Photothermal modulation of human stem cells using light-responsive 2D nanomaterials

James K. Carrowa,1, Kanwar Abhay Singha,1, Manish K. Jaiswala, Adelina Ramireza, Giriraj Lokhandea, Alvin T. Yeha, Tapasree Roy Sarkarb, Irtisha Singha,c,2, and Akhilesh K. Gaharwara,d,e,2

aBiomedical Engineering, College of Engineering, Texas A&M University, College Station, TX 77843; bDepartment of Biology, Texas A&M University, College Station, TX 77843; cDepartment of Molecular and Cellular Medicine, Texas A&M Health Science Center, Texas A&M University, Bryan, TX 77807; dMaterials Science and Engineering, College of Engineering, Texas A&M University, College Station, TX 77843; and eCenter for Remote Health Technologies and Systems, Texas A&M University, College Station, TX 77843

Edited by Catherine J. Murphy, University of Illinois at Urbana–Champaign, Urbana, IL, and approved April 10, 2020 (received for review August 18, 2019)

Two-dimensional (2D) molybdenum disulfide (MoS2) nanomateri- Subsequently, we utilize RNA-seq to characterize the in-depth and als are an emerging class of biomaterials that are photoresponsive unbiased effect of MoS2 on the gene expression program of hMSCs. at near-infrared wavelengths (NIR). Here, we demonstrate the RNA-seq reveals the steady-state expression of the transcriptome ability of 2D MoS to modulate cellular functions of human stem 2 after the MoS2 treatment, as well as the photothermal effect of cells through photothermal mechanisms. The interaction of MoS2 MoS2 at near-infrared wavelengths (NIR). This information assists and NIR stimulation of MoS2 with human stem cells is investigated in identifying the role of MoS and NIR exposure in influencing key using whole-transcriptome sequencing (RNA-seq). Global gene ex- 2 signaling pathways. pression profile of stem cells reveals significant influence of MoS2 and NIR stimulation of MoS2 on integrins, cellular migration, and Biophysical Characterization of MoS2 Nanosheets wound healing. The combination of MoS and NIR light may pro- 2 Bulk MoS is composed of atomically thin S–Mo–S sheets vide new approaches to regulate and direct these cellular func- 2 tions for the purposes of regenerative medicine as well as stacked by short-range van der Waals forces. Bulk MoS2 was cancer therapy. chemically exfoliated following previously reported protocols (3, 18, 19). X-ray diffraction (XRD) of bulk MoS2 showed charac-

two-dimensional (2D) nanomaterials | whole-transcriptome sequencing | teristic peaks at (002), (004), (103), (006), (105), and (008), while ENGINEERING cell–nanoparticles interactions | human mesenchymal stem cells | cell peaks broadened postexfoliation at (002), (100), and (110) as adhesion indexed by Joint Committee on Powder Diffraction File card number 37-1492 (Fig. 1A). The peaks of exfoliated MoS2 in- wo-dimensional (2D) nanomaterials are an emerging class of dicated a hexagonal structure. Using Bragg’s equation [2d sin(θ) Tmaterials with anisotropic layered structure that have shown = nλ], the interlayer spacing [(002) Miller indices spacing] of – distinct physical and chemical properties (1 4). Recently, 2D exfoliated MoS2 is 6.328 Å, which is larger than the interlayer transition metal dichalcogenides (TMDs) such as molybdenum spacing of bulk MoS2 (6.124 Å). The full width at half maximum disulfide (MoS2) have been explored for biomedical applications of peak (002) was 0.089 rad for bulk MoS2, which increased to due to their photothermal characteristics (5–9). Specifically, the 2.493 rad for exfoliated MoS2. The crystallite sizes for bulk and generation of ultrathin sheets via exfoliation of MoS2 enables the formation of direct bandgap nanosheets, which can be utilized Significance for light-based therapies (8, 10–13). In addition, the absence of dangling bonds from terminal sulfur atoms of MoS nano- 2 We demonstrate the ability of two-dimensional transition materials render high physiological stability (6, 10). The photo- metal dichalcogenides (TMDs) such as molybdenum disulfide responsive ability of 2D MoS along with its chemical stability in 2 (MoS ) to modulate and direct cellular functions of human stem biological microenvironments can be exploited for the photo- 2 cells through photothermal modulation. The global gene ex- modulation of cellular functions. Although some studies have – pression profile of stem cells reveals significant influence on investigated cellular compatibility of 2D MoS2 (14 17), no integrins, cellular migration, and wound healing by photo- studies have explored their photomodulation ability. thermal modulation of MoS2. The photostimulation of MoS2 Understanding cellular responses following treatment with 2D may provide new approaches to regulate cellular migration MoS2, with and without photostimulation, will assist in exploring and related functions. their biomedical applications. Although molecular biology tech- niques such as PCR and microarrays can be used to evaluate the Author contributions: J.K.C., K.A.S., I.S., and A.K.G. designed research; J.K.C., K.A.S., effect of this treatment on cells, they are limited in throughput. To M.K.J., A.R., G.L., and I.S. performed research; A.K.G. contributed new reagents/analytic overcome this limitation, “omics” techniques can provide global tools; J.K.C., K.A.S., G.L., A.T.Y., T.R.S., I.S., and A.K.G. analyzed data; and J.K.C., K.A.S., I.S., high-throughput readouts of various cellular and molecular pro- and A.K.G. wrote the paper. cesses and can help discern affected cell–nanomaterials interac- The authors declare no competing interest. tions. Specifically, whole-transcriptome sequencing (RNA-seq) of This article is a PNAS Direct Submission. treated cells can provide an unbiased and global perspective of This open access article is distributed under Creative Commons Attribution-NonCommercial- stimulated cellular and molecular activity as well as pivotal sig- NoDerivatives License 4.0 (CC BY-NC-ND). Data deposition:The data reported in this paper have been deposited in the Gene Ex- naling pathways triggered by exposure to the nanomaterial. RNA- pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. seq can be used for determination and quantification of all GSE141456). expressed transcripts in cells by overcoming the limitations and 1J.K.C. and K.A.S contributed equally to this work. biases of conventional PCR and microarrays analysis. 2To whom correspondence may be addressed. Email: [email protected] or isingh@ Here, we report interactions of exfoliated MoS2 with human tamu.edu. mesenchymal stem cells (hMSCs) by uncovering molecular targets This article contains supporting information online at https://www.pnas.org/lookup/suppl/ and affected signaling pathways. We first examine the interplay doi:10.1073/pnas.1914345117/-/DCSupplemental. of 2D MoS2 with biomolecules under physiological conditions. First published May 27, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.1914345117 PNAS | June 16, 2020 | vol. 117 | no. 24 | 13329–13338 Downloaded by guest on October 3, 2021 Exfoliated Molybdenum Disulfide (MoS ) A Bulk Molybdenum Disulfide (MoS2) 2

