Molybdenum Disulfide Nanoparticles as Multifunctional Inhibitors

Jun 14, 2017 - The complex pathogenic mechanisms of Alzheimer's disease (AD) include the aggregation of β-amyloid peptides (Aβ) into oligomers or fi...
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Molybdenum Disulfide Nanoparticles as Multifunctional Inhibitors against Alzheimer’s Disease Qiusen Han,†,‡ Shuangfei Cai,† Lin Yang,† Xinhuan Wang,† Cui Qi,† Rong Yang,*,†,‡ and Chen Wang*,†,‡ †

CAS Center of Excellence for Nanoscience, National Center for Nanoscience and Technology, UCAS, Beijing 100190, P. R. China Sino-Danish College, Sino-Danish Center for Education and Research, UCAS, Beijing 100190, P. R. China

‡

S Supporting Information *

ABSTRACT: The complex pathogenic mechanisms of Alzheimer’s disease (AD) include the aggregation of β-amyloid peptides (Aβ) into oligomers or fibrils as well as Aβ-mediated oxidative stress, which require comprehensive treatment. Therefore, the inhibition of Aβ aggregation and free-radical scavenging are essential for the treatment of AD. Nanoparticles (NPs) have been found to influence Aβ aggregation process in vitro. Herein, we report the inhibition effects of molybdenum disulfide (MoS2) NPs on Aβ aggregation. Polyvinylpyrrolidone-functionalized MoS2 NPs were fabricated by a pulsed laser ablation method. We find that MoS2 NPs exhibit multifunctional effects on Aβ peptides: inhibiting Aβ aggregation, destabilizing Aβ fibrils, alleviating Aβ-induced oxidative stress, as well as Aβ-mediated cell toxicity. Moreover, we show that MoS2 NPs can block the formation of the Ca2+ channel induced by Aβ fibrils in the cell membrane for the first time. Thus, these observations suggest that MoS2 NPs have great potential for a multifunctional therapeutic agent against amyloidrelated diseases. KEYWORDS: amyloid peptide, MoS2 nanoparticles, pulsed laser ablation, neuronal cytotoxicity, antioxidant activity

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INTRODUCTION Alzheimer’s disease (AD) is one of the most common ageassociated brain diseases characterized by cerebral extracellular amyloid plaques and intracellular neurofibrillary tangles, which triggers an impairment of cognitive dysfunction.1 β-Amyloid peptide (Aβ), such as Aβ42 or Aβ40, originated from the amyloid precursor protein (APP), can form abnormal aggregation and is deemed to play an important role in the pathological process.2−4 Recent strategies for Aβ therapy are the inhibition of Aβ aggregation and dissociation of Aβ fibrils.5 Many agents, such as peptides like KLVFF6,7 and organic small molecules with aromatic groups,8−12 are reported to inhibit Aβ aggregation as well as destabilize preformed Aβ fibers in vitro.13,14 However, many of the inhibitors only showed a mitigated inhibition effect or weak dissociation ability. The therapeutic application is limited by their weak targeting ability and low permeability to the blood−brain barrier (BBB) as well as toxic effects.15 Many recent works have investigated nanoparticles (NPs) as novel agents for inhibiting Aβ aggregation.16−18 Compared to the agents mentioned above, the advantage of NPs mainly includes the promising capacity to penetrate the BBB, which can deliver targeted drugs to diseased brains.19 The underlying mechanism may be receptor-mediated endocytosis followed by transcytosis in brain vessel endothelial cells. Modification of © XXXX American Chemical Society

NPs with targeted ligands or some surfactants that can enable the adsorption of some specific plasma proteins is needed for the receptor-mediated uptake.20−22 Meanwhile, NPs with large surface ratio and surface functionalization can target and adsorb specific peptides or proteins.23,24 Thus, they will own a great role in the fibrillation process with various amyloid related proteins.17 In addition, many NPs have showed excellent biocompatibility and low toxicity.25 Several typical inorganic NPs were investigated widely as efficient inhibitors for amyloid fibrillation,26 including gold NPs,27 magnetic NPs,28,29 and carbon-based nanomaterials.23,24,30−32 Kim et al. have reported that fullerene could specifically interact with KLVFF, the central hydrophobic motif of Aβ peptides, and then inhibit Aβ aggregation at an early stage.23 Mahmoudi et al. also reported that grapheme oxide sheets could delay the Aβ fibrillation by adsorbing amyloid monomers.31 In our previous work, GO/Au nanocomposites greatly reduced aggregation and cytotoxicity of Aβ42.33 The mechanisms of interaction between NPs and amyloid proteins are mainly based on electrostatic attraction and high surface ratio effects.34 Received: March 17, 2017 Accepted: June 6, 2017

