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1 Supporting Information Functionalized Nano-MoS 2 with Peroxidase Catalytic and Near- Infrared Photothermal Activities for Safe and Synergetic Wound Antibacterial Applications Wenyan Yin, a,#, * Jie Yu, a,b,# Fengting Lv, c Liang Yan, a Li Rong Zheng, a Zhanjun Gu, a, *and Yuliang Zhao a,d, * a Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, , China b Key Laboratory of Polymer Science and Technology, School of Science, Northwestern Polytechnical University, Xi an, Shaanxi , China c Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing, , China d Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China, Beijing, , China *Address correspondence to: yinwy@ihep.ac.cn, zjgu@ihep.ac.cn, zhaoyuliang@ihep.ac.cn # These authors contributed equally. 1. Supporting Discussion Calculation of the photothermal conversion efficiency (η) of PEG-MoS 2 NFs Photothermal properties of the PEG-MoS 2 NFs were measured according to the previous reports 1,2. The photothermal conversion efficiency (η) was calculated as follows: 1 ml of the PEG-MoS 2 NFs aqueous dispersion of different concentrations (31.25, 62.5, 125, 250, and 500 ppm) were exposed to 808 nm NIR laser for 10 min, and then the laser was shut off to let the solution cool to room temperature. Heating and cooling temperature patterns of one sample (500 ppm for MoS 2 ) were recorded by FLIR thermal camera. Then, the η value was calculated according to Eq. 1: hs Tmax T I 1 10 Where h is the heat transfer coefficient, S is the sample container surface area, T max is surr A 808 Q 0 (1) 1

2 the steady state maximum temperature, T surr is the ambient room temperature, Q 0 is the baseline energy input by the solvent and sample container without the PEG-MoS 2, I is the laser power, and A 808 is the absorbance of PEG-MoS 2 NFs at 808 nm. The value of hs is calculated from Eq. 2: m C d s hs Where τ s is the characteristic thermal time constant, the mass of the PEG-MoS 2 NFs solution (m d ) in g, and its heat capacity (C d ) was approximated to be 4.2 J g -1 k -1 (heat capacity of water). The heat energy (Q 0 ) of the sample container and solvent without PEG-MoS 2 NFs was measured independently using Eq. 3: Q 0 hs T max The time constant was τ s = s based on the linear fit from the cooling period after 600 s vs. -lnθ as shown in Figure S7d. Accordingly, the photothermal conversion efficiency calculated by Eq. 3 and Eq.1 was η = %. Preparation of bacterial solutions Single colony of Gram-negative ampicillin-resistant Escherichia coli (Amp r E. coli) grown on Luria-Bertani (LB) agar plate and Gram-positive endospore-forming Bacillus subtilis (B. subtilis) grown on Beef-Peptone-Yeast (BPY) agar plate were transferred to 10 ml of LB or BPY broth at 37 o C for 6 h, respectively. The LB culture media contained 50 μg ml -1 of ampicillin. Bacteria were harvested by centrifuging (8000 rpm for 1 min), and then were washed with phosphate buffer saline (PBS, 10 mm, ph=7.4). The supernatant was discarded and the remaining bacteria were resuspended in PBS, and diluted to an optical density of 0.1 at 600 nm (OD600 = 0.1). d T surr (2) (3) 2

3 2. Supporting Figures Figure S1. FE-SEM image of PEG-MoS 2 NFs. 3

4 Figure S2. (a) Raman spectra of bulk MoS 2 and PEG-MoS 2 NFs. (b) XRD pattern of PEG-MoS 2 NFs. XPS spectra of (c) Mo, (d) S, (e) C, and (f) O elements together with their corresponding fitting curves. The presence of C and O elements may be due to the PEG ligands or the negligible oxidation of PEG-MoS 2. 4

5 Figure S3. Distribution of the hydrodynamic diameters of (a) MoS 2 NFs and (b) MoS 2 dispersions in water synthesized in the presence and absence of PEG, respectively. (c) Zeta potential of PEG-MoS 2 NFs and MoS 2 dispersions in water synthesized in the presence and absence of PEG. 5

6 Figure S4. Determination the formation of hydroxyl radical ( OH) at (a) ph = 4.0 and (b) ph = 7.0 conditions by using TA as fluorescent probe. Reaction conditions: 1 μg ml -1 MoS 2, 100 μm H 2 O 2, 0.5 mm TA, 0.1 M acetate buffer (ph = 4.0) and PBS (ph = 7.0) at 30 o C for 12 h. 6

7 Figure S5. Steady-state kinetic assay and catalytic mechanism of PEG-MoS 2 NFs. The velocity ( ) of the reaction was measured using PEG-MoS 2 (33 μg ml -1 ) and TMB (1 mm) under different (a) H 2 O 2, and (b) TMB concentrations. (c-d) Corresponding double-reciprocal plots of peroxidase-like activity of MoS 2 NFs at a fixed concentration of one substrate versus varying concentration of another substrate for (c) H 2 O 2 or (d) TMB. (e-f) Lineweaver-Burk plots of PEG-MoS 2 NFs at each concentration of one substrate versus varying concentration of another substrate. 7