Exfoliated MoS Bulk MoS2 2 (002) (002) (100) (110) (004) (006) (008) Intensity (a.u) Intensity (a.u) (103) (105)

10 20 30 40 50 60 70 10 20 30 40 50 60 70 2 (degrees) 2 (degrees) B C 4 200 110 3 2 105 2 103 1 1 100

Height (nm) 3 0 5 m 1 m 10 nm-1 -1 100 nm 0123 Distance (μm)

D E F G

Bulk Bulk ).u.a( Atomic ( %) Mo 3d Bulk Bulk 5/2 Exfoliated Exfoliated Exfoliated S 2p3/2 Exfoliated Sample Mo S A1g

Mo 3d ).u

yti

Bulk 14.8 % 85.2 % .a sne

(ytisne

t

Exfoliated 22.8 % 77.2 % n S 2p Idezilam 1/2 E1 E 2g tn 1g

Intensity (a.u.) I

J r Raman Intensity (a.u.) 3

oN J2

226 230 234 160 162 164 200 300 400 500 600 650 700 750 800 Binding energy (eV) Binding energy (eV) Raman Shift (cm-1) Wavelengh (nm)

H Photo-thermal stimulation via near infrared (NIR) (λ = 800 nm, 28 Control (water) P = 340 mW/cm2, t = 7 mins) Control (media) Control (no MoS )MMoS (500 g/mL) oS(2 mg/mL) 26 2 2 mg/mL MoS2 (340 mW/cm ) 2 2 mg/mL MoS2 (20 mW/cm ) 24 2 500 g/mL MoS2 (340 mW/cm ) 2 500 g/mL MoS2 (20 mW/cm )

Temperature (°C) Temperature 22

15 mm Thermal Imaging 01234567891011 Time (min) IJ 0 800 40000 * Protein + SDS (-control) * Protein only (+control)

-10 600 30000 Protein + 50 g/ml MoS2 Protein + 100 g/ml MoS * 2 Protein + 150 g/ml MoS 20000 2 -20 400 MoS only * 2

-30 200 Intensity (a.u.) PL

Zeta Potential (mV) 10000 Hydrodynamic diameter (nm) -40 0 0 400 450 500 550 600 400 450 500 550 600 ater PBS PBS W Media Water Media Wavelength (nm) Wavelength (nm)

Fig. 1. Physicochemical characterization of exfoliate 2D MoS2 nanosheets. (A) XRD of bulk and exfoliated MoS2.(B) AFM measures micrometer-sized nanosheets with nanometer thickness, confirming 2D shape. (C) TEM images of ultrathin MoS2 sheets displayed overlapping layers; each individual layer is numbered. Further, electron diffraction corroborated the formation of the 1T phase following lithium intercalation. (D) Atomic composition of bulk and

exfoliated MoS2 determined via elemental analysis. (E) Crystallographic transformations from bulk to exfoliated MoS2 were monitored with XPS analysis with shifts from 2H to 1T phases observed. (F) Raman spectroscopy and (G) photoluminescent measurements likewise indicated structural modifications had occurred, seen through changes in vibrational energy bands and luminescent intensity, respectively. (H) Response to NIR light was determined using an IR

camera and change in temperature over time. (I) Zeta potential and hydrodynamic size of exfoliated MoS2 in water, PBS, and media. (J) ANS assay shows protein structures are intact in the presence of exfoliated MoS2.

