Remote Induction of Cell Autophagy by 2D MoS2 Nanosheets via

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Biological and Medical Applications of Materials and Interfaces

Remote Induction of Cell Autophagy by 2D MoS2 Nanosheets via Perturbing Cell Surface Receptor and mTOR Pathway from Outside of Cell Xiaofei Zhou, Jianbo Jia, Zhen Luo, Gaoxing Su, Tongtao Yue, and Bing Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21886 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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ACS Applied Materials & Interfaces

Remote Induction of Cell Autophagy by 2D MoS2 Nanosheets via Perturbing Cell Surface Receptor and mTOR Pathway from Outside of Cell

Xiaofei Zhou,† Jianbo Jia,‡ Zhen Luo,§ Gaoxing Su,‖ Tongtao Yue,§ Bing Yan*,‡,⊥

† School

of Chemistry and Chemical Engineering, Shandong University, Jinan, 250100,

China ‡ Key

Laboratory for Water Quality and Conservation of the Pearl River Delta, Ministry

of Education, Institute of Environmental Research at Greater Bay, Guangzhou University, Guangzhou 510006, China §

Center for Bioengineering and Biotechnology, State Key Laboratory of Heavy Oil

Processing, College of Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, China ‖ School

of Pharmacy, Key Laboratory of Inflammation and Molecular Drug Targets of

Jiangsu Province, Nantong University, Nantong 226001, China ⊥

School of Environmental Science and Engineering, Shandong University, Jinan

250100, China

To whom correspondence should be addressed. E-mail: [email protected] 1

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Abstract The ability of nanoparticles to induce adverse consequences in human cells rely on their physical shapes. In this aspect, how two-dimensional nanoparticles differ from three dimensional nanoparticles is not well known. To elucidate this difference, combined experimental and theoretical approaches are employed to compare MoS2 nanosheets with 5-layer and 40-layer thickness for their cellular effects and the associated molecular events. At a concentration as defined by nanosheet surface areas (10 cm2/mL), 40-layer nanosheets are internalized by cells, while 5-layer nanosheets mostly bind to cell surface without internalization. Although they alter different autophagy-related genes, a common mechanism is that they both perturb a cell surface protein amyloid precursor proteins (APP) and activate mTOR signaling pathway. Our findings that perturbation of cellular function without nanoparticle internalization has significant nanomedicinal and nanotoxicological significances.

Keywords: MoS2 nanosheets, signal transduction, cell surface receptor, mTOR, autophagy

Introduction Two-dimensional transition metal dichalcogenides (2D TMDCs) have recently emerged as promising materials for important applications, such as catalysis,1 energy storage,2 photoelectronic devices,3-4 carriers for drug and gene delivery,5-8 photodynamic therapy,9-10 and biosensing.11 The increasing usage of 2D TMDCs 2


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increases the possibility of human exposures to TMDCs. For example, biomedical applications of 2D TMDCs require their direct injection into circulation and lung is one of the major target organs for the deposition of 2D TMDCs.12 In addition, the lung may be directly exposed to low concentrations of 2D TMDCs as air-borne particles generated during the production, transportation, usage and disposal of 2D TMDCsbased products. Therefore, exposures of human lung to these nanoparticles and associated health effects need to be addressed.

Two-dimensional TMDCs are known to affect cell behaviors such as cell adhesion, spreading,13 proliferation, differentiation,14 oxidative stress,15 inflammation16 and metabolism.17 How 2D TMDCs perturb cell functions depends on physicochemical properties of these nanomaterials. Thickness is one of the important physicochemical properties of 2D TMDCs that affect their applications18 and possibly their biological effects. Other types of 2D nanoparticles such as polymeric 2D nanodiscs,19 Mg(OH)2 nanoflakes,20 and graphene nanosheets21 tend to bind to cell surface without internalization when interacting with cells at low concentrations. However, due to their strong tendency to aggregate in aqueous solution, the 2D nanoparticles may aggregate easily to form 3D-like particles at higher concentrations and then enter cells through endocytosis.22-23 However, questions are: whether 2D TMDC nanosheets also bind to cell surface without cell uptake and perturb cellular functions at the same time.

3



In this study, we compared interactions between thin (5-layer) or thick (40-layer) 2D TMDCs and normal human bronchial epithelial cells 16HBE and human lung alveolar carcinoma cells A549 at a very low concentration (10 cm2/mL). We found that thin 2D TMDCs mainly bound to cell surface while thick nanoparticles entered cells. Despite they were located at different cellular locations, they both induced cell autophagy, possibly through interactions with amyloid precursor proteins (APP) on cell surface and via perturbing mTOR-dependent pathways.