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(10 Hz, 532 nm, 100 mJ) was used for ablation for 10 min. During the ablation process, the colloidal solution was stirred continuously. Finally, the MoS2 NPs were filtered with 0.45 μm filters and centrifuged at 20000 rpm for 10 min. The morphology of the asprepared MoS2 NPs was characterized by SEM (Hitachi S-4800) and TEM (Tecnai G220). Zeta potential (Malvern) and X-ray diffraction (XRD) (Bruker D8) were also carried out. The concentration of MoS2 NPs used for next assays was measured by ICP-OES (PerkinElmer optima 6300DV). Amyloid Peptide Preparation. Aβ42 (purity 95%) was synthesized by Shanghai Science Peptide Biological Technology Co. Ltd. (China). The peptide purity was determined by high-performance liquid chromatography (HPLC) analysis. Aβ42 was dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) with a concentration of 1 mg/mL and then incubated at room temperature for 14 h. Before use, the HFIP was evaporated under vacuum, and then DMSO was used to resolve the peptide. Fibrillation Experiments. To investigate the inhibition effect of MoS2 on Aβ42, MoS2 NPs with various concentrations (1, 5, 10 μg/ mL, respectively) were incubated with Aβ42 at 37 °C in a swing bed (THZ-D, Huamei). The final concentration of Aβ42 was 20 μM. Meanwhile, ThT fluorescence probe was selected to monitor the fibrillation process. Twenty microliter aliquots from each sample were extracted at different time points and then mixed with 180 μL of ThT (10 μM) in a black 96-well plate. The ThT fluorescence spectra were collected at 485 nm with excitation at 440 nm using a micro plate reader. Characterization of Aβ42 Secondary Structure. The secondary structure of Aβ42 was characterized by circular dichroism (CD) spectra with a JASCO-J810 spectropolarimeter (JASCO, Japan). Aβ42 was dissolved in a 1:1 (v/v) mixture of trifluoroethanol and ultrapure water with or without MoS2 NPs. Typically, spectra were collected from 250 to 190 nm with a quartz cell, the step size was 0.2 nm, and the speed was 50 nm/s. Tyrosine Fluorescence Spectra. Tyrosine fluorescence spectra were used to study the interaction between Aβ42 and MoS2 NPs by using Hitachi FP-4500 fluorescence spectrophotometer. The excitation wavelength was 280 nm (slit width = 5 nm), and the data were collected over 290−350 nm (slit width = 5 nm). Samples were added in a quartz fluorescence cell, and data were recorded at room temperature. AFM and TEM Characterization. The morphology of Aβ42 aggregates was characterized by AFM and TEM. For AFM, samples were dropped to freshly cleaved mica surface and then dried by N2 flow. AFM experiments were performed on Dimension 3100 system with a tapping mode (Bruker). For TEM, 10 μL of sample solution was added on 200 mesh carbon-coated copper grids. Then the sample was negatively stained with 2% phosphotungstic acid for 2 min. Tecnai G220ST electron microscope was operated at 200 kV. Cell Culture. Human neuroblastoma cells (SH-SY5Y, ATCC) were cultured in RPMI 1640 (Gibco) with 15% (v/v) fetal bovine serum (FBS, Gibco) containing 100 μg/mL streptomycin and 100 IU/mL penicillin. BV-2 microglia cells were cultured in DMEM medium (Hyclone) with 10% (v/v) FBS, 1% glutamine, as well as 100 IU/mL of penicillin and 100 μg/mL of streptomycin. Cells were routinely subcultured once every 3 days with a dilution of 1:3 and incubated in a 95% humidified incubator (Thermo) containing 5% CO2 at 37 °C. Cell Viability Assay. BV-2 microglia cells and SH-SY5Y cells (15000 cells/well,100 μL) were seeded in 96-well plates and incubated overnight. Next, SH-SY5Y cells and BV-2 microglia cells were mixed with fresh Aβ42 at different concentrations (2, 5, 10, 20, 30, and 40 μM) with or without MoS2 (2 μg/mL) for 48 h, respectively. The cell viability was evaluated by MTT assay. ROS Measurement. The ROS level induced by Aβ42 was monitored with DCFH-DA probe (Sigma). SY5Y cells (2 × 105 cells/mL) were seeded in a 24-well plate and incubated overnight. Before use, cells were incubated with DCFH-DA (100 μM) for 2 h and then washed with PBS three times. Next, cells were cultured with various concentrations of MoS2 for 1 h. After that, Aβ42 with a concentration of 40 μM was introduced. Four hours later, the