13330 | www.pnas.org/cgi/doi/10.1073/pnas.1914345117 Carrow et al. Downloaded by guest on October 3, 2021 exfoliated MoS2 were calculated using Scherrer equation as the interface of bound charges and diffuse layer, is shifted further ∼120 nm and 3.5 nm, respectively. from the surface, effectively reducing the zeta potential. This The morphology of MoS2 nanosheets was characterized by phenomenon was observed as the zeta potential significantly atomic force microscopy (AFM) and transmission electron mi- dropped to −17.3 ± 0.8 mV after subjecting MoS2 nanosheets to croscopy (TEM). AFM measured layered sheets that were 2 to cell culture media. This indicated that while biomolecules and 7 μm in diameter and ∼3 nm thick (Fig. 1B). The theoretical multivalent ions were strongly interacting with the nanomaterial calculation suggested the S–Mo–S atomic lattice thickness to be surface, the repulsive forces between MoS2 nanosheets are reduced about 6.5 Å (8), which indicated the selected nanoflake in the via charge screening, leading to eventual flocculation at longer magnified AFM image contained three to five sheets of mono- timescales. layer MoS2. TEM images indicated smooth topography and To improve nanosheet dispersions within these aqueous media crystalline structure of MoS2 (Fig. 1C). The selected area elec- environments, bath or probe sonication can be utilized. These tron diffraction (SAED) pattern of monolayer MoS2 confirmed mechanical methods to reduce interactions between sheets may the formation of 1T phase after exfoliation. Characteristic SAED also reduce the effective sheet size. To investigate the effects of peaks agreed with XRD data indicating a hexagonal structure. type and duration of sonication, dynamic light scattering (DLS) The elemental composition of bulk (14.8% Mo and 85.2% S) was utilized on aqueous dispersions of MoS2. Samples lacking and exfoliated MoS2 (22.8% Mo and 77.8% S) was determined sonication displayed relatively large nanosheet sizes with an av- by X-ray photoelectron spectra (XPS) (Fig. 1D). The binding erage hydrodynamic diameter of 6.9 ± 0.6 μm. Following soni- energies (BE) for molybdenum and sulfur core-level electrons cation (2 min), there was a significant reduction in size to 2.6 ± revealed the crystallographic phases associated with bulk MoS2 0.8 μm (bath sonication) and 0.6 ± 0.03 μm (probe sonication). transformed to a few layers (Fig. 1E). The BE for Mo 3d dou- Extending sonication (10 min) further reduced the hydrody- blets (3d5/2 and 3d3/2) were at 228.5 and 231.7 eV for bulk MoS2, namic size to 1.0 ± 0.3 μm (bath sonication) and 0.38 ± 0.02 μm which, after exfoliation, shifted to 228.2 and 231.5 eV, re- (probe sonication). Significant reduction in physical size within spectively, indicating the 2H to 1T phase transformation. Simi- 10 min of sonication has important ramifications for sample larly, the BE for S 2p doublets (2p3/2 and 2p1/2) were 161.4 and preparation prior to cellular exposure. Specifically, even a brief 162.6 eV, which correlate with the 2H phase and shifted to 161.1 sonication to improve dispersions of the nanosheets may result in and 162.3 eV, respectively, following exfoliation indicating physical changes to the MoS2. Smaller MoS2 nanosheet sizes will 1T phase. affect binding dynamics to proteins in the aqueous environment Raman spectroscopy confirmed the vibrational modes associ- and subsequently to the cell membrane. Furthermore, reduction ated with the MoS2 lattices for both bulk and exfoliated sheets in hydrodynamic size as a result of sonication facilitates im- ENGINEERING 1 (Fig. 1F). As expected, the two characteristic modes, E 2g cor- proved uptake of the nanosheets. Hence, we selected probe responding to in-plane vibrations of sulfur atoms with respect to sonication for 10 min for MoS2 (∼400-nm-long and 3- to molybdenum and A1g corresponding to out-of-plane vibration of 4-nm-thick nanosheet) for all future studies. −1 sulfur atoms, had energies of 380.8 and 406.5 cm , respectively, As previously noted, the hydrodynamic size of MoS2 was 1 −1 in bulk form. Upon exfoliation, the E 2g shifted to 376.5 cm influenced by the presence of ions (PBS and media) and protein −1 and the A1g band shifted to 403.9 cm . The relative magnitude (media). The adsorption of protein on the MoS2 nanosheet will of out-of-plane (A1g) vibration was larger than in-plane vibration influence cellular adhesion and internalization due to formation 1 (E 2g) in bulk. After layer exfoliation, the relative magnitude of of protein corona on the nanosheet. To evaluate these changes in 1 in-plane vibration (E 2g) was larger than out-of-plane vibration protein conformation, an 8-anilinonapthalene-1-sulfonic acid (A1g), corroborating more free sulfur atoms. Furthermore, due to (ANS) assay (21) was utilized (Fig. 1J). ANS binds selective the phase change from 2H to 1T, additional vibrational bands cationic amino acid residues (such as arginine and lysine) via such as E1g, J2, and J3 were also observed for the exfoliated MoS2 formation of an ion pair between the positively charged residues sample (20). and the sulphonyl groups of the ANS. The formation of these ion Furthermore, photoluminescent intensity decreased for exfo- pairs results in increased fluorescence emission, and thus liated MoS2 samples due to Li intercalation, resulting in a phase changes in conformation can be detected by measuring the rel- transition from semiconducting 2H to metallic 1T phase ative fluorescence intensity. When a model protein with positive (Fig. 1G). This corroborates the observation of phase transition charge at buffered pH, lysozyme (isoelectric point –11.35) is from 2H to 1T in the Raman spectra. The NIR responsiveness subjected to sodium dodecyl sulfate (SDS), a known disruptor of was evaluated to ensure photothermal effects could be utilized protein structure, a significant change in protein conformation for cell stimulation. A solution of MoS2 (500 μg/mL and 2 mg/ was observed, as indicated by an increase in fluorescence signal. mL) was subjected to NIR light, and the solution temperature However, subjecting protein to MoS2 nanosheets resulted in a was monitored using a thermocouple and IR imaging. After minimal change in fluorescence signal and therefore protein 10 min of exposure to NIR light, the temperature of MoS2 so- conformation. Furthermore, following an increase in the con- lution (2 mg/mL) was increased by ∼5.5 °C and ∼1.5 °C for the centration of MoS2 nanosheet from 50 to 150 μg/mL, no signif- high- (340 mW/cm2) and low- (20 mW/cm2) power NIR light, icant impact on fluorescent signal was observed, indicating respectively (Fig. 1H). For low concentration of MoS2 (500 μL/mL) maintenance of native protein structure over a range of relevant and water, no significant change in temperature was observed. This concentrations. The sequestering proteins with intact native demonstrated that photothermal effects could be expected within structure would enable clustering of receptors on the cell a local cellular environment following NIR exposure to exfoliated membrane and stimulate downstream intracellular pathways MoS2 nanosheets. (22, 23).

Physicochemical Stability of MoS2 Nanosheets Cellular Interaction of MoS2 Nanosheets

The aqueous stability of exfoliated MoS2 nanosheets was eval- As soon as MoS2 is subjected to physiological fluids, protein uated in deionized water (diH2O), phosphate-buffered saline corona formed on its surface due to high surface area and charge (PBS), and cell culture media containing fetal bovine serum. characteristics. We used gel electrophoresis to observed forma- MoS2 nanosheets were stable in diH2O and PBS and showed tion of protein corona. Most of serum protein was adsorbed on strong negative zeta-potential measurements of −32.7 ± 1.1 mV MoS2 and no significant difference in protein adsorption was and −29.2 ± 1.7 mV for diH2O and PBS, respectively (Fig. 1I). observed due to NIR treatment of MoS2. The colloidal stability After the addition of protein, however, the surface of shear, or of MoS2 in physiological conditions will enable significant

Carrow et al. PNAS | June 16, 2020 | vol. 117 | no. 24 | 13331 Downloaded by guest on October 3, 2021 interactions of MoS2 nanosheets with the cell membrane. It is these nanomaterials following introduction into the media. In- expected that due to 2D characteristics, MoS2 can become in- terestingly, scanning electron microscopy (SEM) images of the ternalized by cells (Fig. 2A). We investigated cellular compati- cell body depicted homogeneous coverage of submicron-sized bility of MoS2 by determining the half inhibitory concentration material across the membrane. The uniform coverage of MoS2 (IC50) using metabolic activity assay. MoS2 nanosheets showed on the cell surface is expected to improve the likelihood of high cytocompatibility at lower concentrations (<100 μg/mL) photothermal activation of cells. However, a slight decrease in after 7 d of culture (Fig. 2B). The IC50 of MoS2 was observed to cell spreading was observed due to MoS2 exposure (100 μg/mL) be ∼400 μg/mL. over a period of 72 h (SI Appendix, Fig. S1). Bright-field imaging indicated substantial binding of MoS2 To determine the uptake mechanism of MoS2 nanosheet, an in- nanosheets (100 μg/mL) to cell surfaces (Fig. 2C). Electron mi- hibition assay was performed using endocytosis pathway-specific in- croscopy was used to evaluate the extent of membrane coating by hibitors (chlorpromazine hydrochloride to inhibit clathrin-mediated,

MoS nanosheet (Adhesion to cell membrane and internalization via macropinocytosis) A 2 B IC50~ 400 g/mL Plasma 100 protein MoS2 with Plasma protein corona Macropinocytosis membrane 75 protrusion 50 Nucleus

Cytosol 25 Cell viability (%)

0 101 102 103

MoS2 concentration ( g/mL) C Bright field image SEM images D 120 ** ** No Inhibitor * 100 * Inhibitor of calveolar-mediated uptake Inhibitor of clathrin-mediated uptake 80 Inhibitor of macropinocytosis 60

40 300 µm 50 µm10 µm

Normalised Uptake (%) 20

3 hours24 hours 72 hours 0 MoS2(25 g/mL) MoS2_NIR E

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u 60 50 40 e (%) cells 0.5 v 40 ll Pop Li Ce Cell Population (% G1 20 25 Live 20 Cell viability (fraction)

