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Reduced Graphene Oxide Functionalized with Gold Nanostar Nanocomposites for Synergistically Killing Bacteria through Intrinsic Antimicrobial Activity and Photothermal Ablation Yonghai Feng, Qingyu Chen, Qing Yin, Guoqing Pan, Zhigang Tu, and Lei Liu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00608 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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ACS Applied Bio Materials

Reduced Graphene Oxide Functionalized with Gold Nanostar Nanocomposites for Synergistically Killing Bacteria through Intrinsic Antimicrobial Activity and Photothermal Ablation Yonghai Feng,† Qingyu Chen,† Qing Yin,‡ Guoqing Pan,† Zhigang Tu,§ Lei Liu†,* †

Institute for Advanced Materials, School of Materials Science and Engineering,

Jiangsu University, Zhenjiang 212013, China ‡

Department of Clinical Laboratory, Affiliated Hospital of Jiangsu University,

Zhenjiang 212001, China § Institute

of Life Science, Jiangsu University, Zhenjiang 212013, China

ABSTRACT The exploration of multifunctional photothermal agent is important for antibacterial photothermal lysis, which has emerged as an effective approach to address the problem of pathogenic bacteria infection irrespective of the drug resistant effect. In the present work, a 2D reduced graphene oxide supported Au nanostar nanocomposite (rGO/AuNS) was prepared by the seed mediated growth method for synergistically killing multidrug resistant bacteria. Owing to the prickly and sharp-edge nanostructure, the rGO/AuNS displayed superior antibacterial activity probably due to the damaging of the cell walls or membranes. The cell viability of MRSA was as low as 32% when the MRSA were incubated with rGO/AuNS for 180 min in the absence of light. The 2D structure of the rGO/AuNS facilitated the strong binding affinity towards bacteria. Upon the 808 nm NIR laser irradiation, significant enhancement in

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bactericidal efficiency (complete death) was obtained due to the localized hyperthermal effect of rGO/AuNS. Moreover, the RGO/AuNS displayed promising biocompatibility. It indicates that the rGO/AuNS can be an alternative and effective dual functional photothermal agent for synergistically killing the multidrug resistant bacteria. KEYWORDS:

Reduced

Graphene

Oxide,

Gold

nanostars,

Antibacterial

photothermal lysis, Prickly nanostructure, Multidrug-resistant bacteria

1. INTRODUCTION The pathogenic bacterial contaminations and infections are always the great challenge for the healthcare all over the world.1 Especially, traditional antibiotic drugs show less effectiveness for fighting against the widespread infectious diseases caused by the multidrug-resistant (MDR) bacteria.2 To date, for the aim of antibacterial or bacteria-killing purpose, much effort has been focused on the development of new techniques such as photocatalysts,3 low temperature plasma,4 photodynamic therapy (PDT),5 and photothermal therapy (PTT),6 and novel antibacterial materials such as strong oxidant,7 supramolecular complex8 and noble metal nanomaterials.9 Among them, the photothermal therapy in particular emerges as an attracting effective, real-time, and precise therapy approach, in which photothermal agent plays the main role that can interact with bacteria, absorb light and generate heat to kill the bacteria through thermal ablation, and near-infrared (NIR) light can be used as promising light source that has high penetration depth in biological tissue and no significant harm to

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the tissue.10-11 Novel photothermal nanomaterials with strong NIR absorption, good bacterial binding affinity and good biocompatibility has drawn great attention for the application in the photothermal lysis of pathogenic micro strains, including noble metal nanoparticles, metal oxide or sulfides, carbon-based nanomaterials, and even organic materials.9, 12-13 Gold has many biomedical applications due to the unique optical property and good biocompatibility.14-19 Recently, gold have attracted more attention in photothermal therapy of bacteria or cancer compared to other noble metal nanostructures such as Ag nanoshell,20 Pt nanocubes,21 or Pd nanodots22 due to the controllable localized surface Plasmon resonance (LSPR) and the uniform nanostructures.23 The unique photothermal therapy has been proved by many gold nanostructures with different morphology,

including

nanospheres,

nanorods,

nanoshells,

nanocages

and

nanostars.24-28 Generally, the synthesis of special Au nanostructures (like Au nanorods) with strong NIR absorption requires a lot of organic surfactants (like CTAB) to template and stabilize, most of which are very toxic.29 They have to be modified with biomolecules or biocompatible polymers to enhance the interaction with bacteria and reduce the cytotoxicity before use, which however meanwhile reduces their photothermal conversion efficiency and stability, and increases the preparation cost. Moreover, most Au nanostructures themselves have non antibacterial activity, which may surfer the problem that residual bacteria surviving from photothermal therapy can reproduce easily in the absence of light irradiation. Therefore, the development of Au based photothermal nanomaterials with intrinsic antibacterial activity is