However, the inhibition of Aβ aggregation or destabilization of Aβ fibrils is not sufficient. The pathogenesis of AD also includes elevating the ROS level and disruption the Ca2+ homeostasis mediated by Aβ, both of which may lead to neuron injury and play an important role in AD.35 Therefore, an inhibitor with multifunctional performance toward AD is desirable. MoS2 NPs, which have hollow cage structures and are wellknown as inorganic fullerene-like nanoparticles, are utilized as self-lubricating coatings and additives to fluid lubricants due to their superior tribological properties. Recently, the potential medical applications of MoS2 NPs have drawn more and more interest, such as coatings for artificial joints or orthodontic wires.36 Tian et al. have demonstrated that 2D MoS2 glycosheets could produce ROS in certain cell lines by a targeted manner.37 Transition metal oxides and dichalcogenides have also been designed as functional materials for the fabrication of composite structures with biological ligands for disease diagnosis and therapy.38 Meanwhile, ultrasmall MoS2 NPs (2 nm) with attractive electrocatalytic activity for reduction of H2O2 have been constructed.39 Zhang et al. have reported that ultrasmall (sub-5 nm) cysteine-protected MoS2 dots with highly catalytic activity can contribute in cleaning up the accumulated free radicals, repairing DNA damages and recovering all vital chemical and biochemical indicators, showing the unique role as free radical scavengers.40,41 IFMoS2 also could induce low levels of the pro-inflammatory cytokines, such as IL-6, IL-8, and IL-1β, and TNF-α in human bronchial cells (NL-20) and activate antioxidant response.42 Moreover, MoS2 NPs were no apparent toxic effect in vitro, even at high concentrations up to 100 μg/mL.47 Thus, the potential advantage of MoS2 NPs in the treatment of AD will be not only the inhibition effects on Aβ aggregation but also the beneficial rules in scavenging the radical species in AD.43−45 However, the interactions between MoS2 NPs and Aβ have not been reported to our knowledge. Hence, the main aim of this work is to study the effect of MoS2 NPs on the Aβ fibrillation process and the clearance of ROS mediated by Aβ. Herein, MoS2 NPs are fabricated by a pulsed laser ablation (PLA) method. PLA has been developed to fabricate various nanomaterials with special structure and unique morphologies, including noble metal nanomaterials, semiconductor nanoparticles, and nanocomposites. PLA method can also achieve kinds of functionalized nanostructures with novel properties through one-step formation, which may be applicated in biosensors and biomedicine fields.46 In our previous work, we have fabricated MoS2 NPs with homogeneous morphology by PLA in water, which showed less cytotoxicity.47 However, pure MoS2 NPs in water may aggregate. Thus, polyvinylpyrrolidone (PVP) was introduced to functionalize MoS2 NPs during PLA process. The as-prepared MoS2 NPs exhibit remarkable capability to inhibit Aβ fibrillation and to scavenge Aβ-induced ROS. The blocking effect of MoS2 NPs on calcium channel formation mediated by Aβ is also reported for the first time. Our results show that MoS2 NPs have a great potential for multifunctional therapy against amyloid-related diseases.

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EXPERIMENTAL SECTION

Synthesis of MoS2 NPs. Synthesis of MoS2 NPs was studied by PLA in PVP solution. Briefly, the MoS2 powder (Sigma) was first pressed into a target pellet with diameter of 15 mm by using the tablet machine. Then the pellet was fixed at the bottom of a quartz tube containing 7 mL of PVP solution (72 μM). A Nd:YAG pulsed laser B