0.0 0 0 0 2 2 2 2 2 2 IR S S Cs IR IR N o N _ Mo S MoS _N MSCs s _M s_ _ 2 hMSCs hMSCs hMSCs h C s R_ hM C s S C I C o MS N MS M h MS _ h MS _ hMSCs_MoS hMSCs_MoS hMSCs_MoS h Cs h Cs S hMSCs_Mo(50%) hMSCs_Mo(50%) MS hMSCs_Mo(100%) hMSCs_Mo(100%) hM h

Fig. 2. Cellular interactions of 2D MoS2 with hMSCs. (A) A protein corona forms when 2D MoS2 is suspended in media and adheres to the cell surface and is subsequently internalized by cells. (B)IC50 of MoS2 determined using metabolic assays. (C) Cellular internalization of MoS2 determined using optical, electron, and fluorescence microscopy. (D) MoS2 adhere to cell membrane and internalize via micropinocytosis and clathrin-mediated endocytosis. (E) Effect of MoS2 on metabolic activity over 7 d. (F) Effect of Mo and MoS2 nanosheet at equivalent molarities on cell viability (day 3 and day 7 on the left and right, respectively). (G) Effect of MoS2 on cell cycle. (H) Cell viability when treated with NIR, MoS2, and MoS2_NIR. *P < 0.05; not significant (n.s.) > 0.05.

13332 | www.pnas.org/cgi/doi/10.1073/pnas.1914345117 Carrow et al. Downloaded by guest on October 3, 2021 nystatin to inhibit caveolar-mediated, and wortmannin to inhibit (26 ± 4% inhibition, P < 0.01, one-way ANOVA). No significant macropinocytosis) (Fig. 2D). The results indicated that the pri- difference was observed using caveolar inhibitor as 4 ± 2% in- mary mechanisms of nanosheets uptake were clathrin (16 ± 3% hibition was observed. It is also important to note that adhered inhibition, P < 0.05, one-way ANOVA) and macropinocytosis MoS2 nanosheets may increase side scattering, the metric utilized

15 A r: 0.99 C Differentially expressed genes (DEG) hMSCs_NIR 10

5 (hMSCs_NIR_2 fpkm) 2 0 Log 0 5 10 15 2 Log2(hMSCs_NIR_1 fpkm)

15 r: 0.99 1 NIR Row-scaled z-score _2 fpkm) hMSCs_MoS2 2 10 hMSCs (25 µg/mL) (control) 5 0 (hMSCs_MoS MoS2 2 0

Log 0 5 10 15 Log (hMSCs_MoS _1 fpkm) 2 2 −1 15 r: 0.98 15 r: 0.98 10 hMSCs_MoS _NIR 2 −2

MoS _NIR_2 fpkm) 10 2 2 5 +NIR (hMSCs_2 fpkm) 2 ENGINEERING 5 Log 0 0 5 10 15 (hMSCs_MoS

2 0 Log (hMSCs_1 fpkm) 2 0 5 10 15 Log

Log2(hMSCs_MoS2_NIR_1 fpkm)

B hMSCs_NIR vs hMSCs hMSCs_MoS2 vs hMSCs e g FoldChange FoldChan 2 2 Log Total (10996) Log Total (10996) High (1) High (27) Low (1) Low (76) −6 −4 −2 0 2 4 6 −6 −4051015 −2 0 2 4 6 0 5 10 15

Log2 (Mean of normalized counts) Log2 (Mean of normalized counts)

hMSCs_MoS2_NIR vs hMSCs Venn diagram showing overlaping genes (FDR-adjusted P < 0.05) _1 _2 2 2 NIR (2 gene) _NIR_2 _NIR_1 46 2 2 hMSCs_2 hMSCs_1 hMSCs_NIR_2 hMSCs_NIR_1 hMSCs_MoS hMSCs_MoS MoS MoS and MoS _NIR MoS _NIR

FoldChange 2 2 2 2 2 (23 genes) (80 genes) (77 genes) hMSCs_MoS hMSCs_MoS

Log Total (10996) High (41)

−6 −4 −2 0 2 Low (116) 051015

Log2 (Mean of normalized counts)

Fig. 3. Understanding the effect of NIR, MoS2, and MoS2_NIR on hMSCs using RNA-seq. (A) Pearson correlation coefficient of expressed genes between replicates of different conditions (r = 0.98 for hMSC controls, r = 0.99 for hMSCs_MoS2, r = 0.99 for hMSCs_NIR, and r = 0.98 for hMSCs_MoS2_NIR). (B) MA plot highlighting differentially regulated genes (FDR-adjusted P < 0.05) of the treatment groups against the untreated hMSCs and extent of expression change (gray: all of the expressed genes, red: up-regulated genes, blue: down-regulated genes). Venn diagram indicating differential genes overlap between pairwise comparison of treatment groups. (C) Heat map of row normalized FPKM z-scores of differentially expressed genes across all pairwise comparisons against the untreated hMSCs (red: up-regulated, blue: down-regulated).