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imperative. We inspired from the hierarchical TiO2 nanorod spheres that displayed the intrinsic antibacterial activity in absence of light due to their sharp outward spikes easily piercing and penetrating the walls of bacteria.30 Therefore, the fabrication of prickly Au nanostructures will be possible an alternative strategy to endow the Au nanostructure with intrinsic antibacterial activity. From among the Au nanostructures, the gold nanostar (AuNS) is a new type of anisotropic gold nanoparticles with special hybridization of plasmons focalized at the core and the spikes, showing good NIR absorption,31 which could be used as a good antibacterial photothermal agent. However, pure AuNS particles are hard to be stabilized and easily aggregate, which significantly affects their light absorption.32 Wang et al. recently reported that the combining rGO nanosheets were able to well disperse and stabilize the AuNS particles, and their composites displayed enhanced SERS detection and could be used as good drug delivers.33 Moreover, due to the nanosheet structure and sharp edge, the GO and rGO are reported to have superior antibacterial property, possible through the oxidative stress or physical disruption interaction.34 Previous studies have shown that the composite of AuNPs with carbon nanomaterials (eg. carbon nanotube, GO, rGO, etc.) can improve the photothermal therapy efficiency compared to the single composition.35-37 Thus, the fabrication of AuNS and rGO nanocomposites will provide an alternative photothermal agent for the synergistic killing of bacteria, especially for the MDR bacteria. Herein, we prepared the AuNS and rGO nanocomposites (rGO/AuNS) through the

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seed mediated growth method.33 The AuNS in situ grew on the surfaces of rGO nanosheets, of which the spike number could be tuned by changing the amount of rGO, and thus leading to the variation of the LSPR absorption bands of rGO/AuNS. By optimization of the rGO amount, we could obtain the best rGO/AuNS with optimal morphology and photothermal conversion efficiency. We predicted that owing to the prickly nanostructures of AuNS and the softness and sharp edges of 2D rGO nanosheet, the rGO/AuNS exhibited superior antibacterial activity towards the MDR bacteria (Methicillin-resistant Staphylococcus aureus, MRSA) in the absence of light. Upon 808 nm laser irradiation, the rGO/AuNS could induce hyperthermal effect to completely kill the MRSA. Meanwhile, the cytotoxicity of rGO/AuNS was evaluated and the synergistic antibacterial mechanism of rGO/AuNS towards MDR bacteria was proposed. 2. EXPERIMENTAL 2.1. Materials Chloroauric acid (HAuCl4·3H2O), Graphene oxide stock solution (2 mg mL‒1) L-ascorbic acid (AA), sodium citrate, and hydrochloric acid were obtained from Sinopharm Chemical Reagent Co., Ltd; Silver nitrate (AgNO3) was purchased from Tianjin Damao Chemical Reagent Factory. Milli-Q water (18 MU cm‒1) was used for all solution preparations. 2.2. Synthesis of rGO Supported Au seeds (rGO/AuS) To prepare rGO/AuS, 2.5 mL of graphene oxide aqueous suspension (0.08 mg mL‒1) was mixed with 4 µL of HAuCl4 solution (158.3 mM) and the resultant solution

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was stirred moderately in a 5 mL glass bottle for 30 min to promote the electrostatic interaction between Au ions and the surface of graphene oxide. Meanwhile, the suspension was heated to 85 oC. Then 25 µL of sodium citrate (1.0 M) was added dropwise and kept at 85 oC for 1 h under agitation to form rGO/AuS. 2.3. Preparation of rGO/AuNS Nanocomposites For the synthesis of rGO/AuNS nanocomposites, 2 mL of HAuCl4 solution (0.25 mM) was mixed with different amounts of the freshly prepared rGO/AuNPs seed suspension (the amount of added seed suspension tuned by changing the R value: mole ratio of rGO to Au3+) through the way of inversion in a test tube. Then, 1 µL of 1 M HCl, 12 µL of silver nitrate (10 mM) and 10 µL of ascorbic acid (100 mM) were added in sequence quickly. 2.4. Characterization of rGO/AuNS A UV-Vis-NIR spectrometer (UV-1800PC) was observed to record the optical absorption spectra. Raman spectra were recorded by Laser Raman Spectrometer (DXR, ThermoFisher) with the excitation wavelength of 532 nm and the power intensity of 5mW. Atomic force microscopy (AFM) images were obtained by an atomic force microscope (MultiMode VIII SPM, Bruker). Transmission electron microscopy (TEM) were performed on a microscopy (Tecnai 12) operated at an acceleration voltage of 120 KV. As for the samples of bacteria, the copper grid was placed on the tweezers and dyed by 10 µL 1% bis(acetato)dioxouranium for 2 min, and rinsed by water for 2 min. Scanning electron microscopy (SEM) were performed on a scanning electron microscope (JSM 7001F) operated at an acceleration voltage of