Materials and methods Reagents and antibodies. MoS2 nanosheets with 5-layer and 40-layer MoS2 (MoS2-5 and MoS2-40 respectively) thickness were obtained from XFNANO (Nanjing, Jiangsu, China). Rapamycin, bafilomycin A1, FITC-BSA were purchased from Sigma-Aldrich (St Louis, MO, USA). Primary antibodies against LC3B, p-mTOR, mTOR, APP, IGF-1 and DAPK1 were purchased from Cell Signaling Technology (Boston, MA, USA). Primary antibody β-actin was purchased from Abgent (San Diego, CA, USA). Guava Nexin Reagent, Western blot luminescence reagent and PVDF membranes were purchased from Millipore (Billerica, MA, USA).

Characterization of MoS2-5 and MoS2-40 nanosheets. The thickness and size of MoS2-5 and MoS2-40 were characterized employing atomic force microscopy (AFM). For zeta potential and hydrodynamic diameter analysis, MoS2-5 and MoS2-40 were diluted in cell culture medium supplemented with 10% FBS or ultrapure water (18.2 4


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MΩ). The zeta potential and hydrodynamic diameter were measured using Malvern Nano Z Zetasizer (Nano ZS90, Malvern, U.K.).

LC3-GFP U87 cell line. The autophagy reporter cell line LC3-GFP U87 was constructed by stably transfected the LC3-GFP to human astrocytoma U87 cells.24 In these cells, autophagosomes can be directly observed as fluorescent punctuates when LC3-I is conjugated to phosphatidylethanolamine to form the LC3-II on the surface of autophagosomes. Autophagy can be easily identified by counting fluorescent punctuates.

Cell culture. The LC3-GFP U87 cells were cultured in DMEM (Gibco) culture medium. Human bronchial epithelial cells 16HBE were cultured in MEM (Gibco) culture medium. Human alveolar epithelial cells A549 were cultured in 1640 (Gibco) culture medium. All the culture mediums were supplemented with 10% fetal bovine serum (Clark), 100 U/mL of penicillin and 100 μg/mL streptomycin (Gibco). The three cell lines were cultured at 37 °C with 5% CO2.

Cellular localizations of MoS2 nanosheets determined by confocal laser scanning microscopy (CLSM). 16HBE cells and A549 cells were respectively incubated with FITC-BSA labeled MoS2-5 and MoS2-40 (10 cm2/mL) for 12 h. Then cells were fixed with 4% paraformaldehyde (PFA) for 1 h under room temperature, followed by PBS washing. The images were analyzed using Laser Scanning Microscope LSM 710 (Carl 5



Zeiss GmbH, Germany).

Cellular localizations of MoS2 nanosheets measured by transmission electron microscopy (TEM). 16HBE and A549 cells were treated with MoS2-5 or MoS2-40 (10 cm2/mL) for 12 h. Cells were then fixed in 3% glutaraldehyde (pH 7.4) under room temperature and rinsed. Next, cells were then stained with 2% aqueous uranyl acetate solution and dehydrated through alcohol of concentration gradient. Cells were embedded in 100% Epon after treatments with propylene oxide and a series of propylene oxide/Epon dilutions. The ultrathin sections were cut using a LKB-V ultramicrotome, and the images were acquired by JEOL-1200EX using MORADA-G2.

Cellular quantification of MoS2 nanosheets by inductively coupled plasma mass spectrometry (ICP-MS). After treatment with MoS2-5 or MoS2-40 (10 cm2/mL) for 12 h, cells were washed once (to measure amounts of nanosheets adhering to cell surface) or for six times (the nanosheets adhering on cell surface were completely washed away, thus only measure the internalized nanosheets). Then cells were detached by trypsin-EDTA solution. After counting the cell number, cells were lysed with aqua regia overnight. The total amount of Mo was determined by ICP-MS. The amounts of surface-bound nanosheets were obtained by the difference between the two values.

Quantification of autophagosomes. The LC3-GFP U87 cells were seeded on confocal culture dish. After various treatments, cells were fixed with 4% paraformaldehyde. The 6


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fluorescent images of cells were acquired using the laser scanning confocal microscope (LSM 700, Zeiss, Germany). The number of fluorescent punctuates in at least 30 cells was counted to quantify cell autophagy. Three independent experiments were carried out.