Carrow et al. PNAS | June 16, 2020 | vol. 117 | no. 24 | 13333 Downloaded by guest on October 3, 2021 to monitor uptake, which may inflate uptake values and result in respectively. Interestingly, our previous work investigating 2D relatively modest inhibition using pathway-specific inhibitors. nanomaterial treatment of hMSCs had demonstrated a higher Long-term studies indicate that hMSCs readily proliferated over number of DEGs (28), indicating that the chemical and physical a course of 7 d in the presence of various concentration of MoS2, characteristics of MoS2 affects a more selective group of cellular > indicating cytocompatibility even at higher concentrations ( IC50) pathways. The DEGs in hMSCs_MoS2 and hMSCs_MoS2_NIR (Fig. 2E). showed a decent overlap (80 genes). Earlier studies have mentioned that MoS2 degrades in physi- The hierarchical clustering of DEGs highlights the concor- ological conditions particularly in the presence of peroxidases dance of effect of MoS2 and MoS2_NIR treatments compared to (24). The effect of Molybdenum (Mo) was evaluated on cell vi- untreated control (hMSCs) and hMSCs_NIR (Fig. 3C) across ability by directly exposing 50 and 100% molar concentration of samples. Overall, our results indicated that the effect of MoS2 on Mo to cells (Fig. 2F). A significant reduction in cell viability was hMSCs is relatively moderate compared to previously reported observed due to Mo exposure, while a molar equivalent of MoS2 2D nanomaterials (28). We suspect that the relatively large size did not impact cell viability, indicating that Mo is not released and chemical stability of MoS2 nanomaterials limited the cell– from MoS2 in physiological conditions. Other indicators of cel- nanomaterials interaction. However, our study suggests that lular viability such as cell cycle showed no change as result of NIR-stimulated MoS2 nanomaterials may be used to regulate MoS2 exposure (Fig. 2G). These results were used to determine gene expression on demand. the concentration of MoS2 nanosheets (25 to 100 μg/mL) for subsequent photothermal activation. Photothermal Modulation of Stem Cell Gene Expression To further establish the cytocompatibility of MoS2, cellular In order to determine the pathways significantly impacted by apoptosis after exposure to MoS2 was measured using Annexin V MoS and MoS _NIR exposure, we utilized a variety of gene set μ 2 2 assay. The cells were treated with MoS2 nanosheets (25 g/mL) enrichment techniques including GOStats (29) and Cytoscape for a period of 24 h with and without NIR exposure. The data (30, 31). Gene ontology (GO) enrichment analysis identifies indicated that there was no significant difference in live cell cellular functions, processes, and components that show en- populations between control and treated cells, irrespective of richment for DEGs affected by nanomaterials and NIR treat- NIR treatment (Fig. 2H). The increase in NIR intensity (340 ment. While MoS treatment alone showed enrichment for 106 mW/cm2) can be used for photothermal therapy, which will be 2 GO terms (P < 0.05), MoS2_NIR treatment resulted in 218 GO useful for cancer treatment. Specifically, cells in the selected terms (P < 0.05). Focusing largely on GO terms with high sig- region can be remotely ablated by focusing NIR light (SI Ap- nificance (P value < 0.05), we encountered key terms specific to pendix, Fig. S2). However, here we have used low-intensity NIR 2 extracellular matrix organization (GO:0030198) as well as an- (20 mW/cm ) exposure for photothermal modulation. giogenesis (GO:0001525) and wound healing (GO:0042060) Global Transcriptomic Profile of Stem Cells Treated with (Fig. 4A). REVIGO analysis (32), a data management technique MoS Nanosheets to reduce redundancies among terms from clustered genes, re- 2 fined enrichments within the MoS /NIR treatment group to The changes in the steady-state expression levels of cellular 2 macromolecule metabolism, extracellular matrix (ECM) orga- transcriptome after treatment with external agents such as nization, response to stimulus, wound healing, and regulation of nanoparticles and radiation can be determined using RNA-seq motility and proliferation (SI Appendix, Fig. S3). These reduced (25). To investigate the effect of NIR (20 mW/cm2), MoS (25 2 terms correlated well with our GO terms of high significance and μg/mL), and NIR stimulation of MoS2 (MoS2_NIR), hMSCs were cultured under four different conditions including un- demonstrated a clarified progression of cellular events following treated hMSCs as control (Fig. 3A). A lower concentration of nanomaterial treatment and NIR stimulation. MoS (25 μg/mL) was selected to avoid molecular perturbation We likewise created a gene network from coexpression data 2 to examine clusters of these differentially expressed genes due to cellular stress. After 7 d of treatment, the expressed < messenger RNA (mRNA) of treated cells were collected and (FDR-adjusted P 0.05) using Cytoscape (30, 31). These clus- sequenced (Materials and Methods and SI Appendix, Materials ters grouped genes based on known pathways and signaling and Methods). This early-stage time point was utilized to uncover function, thereby generating a visualization of genetic interde- initial molecular changes, as well as to capture a snapshot of pendencies among multiple cellular processes (Fig. 4B). Out of the pool of genes identified from differential analysis, eight clear cell–nanomaterial interactions as MoS2 remained on the cell surface up until 7 d. clusters of cell function and behavior emerged pertaining to – Using RNA-seq aligner (26), the sequenced reads were angiogenesis, vascular development, cell cell interactions, ion aligned to the reference genome (hg38). The gene expression homeostasis, stress signaling, ECM organization, nucleic acid levels for every gene in every sample was normalized to obtain homeostasis, and protein binding/intracellular signaling. Specif- fragments per kilobase of transcript per million reads (FPKM). ically, for MoS2-treated hMSCs, there were clear dependencies The correlation of expressed gene levels (FPKM) for replicate between genes expressed within angiogenic/vascular develop- samples subjected to different conditions indicated a high degree ment clusters with that of the extracellular matrix. of consistency (r = 0.98 for hMSC controls, r = 0.99 for To evaluate the activation of stress related pathways such hMSCs_MoS2, r = 0.99 for hMSCs_NIR, and r = 0.98 for mitogen-activated protein kinase cascade due to NIR, MoS2, and hMSCs_MoS2_NIR). The read count for gene expression was MoS2_NIR treatment, we evaluated production of phospho- modeled by generalized linear models (GLMs) (27), revealing MEK1/2. MEK1/2 are dual-specificity protein kinases that me- significant gene expression changes between different treatment diate the phosphorylation of tyrosine and then threonine in conditions compared to untreated hMSCs (Fig. 3B and Dataset ERK1 or ERK2. As no significant production of pMEK1/2 was S1). NIR exposure to hMSCs (hMSCs_NIR) displayed minimal observed, treatment with NIR, MoS2, and MoS2_NIR did not effect on the gene expression program, as only two differentially result in activation of cellular stress-related signaling pathways expressed genes (DEGs, false discovery rate [FDR]-adjusted P < (SI Appendix, Fig. S4). We also evaluated the differentiation 0.05) were observed. Meanwhile application of MoS2 and ability of hMSCs after NIR, MoS2, and MoS2_NIR treatment; MoS2_NIR resulted in more robust changes in the gene ex- however, no significant change in expression of early osteogenic pression program of hMSCs. Specifically, hMSCs treated with marker such as alkaline phosphate was observed (SI Appendix, MoS2 and MoS2_NIR showed significant (FDR-adjusted P < Fig. S5). Overall, these results indicate that NIR, MoS2, and 0.05) change in their gene expression profile, 103 and 157 genes, MoS2_NIR treatment do not cause any cellular stress.