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10 kV to characterize the morphology of bacteria. Zeta potential of the samples was measured by using a Zetasizer (Nnao ZS90) at a temperature of 25 oC. The concentrations of Au were detected with an ICP spectrometer (VISTA-MPX). 2.6. Measurement of Photothermal Conversion Efficiency of rGO/AuNS The photothermal conversion performance of rGO/AuNS was measured by the following method. Briefly, rGO/AuNS suspensions were diluted to different concentrations (0.26, 0.13, 0.0265, and 0.0325 mM) in water. 200 µL of rGO/AuNS suspensions in a 96-well plate was irradiated with an 808 nm NIR laser (ENS-808FC-C10, Shenzhen LEO-Photoelectric Co., Ltd) at the power 3.0 Wcm‒2 for 10 min. The temperature evolution was measured using an infrared camera provided by Brit IR, Wuhan Gold Infrared Co., Ltd in real time. The suspension of deionized water was recorded as a control. 2.7. Bacteria culture Methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300) provided by Affiliated Hospital of Jiangsu University, Staphylococcus aureus (S. aureus, ATCC25923) and Escherichia coli (E. coli, ATCC25922) provided by Institute of Life Science of Jiangsu University were selected as the model of pathogenic bacteria,. Initially, bacteria samples placed in 15 mL Screw cap centrifuge tubes was incubated in 5 mL Luria Bertani (LB) nutrient broth (10 g L‒1 peptone, 5 g L‒1 yeast extract, 10 g L‒1 NaCl, stored at 4 oC when not used) at 37 oC for 10‒12 h in a Digital display constant temperature oscillator (QYC-2012C) at 220 rpm. Then, discarded the supernatant by centrifuging at 4000 rpm for 1 min and washed with equal PBS

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buffered solution twice. OD600 was used to make sure the concentrations of resuspended bacterial solution and the resultant bacterial concentration of approximately ~106 CFU mL‒1. For safety considerations, the solution of LB and PBS buffered need to sterilize for 20 min at 121 oC and 101 kPa in High-Pressure Steam Sterilization Pot (LDZM, shanghai Shen An medical Co., Ltd). 2.8. Photothermal bactericidal property of the rGO/AuNS To explore the photothermal bactericidal property, the bacterial suspension were incubated in the presence of various concentrations of rGO/AuNS suspensions with a continuous wavelength laser of 808 nm and the power of 3.0 W cm‒2 for 6 min in 96-well plates. Then, the mixture were diluted about 1000-fold and 100 μL of bacterial solution of each sample were transferred to an agar medium, which was cultured at 37 oC for 24 h. For the survival percentage of microbial strains, the photothermal bactericidal performance was evaluated by the colony number for each agar plate. 2.9. Cytotoxicity assay of rGO/AuNS Because biocompatibility is a critical factor for the practical application, Human umbilical vein endothelial cell lines (HUVEC) provided by Institute of Life Science of Jiangsu University was used as models to analyze the cytotoxicity effects of rGO/AuNS. Briefly, HUVEC cells was grown in a 96-well plate at a concentration of 5×103 cells per well 100 μL of culture medium for 24 h at 37 oC, 5% CO2. After that, removed the supernatant and incubated with different concentrations of rGO/AuNS (0.26 mM-50.0 μg mL-1, 0.13 mM-25.0 μg mL-1, 0.065 mM-12.5 μg mL-1, 0.0325

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mM-6.3 μg mL-1) for 48 h, respectively. For the cytotoxicity analysis, 10 µL MTT (5 mg mL−1) was added and incubated for 1.5 h. After this, removed all the solution and washed each well with culture solution. Finally, each well was added 100 µL of dimethylsulfoxide (DMSO) and measured the absorbance at 550 nm determined by a microplate reader (SYNERGY H4). 2.10. Live/Dead Staining Test MRSA selected as the model bacteria were grown overnight and washed with equal saline twice to make sure the OD600 was 1.3. Then, the suspension of MRSA was incubated with rGO/AuNS0.02 in the absence or presence of NIR irradiation. The equal propidium iodide and Syto9 (L-7012, LIVE/DEAD BacLight Bacterial Viability Kit, Molecular Probes, OR, USA) was added into saline. The supernatant of MRSA and rGO/AuNS0.02 by centrifuging at 4000 rpm for 1 min was discarded and replaced with equal LIVE/DEAD BacLight Bacterial Viability Kit for 15 min in the dark and washed with saline again. 3. RESULTS AND DISCUSSION 3.1. Characterization of rGO/AuNS nanocomposites The rGO/AuNS nanocomposites were prepared by the seed-mediated growth method, as shown in Figure 1a. The rGO nanosheet supported Au seeds (AuNPs) were first prepared by the reduction of Au3+ ion absorbed on the surface of rGO nanosheets due to the existence of abundant negative charged oxygen-containing functional groups on rGO.38 Then, the AuNPs anchored on rGO was used as seeds to grow into the AuNS after being added into the growing solution containing HAuCl4,