Immunoblotting. Treated cells were harvested and lysed using cell lysis buffer (FNN0011, Invitrogen) supplied with protease inhibitor cocktail (P8340, SigmaAldrich) and phenylmethylsulfonyl fluoride (P7626, Sigma-Aldrich). Protein samples were loaded onto SDS-PAGE. Then the proteins were separated and transferred onto PVDF membrane. 5% w/v nonfat dry milk in TBST (TBS with 0.05% Tween-20) was used to block the membrane. After washing with TBST, the membrane was incubated with the primary antibody (1:1500, in 5% BSA or nonfat dry milk in TBST) at 4 °C overnight. After washed for three times with TBST, the membrane was incubated with secondary antibody (1:3000, in 5% BSA or nonfat dry milk in TBST) for 1 h at room temperature. Then the membrane was washed for another three times with TBST and incubated with a luminescent reagent. The protein bands emerged and band intensity was quantified by ImageJ.

PCR array analysis. After incubating with MoS2-5 or MoS2-40 (10 cm2/mL) for 12 h, the 16HBE cells were lysed using Trizol (Invitrogen, CA, USA), and total RNA was extracted. Approximately 1.5 μg of RNA was used for cDNA synthesis using SuperScript III Reverse Transcriptase (Invitrogen). The cDNA was added to wells of 7



an autophagy-specific PCR array plate (PAHS-084A, Qiagen) with autophagy-specific gene or housekeeping gene primer mix pre-loaded in each well. The PCR plate was subjected to the two-step RT2 PCR program (95 °C for 10 min, 95 °C for 15 s and 60 °C for 60 s). The mRNA expressions of 84 autophagy-specific genes were quantified by ΔΔCt method.

Dissipative particle dynamics simulation. To elucidate how the thickness of MoS2 nanosheet regulates its interaction state with the cellular membrane, we adopted dissipative particle dynamics (DPD) simulations. The DPD method, which is coarsegrained simulation technique with hydrodynamic interaction, was first introduced to simulate hydrodynamic behavior of complex fluids25-27 and proved to be useful in study of the mesoscale behavior of lipid membranes,28-30 especially the nanoparticlemembrane interaction.31-33 The elementary units of DPD simulations are soft beads whose dynamics are governed by Newton’s equation of motion, similar to the MD method. The inter-bead force exerted on each bead is composed of conservative, dissipative, and random force. In the pure membrane system, the value of interaction parameters is determined by the hydrophobicity of the two beads. For any two beads of the same type, the parameter was set to 25. For two beads of different types, the parameter was 25 if the two beads are both hydrophobic or both hydrophilic. If one bead is hydrophobic and the other is hydrophilic, the parameter is set to 80. To describe the binding interaction between nanosheets and the membrane, the corresponding interaction parameter was set to 5. Note that although some atomistic details were 8


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sacrificed during the coarse-graining procedure, the essential features of the system can be reproduced by the model and the parameter set. In this work, we used the N-varied DPD simulation method,29 a particular variant of the DPD method, in which the targeted membrane surface tension can be precisely controlled by monitoring the lipid density in the boundary region, which plays a role as a reservoir of lipids. Detailed description of the simulation method can be found elsewhere.34-35

Results and discussion Characterization of MoS2-5 and MoS2-40 Nanosheets To explore how the thickness of 2D nanoparticles affects nanoparticle-cell interactions, MoS2 samples with similar size distribution but different thickness were analyzed. The morphology of MoS2-5 and MoS2-40 (Table 1) was first characterized by atomic force microscopy (AFM). AFM showed an average thickness of MoS2-5 to be 4.6 ± 0.8 nm, and MoS2-40 to be 40.1 ± 8.6 nm. The size distribution analysis showed that most nanosheets ranged from 100 to 250 nm for both MoS2-5 and MoS2-40. Therefore biological effects of MoS2-5 and MoS2-40 were investigated in this work.

MoS2-5 and MoS2-40 nanosheets carried negative charges as shown by their zeta potentials (Table 1). In water, the zeta potentials of MoS2-5 and MoS2-40 are -44.7 ± 4.1 and -41.3 ± 0.8 mV. In cell culture medium containing 10% fetal bovine serum (FBS), the zeta-potentials of MoS2-5 and MoS2-40 were shifted to -10.2 ± 0.6 and -10.3 ± 0.9 mV, indicating significant protein adsorption on their surfaces. As an indication 9



of suspension uniformity, the hydrodynamic diameters of MoS2-5 and MoS2-40 showed values of 164.7 ± 1.6 and 169.2 ± 9.6 nm in water, and 211.2 ± 33.3 and 225.1 ± 17.6 nm in cell culture medium with 10% FBS (Table 1). These data showed that MoS2-5 and MoS2-40 were suspended well in aqueous solution or biological medium.

Table 1. Characterization of MoS2-5 and MoS2-40.

10


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In AFM characterization of nanosheets, a concentration of 10 cm2/mL was used for MoS2 nanosheets. Our characterization results showed that at this concentration the difference between two nanosheets was the most evident, i.e. MoS2-40 was eight times thicker than MoS2-5. Therefore, 10 cm2/mL was an appropriate concentration to investigate the effect of thickness on cellular location and cell perturbation.