13334 | www.pnas.org/cgi/doi/10.1073/pnas.1914345117 Carrow et al. Downloaded by guest on October 3, 2021 hMSCs_MoS _NIR vs hMSCs hMSCs_MoS _NIR vs hMSCs AB2 2 Matrix Interactions MYH10 EXTL1 CRYAB FHL1 Cell-matrix adhesion (GO:0007160) SORT1 LMCD1 Cell-substrate adhesion (GO:0031589) SCUBE3 CKB Cell-substrate junction assembly (GO:0007044) DMPK CNN1 Cell junction assembly (GO:0034329) LMOD1 Vascular ACTA2 Cell junction organization (GO:0034330) TPM2 Development SYNPO2 ALDH1B1 BST1 DKK1 THBS1 Integrin-mediated signaling (GO:0007229) ACTG2 MYH9 PDGFRB PHLDA1 DACT1 Cell adhesion mediated by integrin (GO:0033627) SERPINB2 GPC4 RCAN1 SIRPA MGST1 IGSF23 Adherens junction assembly (GO:0034333) STEAP1 MAP3K7CL TEK Cell-Cell MLPH PPP1R14A DCBLD2 Substrate-dependent cell migration (GO:0006929) Interactions LRRC17 LDB3 LBH Angiogenesis Cell-substrate adherens junction (GO:0007045) DUSP6 FZD7 CCL2 ADAMTS7 0246 GPNMB FLG PLA2G4A NPPB -log (p-value) KISS1 10 ESM1 HSPB7 SAA1 Angiogenesis IL8 L1CAM Wound healing (GO:0042060) Response to wounding (GO:0009611) ITPR3 IDH2 Blood vessel development (GO:0001568) CTSC PXDN Vasculature development (GO:0001944) HMOX1 SLC2A1 PFKFB4 FSTL1 Cardiovascular system development (GO:0072358) PCDH10 FTH1 MEGF6 PPME1 IGFBP5 Angiogenesis (GO:0001525) STK38L CPNE7 Ion SCD5 OSBPL10 Blood vessel morphogenesis (GO:0048514) Homeostasis PCSK7 KRT7 AKR1C3 TENM2 NREP ALS2CL Sprouting angiogenesis (GO:0002040) Protein Binding/ BDKRB1 LIMCH1 IFFO2 Regulation of blood vessel (GO:0097746) ABCA3 Intracellular Events Endothelial cell migration (GO:0043534) ANXA10 CLIC3 TPM1 KRT14 TNFSF4 PODN Regulation of blood vessel size (GO:0050880) MASP1 EFHD1 TMEM171 PLTP JUP 0246810 LXN CDKN2B -log (p-value) RARRES1 SCG5 10 NXPH3 IFIT1 COL5A1 Vasculature Development IFI44 LPXN SPARC HAPLN3 GREM1 COL4A2 Collagen catabolic process (GO:0030574) CFB CTPS1 TNS1 TINAGL1 Collagen metabolic process (GO:0032963) Stress CD248 NID2 AKR1B10 HAPLN1 ITGA11 HMGA1 Collagen fibril organization (GO:0030199) Signaling Muscle cell differentiation (GO:0051146) TSLP PVR FBN1 COL12A1 Muscle system process (GO:0003012) APLP1 DPYSL4 RFX8 AKR1C1 NQO1 Supramolecular fiber organization (GO:0097435) C1orf198 TUFT1 COL16A1 Muscle cell differntiation (GO:0042692) CSF2RB Extracellular Matrix LIMS2 COL11A1 TP53I11 Nucelic Acid Muscle contraction (GO:0006936) Organization AGRN COL1A1 Homeostasis Muscle structure development (GO:0061061) Myofibril assembly (GO:0030239) FN1 COL4A1 Smooth muscle cell prolfieration (GO:0048659) DAG1 ITGA7 Actomyosin structure organization (GO:0031032) COL5A2 COL5A3 Muscle cell development (GO:0055002) COL1A2 FNDC1 CTSK COL8A2 Muscle tissue development (GO:0014706) ACAN TFPI2 ELNLAMA5 SPP1

0 5 10 15 ENGINEERING

-log10 (p-value)

C Wound healing (GO:0042060) D 0 hour 12 hours 24 hours 48 hours LBH COL1A1 CCL2 TFPI2 5

hMSCs SPARC 4 COL5A1

3 FZD7 hMSCs_MoS FN1 2 HMOX1 DCBLD2 (p-adjust)

10 2 PDGFRBTHBS1 SERPINB2 TPM1 MYH9 SAA1 COL1A2 ITPR3 -Log DAG1 MCAM hMSCs_NIR 1

0 hMSCs_MoS _NIR −20 2 2

Log2(hMSCs_MoS2_NIR/hMSCs)

48 hours 100 Pm E 150 HUVECs *** ** 100 Cells 120 Cells_NIR m) P

%) Cells_MoS Invasion 80 2 90 Cells_MoS2_NIR HUVECs_MoS2

60 60 Invasion Depth ( 30 40

HUVECs_MoS2_NIR

2 20 Wound Healing Coverage ( Healing Wound _NIR 2 HUVECs

100 Pm HUVECs_MoS 0 HUVECs_MoS hMSCs hSMCs MCF-7

Fig. 4. Functional effect of NIR, MoS2, and MoS2_NIR on cells. (A) GO terms obtained by gene enrichment analysis of DEGs (FDR-adjusted P < 0.05) due to MoS2_NIR treatment. (B) GeneMANIA analysis produced a network of coexpression interactions for differentially expressed genes within the hMSCs_MoS2_NIR population (red: up-regulated, blue: down-regulated; size increases with significance). (C) A volcano plot for the wound healing (GO:0042060), gray: all of the expressed genes, blue: genes associated with the GO term with no significant change in expression, red: genes associated with the GO term that show significantly

differential expression due to treatment. (D) Scratch assay used to determine the effect of NIR, MoS2,andMoS2_NIR on migration, cell proliferation, and wound healing of hMSCs, hSMCs, and MCF-7. The dotted lines represent scratch generated using pipette tip. The cell migration across the wound area was determined by

imaging the sample after 12, 24, and 48 h. (E) Three-dimensional invasion assay demonstrating ability of HUVECs treated with MoS2 and MoS2_NIR to invade collagen gel.