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Figure 1. (a) SEM image, (b) HRTEM image, (c) HAADF−STEM image, elemental maps for Mo and S (scale bar: 200 nm), (d) XRD of MoS2 NPs and (e) ThT fluorescence signal of Aβ42 exposed to various concentrations of MoS2 NPs. fluorescence intensity (indicating ROS level) was collected by a microplate reader with excitation/emission at 485/530 nm. All of the procedures were performed without exposure to light. Intracellular Free Calcium Assay. SY5Y cells were plated in 24well plates at a density of 4 × 105 cell/mL for 14 h. Then the cells were mixed with Aβ42 alone or Aβ42 plus the MoS2 NPs with different concentrations for another 12 h. Next, the cells were washed and incubated at 37 °C in 1640 serum-free medium containing 5 μM Fluo3 AM (sigma) for 1 h. Fluorescence intensity from Fluo-3 AM was captured by flow cytometry and confocal spectroscopy with excitation wavelength at 488 nm. Electron-Spin Resonance. The 75 μL samples were prepared by adding 5 μL of 20 mM FeSO4, 10 μL of 3% H2O2, 20 μL of 0.1 M DMPO, and 20 μL of 0.2 M NaAc buffer (pH 3.0) into a plastic tube in the presence or absence of 20 μL of MoS2 (10 μg/mL), respectively. Then the as-prepared sample was transferred to a quartz capillary tube and put in the ESR cavity. DMPO as a trapping agent was adopted to trap the •OH radicals to form the DMPO−•OH spin adduct. The ESR spectra were collected on ESR 300E (Bruker). ANS Fluorescence Assay. The ANS−Na fluorescent probe was selected to study the binding sites between MoS2 NPs and Aβ42. Samples (150 μL) were mixed with 3 μL of ANS solution (10 mM) for 3 min. The fluorescence intensity was measured with an excitation wavelength at 400 nm (slit width = 5 nm), and the data were collected over 450−650 nm (slit width = 10 nm). Statistical Analyses. All experiments are means of triplicates. All of the data are expressed as mean standard deviation. Statistical

analysis were carried out with SPSS with a paired t-test. The difference was considered statistically significant when P < 0.05.

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RESULTS AND DISCUSSION In this work, a facile strategy to fabricate MoS2 NPs by pulsed laser ablation (PLA) in polyvinylpyrrolidone (PVP) solution was developed. The PLA method is known to be versatile to fabricate pure nanoparticles in water without chemical residues. Yet aggregation may occur, which seems adverse to biomedicine application. Therefore, PVP solution was adopted to anchor on the surface of MoS2 NPs during the PLA process. Excess unbounded PVP was removed by centrifugation and repeated washing. The morphologies of as-prepared MoS2 NPs were characterized by SEM (Figure 1a). MoS2 NPs showed a spherical shape with an average diameter of 100 nm. The typical highresolution TEM (HRTEM) image of MoS2 NPs was shown in Figure 1b. The layered structure is quite clear. To further investigate the nanostructure of MoS2 NPs, the element distribution of Mo and S in the sample was studied with a high-angle, annular dark-field scanning transmission electron microscope (HAADF-STEM). Figure 1c showed the representative STEM image as well as corresponding elemental maps, respectively. Mo and S elements were identified in the C

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Figure 2. AFM and TEM images of Aβfibrils alone (a and e) and in the presence of MoS2 NPs (concentrations are 1 (b and f), 5 (c and g), and 10 μg/mL (d and h)). Scale bar (a, b, c, d) is 2 μm, and scale bar (e, f, g, h) is 200 nm, respectively.

Figure 3. (a) Tyrosine fluorescence quenching effects. (b) CD spectra and (c) ANS fluorescence signal of Aβ42 with or without MoS2 NPs (1, 5, and 10 μg/mL).

first few hours and reached a plateau after 12 h. The Aβ42 fibrillation followed a typical nucleation growth mechanism, and the process can be divided into three steps, including a lag phase, exponential growth, and an equilibrium phase. Upon introduction of MoS2 NPs, the increase of ThT fluorescence intensity became slower, indicating that the Aβ fibrillation process was suppressed. The inhibition effects exhibited dose dependence, as shown in Figure 1e. When Aβ42 was exposed to MoS2 NPs with various concentrations (1, 5, 10 μg/mL), the fluorescence intensities were reduced by approximately 20%, 40%, and 60%, respectively, which could be ascribed to the increase of surface areas of MoS2 NPs presented in solution. We reason that when incubated with MoS2 NPs, Aβ monomers or oligomers were interacted and adsorbed on the surfaces of MoS2 NPs, which led to delay the nucleation process and exhibited an inhibition effect. The influence of MoS2 NPs on ThT fluorescence was evaluated. As shown in Figure S4, due to the relatively low concentration used in the assay, no apparent fluorescence quenching can be seen. In addition, the influence of PVP on Aβ fibrils was also investigated, and the results are shown in Figure S5. Because the mature fibrils are involved in the pathogenesis, the dissociation effect of MoS2 NPs on the preformed fibrils was further tested (Figure S6). The Aβ mature fibrils were achieved by incubating Aβ42 monomers at 37 °C for 72 h. Then the mature fibrils were mixed with MoS2 NPs (5 μg/mL) to form a complex. Because the amount of fibrils was proportional to ThT fluorescence intensity, the destabilization