Thickness-Dependent Cellular Internalization of Nanosheets The cellular localization of nanoparticles may affect their perturbations to cellular functions and cell fate. To explore the relationship between the thickness of MoS2 nanosheets and their cellular interactions, we first investigated cell internalization of nanoparticles using three methods: confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS).

FITC-bovine serum albumin (BSA)-labeled MoS2-5 (FITC-BSA-MoS2-5) or MoS2-40 (FITC-BSA-MoS2-40) were incubated with 16HBE cells or A549 cell first and then cells were analyzed by CLSM. As shown in Figure 1 and Figure S1, most FITC-BSAMoS2-5 nanosheets were adsorbed to the cell membranes of 16HBE cells (Figure 1a-c) or A549 cells (Figure S1a-c). On the other hand, most FITC-BSA-MoS2-40 nanosheets were internalized by 16HBE (Figure 1d-f) or A549 cells (Figure S1d-f).

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Figure 1. Cell binding or internalization of MoS2-5 or MoS2-40 nanosheets in 16HBE cells. (a-f) CLSM images of 16HBE cells after being treated with FITC-BSA labeled MoS2-5 or FITC-BSA labeled MoS2-40 at a concentration of 10 cm2/mL for 12 h. Green: MoS2-5 or MoS2-40 nanosheet. (g-h) TEM images of sections of 16HBE cells showing that MoS2-5 nanosheets bound to surface while MoS2-40 nanoparticles are internalized 12 h after incubation with MoS2-5 (g) or MoS2-40 (h) at a concentration of 10 cm2/mL. (i) The average fraction of nanosheets internalized by 16HBE cell or binding on cell surface after MoS2-5 or MoS2-40 treatment as determined by ICP-MS. Data were shown as mean ± s.d. (n = 3). *P < 0.05.

We also analyzed the cellular localization of MoS2-5 and MoS2-40 nanosheets using TEM. Most MoS2-5 nanosheets bound to cell membranes outside of 16HBE (Figure 12


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1g) and A549 cells (Figure S1g). On the other hand, MoS2-40 nanosheets were more frequently observed inside of 16HBE (Figure 1h) or A549 cells (Figure S1h), which is consistent with CLSM observations. The semi-quantitative analysis by counting particles in 41 cells in 34 TEM images showed that about 70% of MoS2-5 particles were outside of 16HBE cells. On the other hand, about 80% of MoS2-40 nanosheets were internalized by 16HBE cells (Figure S2a). Similar trend was observed in A549 cells (Figure S2b).

Above results, although gave some direct observations, could not offer any quantitative evaluation. To further quantitatively assess cell internalization of MoS2 nanosheets, we determined the cellular internalization and membrane binding of MoS2-5 or MoS2-40 in 16HBE cells and A549 cells by quantifying total amount of Mo before or after washing away membrane bound nanosheets using ICP-MS analysis. Quantitative analysis results showed that more than 80% of MoS2-5 nanosheets bound to surface, while about 80% of MoS2-40 nanosheets were internalized by 16HBE (Figure 1i) or A549 cells (Figure S1i).

Two dimensional nanoparticles were seldom internalized by cells at low concentrations.19-21 However, some studies reported that 2D nanoparticles were internalized by cells. Two dimensional TMDCs such as MoS2 and WS2 nanoparticles were reported to be taken up by various cancer cells at relatively high concentrations (34-100 μg/mL),6,

10, 36-37

at which concentrations aggregation of MoS2 nanosheets 13



occurred and 3D nanoaggregates formed. To confirm this speculation, we examined effect of nanosheets concentration on nanosheets thickness and their cellular internalization. As the concentration was increased from 10 to 40 cm2/mL, the thickness of MoS2-5 increased from ~ 4.6 nm (Table 1) to 20-40 nm (Figure 2a,b), indicating the aggregation tendency of MoS2-5 at higher concentrations. Furthermore, the concentration increase of MoS2-5 also increased the cell internalization of nanosheets in 16HBE cells (Figure 2c,d). The energy optimization aspects of thickness-dependence of cell uptake of 2D nanosheets were also investigated next by computational modeling studies.

Figure 2. Increased cell internalization of MoS2-5 caused by aggregation at high concentration. (a) Representative AFM topography of aggregated MoS2-5 at high 14


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concentration of 40 cm2/mL. (b) Analysis of the height of aggregated MoS2-5 through AFM at concentration of 40 cm2/mL. (c) Cellular internalization of MoS2-5 or aggregated MoS2-5 nanoparticles after incubated with 16HBE cells under 10 or 40 cm2/mL for 12 h. Data were shown as mean ± s.d. (n = 3). *P < 0.05. (d) Scheme showing

concentration-dependent

MoS2-5

nanosheets

aggregation

and

cell

internalization.