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AgNO3, and ascorbic acid.

Figure 1. Schematic illustration of preparation of graphene-supported gold nanostar nanocomposite (rGO/AuNS) (a); AFM images of rGO (b) and rGO/AuNS (c); Normalized Raman scattering spectra recorded 532 nm laser excitation for rGO/AuNS0.02, AuNS, and rGO (d); TEM images of AuNS (e), rGO/AuNS0.02 (f),

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rGO/AuNS0.04 (g), and rGO/AuNS0.08 (h), and HRTEM image of AuNS of rGO/AuNS0.02 (insert of Figure 1f); XPS spectra of rGO/AuNS0.02 and high resolution XPS spectrum of Au4f (insert) (i); and UV-Vis-NIR spectra for rGO/AuNS (j). Figure 1b shows the AFM image of the rGO, displaying that the rGO was nanosheet with a thickness of 2 nm. Figure 1c shows the AuNS particles were well immobilized and dispersed on the surface of rGO nanosheets after the in situ growth of AuNS on rGO. As shown in Figure 1d, the Raman spectrum of the as-prepared rGO/AuNS nanocomposites has two distinctive characteristic peaks of graphene at 1340 cm‒1 (D band) and 1580 cm‒1 (G band), respectively, along with two distinctive Raman shift peaks at 2610 cm‒1 and 2900 cm‒1, ascribing to the 2D band and the D+D’ band of rGO, respectively.39 However, there is no significant characteristic peak of graphene for the single AuNS (without graphene). As shown in the TEM images of rGO/AuNS nanocomposites (Figure 1e‒h), there were visible wrinkles surrounding the AuNS particles while no wrinkles were observed in the TEM image of single AuNS particles (Figure 1e). The results indicate that rGO/AuNS nanocomposites were successfully synthesized in our experimental conditions. Interestingly, the morphology of the rGO/AuNS nanocomposites can be tuned through simply changing the ratio of rGO concentration to Au3+ ion concentration (R value). Figure 1e shows single AuNS particles can be prepared without the addition of rGO/AuNPs into the growth solution, showing an average particle size of 127.4 nm and an average spike number of 17 for an individual AuNS particle. When adding the rGO/AuNPs into the growth solution with the R value (mole ratio of rGO to Au) of

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0.02, rGO/AuNS0.02 nanocomposites with the average particle size of 78.3 nm and the average spike number of 13 were obtained (Figure 1f). Increasing the R value to 0.04, the average particle size and the average spike number of rGO/AuNS0.04 nanocomposites decreased to 50.8 nm and 8, respectively (Figure 1g). Further increasing the R value to 0.08, smaller-sized rGO/AuNS nanocomposites (36.4 nm) almost without spike growing were observed (Figure 1h). For the rGO/AuNS nanocomposites, increasing the R value led to the formation of smaller particle size and fewer spike number of AuNS particles, probably due to the secondary nucleation and seed oxidation depend on the R value.31 The HRTEM image (insert of Figure 1f) shows the lattice fringes of Au nanoparticles were examined to be ca. 0.23, which can be ascribed to the Au (1 1 1) facets of metallic Au nanoparticles. Figure 1i shows the XPS spectrum of rGO/AuNS0.02 had the peaks of C, O and Au elements, indicating the rGO/AuNS0.02 are composed of C, O, and Au elements. The high resolution XPS spectrum of Au4f in rGO/AuNS0.02 shows there were two peaks presenting at 87.6 and 83.8 eV, respectively, which could be assigned to the metallic Au.40,41 The UV-vis spectra of rGO/AuNS nanocomposites are shown in Figure 1j. For the spectra of single AuNS particle, rGO/AuNS0.02, rGO/AuNS0.04, and rGO/AuNS0.08 nanocomposites, there was one broad plasmonic absorbance band, appearing at 855 nm, 805 nm, 735 nm, and 550 nm, respectively. It is clear that the morphology of rGO/AuNS nanocomposites significantly affected their LSPR band position and intensity. Reducing the particle size and the spike number could lead to the blue shift of the absorbance band of AuNS