MoS2 NPs. Raman spectra of MoS2 NPs and 2H-MoS2 powder are shown in Figure S1, which were obtained by using the 514 nm He−Ne laser. All of the peaks were in good accordance with the previously reported MoS2 Raman studies.48 The functionalization of PVP anchored on the surface of MoS2 NPs was verified by FTIR spectroscopy. As a reference, the FTIR spectrum of PVP alone was also studied. As shown in Figure S2, PVP-conjugated MoS2 NPs exhibited three characteristic peaks at 1630, 1420, and 1280 cm−1, which can be attributed to CO, C−H2, and C−N stretching, respectively. The results matched well with pure PVP as reported in the literature,49 indicating that PVP was successfully conjugated to the surface of MoS2 NPs. To determine the crystalline structure and purity, the asprepared MoS2 NPs were measured by XRD. As shown in Figure 1d, diffraction peaks at 2θ values of 14.5°, 39.4°, 44.2°, and 49.8° can be assigned to the (002), (103), (006), and (105) crystal planes of a MoS2 hexagonal structure, respectively (ICDD-JCPDS card no. 37-1492). The zeta potential of MoS2 NPs was −43.5 mV, which can form stable dispersions, illustrating that the MoS2 NPs exhibit relatively high colloidal stability, as shown in Figure S3. Next, the inhibition effect of MoS2 on Aβ42 aggregation was evaluated. A thioflavin T (ThT) fluorescence probe was selected to monitor the aggregation kinetics of Aβ42 with or without MoS2 NPs. ThT can bind with a β-sheet structure specifically and emit fluorescence at 485 nm by excitation at 440 nm. When fresh Aβ42 alone was cultured at 37 °C, the ThT fluorescence signal was increased significantly within the D

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the fluorescence intensity was not quenched with various concentrations, as shown in Figure.S7. Next, circular dichroism (CD) analysis was applied to study the conformation transition of Aβ42 during aggregation. As shown in Figure 3b, the CD spectra of Aβ42 alone showed one positive peak around 196 nm and one negative valley at 215 nm, corresponding to a typical β-sheet structure, which is in accordance with the previous reports.9 However, after introduction of MoS2 NPs with various concentrations, the representative negative valley at 215 nm disappeared gradually, especially at higher concentration. The effect indicated that the ratio of β-sheet structure was much less than that of Aβ42 alone and secondary structures became random coils or amorphous aggregates. We then explored the effect of MoS2 NPs on modulating Aβmediated cellular toxicity. SH-SY5Y cells were selected, and the methyl thiazolyl tetrazolium (MTT) assay was carried out to determine the relative viabilities. As shown in Figure 4, after 48

effect was measured by quantification ThT fluorescence intensity at different time intervals. The reduction of ThT fluorescence was detected after 2, 4, and 6 h incubation, which showed concentration and time dependence, revealing that MoS2 NPs efficiently induced the destabilization of preformed fibrils. AFM and TEM were also employed to study the inhibition effects of MoS2 NPs on the morphology of Aβ42 aggregates. Solutions of fresh Aβ42 (20 μM)were incubated with MoS2 NPs at 37 °C for 7 days. Aβ42 alone could form long and unbranched fibrils. In contrast, after addition of MoS2 NPs, fibers became short and rare. As the MoS2 NPs concentration increased, the inhibitory effect enhanced. In particular, spheres with heights of 3−5 nm were formed when the MoS2 NP concentration reached to 10 μg/mL, as shown in Figure 2. The results above further supported the ThT assay and also indicated that MoS2 could efficiently affect the elongation rate of Aβ fibrillation, finally hindering the Aβ fibril formation. It is known that NPs can adsorb Aβ42 peptide onto their surface and bury the tyrosine residue of Aβ42 peptide in the hydrophobic environment, which may quench the intrinsic fluorescence of tyrosine. To investigate whether MoS2 NPs can interact with Aβ42, tyrosine fluorescence emission spectra were monitored with or without MoS2 NPs. As shown in Figure 3a, when Aβ42 was incubated with MoS2 NPs, the fluorescence signal exhibited a significant quenching at 310 nm as a dosedependent manner. Compared to Aβ42 alone, the fluorescence intensity decreased almost 50% when MoS2 NPs concentration was up to 10 μg/mL. The results illustrated that MoS2 NPs could adsorb Aβ42 on the surface and lower the concentration of free monomers in solution. The binding constant (KA) of the MoS2 NPs to Aβ42 aggregates was also investigated by monitoring the quenching of tyrosine fluorescence and then calculated as shown in eq 1: lg[(F0 − F )/F ] = lg KA + n lg[Q ]

