda,

G.; Chhowalla, M. Enhanced catalytic activity in strained

chemically exfoliated WS2 nanosheets for hydrogen

evolution. Nature Materials 2013, 12, 850-855.

[37] Elías, A. L.; Perea-López, N.; Castro-Beltrán,

A.; Berkdemir, A.; Lv, R.; Feng, S.; Long, A. D.; Hayashi,

T.; Kim, Y. A.; Endo, M.; Gutiérrez, H. R.; Pradhan, N.

R.; Balicas, L.; Mallouk, T. E.; López-Urías, F.; Terrones,

H.; Terrones, M. Controlled synthesis and transfer of

large-area WS2 sheets: from single layer to few layers.

ACS Nano 2013, 7, 5235-5242.

[38] Bhandavat, R.; David, L.; Singh, G. Synthesis of

Surface-Functionalized WS2 Nanosheets and Performance

as Li-Ion Battery Anodes. J. Phys. Chem. Lett. 2012, 3,

1523.

[39] Zhu, C.; Zeng, Z.; Li, H.; Li, F.; Fan, C.; Zhang, H.


12 Nano Res.

Single-layer MoS2-based nanoprobes for homogeneous

detection of biomolecules. J. Am. Chem. Soc. 2013, 135,

5998-6001.

[40] Heckl, WM.; Smith, D. P.; Binnig, G.; Klagges,

H.; Hänsch, T. W.; Maddocks, J. Two-dimensional

ordering of the DNA base guanine observed by scanning

tunneling microscopy. Proc. Natl. Acad. Sci. U.S.A. 1991,

88, 8003-8005.

[41] Moses, P. G.; Mortensen, J. J.; Lundqvist, B. I.; Norskov, J.

K. Density functional study of the adsorption and van der

Waals binding of aromatic and conjugated compounds on

the basal plane of MoS(2). J. Chem. Phys. 2009, 130,

104709.

[42] Füchtbauer, H. G.; Tuxen, A. K.; Moses, P. G.; Topsøe,

H.; Besenbacher, F.; Lauritsen, J. V. Morphology and

atomic-scale structure of single-layer WS2 nanoclusters.

Phys. Chem. Chem. Phys. 2013, 15, 15971-15980.

[43] Geng, J.; Li, M.; Ren, J.; Wang, E.; Qu X.

Polyoxometalates as inhibitors of the aggregation of

amyloid β peptides associated with Alzheimer's disease.

Angew. Chem. Int. Ed. 2011, 50, 4184-4188.

[44] Vathsala, K.; Venkatesha, T. V.; Ranganatha, S.; Kumar, M.

K. P. Zn-WS2 Nanocomposite Coatings on Mild Steel:

Electrochemical Aspects. Synth. React. Inorg. M. 2012,

42, 779-785.

[45] Chou, S. S.; Kaehr, B.; Kim, J.; Foley, B. M.; De,

M.; Hopkins, P. E.; Huang, J.; Brinker, C. J.; Dravid, V. P.

Chemically exfoliated MoS2 as near-infrared

photothermal agents. Angew. Chem. Int. Ed. 2013, 52,

4160-4164.

[46] Liu, T.; Wang, C.; Gu, X.; Gong, H.; Cheng, L.; Shi, X.;

Feng, L.; Sun, B.; Liu, Z. Drug Delivery with PEGylated

MoS2 Nano-sheets for Combined Photothermal and

Chemotherapy of Cancer. Adv. Mater. 2014, 26,

3433-3440.

[47] Liu, T.; Shi, S.; Liang, C.; Shen, S.; Cheng, L.; Wang, C.;

Song, X.; Goel, S.; Barnhart, T. E.; Cai, W.; Liu, Z. Iron

oxide decorated MoS2 nanosheets with double

PEGylation for chelator-free radiolabeling and

multimodal imaging guided photothermal therapy. ACS

Nano 2015, 9, 950-960.

[48] Kogan, M. J.; Bastus, N. G.; Amigo, R.; Grillo-Bosch,

D.; Araya, E.; Turiel, A.; Labarta, A.; Giralt, E.; Puntes, V.