Carrow et al. PNAS | June 16, 2020 | vol. 117 | no. 24 | 13335 Downloaded by guest on October 3, 2021 MoS2 and NIR Exposure Suppresses Cell Migration by transduction and functions such as cellular migration, differenti- Modulating Cellular Adhesion ation, proliferation, and apoptosis. One of the potential ap- By evaluating the genes comprising highly significant GO terms, proaches to investigate the interaction between MoS2 nanosheets we may identify genes responsible for observable changes in cell and integrin molecules is to quantify the amount of integrin beta-1 behavior. Among the cluster of genes related to wound healing (CD29) using flow cytometry. Beta-1 integrins recognize the se- – – (GO:0042060), we noted the majority of significantly DEGs quence R G D in a wide array of ligands and can form hetero- dimers with integrin alpha-7. Integrin alpha-7/beta-1 is known to between control cells and those receiving MoS2_NIR were down- regulated (14 out of 20) (Fig. 4C). Among these, the limb bud regulate cell adhesion, matrix deposition, cell motility, and an- and heart development gene (LBH) was down-regulated after giogenesis. The flow cytometry data showed a significant decrease in unbound integrin beta-1 levels in cells treated with MoS cells were introduced to MoS2 and MoS2_NIR (log2fold: −1.799 2 and −2.846) (SI Appendix, Fig. S6). LBH plays a prominent role nanosheets (Fig. 5B and SI Appendix,Fig.S8), thus indicating that in cardiogenesis as a transcriptional activator. In addition, LBH nanosheets inhibit integrin function. Although the flow experi- interacts with a multitude of genes implicated in a variety of ments support our primary hypothesis that integrin signaling is processes, including vascular development and transcriptional involved, antibody blocking and knock-down studies are appro- events inside the nucleus. priate methods to further confirm this hypothesis. Due to this significant shift in signaling and impact on cell To identify a potential mechanism by which MoS2 nanosheets machinery, we investigated the ability of cells to migrate in a regulate cellular motility, we looked at another important GO simulated wound-healing experiment. Compared to control term that showed a high number of differentially expressed populations or those receiving NIR exposure alone, hMSCs ex- genes: cell adhesion (GO:0007155) (Fig. 5C). A large number of genes in this GO term were down-regulated (23 of 28) due to posed to MoS2 and MoS2_NIR migrated into a simulated wound at significantly reduced rates (Fig. 4D and SI Appendix, Fig. S7). MoS2_NIR treatment compared to control. Some of the prom- We further validated this effect on two different cells, human inent genes in this GO term are related to extracellular matrix proteins (COL11A1, COL4A1, and FN1), cell–ECM adhesion smooth muscle cells (hSMCs) and Michigan Cancer Foundation- receptors (ITGA11, ITGA7, IGFBP2, IGFBP5, NID2, and 7 (MCF-7) breast cancer cells, with both types displaying sig- HAPLN3), and cytoskeletal rearrangement (ACTA2, ACTG2, nificantly decreased migration following MoS and MoS _NIR 2 2 MYH9, and MYH10) (SI Appendix, Fig. S9). In particular, the treatment. This indicated that the reduction in cell migration due down-regulation of integrin subunits (ITGA7, log fold: −1.772 to MoS and MoS _NIR was not cell-type-specific and utilized 2 2 2 and ITGA11, log fold: −1.405) in conjugation with the cyto- universal cell machinery that is affected by MoS . To further 2 2 skeletal protein (ACTA2, log fold: −1.506) indicates that MoS strengthen this hypothesis, we performed three-dimensional in- 2 2 nanosheets may be interfering with surface receptor-mediated vasion assay to determine the effect of MoS2 nanosheets and focal adhesion formation (Fig. 5D). NIR stimulation on human umbilical vein endothelial cells To further validate this hypothesis, we quantified the protein (HUVECs) migration within a collagen matrix (33). HUVECs synthesis of integrin alpha-7 (ITGA7), focal adhesion kinase treated with MoS2 and MoS2_NIR showed significant decrease (FAK), smooth muscle actin (ACTA2), vinculin, and paxillin in invasion ability as determined by the invasion depth as com- (Fig. 5E). FAK, vinculin, and paxillin are known to play key roles pared to control (Fig. 4E). in integrin-dependent cellular adhesion, speeding, and motility All these experimental results validated our observations from (36, 37). The results indicate the ITGA7 synthesis was significantly RNA-seq data. Specifically, GO term enrichment analysis in- decreased in cells treated with MoS2 and MoS2_NIR. In- dicated highly related terms, including regulation of cell motility terestingly, no significant difference was observed due to photo- (GO:2000145) and negative regulation of locomotion (GO:0040013). thermal modulation on ITGA7 production. The trend in ITGA7 Within these GO terms for the MoS2_NIR group, genes like expression in different conditions is similar to results observed in PODN (log2fold: −1.526), GREM1 (−1.232), CCL2 (1.651), and wound-healing experiments, indicating the influence of MoS2 and HMOX1 (1.320) were all significantly differentially expressed. MoS2_NIR on cellular migration. The expression of other proteins Each of these markers plays a distinct role in ECM binding or involved in focal adhesion such as FAK, vinculin, paxillin, and cytoskeletal remodeling during cell locomotion. These genes, ACTA2 showed significant decrease in expression in MoS2_NIR among others, may drive the changes in cell motility following compared to control (hMSCs). To further visualize the effect of MoS _NIR treatment. For example, GREM1 is known to stim- 2 MoS2 nanosheets on focal adhesion and subsequent cellular ulate cellular motility and induce potent angiogenic response spreading, the cellular area was measured after treating with MoS2 in vivo (34, 35). Due to its role in breast cancer motility, invasion, nanosheets (SI Appendix,Fig.S1). We noted a gradual decrease in and angiogenesis, GREM1 is considered an attractive molecular cellular spreading with an increase in MoS2 concentration, in- target in cancer therapeutics. Suppression of GREM1 expression dicating reduced integrin-mediated cell adhesion. due to MoS2_NIR treatment emphasizes its therapeutic poten- tial in cancer. In addition, MoS2_NIR treatment upregulated Conclusions HMOX1, which has shown to inhibit migration and cell invasion Chemically exfoliated MoS2 is an emerging biomaterial with in breast cancer. It is important to note that spatial rearrange- unique photoresponsive ability. We investigated the biological ment of cells is necessary for angiogenesis. Based on these re- interactions of MoS2 nanosheets with proteins and cells. The sults, we therefore believe that MoS2 and MoS2_NIR treatment high surface area of MoS2 nanosheets facilitates protein ad- may limit the angiogenic potential of cells involved in vascular sorption and cellular adhesion/internalization. Once localized to development or wound-healing responses by modulating cellular the cell surface, the MoS2 nanosheets can be exploited with NIR motility. light to generate thermal stimulation. The effect of MoS2 nanosheets and subsequent stimulation with NIR light were in- MoS2 and Concurrent NIR Exposure Modulate Integrin vestigated via RNA-seq to provide a global and unbiased snap- Signaling shot of gene expression of the cells. No significant effect of NIR The reduction in cell motility due to MoS2 and MoS2_NIR exposure alone was observed on gene expression of hMSCs. treatment might be affected by integrin signaling (Fig. 5A). However, treatment with MoS2 nanosheets and subsequent stim- Integrin belongs to a glycoprotein family consisting of a non- ulation with NIR light influenced ∼103 and 157 genes in hMSCs, covalently linked α-andβ-subunits. They form heterodimers upon respectively. Most of these genes were related to cellular migra- interactions with ECM components and play critical roles in signal tion and wound healing. Further validation using a wound-healing

13336 | www.pnas.org/cgi/doi/10.1073/pnas.1914345117 Carrow et al. Downloaded by guest on October 3, 2021 A MoS inhibit B 2 1.5 integrin binding 120 hMSCs_MoS2 ECM ** hMSCs 1.2 90

Integrins 0.9 (ITGA7) 60 0.6 Counts (a.u) FAK 30 FAK 0.3 Paxillin Normalized intesity (a.u.)