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particle through simply tuning R value. The rGO/AuNS0.08 shows weak absorption in NIR region could be due to the morphology of Au nanostructure of rGO/AuNS0.08 with dominated spherical structure, which usually had weak NIR absorption. Table 1 Physical and chemical properties of rGO/AuNS nanocomposites

Samples AuNS rGO/AuNS0.02 rGO/AuNS0.04 rGO/AuNS0.08

The size of rGO/AuNSa Size Spike (nm) number 127.4 17 78.3 13 50.8 8 36.4 0

λmax (nm)

Zeta Potentials (mV)

η valuec (%)

Maximum Temperaturesd (oC)

855 805 735 550

-1.1 -24.2 -22.5 -27.0

16.5 22.6 21.8 21.2

61 73.5 71.6 53.1

b

aThe

particle sizes of rGO/AuNS was calculated by TEM.

bThe

LSPR peaks of rGO/AuNS were measured according to the UV-vis-NIR spectra.

cThe

photothermal conversion efficiency (η value) of rGO/AuNS were calculated by

measuring through the Roper's calculating method.42 dThe

maximum temperatures of rGO/AuNS were measured under NIR irradiation for

6 min. 3.2. Photothermal evaluation of rGO/AuNS nanocomposites

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Figure 2. The photothermal conversion efficiency of different rGO/AuNS aqueous solution (a) and different rGO/AuNS0.02 concentrtions (b) under NIR laser irradiation for 10 min at the power density of 3 W cm‒2; (c) Photothermal effect of 0.26 mM rGO/AuNS0.02 being irradiated with different power density and shutting off the laser; (d) Time constant for heat transfer from the system is determined to be s=119.7 s by applying the linear time data from the cooling period (after 360 s) of versus negative natural logarithm of driving force temperature. The photothermal performance of different rGO/AuNS nanocomposites is shown in Figure 2a under continuous irradiation of an 808 nm laser with a power density of 3 W cm‒2 for 10 min. The temperatures of the aqueous dispersions containing AuNS, and rGO/AuNS0.02, rGO/AuNS0.04, and rGO/AuNS0.08, respectively, at the concentration of 50 μg mL‒1, increased from room temperature to the maximum temperatures of 61, 73.5, 71.6, and 53.1oC, indicating the rGO/AuNS0.02 had the best 14


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photothermal performance owing to the strong NIR absorption features and the location (805 nm) of maximum absorption wavelength, which could rapidly and efficiently convert the 808 nm wavelength laser energy into heat energy. To further study the photothermal capability of rGO/AuNS0.02, the temperature change of rGO/AuNS0.02 suspension with different concentrations was monitored as a function of time and NIR laser irradiation power. As shown in Fig. 2b, the increase of temperature was obvious concentration-dependent of AuNS. The temperature of suspension is also increased significantly with the increment of output power of the laser (Figure 2c). In the case of deionized water without AuNS as a control, the temperature elevation only reached 28.6 oC under the same irradiation condition. A quantitative investigation of the photothermal performance of rGO/AuNS0.02 was carried out. Temperature vs. time curves were recorded for rGO/AuNS0.02 upon irradiation for 6 min following by natural cooling to room temperature as shown in Figure 2c, which were used to determine the rate of heat transfer from the system. As shown in Figure 2d, the time constant (τs) for heat transfer from the system was determined to be 119.7 by applying the linear time data from the cooling period of Figure 2c (3 W) versus negative natural logarithm of driving force temperature. Through the Roper's calculating method

42

(the detail description can be seen in the

supporting information), the photothermal conversion efficiency (η value) was calculated to be 22.6 %, which was higher than that of AuNS (16.5 %), rGO/AuNS0.04 (21.8 %), and rGO/AuNS0.08 (21.2 %).

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3.3. Cytotoxicity and photothermal bactericidal efficiency of rGO/AuNS nanocomposites Because biocompatibility is a critical factor for biomedicine application, the human umbilical vein endothelial cell lines (HUVEC) was used as a model normal cell to evaluate the cytotoxicity of rGO/AuNS nanocomposites. As shown in Figure 3a, all the cell viabilities were still higher than 80% even at the high concentration of 50 μg mL‒1 for all the nanomaterials incubated for 48 hours with different concentrations (10‒50 μg mL‒1) of rGO/AuNS. The result indicated the rGO/AuNS displayed excellent biocompatibility at these concentrations.