Computer Simulation Indicates that Cell Surface Binding of MoS2-5 is Favored To understand at the molecular level how the nanosheet-cell membrane interaction influenced by properties of both the nanosheet and the membrane, we performed dissipative particle dynamics (DPD) simulations. The lateral size of all nanosheets was fixed to 13 × 13 nm2 due to technical limitation of computation, with the layer number varying from 2 to 16. All the nanosheets were initially positioned 1.0 nm above the membrane of different surface tensions. The system setup and coarse-grained models used in our simulations are illustrated in Figure S3.

In the first set of simulations, nanosheets of different layer numbers were horizontally positioned above plasma membranes of different surface tensions. After 5 μs simulation for each case, five interaction states were identified, depending on both the nanosheet thickness and the membrane surface tension, as summarized in the phase diagram (Figure S4). For few layered nanosheets, they were found to keep adhering on the membrane of moderate surface tensions (Figure 3a). The membrane wrapping on the 15



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nanosheet, which is responsible for the cellular uptake, became energetically unfavorable due to the sharp edge of nanosheets. When the membrane was under a negative surface tension, a different interaction pathway was identified (Figure 3b), i.e., the nanosheet tilted to allow the membrane protrude from the wrapping front and finally wrap the nanosheet from the top surface (Figure 3c,d).35 The final equilibrium position of the nanosheet was found to keep lining at the membrane midplane (Figure 3e), suggesting that the nanosheet was trapped at the membrane with failed internalization.

Figure 3. Few layered nanosheets preferred to bind on the plasma membrane surface with failed internalization. (a) Time sequence of typical snapshots showing thin nanosheet binding on the membrane of a higher surface tension (

 LNPA

= 1.4). (b)

Time sequence of the typical snapshot showing that the thin nanosheet can be wrapped by the membrane of a lower surface tension (

 LNPA

= 1.6), but trapped at the

membrane with failed internalization. (c-e) Time evolutions of the nanosheet rotational angle, the membrane wrapping percentage, and the distance between the nanosheet 16


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center and the membrane center under both higher and lower surface tensions.

For thick nanosheets, they were experimentally found to enter cells readily through endocytosis (Figure 1h, Figure S1h). Here, when positioned an eight-layered nanosheet above a plasma membrane of a higher surface tension, only partial membrane wrapping was achieved in the finite simulation time (Figure 4a). However, when the membrane was under a lower surface tension, the wrapping was facilitated and accomplished in an endocytic pathway (Figure 4b,c). Even under a higher surface tension, the final position of nanosheet was below the membrane center and kept moving downwards as the simulation proceeded, suggesting that thick nanosheets can be finally wrapped by the membrane to achieve the internalization (Figure 4d).

Figure 4. Multi layered nanosheets were readily wrapped by the plasma membrane in an endocytic pathway. (a) Time sequence of typical snapshots showing partial wrapping 17



achieved in the finite simulation time under a higher membrane surface tension (

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 LNPA

= 1.4). (b) Endocytic wrapping of the multi layered nanosheet by the membrane under a lower surface tension (

 LNPA

= 1.7). (c, d) Time evolutions of the wrapping

percentage and the distance between the nanosheet and the membrane of two different surface tensions.

In real systems, nanosheets rotate randomly before encountering the cell, thus raising an important question of whether or how the initial orientation of nanosheets influences interactions with the cell membrane. To test it, nanosheets of different layer numbers were positioned on the membrane with the nanosheet plane in parallel with the membrane normal direction. Our simulation results showed that the final interaction state was nearly unaffected by the initial orientation of nanosheets (Figure S5), i.e., thin nanosheets preferred to adhere onto or be trapped at the membrane with failed internalization, while thick nanosheets are readily wrapped by the membrane to achieve internalization, in good agreement with our experimental findings.

Therefore, both experimental findings (Figure 1, Figure 2, Figure S1-2) and theoretical predictions (Figure 3, Figure 4, Figure S3-5) converged to a conclusion that cell surface binding and cellular internalization of nanosheets depend on thickness of nanosheets. Previous studies have shown that negatively charged NPs should not be easily internalized by cells because of the repellent interactions with negatively charged lipids on cell membranes.38 MoS2-5 nanosheets used in this work were negatively charged. 18


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We speculated that the electrostatic repulsion from cell membranes may further inhibit the cellular uptake of MoS2-5. In addition to low concentration and thin sheet feature, surface charge may be another reason for the lower cellular internalization of MoS2-5 nanosheets. The next question is how the different modes of cell binding by thin and thick MoS2 nanosheets affect cell functions and cellular signaling events.