Figure 4. Protection effects of MoS2 NPs on Aβ42-mediated cytotoxicity of SY5Y cells.

(1)

Here, F0 and F represent the fluorescence intensity value without or with MoS2 NPs, KA refers the binding constant between Aβ and MoS2 NPs, n represents the binding site number, and Q is the concentration of MoS2 NPs.50 The value of KA was 1.614 × 103 L·mol−1 according to eq 1, which indicated that a relatively strong interaction force between Aβ and MoS2 NPs was formed. ANS (l-anilinonaphthalene-8-sulfonate) was used as a “hydrophobic probe” to further detect the binding regions between Aβ42 and MoS2 NPs, which are usually utilized to recognize hydrophobic domain in proteins.51,52 The fluorescence intensity of ANS was highly enhanced when bound to Aβ42 with an emission spectrum peak at 496 nm, owing to the high hydrophobicity of Aβ42 fibrils. Upon addition of MoS2 NPs, ANS fluorescence intensity decreased significantly, which was also concentration dependent, and the lower fluorescence intensity was monitored when incubated with higher concentration of MoS2 NPs (Figure 3c). The results indicated that MoS2 NPs could interact with the hydrophobic region of Aβ42 and avoid the ANS probe to bind at the same site. It is known that the hydrophobic fragments in Aβ42, such as Aβ33− 42, play important roles in the β-sheet structure formation through the hydrophobic interactions, so the inhibitory effect induced by MoS2 NPs can be also attributed to the interactions between MoS2 NPs and the hydrophobic region of Aβ42. The influence of MoS2 NPs on ANS fluorescence was further investigated. When ANS was incubated with MoS2 NPs alone,

h incubation, Aβ42 alone triggered a reduction of ∼60% in cell viability compared to the negative control. With 2 μg/mL MoS2 NPs, the value of cell proliferation was increased. For instance, the cell viability was down to 40% when Aβ42 concentration was up to 40 μM, which increased to 60% when MoS2 NPs were added. Similarly, the reduced toxicity on BV2 microglia cells was also obtained, as shown in Figure S8. At the same time, MoS2 NPs alone showed no apparent effects on cell growth and proliferation at the same concentration (Figure S9). Thus, MoS2 NPs could modulate the Aβ aggregation process in vitro and also decrease Aβ-induced cell toxicity, as schematically illustrated in Figure 5.

Figure 5. Schematic illustration of MoS2 NPs inhibiting Aβ42 aggregation. MoS2 NPs can act as a novel therapeutic agent to modulate the Aβ aggregation process and finally reduce cell toxicity mediated by Aβ. E

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Figure 6. (a) ESR spectra of •OH species in the systems of H2O2−FeSO4−MoS2 NPs−DMPO and H2O2−FeSO4−H2O−DMPO. (b) Effect of MoS2 NPs on scavenging intracellular ROS induced by Aβ42.