F. Nanoparticle-mediated local and remote manipulation

of protein aggregation. Nano Lett. 2006, 6, 110-115.

[49] Adura, C.; Guerrero, S.; Salas, E.; Medel, L.; Riveros,

A.; Mena, J.; Arbiol, J.; Albericio, F.; Giralt, E.; Kogan,

M. J. Stable conjugates of peptides with gold nanorods

for biomedical applications with reduced effects on cell

viability. ACS Appl. Mater. Interfaces 2013, 5, 4076-4085.

[50] Li, M.; Yang, X.; Ren, J.; Qu, K.; Qu, X. Using graphene

oxide high near-infrared absorbance for photothermal

treatment of Alzheimer's disease. Adv. Mater. 2012, 24,

1722-1728.

[51] Rozhkova, E. A.; Ulasov, I.; Lai, B.; Dimitrijevic, N.

M.; Lesniak, M. S.; Rajh, T. A high-performance nanobio

photocatalyst for targeted brain cancer therapy. Nano Lett.

2009, 9, 3337-3342.

[52] Krol, S.; Macrez, R.; Docagne, F.; Defer, G.; Laurent,

S.; Rahman, M.; Hajipour, M. J.; Kehoe, P.

G.; Mahmoudi, M. Therapeutic benefits from

nanoparticles: the potential significance of nanoscience in

diseases with compromise to the blood brain barrier.

Chem. Rev. 2013, 113, 1877-1903.

[53] Joensen, P.; Frindt, R. F.; Morrison, S. R. Single-layer

MoS2. Mater. Res. Bull. 1986, 21, 457-461.

[54] Eda, G; Yamaguchi, H.; Voiry, D.; Fujita, T.; Chen,

M.; Chhowalla, M. Photoluminescence from chemically

exfoliated MoS2. Nano Lett. 2011, 11, 5111-5116.

[55] Matte, H. S.; Gomathi, A.; Manna, A. K.; Late, D. J.; Datta,

R.; Pati, S. K.; Rao, C. N. MoS2 and WS2 analogues of

graphene. Angew. Chem. Int. Ed. 2010, 49, 4059-4062.

[56] Yu, H.; Li, Meng.; Liu, G.; Geng, J.; Wang, J.; Ren,

J.;Zhao, C.; Qu, X. Metallosupramolecular complex

targeting an α/β discordant stretch of amyloid β peptide.

Chem. Sci. 2012, 3, 3145-3153.

[57] Geng, J.; Li, M.; Wu, L.; Ren, J.; Qu, X. Liberation of

copper from amyloid plaques: making a risk factor useful

for Alzheimer's disease treatment. J. Med. Chem. 2012,

55, 9146-9155.


13 Nano Res.

[58] Skaat, H.; Chen, R.; Grinberg, I.; Margel, S. Engineered

polymer nanoparticles containing hydrophobic dipeptide

for inhibition of amyloid-β fibrillation.

Biomacromolecules 2012, 13, 2662-2670.

[59] Cabaleiro-Lago, C.; Quinlan-Pluck, F.; Lynch, I.; Dawson,

K. A.; Linse, S. Dual effect of amino modified

polystyrene nanoparticles on amyloid β protein

fibrillation. ACS Chem. Neurosci. 2010, 1, 279-287.

[60] Evers, F.; Jeworrek, C.; Tiemeyer, S.; Weise, K.; Sellin,

D.; Paulus, M.; Struth, B.; Tolan, M.; Winter, R.

Elucidating the mechanism of lipid membrane-induced

IAPP fibrillogenesis and its inhibition by the red wine

compound resveratrol: a synchrotron X-ray reflectivity

study. J. Am. Chem. Soc. 2009, 131, 9516-9521.

[61] Seeliger, J.; Evers, F.; Jeworrek, C.; Kapoor, S.; Weise,

K.; Andreetto, E.; Tolan, M.; Kapurniotu, A.; Winter ,R.