Cell Autophagy Induction by Both Thin and Thick Nanosheets at Non-Lethal Concentration To evaluate the interactions between MoS2 nanosheets and cells, we first tested cytotoxicity induced by MoS2-5 or MoS2-40 at various concentrations. Our results showed that both MoS2-5 and MoS2-40 exhibited very low cytotoxicity at a concentration of 10 cm2/mL (equivalent to 0.025 mg/mL for MoS2-5 and 0.2 mg/mL for MoS2-40) in 16HBE cells (Figure S6a). To test whether the sharp edges of MoS2-5 or MoS2-40 caused physical damage to cell membranes during nanosheets/cell interactions, a lactate dehydrogenase (LDH) release assay was performed. The results, however, showed that both MoS2-5 and MoS2-40 did not induce LDH release at such a low concentration (10 cm2/mL) (Figure S6b). Two dimensional nanosheets may rotate randomly before encountering cell membranes in multiple orientations. Hypothetically, when nanosheets interact with cell membranes in vertical orientation, physical damage of cell membrane may be induced by the sharp edges of nanosheets. However, in this work, MoS2 nanosheets did not induce obvious physical damage to cell membranes. Our computational simulation results showed that the final interaction state was 19



unaffected by initial orientation of nanosheets when they encounter cells, i.e., despite different initial orientations, both the thin and thick MoS2 nanosheets interact with cell membrane in parallel orientation rather than vertical orientation due to energy reasons.

To test whether these nanosheets affected regular cell functions at this concentration, we also examined cell cycle perturbation and cell apoptosis. Comparing with controls, MoS2-5 or MoS2-40 induced no obvious cell cycle arrest (Figure S6d), no obvious early apoptosis, late apoptosis, or necrosis (Figure S6c) at the nanosheets concentration of 10 cm2/mL in 16HBE cells.

Although most cell functions were not affected at a low concentration, the interactions of nanosheets with cells might cause a stressful condition and induce cell autophagy. To assess cell autophagy induced by MoS2-5 or MoS2-40, an autophagy-reporting cell line, LC3-GFP U87 was used.24 The cell culture medium induced a basal level of autophagy represented by an average of five punctuates per cell by counting 54 cells, while MoS2-5 or MoS2-40 induced 18-25 punctuates per cell counting from at least 81 cells for each group, indicating an enhanced autophagy (Figure 5a, Figure S7). To verify that autophagy induction by MoS2-5 and MoS2-40 was not limited to one cell line, we also examined nanosheet-induced autophagosomes formation in 16HBE cells using TEM. Result confirmed that MoS2-5 and MoS2-40 also induced cell autophagy in 16HBE cells (Figure 5b). We also determined levels of LC3-II proteins quantitatively in 16HBE cells. The protein levels of LC3-II in cells exposed to MoS2-5 or MoS2-40 at 20


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the concentration of 10 cm2/mL were 2.5 or 2.9 times of basal LC3-II levels in normal cells treated with cell culture medium (Figure 5c,e,g,h). The obvious enhancement of LC3-II bands confirmed that both MoS2-5 (Figure 5c,g) and MoS2-40 (Figure 5e,h) enhanced autophagy in 16HBE cells. Previous report showed that impurities such as lithium ion used in nanosheet preparation could induce cell autophagy through mTORindependent pathway.39 To exclude such possibilities, we tested autophagy induction by supernatant of nanosheets suspension. Our results showed that no increase in LC3II protein concentration (Figure S8a,b), suggesting that cell autophagy induction was not triggered by supernatant or impurities in nanosheets suspensions.

Another possibility is that the accumulation of LC3-II may be a result of the impaired degradation of autophagosomes, not really autophagy. To exclude this possibility, we carried out autophagy flux assay. A specific vacuolar-type H+-ATPase inhibitor, bafilomycin A1, was used to inhibit the lysosomal degradation of autophagosomes. Our data showed that both MoS2-5 and MoS2-40 induced a 23% and 25% higher formation of LC3-II in bafilomycin A1 treated-cells than cells only treated with bafilomycin A1 without nanoparticles (Figure 5d,f, Figure S9a,b). These results indicated that both MoS2-5 and MoS2-40 actually triggered autophagy induction, rather than just blocked the degradation of autophagosomes.