Figure 3. Cytotoxicity of rGO/AuNS with different R value on Human Umbilical Vein Endothelial Cells (HUVEC) at different concentrations (a). Bacteria viability of S.aureus exposed to NIR laser irradiation (3 W cm‒2) for 6 min after incubating with different rGO/AuNS (b). Bacteria viability of S.aureus (c), and E.coli (d) exposed to

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NIR laser irradiation (3 W cm‒2) for 6 min after incubating with different concentrations of rGO/AuNS0.02. Figure 3b shows the photothermal antibacterial performance of rGO/AuNS nanocomposites. When gram-positive bacterium S. aureus incubated with AuNS, rGO/AuNS0.02, rGO/AuNS0.04, and rGO/AuNS0.08 suspensions, respectively, at the concentration of 25 μg mL−1 and with the NIR laser irradiation of 3 W cm2 for 6 min, the temperature of the suspensions reached about 52, 61, 55, and 48 oC, respectively, with the bacteria viabilities of S. aureus of 42%, 3%, 8%, and 98% compared to the control experiment. In the absence of NIR laser irradiation, the S. aureus bacteria (the viabilities higher than 84%) cannot be killed directly by the rGO/AuNS nanocomposites under these conditions. Compared to AuNS and rGO/AuNS0.08, the rGO/AuNS0.02 and rGO/AuNS0.04 could elevate the temperature of the suspension higher than 55 oC in 6 min upon NIR laser irradiation due to their higher photothermal conversion efficiency, and thus displayed a much higher bactericidal efficiency. The results indicated the death of the bacteria originated from the hyperthermal effect generated by rGO/AuNS nanocomposites, and 55 oC could be the critical temperature of suspension for the fast and effective killing of bacteria. As the rGO/AuNS0.02 nanocomposite exhibited the highest photothermal bactericidal efficiency, it was selected as the optimal photothermal agent for the following experiments. Figure 3c and d shows the concentration effect of rGO/AuNS0.02 on the photothermal bactericidal efficiency of both gram-positive bacterium S. aureus and gram-negative bacterium E. coli, respectively. Without NIR laser irradiation,

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increasing the rGO/AuNS0.02 concentration from 12.5 μg mL‒1 to 50 μg mL‒1 led to the viabilities of S. aureus decreasing from 98% to 78 %, and E. coli from 99 % to 67 %, respectively, indicating that high rGO/AuNS0.02 concentration of 50 μg mL‒1 are able to inhibit the growth of bacteria after incubating for 10 min, especially the growth of E. coli. It implied that the rGO/AuNS0.02 nanocomposite could have promising intrinsic antibacterial activity, which could be attributed to its membrane breaking effect because of the sharpness of 2D graphene nanosheet and the prickly structure of Au nanostars. Upon NIR laser irradiation for 6 min, the temperatures of the bacterial suspensions with the rGO/AuNS0.02 concentrations of 12.5, 25, and 50 μg mL‒1, were raised to 48, 61, and 70 oC, respectively, along with the bacterial viabilities of 42%, 3%, and 0% for S. aureus, and 60%, 2%, and 0% for E. coli. It is elucidated that the increment of the rGO/AuNS concentration favored high photothermal bactericidal efficiency due to that the bacteria could be killed more easily at the high temperature. 3.4. rGO/AuNS nanocomposites for photothermal lysis and inhibition of MRSA As the MDR bacteria such as MRSA are more dangerous than those common pathogenic bacteria, and the current and traditional antibiotics show less effectiveness on killing these MDR bacteria, it is of great significance to find alternative and safer way to destroy these bacteria, irrespective of their drug resistance. Thanks to the high photothermal conversion efficiency, the rGO/AuNS0.02 was able to kill the MRSA completely when they were treated by the NIR laser irradiation for 6 min at concentration of 50 μg mL‒1 (Figure 4a). The photothermal bactericidal efficiency of

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rGO/AuNS0.02 towards MRSA depended on the dose of the rGO/AuNS0.02. The MRSA could not be killed at the low rGO/AuNS0.02 concentration of 12.5 μg mL‒1 due to the small temperature elevation of the solution. Increasing the rGO/AuNS0.02 concentration to 25 μg mL‒1, the photothermal bactericidal efficiency significantly increased due to the high temperature elevation of the solution. However, there were still living MRSA (the viability was about 5%). Interestingly, when this NIR-treated MRSA suspension was kept in the dark for 180 min, it was found the viability of MRSA decreased from 5% to 0%, meaning the residual living MRSA was killed completely. Here, the non-intrinsic antibacterial gold nanorods modified with mPEG5000-SH (AuNRs@PEG) was used for comparison. The viability of MRSA over AuNRs@PEG treated with the NIR laser irradiation for 6 min was about 7%, which however was not changed after being kept in the dark for 180 min. It demonstrated the rGO/AuNS0.02 was able to kill the residual MRSA completely due to its special nanostructure that can damage the bacterial cell membrane,43 which is very significant for photothermal therapy.