Cross-amyloid interaction of Aβ and IAPP at lipid

membranes. Angew. Chem. Int. Ed. 2012, 51, 679-683.

[62] Mossuto, M. F.; Bolognesi, B.; Guixer, B.; Dhulesia,

A.; Agostini, F.; Kumita, J. R.; Tartaglia, G. G.; Dumoulin,

M.; Dobson, C. M.; Salvatella, X. Disulfide bonds reduce

the toxicity of the amyloid fibrils formed by an

extracellular protein. Angew. Chem. Int. Ed. 2011, 50,

7048-7051.

[63] Saha, A.; Mondal, G.; Biswas, A.; Chakraborty, I.; Jana,

B.; Ghosh, S. In vitro reconstitution of a cell-like

environment using liposomes for amyloid beta peptide

aggregation and its propagation. Chem. Commun. 2013,

49, 6119-6121.

[64] Guo, L.; Salt, T. E.; Luong, V.; Wood, N.; Cheung,

W.; Maass, A.; Ferrari, G.; Russo-Marie, F.; Sillito, A.

M.; Cheetham, M. E.; Moss, S. E.; Fitzke, F. W.; Cordeiro,

M. F. Targeting amyloid-beta in glaucoma treatment. Proc.

Natl. Acad. Sci. U.S.A. 2007, 104, 13444-13449.

[65] Zhang, M.; Mao, X.; Wang, C.; Zeng, W.; Zhang, C.; Li,

Z.; Fang, Y.; Yang, Y.; Liang, W.; Wang, C. The effect of

graphene oxide on conformation change, aggregation and

cytotoxicity of HIV-1 regulatory protein (Vpr).

Biomaterials 2013, 34, 1383-1390.

[66] Delcea, M.; Sternberg, N.; Yashchenok, A. M.; Georgieva,

R.; Bäumler, H.; Möhwald, H.; Skirtach A. G.

Nanoplasmonics for dual-molecule release through

nanopores in the membrane of red blood cells. ACS Nano

2012, 6, 4169-4180.

[67] Leyva-Gómez, G.; Cortés, H.; Magaña, J. J.; Leyva-García,

N.; Quintanar-Guerrero, D.; Florán, B. Nanoparticle

technology for treatment of Parkinson's disease: the role

of surface phenomena in reaching the brain. Drug Discov.

Today 2015, doi: 10.1016/j.drudis.2015.02.009.

[68] Bramini, M.; Ye, D.; Hallerbach, A.; Raghnaill, M. N.;

Salvati, A.; Åberg, C.; Dawson, K. A. Imaging approach

to mechanistic study of nanoparticle interactions with the

blood-brain barrier. ACS Nano 2014, 8, 4304-4312.

[69] Hemley, R. J.; Mao, H.; Shen, G.; Badro, J.; Gillet, P.;

Hanfland, M.; Häusermann, M. X-ray Imaging of Stress

and Strain of Diamond, Iron, and Tungsten at Megabar

Pressures. Science 1997, 276, 1242-1245.

[70] Smith, M. F.; Gilland, D. R.; Coleman, R. E.; Jaszczak, R.

J. Quantitative imaging of iodine-131 distributions in

brain tumors with pinhole SPECT: a phantom study. J.

Nucl. Med. 1998, 39, 856-864.

[71] Cheng, L.; Liu, J.; Gu, X.; Gong, H.; Shi, X.; Liu,

T.; Wang, C.; Wang, X.; Liu, G.; Xing, H.; Bu, W.; Sun,

B.; Liu, Z. PEGylated WS(2) nanosheets as a

multifunctional theranostic agent for in vivo dual-modal

CT/photoacoustic imaging guided photothermal therapy.

Adv. Mater. 2014, 26, 1886-1893.

[72] Dong, K.; Liu, Z.; Liu, J.; Huang, S.; Li, Z.; Yuan, Q.; Ren

J.; Qu X. Biocompatible and high-performance amino

acids-capped MnWO4 nanocasting as a novel

non-lanthanide contrast agent for X-ray computed

tomography and T(1)-weighted magnetic resonance

imaging. Nanoscale 2014, 6, 2211-2217.