8 Table S1 The Michaelis-Menten (K m ) constant and maximum reaction rate (V max ) of MoS 2 NFs. Catalyst Substance K m (mmol L -1 ) V max (mol L -1 S -1 ) MoS 2 TMB MoS 2 H 2 O HRP 3 TMB HRP 3 H 2 O

9 We choose the TMB and H 2 O 2 as substrates to evaluate the enzymatically activity of the nano-mos 2 with or without PEG coating. It can be found that PEG could passivate nanoparticles a little (decreasing 29.8 % compared to bare MoS 2 ) as shown in Figure S6a-b, but PEG-MoS 2 nanoparticles are good enough to catalyze TMB in the presence of H 2 O 2 (10 mm). Moreover, without PEG coating, the bare MoS 2 nanoparticles tend to aggregate soon in bio-fluids (Figure S6c), which greatly limited their bio-medical applications. While coating with PEG, the as-prepared MoS 2 NFs show well biocompatibility and water solubility, favoring its bio-medical application. After weighting the pros and cons, we use PEG coating the surface of MoS 2. Figure S6. The peroxidase-like catalytic activities of PEG-MoS 2 NFs (33 μg ml -1 ) and MoS 2 (33 μg ml -1 ) without PEG. (a) Photos of the catalytic system reacted with TMB (1mM) and H 2 O 2 (10 mm ) after 5 min at 25 o C, and (b) the corresponding absorbance value for each group. The control group was TMB+H 2 O 2. (c) Stability tests of PEG-MoS 2 NFs (i) and bare MoS 2 (ii) in PBS solutions (concentration: 600 µg ml -1 ). 9

10 Figure S7. (a) Temperature changes based on the PEG-MoS 2 NFs concentration under 808-nm laser irradiation. (b) Temperature change plot over a period of 600s irradiation versus PEG-MoS 2 NFs concentration. (c) The photothermal effect of PEG-MoS 2 NFs aqueous solution under 808-nm NIR laser irradiation for 600 s and then turn off the NIR laser for 600 s. (d) The cooling time plot after 600 s versus the negative natural logarithm of driving force temperature (-lnθ) with τ s = of the concentration of 500 ppm. 10

11 Figure S8. Relative bacterial viabilities of ampicillin drug-resistant E. coli treated with different concentrations of (a) H 2 O 2, (b) PEG-MoS 2, and (c) PEG-MoS 2 +H 2 O 2 when kept H 2 O 2 concentration at 100 μm. 11

12 Figure S9. The photos of six plates seeded with 1x10 5 CFU of Amp r E. coli cultured in LB broth at (a) 37 o C and (b) 50 o C for 12 h. Three plates were used for each temperature. The results show that the direct heating of Amp r E. coli cultured in LB in the incubator at 50 C did not cause the bacterial death. 12

13 Figure S10. Photographs for the color change after GSH oxidation with different concentrations of PEG-MoS 2 NFs at different time intervals determined by Ellman s assay. 13

14 Figure S11. Cell viabilities of (a) HeLa cells and (b) HUVEC cells treated with different concentrations of PEG-MoS 2 NFs for 24 h. (c) Change in body weight obtained from mice injected with PEG-MoS 2 NFs (n =4, dose = 15 mg kg -1, Test group) and without injection (n = 4, Control). 14

15 Figure S12. Wound sizes of relative area versus initial area at 0, 2, 5, and 17 days. Error bars represent the standard deviation of three mice. 15

16 Figure S13. (a) Photographs of bacterial colonies obtained from wound tissue treated with Amp r E. coli after exposed to (I) Control (PBS), (II) H 2 O 2, (III) MoS 2, (IV) MoS 2 +NIR, (V) MoS 2 +H 2 O 2, and (VI) MoS 2 +H 2 O 2 +NIR. (b) Relative bacteria viabilities determined by plate counting method normalized to the control group. 16

17 3.References [1] Yu, J.; Yin, W.; Zheng, X.; Tian, G.; Zhang, X.; Bao, T.; Dong, X.; Wang, Z.; Gu, Z.; Ma, X.; Zhao, Y. Smart MoS 2 /Fe 3 O 4 Nanotheranostic for Magnetically Targeted Photothermal Therapy Guided by Magnetic Resonance/Photoacoustic Imaging. Theranostics 2015, 5, [2] Yin, W.; Yan, L.; Yu, J.; Tian, G.; Zhou, L.; Zheng, X.; Zhang, X.; Yong, Y.; Li, J.; Gu, Z.; Zhao, Y. High-Throughput Synthesis of Single-Layer MoS 2 Nanosheets as a Near-Infrared Photothermal-Triggered Drug Delivery for Effective Cancer Therapy. ACS Nano 2014, 8, [3]Lin,T.; Zhong, L.; Guo, L.; Fu, F.; Chen, G. Seeing Diabetes: Visual Detection of Glucose Bas ed on the Intrinsic Peroxidase-like Activity of MoS 2 Nanosheets. Nanoscale 2014, 6,

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