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Figure 4. (a) Bacteria viability of MRSA exposed to NIR laser irradiation (3 Wcm−2) for 6 min after incubating with and different concentrations of rGO/AuNS0.02; (b) Bacteria viability and (c) Colony images of MRSA treated by NIR laser irradiation for 6 min after incubating with rGO/AuNS0.02 (0.13 mM) and AuNRs@PEG (0.2 mM), and thereafter kept in the absence of NIR for 180 min. In order to clarify the intrinsic antibacterial activity of rGO/AuNS nanocomposites, their antibacterial performance towards MRSA was investigated in the absence of

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light, as shown in Figure S3. When 50 μg mL‒1 of rGO/AuNS0.02 nanocomposite was incubated with MRSA, the viabilities of MRSA decreased from ~100% to 34% with prolonging the incubation time from 10 min to 180 min, lower than those for rGO/AuNS0.04 (37%) and rGO/AuNS0.08 (50%). It could be attributed to that the rGO/AuNS0.02 had more sharp spikes, which could penetrate and damage the cell membrane more seriously. However, the viability of MRSA for the single AuNS (45%) was higher than that for rGO/AuNS0.02 (34%) even though the AuNS had more sharp spikes than the rGO/AuNS0.02. It was found that pure rGO nanosheets displayed good antibacterial activity towards MRSA (its viability was 47% at 180 min) at the concentration of 50 μg mL‒1 due to its strong bacterial adhesion property and its sharp edges. Therefore, it revealed that the enhanced antibacterial activity of rGO/AuNS0.02 nanocomposite could be originated from the bacterial adhesion and sharp edge of rGO nanosheet in contrast to single AuNS. 3.5. Antibacterial mechanism of rGO/AuNS nanocomposites In order to investigate the antibacterial mechanism of rGO/AuNS nanocomposites, the MRSA was selected as the model bacteria and the rGO/AuNS0.02 nanocomposite was taken as the optimal photothermal nanoagent. To understand the antibacterial mechanism of rGO/AuNS0.02 nanocomposites, the interaction between rGO/AuNS0.02 and bacteria was first investigated by TEM and SEM analysis, as shown in Figure 5. Figure 5b shows the rGO/AuNS0.02 nanocomposites dispersedly decorated on the surface of MRSA after incubating for 10 min. The enlarged TEM image (insert image of Figure 5b) clearly displays the spikes of rGO/AuNS0.02 nanocomposites penetrated 21



into the cell membrane of MRSA along with the rGO nanosheets covering the cell membrane, indicating the rGO/AuNS0.02 nanocomposites had strong interaction with MRSA. In the case of the single AuNS nanoparticles (Figure 5c), only a few of them anchored on the surface of the MRSA but most aggregated together, meaning that the interaction between them was relative weak. The surfaces of the rGO/AuNS0.02 nanocomposites were negatively charged (Zeta potential value, Table 1), similar to that of MRSA, so the strong interaction between rGO/AuNS0.02 nanocomposites and MRSA could not attribute to the electrostatic attraction. As shown in Figure 5d, the rGO nanosheets favored the strong adhesion towards MRSA, covering the whole cell, which caused the strong inhibition of bacterial growth (Figure 4a), probably due to the 2D soft structure and the surface having abound of active groups,44 for bacterial binding, and the excellent capacity of GO or rGO extracting phospholipids.45 Thus, it indicates that the strong interaction between rGO/AuNS0.02 and MRSA could be due to the strong bacterial adhesion of rGO nanosheets of the nanocomposites, facilitating the prickly AuNS breaking the bacterial cell membrane. It can be proved that the lactate dehydrogenase (LDH) level of MRSA incubated with rGO/AuNS0.02 for 3 h in the absence of light was higher than the control bacteria (Figure S10), meaning cell membrane damage occurred. The SEM image (Figure 5f) also presented that the surfaces of MRSA were anchored with a number of particles and became rather wrinkled, after the incubation with rGO/AuNS0.02 nanocomposites, which was different from the morphologies of MRSA incubated with the single AuNS nanoparticles (smooth surfaces decorated