Nano Res.

Electronic Supplementary Material




Meng Li, Andong Zhao, Kai Dong, Wen Li, Jinsong Ren and Xiaogang Qu1()

Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)

Materials and Chemicals.

WS2 powder, n-butyllithium and hexane were purchased from Aladdin. All other chemicals were

purchased from Sigma-Aldrich and used as supplied. Bovine Serum Albumin (BSA) was obtained

from Sangon Biotechnology (Shanghai, China). Nanopure water (18.2 MΩ; Millpore Co., USA)

was used for the preparation of all solutions throughout the experiments.

Characterization of Interaction of WS2 with Aβ40 and BSA Proteins

A 200 μL amount of BSA solution (1 mgmL-1) was mixed with 80μg WS2, and then the mixture was

incubated for 6 h at 37 °C. For Aβ40, the WS2 solution (80 μg/mL, 100μL) was mixed with Aβ40

monomer solution (200 μM, 100μL) at 37 °C for 6 h. Finally, the nanosheets were centrifuged and

washed with a Tris buffer. All of the supernatant was collected for UV/vis absorbance measurement.

Through calculation of the difference of absorbance at 276 nm between the Aβ40 or BSA solution

before and after incubation, the Aβ40 or BSA bound efficiency was determined as 1.89 g/g WS2 or

0.63 g/g WS2, respectively.


Nano Res.

Permeability measurements

Liposome was prepared using egg phosphatidylcholine (egg-PC) and cholesterol with molar

fractions of 55:45. The large unilamellar vesicles (LUVs) were obtained from the multilamellar

vesicles through extruded ten times through two stacked polycarbonate membrane filters (pore size:

400 nm and 100 nm) using a Mini-Extruder (Avanti). 5(6)-carboxyfluorescein (5(6)-CF) was

encapsulated into liposome as a solute marker, and the 5(6)-CF leakage from the liposome was

measured by using JASCO FP-6500 spectrofluorometer. For inhibition experiment, Aβ40 peptide

(50 μM) was incubated with or without WS2 (40 μgmL-1) at 37 °C for 7 days. Then the aged Aβ40

with the final concentration of 10 μM were added directly to incubate with liposome. While for the

disaggregation experiment, the sample of aged Aβ40 with the final concentration of 10 μM was

mixed with WS2 (8 μgmL-1), which followed by irradiated using an 808 nm NIR laser at a power

density of 0.5 Wcm-2 for 5 min. Immediately, after the irradiation, the liposome were treated with

the irradiated samples.

Cytotoxicity assay

For the inhibition experiment, Aβ40 peptide (50 μM) was incubated with or without different

concentrations of WS2 (20 μgmL-1 or 40 μgmL-1) at 37 °C for 7 days. PC12 cells (rat

pheochromocytoma, American Type Culture Collection) were cultured in IMDM (Iscove-modified

Dulbecco medium, Gibco BRL) medium supplemented with 5% FBS, 10% horse serum in a 5%

CO2 humidified environment at 37 °C. For the MTT (Sigma-Aldrich) assay, cells were plated at a

density of 10 000 cells per well on 96-well plates for 24 h, and then Aβ40 peptides (50 μM) that had

been aged with or without various concentrations of WS2 (20 μgmL-1 or 40 μgmL-1) were


Nano Res.

dispensed into the PC12 cells. The final concentration of Aβ40 treated with cells was 5 μM. For the

disaggregation experiment, the IMDM medium was replaced by a dispersion of WS2 (2 μgmL-1 or 4

μgmL-1) in IMDM medium. Aged Aβ40 (Final concentration was 5 μM.) was added to the cells

immediately. Afterwards, the plates were irradiated using an 808 nm NIR laser at a power density of

0.5 Wcm-2 for 5 min. As a control experiment, the PC12 cells containing WS2-Aβ40 was kept in the

dark during the same time. After 48 h of incubation, the cells were treated with 10 μL of MTT (5

mgmL-1 in PBS (phosphate buffered saline, 10 mM, pH 7.4)) for 4 h at 37 °C and were then lysed in