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Figure 5. Autophagy induction by MoS2-5 or MoS2-40. (a) Cell autophagy induction by MoS2-5 or MoS2-40 was evaluated using LC3-GFP U87 cells. Representative CLSM images of LC3-GFP U87 cells after incubation with MoS2-5 or MoS2-40 at a concentration of 10 cm2/mL are shown. Cells incubated with cell culture medium or rapamycin (10 μM) for 12 h was used as negative or positive controls. The scale bar is for 5 μm. (b) TEM images of 16HBE cells showing autophagosomes formation after 22


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treatments of cells with MoS2-5 or MoS2-40 at a concentration of 10 cm2/mL. The scale bar is for 500 nm. (c, e) Dose-dependent LC3-II formation in 16HBE cells after treatment with MoS2-5 (c) or MoS2-40 (e) as determined by Western blotting against LC3B antibody. Cells treated with rapamycin (10 μM) for 12 h was used as the positive control. (d, f) Autophagy flux enhancement in 16HBE cells after treatment with MoS2-5 (d) or MoS2-40 (f) at a concentration of 10 cm2/mL with or without treatment by bafilomycin A1 (100 nM) for 4 h. (g, h) Dose-dependent LC3-II formation in 16HBE cells quantified by the ratio of band intensities of LC3-II over β-actin using ImageJ. Data were shown as mean ± s.d. (n = 3). *P < 0.05.

While thick MoS2-40 nanosheets were mostly endocytosed by cells (80%), more than 80% of thin MoS2-5 nanosheets were found only bound to cell surface (Figure 1i). Despite the huge difference on their cellular localization, MoS2-5 and MoS2-40 induced autophagy at a comparable level (Figure 5g,h). This strongly indicated that while thick MoS2-40 nanosheets induced an endocytosis-mediated autophagy in 16HBE cells, the thin MoS2-5 nanosheets induced cell autophagy without internalization, probably by perturbing cell surface receptors. Nanoparticle can bind to cell membrane receptors and perturb cell functions during cell uptake of these nanoparticles.40-42 For example, 2D MoS2 nanosheets tagged with a k-opioid receptor agonist activated k-opioid receptor while being internalized by cells.43 Konjac glucomannan-modified SiO2 nanoparticles also perturbed cell functions without being internalized by cells when endocytosis

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inhibitors were used.44 Then the next question is how MoS2-5 nanosheets activate cell autophagy by binding only to cell membranes?

Autophagy Induction by Both MoS2-5 and MoS2-40 by Modulating Cell Surface Receptors and mTOR-Dependent Pathway Autophagy induction can be divided into two categories: mTOR-dependent and mTORindependent autophagy.45 To explore whether MoS2-5 and MoS2-40 induced autophagy through modulating mTOR signaling pathway, the activation of mTOR was examined. Results showed that both MoS2-5 and MoS2-40 inhibited the phosphorylation of mTOR in a dose-dependent manner (Figure 6a,b, Figure S10a,b), suggesting they induced autophagy via mTOR-dependent pathway.

To further examine the possible mechanisms of MoS2-5- and MoS2-40-induced autophagy, we analyzed the expression of 84 autophagic genes using autophagy specific RT2 Profiler PCR Array (Qiagen, Germantown, USA). The mRNA expression levels of the 84 genes in 16HBE cells after treatment with MoS2-5 or MoS2-40 were compared to those after treatment with cell culture medium. Even though MoS2-5 and MoS2-40 enhanced cell autophagy to a similar level, analyses showed that two nanosheets activated some common (APP and TMEM74) and some different (IGF-1, DAPK1, ULK1 etc.) autophagy-related genes (Figure 6c,d, Table S1). In addition to our findings that both MoS2-5 and MoS2-40 induced mTOR-dependent autophagy, we further constructed molecular interaction networks using Cytoscape software on the 24


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basis of KEGG database.46 Many genes, such as mTOR, IGF-1 and APP for MoS2-5, and mTOR, APP, MAPK8, and ULK1 for MoS2-40 showed much higher interdependence among all the significantly changed genes (Figure 6e,f), suggesting their central roles in regulating autophagy.

Nanoparticles may regulate cell functions by interacting with cell surface proteins. For example, carbon nanotubes perturbed several cell functions by interacting with insulin growth factor-1 receptor,24 bone morphogenetic protein receptor 247-48 and mannose receptor49. 2D graphene oxide nanosheets interacted with various surface proteins such as toll-like receptor 4 (TLR4)/TLR9,50 TGF-β receptor51 and integrin,52 triggering cellular autophagy,50 necrosis,53 apoptosis,51 as well as perturbation of plasma membrane and cytoskeletal meshwork52 in different cells.