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with AuNS aggregates, Figure 5g), and from those incubated with single rGO nanosheets (wrinkled surfaces without AuNS loading, Figure 5h). Thanks to such strong interaction with MRSA, the number of rGO/AuNS0.02 nanocomposites anchored on MRSA increased with increasing the incubation time from 10 min to 180 min (Figure S8, SEM images and Fluorescence microscopy images), which would facilitate the penetration of the spikes of AuNS into the cell membrane and the coverage of the bacteria surface by rGO nanosheets of the nanocomposites and finally triggered the membrane breaking or the inhibition of bacteria growing, contributing to the excellent antibacterial activity of rGO/AuNS0.02 nanocomposites. Moreover, due to the strong LSPR effect, under the 808 nm NIR laser irradiation, the rGO/AuNS0.02 nanocomposites could absorb the optical energy and convert it into heat limitedly surrounding the bacteria, and thus were able to ablate the bacteria through the hyperthermal effect, finally leading to the death of the MRSA, as described in Figure 6.

Figure 5. TEM images of MRSA (a), MRSA incubated with rGO/AuNS0.02 (b) and 23



AuNS (c) after 808 nm NIR irradiation for 6 min, and MRSA incubated with rGO (d); SEM images of MRSA (e), MRSA incubated with rGO/AuNS0.02 (f) and AuNS (g) after 808 nm NIR irradiation for 6 min, and MRSA incubated with rGO (h). The laser power density was 3 W cm−2. Fluorescence microscopy images of MRSA (i), (j) and (k) and MRSA exposed to NIR laser irradiation for 6 min after incubating with 0.26 mM rGO/AuNS0.02 (l), (m) and (n), which were treated with LIVE/DEAD BacLight Bacterial Viability Kit. The scale bar in the figure is 100 μm. To make sure the damage of the cell membrane, the photothermal treated and control bacteria were subjected to Live/Dead viability (L-7012, LIVE/DEAD BacLight Bacterial Viability Kit, Molecular Probes, OR, USA) analysis (Figure 5i-l) and the LDH level analysis (Figure S10). For the normal bacteria, the green fluorescence of the Syto9 probe indicates bacterial cells with an intact membrane. For the bacteria treated with rGO/AuNS0.02 nanocomposites, the fluorescence microscopy images show red fluorescence of Propidium Iodide (PI). This proved that the kinetics of both outer membrane permeation and inner membrane depolarization was changed by the hyperthermal effect. Figure S10 shows the LDH level of MRSA incubated with rGO/AuNS0.02 under NIR irradiation for 6 min was significantly higher than the control MRSA, meaning the cell membrane of MRSA was destroyed. Therefore, it can be seen that the rGO/AuNS nanocomposites can be used as an alternative antibacterial photothermal nanoagent for the killing of MDR bacteria.

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Figure 6. Schematic illustration of mechanism of rGO/AuNS triggered intrinsic sterilization and antibacterial photothermal lysis. 4. CONCLUSIONS In summary, a highly effective photothermal agent, reduced graphene oxide/Au nanostar (rGO/AuNS) nanocomposite, was prepared by in situ chemical reduction through the electrostatic interaction between negative charges graphene oxide and positively charged gold nanoparticles. The alternative structure not only improved the photothermal conversion compared to the pure rGO nanosheets and AuNS, but also displayed promising intrinsic antibacterial activity and significantly enhanced the interaction with bacteria. The rGO/AuNS presented high efficiency in inhibition and photothermal lysis of MRSA, which depended on the amount of rGO, the concentrations of rGO/AuNS, and the treatment time. The low cytotoxicity of rGO/AuNS was anticipated to be extremely biocompatible photothermal material for the killing of drug-resistance bacteria. This work presented a promising potential 25



strategy relied on the graphene/Au-based nanohybrids for killing multidrug resistant bacteria. Supporting Information Photothermal conversion efficiency of rGO. Photothermal effect of rGO/AuNS. SEM image of bacteria colony incubation with rGO/AuNS. Bacteria viability and images of bacteria colony incubation with rGO/AuNS. LDH levels of control bacteria and NIR treated bacteria.

AUTHOR INFORMATION Corresponding Author *E-mail:

[email protected] (L. Liu)

Tel.: +86-(0)511-88787591; Fax: +86-(0)511-88791800 Present Addresses †Institute

for Advanced Materials, School of Materials Science and Engineering,

Jiangsu University, Zhenjiang 212013, China Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21606112, 21573097, 51503087), China Postdoctoral Fundation Committee (No. 2016M600372), Natural Science Foundation of Jiangsu Province (No. BK20160503), Post Doctoral Fund of Jiangsu Province (No. 1601022A), Natural

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Graphic Abstract 756x461mm (96 x 96 DPI)


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Reduced Graphene Oxide Functionalized with ... - ACS Publications

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