DMSO for 10 min at room temperature in the dark. Absorbance values of formazan were

determined at 570 nm with 630 nm as the background absorbance using a Bio-Rad model-680

microplate reader. For WS2 themselves, cells were plated at a density of 10000 cells per well on

96-well plates for 24 h, and then different concentrations of WS2 nanosheets were dispensed into the

PC12 cells. Afterwards, the plates which were used for determining the cell viabilities of WS2 under

NIR irradiation were irradiated using an 808 nm NIR laser at a power density of 0.5 Wcm-2 for 5

min. After that, the cells were continuously incubated with 48h. To rule out the effect of WS2 on

MTT, after incubation cells for 48h, the medium was replaced by the mixture of WS2 and MTT

buffer and incubated for 4 h. Then the medium was extracted and replaced with 100 μL DMSO to

dissolve the formazan salt. Absorbance values of formazan were determined at 570 nm with 630 nm

as the background absorbance using a Bio-Rad model-680 microplate reader.

Handling of Mice

S4880202 normal mice were chosen as test animals, in a weight range of 20-25 g (8-12 weeks old)

and random in sex. All animal studies were conducted in accordance with the principles and


Nano Res.

procedures outlined in “Regulations for the Administration of Affairs Concerning Laboratory

Animals”, approved by the National Council of China on October 31, 1988, and “The National

Regulation of China for Care and Use of Laboratory Animals”, promulgated by the National

Science and Technology Commission of China, on November 14, 1988 as Decree No. 2. Protocols

were approved by the Committee of Jilin University Institutional Animal Care and Use. After

intraperitoneal injection of 600 μL WS2 (100 μgmL-1), the cerebrospinal fluid (CSF) were collected

immediately and diluted in 0.5 mL water. The mouse that treated without WS2 was used as the

control group. The tungsten content of the samples was measured by ICP-MS (Varian 720-ES). The

data points shown are the mean values ± SEM from three independent experiments (Three mice

were used for groups treated with WS2 and three mice for control group.).

Cerebrospinal fluid (CSF) of mice collection

CSF was collected under anesthesia using a glass pulled micropipette after exposure of the cisterna

magna, taking care not to contaminate the CSF with blood. Two to 10 μL was routinely collected.

The CSF was immediately diluted 1:10 in 1%

3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate (CHAPS) in PBS with protease

inhibitors (Roche Diagnostics, Mannheim, Germany) before freezing in liquid nitrogen and storage

at -80 °C.

Address correspondence to [email protected]


Nano Res.

Figure S1 The influence of WS2 nanosheets on the fluorescence of ThT with or without NIR

irradiation. The concentration of WS2 was 100 μg/mL.


Nano Res.

Figure S2 Zeta potential of the WS2 nanosheets in the absence (A) or presence (B) of Aβ40. The

concentrations of Aβ40 and WS2 nanosheets were 10 μM and 20 μg/mL, respectively.


Nano Res.

Figure S3 AFM images and height profiles of the corresponding AFM topographies of typical

nanosheets of WS2 incubated with different concentration of Aβ40. (A) WS2 nanosheet with Aβ40 at

the concentration of 0.25 mmol/g; (B) WS2 nanosheet with Aβ40 at the concentration of 2.5 mmol/g.

The change of height indicated that the Aβ40 monomers could selectively adsorb on the surface of

WS2 nanosheets.


Nano Res.

Figure S4 (A) Fluorescence titration of Aβ40 (3 μM) with various concentrations of WS2 nanosheets

in 10 mM Tris buffer (pH 7.4). The excitation wavelength was 278 nm and the emission intensity at

306 nm was used for analysis. (B) The UV/Vis spectrum of the Aβ40 monomers before incubation

(black) and after incubation (red) with WS2 nanosheets.


Nano Res.