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Figure 6. Perturbation of autophagy related genes, proteins and signaling network by MoS2-5 or MoS2-40. Immunoblot assay indicated that the phosphorylation of mTOR (p-mTOR) was decreased by MoS2-5 (a) or MoS2-40 (b). PCR array analysis of the expression of autophagy-related genes in 16HBE cells after MoS2-5 (c) or MoS2-40 (d) (10 cm2/mL) treatment for 12 h compared to culture medium treatment. (e, f) Interaction network analysis between mTOR and autophagy related genes altered by the treatment of MoS2-5 (e) or MoS2-40 (f) using Cytoscape software.

Amyloid precursor protein (APP) is an integral membrane protein that plays a central role in the pathogenesis of Alzheimer's disease.54 Downregulation of APP by siAPP transfection resulted in a reduced extracellular regulated protein kinases activation,55 thereby suppressing the phosphorylation of mTOR and enhancing cell autophagy.56 Here, we found that both MoS2-5 and MoS2-40 downregulated the expressions of APP at levels of gene and protein (Figure 6c,d, Figure 7a,c, Figure S11a,c), resulting in an mTOR-dependent cellular autophagy, as shown by decreased phosphorylation of mTOR (Figure 6a,b, Figure S10a,b). Insulin-like growth factor-1 (IGF-1) is another cell surface protein that was downregulated by MoS2-5 but not by MoS2-40 (Figure 7b, Figure S11b). Carbon nanotubes downregulated IGF-1 at levels of gene and protein and suppressed phosphorylation of mTOR inducing autophagy.24, 57-59 Therefore, it is likely that MoS2-5 might suppress IGF-1 expression by interacting with IGF-1 receptor on cell surface.

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Figure 7. Probable interactions between MoS2 nanosheets and cell surface proteins and possible perturbations of autophagy related cell signaling. Immunoblot assay indicated that MoS2-5 inhibited APP (a) and IGF-1 (b), and MoS2-40 inhibited APP (c). (d) A signaling scheme showing current understanding of probable mechanisms for MoS2-5or MoS2-40-induced autophagy.

Besides cell surface proteins APP and IGF-1, several genes involved in the upstream regulation of autophagy signaling pathway were also identified, including BNIP3 for MoS2-5, and DAPK1 and ULK1 for MoS2-40. BNIP3 is a member of the BH3-only subfamily of Bcl-2 family proteins.60 The upregulation of BNIP3 by MoS2-5 might promote autophagy by disrupting the Bcl-2–BECN1 complex and activating BECN1.60 Meanwhile, death-associated protein kinase (DAPK1) is normally a mediator of programmed cell death.61 It is mainly expressed in the cytoplasm and induces cell autophagy through multiple signaling pathways, including inhibition of the phosphorylation of mTOR.62-65 The intracellular MoS2-40 enhanced the expression of 28


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DAPK1 at levels of gene and protein (Figure S12, Table S1), thus enhancing cell autophagy via inhibiting the phosphorylation of mTOR and activating ULK1, a key protein involved in the formation of autophagosomes.66

The PCR array data also showed that several Atg family genes involved in the phosphatidylethanolamine conjugation of LC3 were also altered by MoS2 nanosheets exposures. For example, MoS2-5 upregulated the expression of Atg10 (Figure 6c, Table S1), promoting the elongation of isolation membranes. Meanwhile, MoS2-40 downregulated the expression of Atg4 (Figure 6d, Table S1), resulting in an enhanced transformation of LC3-I to LC3-II.67 A summary of cell perturbations by MoS2 nanosheets is shown in Figure 7d.

Concluding remarks Cellular responses to outside invasions are demonstrated by thin 2D MoS2 nanosheets as revealed in this work. At a very low concentration, about 80% of nanosheets bind to cell surface without internalization, while have evidently perturbed cellular signaling networks and activated cell autophagy. The autophagy is induced by direct interactions between nanosheets and cell surface receptors including protein APP and perturbations of mTOR pathway and multiple cellular signaling proteins. Furthermore, proteins involved in different steps of signaling networks are all susceptible to perturbations. This finding reveals superior ability of human cells in their emergent responses when

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foreign nanoparticles are still outside of cells. It also shows that potential toxicity from 2D nanomaterials is a real threat at even very low concentrations.

Supporting Information Data of experimental and computational results on cell binding and internalization analysis, cytotoxicity and autophagy induction by MoS2 nanosheets or the supernatants, quantification of autophagy related proteins, alteration of 84 autophagic genes by PCR array analysis are provided. This Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org.

Acknowledgments We thank Chang Liu for the technical assistance. This work was supported by the National Key Research and Development Program of China (2016YFA0203103), the National Natural Science Foundation of China (91543204 and 91643204) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030401).

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Remote Induction of Cell Autophagy by 2D MoS2 Nanosheets via

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