Vinculin 0 0.0 4 5 6 10 10 10 2 CD29 Alexa Fluor® 647 Actin Actin hMSCs (ACTA 2) (ACTA 2) hMSCs_MoS

RPM RPM Cell adhesion (GO:0007155) ITGA7 ACTA2 CD60 500 COL16A1 GPNMB CXCL8 5 ITGA7 NID2 CCL2 COL1A1 ITGA11 SPP1 hMSCs 0 0 60 500

FBN1 0 0 4 COL12A1 COL5A1 60 500 PCDH10 TENM2 HAPLN1 hMSCs_NIR 0 0 HAPLN3 60 500 JUP 3 FZD7 TINAGL1 COL5A3 FN1 0 0 BST1 60 500 ENGINEERING

(p-adjust) APLP1LIMS2

10 TEK SIRPA 0 0 2 COL8A2 THBS1 PVR hMSCs_MoS2 MYH10 TPM1 60 500 MYH9 -Log GREM1 LPXN TNFSF4 SAA1 DAG1 JAM2 PLXND1 0 0 MCAM COL8A1 60 500 1 IGFBP2

hMSCs_MoS _NIR 0 0 2 60 500

0 0 0 −2 0 2

Log2(hMSCs_MoS2_NIR/hMSCs) 5 kb 5 kb E NIR -+ -+ NIR -+-+ MoS -+-+MoS -+-+

ITGA7 Vinculin

FAK Paxillin

ACTA2 GAPDH

GAPDH

1.5 1.5 1.5 1.5 2.0 * * * * *** 1.5 1.0 1.0 1.0 1.0

1.0

0.5 0.5 0.5 0.5 0.5 Normalised Intensity (FAK/GAPDH) Normalised Intensity (ITGA7/GAPDH) Normalised Intensity (Paxillin/GAPDH) Normalised Intensity (ACTA2/GAPDH) 0.0 0.0 0.0 0.0 Normalised Intensity (Vinculin/GAPDH) 0.0 2 2 2 2 2 _NIR _NIR _NIR _NIR _NIR hMSCs 2 hMSCs 2 hMSCs 2 hMSCs 2 hMSCs 2

hMSCs_NIR hMSCs_NIR hMSCs_NIR hMSCs_NIR hMSCs_NIR hMSCs_MoS hMSCs_MoS hMSCs_MoS hMSCs_MoS hMSCs_MoS

HMSCs_MoS HMSCs_MoS HMSCs_MoS HMSCs_MoS HMSCs_MoS

Fig. 5. Role of integrin expression on cell adhesion due to NIR, MoS2, and MoS2_NIR treatment. (A) Schematic showing the potential interaction between MoS2 nanosheets and integrin molecules at the cell surface. (B) Quantification of unbound integrin beta-1 (CD29) in cells treated with and without MoS2 nanosheets. (C) A volcano plot for the cell adhesion (GO:0007155), gray: all of the expressed genes, blue: genes associated with the GO term with no sig- nificant change in expression, red: genes associated with the GO term that show significant difference in expression due to treatment. (D) Gene track showing

normalized mRNA expression of integrin alpha-7 (ITGA7) and smooth muscle alpha-2 actin (ACTA2) for hMSCs, hMSCs_NIR, hMSCs_ MoS2, and hMSCs MoS2_NIR. (E) Protein expression level of ITGA7, FAK, vinculin, paxillin, and smooth muscle actin (ACTA2) determined using Western blot for hMSCs, hMSCs_NIR, hMSCs_ MoS2, and hMSCs MoS2_NIR. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is internal control. *P < 0.05, ***P < 0.001.

Carrow et al. PNAS | June 16, 2020 | vol. 117 | no. 24 | 13337 Downloaded by guest on October 3, 2021 assay, integrin signaling, and formation of focal adhesion kinase (hg38) using a RNA-seq aligner, Spliced Transcripts Alignment to a Reference (STAR) (26). Expression of a gene was determined by counting the number of confirmed the role of MoS2 and photothermal modulation on uniquely mapped reads overlapping the coding exons normalized by gene cellular functions. Overall, the combination of MoS2 and NIR light may provide a novel tool to modulate cellular activity length in FPKM. Genes expressed with >1 FPKM in at least half of the for potential applications in regenerative medicine and cancer samples of any condition were only used for the analysis. The gene ex- therapeutics. pression read counts were modeled as a negative binomial distribution in GLMs (27) to determine the DEGs. All of the analysis was performed using R. Materials and Methods The GO enrichment analysis was done using GOStats Bioconductor package See SI Appendix for detailed materials and methods. Detailed methods for (29). For network formation (Cytoscape) (31) and GO term enrichment < nanoparticle characterization, in vitro studies, and RNA-seq can be found in (GeneMANIA and ClueGO), only genes with FDR-adjusted P 0.05 were SI Appendix, Materials and Methods. selected. REVIGO (32) was performed to visualize GO clustering. Western analysis was performed using the iBind system (iBind; Invitrogen).

Synthesis and Characterization. The bulk MoS2 was chemically exfoliated following previously reported protocols to obtain MoS2 nanosheets (3, 18, Statistical Analysis. Determination of statistical significance between multiple 19). The structure of MoS2 nanosheets were evaluated using XRD (Bruker D8 groups was completed via ANOVA with the Tukey method. Significant P values Advanced), AFM (Bruker Dimension Icon Nanoscope), TEM (JEOL JEM-2010), were considered <0.05 unless otherwise noted. All analysis was completed in XPS (Omicron XPS system with Argus detector), Raman spectroscopy (Lab- GraphPad Prism. Ram HR confocal Raman microscope; Horiba Inc.), DLS (Zetasizer Nano ZS, Malvern Instrument), and zeta potential. Cellular interactions of MoS2 Data Availability. The data reported in this paper have been deposited in the nanosheet was determined using hMSCs (obtained from Tissue Culture Core, Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/ Institute of Regenerative Medicine, Texas A&M University Health Science geo (accession no. GSE141456). Center). The following in vitro studies were performed: metabolic activity (Alamar Blue assay), cell cycle (propidium iodide), apoptosis assay (Annexin ACKNOWLEDGMENTS. A.K.G. acknowledges financial support from the V), and cytosketal staining (actin-phalloidin and nucleus-DAPI staining). National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health Director’s New Innovator Award (DP2 EB026265). We RNA-Seq and Analysis. RNA-seq was performed on hMSCs-treated with NIR thank Dr. Holly Gibbs for her assistance with experiments utilizing the NIR 2 (20 mW/cm ), MoS2 nanosheet, and both, using a Nova seq platform (Illu- laser. The content is solely the responsibility of the authors and does not mina Nova sEq. 6000) utilizing TruSeqRNA preparation and 75 paired-end necessarily represent the official views of the funding agency. Some of the read length. Sequenced reads were aligned to the human reference genome schematics are prepared using https://biorender.com/.

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