Figure S5 (A) The morphology of MoS2. (B) The morphology of GO. (C) The kinetics of Aβ40 with

these three different nanomaterials. The concentration of Aβ was 50 μM and the concentrations of

WS2, MoS2 and graphene nanosheets were all kept at 40 μg/mL. (D) The dose-dependent inhibition

effects on Aβ40 aggregation of WS2, MoS2 and GO. The concentration of Aβ40 was 50 μM. (E) CD

spectra of Aβ40 in the absence or presence of WS2 nanosheets and GO. The concentration of Aβ

was 50 μM and the concentrations of WS2 and graphene nanosheets were kept at 40 μg/mL. The

samples were measured in 10 mM Tris buffer (pH 7.3) after incubation at 37 for 7 days.


Nano Res.

Figure S6 AFM images of Aβ40 in the absence (A) or presence (B) of WS2 nanosheets. The

concentration of Aβ40 and WS2 nanosheets were 50 μM and 40 μg/mL. (C) CD spectra of Aβ40 at

the beginning with or without WS2.


Nano Res.

Figure S7 Absorbance profile of different concentrations of ceWS2.


Nano Res.

Figure S8 Temperature change curves of the WS2 nanosheets solution and the water exposed to the

808 nm laser at a power density of 0.5 W/cm2 (A), 1 W/cm2 (B) and 2 W/cm2 (C).


Nano Res.

Figure S9 Plot of the fluorescence intensity against irradiation time. The excitation wavelength was

444 nm, and the emission intensity at 480 nm was used for analysis.


Nano Res.

Figure S10 The morphology of Aβ40 aggregates was analyzed by AFM images. (A) Aβ40 fibrils. (B)

Aβ40 fibrils in absence of WS2 nanosheets under the laser irradiation. The concentration of Aβ40

was 50 μM.


Nano Res.

Figure S11 (A) The UV/Vis spectrum of BSA before incubation (black) and after incubation (red)

with WS2 nanosheets. (B) AFM images (left) and height profiles (right) of the corresponding AFM

topographies of typical nanosheets of WS2 incubated with BSA. The amount of BSA adsorbed on

the surface of WS2 nanosheets was 0.63 g/g WS2.


Nano Res.

Figure S12 (A) Fluorescence titration of Aβ40 (3 μM) with various concentrations of WS2-BSA

nanosheets in 10 mM Tris buffer (pH 7.4). The excitation wavelength was 278 nm and the emission

intensity at 306 nm was used for analysis. (B) The influence of WS2-BSA nanosheets on Aβ

aggregation monitored by ThT assay in Tris buffer. The concentration of Aβ40 was 50 μM. AFM

images of Aβ40 in the absence (C) or presence (D) of WS2-BSA nanosheets. The concentration of

Aβ40 and WS2-BSA nanosheets were 50 μM and 40 μg/mL.


Nano Res.

Figure S13 Effect of WS2 nanosheets on PC12 cell viability determined by MTT method.


Nano Res.

Figure S14 The effect of WS2 on MTT. After incubation cells for 48h, the medium was replaced by

the mixture of WS2 and MTT buffer and incubated for 4 h. Then the medium was extracted and

replaced with 100 μL DMSO to dissolve the formazan salt. Absorbance values of formazan were

determined at 570 nm with 630 nm as the background absorbance using a Bio-Rad model-680

microplate reader.

Figure S15 (A) Measurements of 5(6)-CF leakage from liposome induced by Aβ40 aggregates (10

μM) in the presence of WS2 (8 μg/mL) with or without NIR irradiation at 4 h, 12 h and 24 h


Nano Res.

incubation time. (B) The influence of WS2 nanosheet on the fluorescence of 5(6)-CF. The

concentration of 5(6)-CF was 5 μM. The concentrations of Aβ40 aggregates and WS2 were 10 μM

and 8 μg/mL, respectively.

Chemically exfoliated WS nanosheets can efficiently ... · 300 ml of deionized H 2 O was added to...

Documents

Transcript of Chemically exfoliated WS nanosheets can efficiently ... · 300 ml of deionized H 2 O was added to...