Reactive oxygen species (ROS) have been well-known to damage human tissue through protein oxidation and crosslinking, lipid peroxidation, as well as DNA cleavage process. Meanwhile, the human brain is particularly prone to ROS damage owing to poor catalase activity, high content of oxidizable substrates, and low iron-binding capacity.13 Here, we demonstrated that the intracellular ROS level induced by Aβ42 could be reduced by administration of MoS2 NPs for the first time. First, we used the electron spin resonance (ESR) spectrum to confirm the ROS-scavenging ability of MoS2 NPs. Without MoS2 NPs, a characteristic peak with intensity ratio of 1:2:2:1 for the DMPO−•OH adduct was shown, indicating that FeSO4 could decompose H2O2 into •OH radicals. However, the signal intensity of the H2O2−FeSO4− MoS2NPs system was much lower than that of H2O2−FeSO4, which illustrated that MoS2 NPs exhibited good •OH radical clearance ability as shown in Figure 6a. Then a DCFH−DA fluorescence probe was used to monitor the intracellular ROS level. In the absence of MoS2 NPs, strong fluorescence intensity triggered by Aβ42 was shown, indicating a high level of ROS was generated. However, upon incubation with MoS2 NPs, the ROS level decreased with increasing NPs concentrations from1 to 10 μg/mL, as shown in Figure 6b. Another neurotoxic mechanism was proposed where Aβ oligomers could directly incorporate into neuronal cell membranes and form Ca2+ channels in the target neurons. The disturbing of calcium homeostasis can also induce a selfamplifying cascade of ROS and Ca2+-mediated degenerative processes. Thus, blocking of the Aβ-formed calcium channel may be also an effective way to protect neuronal cells. To investigate whether MoS2 NPs could modulate Aβmediated intracellular calcium change, we selected Fluo-3 AM as a calcium-sensitive probe to evaluate the calcium level. As shown in Figure 7a, in the absence of MoS2 NPs, intracellular calcium accumulation induced by Aβ was significantly high. After introduction of MoS2 NPs, the calcium level decreased in a dose-dependent manner, which was indicated by the signal intensity of Fluo-3 arrested in the cells. The result can be confirmed by confocal fluorescent microscopy as shown in Figure 8. In DAPI-stained SY5Y cells, the nucleus appeared bright blue and the Fluo-3 fluorescence signal was green, which was hard to see after incubation with MoS2 NPs. The reason could be that MoS2 NPs adsorb Aβ42 monomers or oligomers onto their surfaces and block the formation of calcium channel, finally reducing the calcium accumulation, as schematically illustrated in Figure 7b. Therefore, MoS2 NPs can act as a calcium channel blocker and

Figure 7. (a) Intracellular calcium level induced by Aβ42 or Aβ42 with MoS2 NPs at various concentrations. (b) Schematic illustration the intracellular calcium change induced by Aβ42 with or without MoS2 NPs.

Figure 8. Intracellular calcium change induced by Aβ42 with or without MoS2 NPs.

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maintain Aβ-induced calcium homeostasis, which is an effective therapeutic for AD.

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CONCLUSIONS In summary, we have fabricated MoS2 NPs by a PLA method in PVP solution and demonstrated their applications as multifunctional inhibitors to reduce Aβ mediated cytotoxicity. The MoS2 NPs have been characterized by TEM, SEM, and XRD. The NPs also showed good biocompatibility and stability. The efficient inhibition of MoS2 NPs on Aβ42 assembly was investigated by ThT fluorescence and AFM and TEM images. Moreover, the interactions between MoS2 NPs and Aβ were monitored by ANS and tyrosine intrinsic fluorescence. CD spectra showed that MoS2 NPs could induce the conformational transition, from β-sheet to random coil, which is less prone to fibrillation. According to the results above, the reduction effect of MoS2 NPs on Aβ-mediated neurotoxicity was observed. Furthermore, the protection effect was also ascribed to the antioxidant activity of MoS2 NPs. Blocking of the Aβ-formed calcium channel and maintaining the calcium homeostasis by MoS2 NPs were also observed for the first time. Thus, these observations suggest that MoS2 NPs have great potential for multifunctional therapy against Alzheimer’s disease. This work provides new insights into the design and construction of potent NP-based therapeutic strategies for amyloid-related diseases.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b03816. List of characterizations of Raman spectra, FTIR spectra, and zeta potential of MoS2 NPs; influence of PVP on the Aβ aggregation process; influence of MoS2 NPs on ThT, ROS, and ANS; dissociation effect of MoS2 NPs on Aβ42 fibrils; protection effects of MoS2 NPs on Aβ42-induced cytotoxicity of BV2 cells and cell toxicity of MoS2 NPs (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Qiusen Han: 0000-0002-3473-4614 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Our work was supported by the National Key Research and Development Program from the Ministry of Science and Technology of China (2016YFC0207102), National Natural Science Foundation of China (21503053, 21573050, 21501034), and the Chinese Academy of Sciences (XDA09030303).

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DOI: 10.1021/acsami.7b03816 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX