Synergistic Bacteria Killing through Photodynamic and Physical

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Synergistic Bacteria-Killing through Photodynamic and Physical Actions of Graphene Oxide/Ag/Collagen Coating Xianzhou Xie, Congyang Mao, Xiangmei Liu, Yanzhe Zhang, Zhenduo Cui, Xianjin Yang, Kelvin Wai Kwok Yeung, Haobo Pan, Paul K Chu, and Shuilin Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06702 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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ACS Applied Materials & Interfaces

Synergistic Bacteria-Killing through Photodynamic and Physical Actions of Graphene Oxide/Ag/Collagen Coating

Xianzhou Xie a, Congyang Mao a, Xiangmei Liu a, Yanzhe Zhang a, Zhenduo Cui b, Xianjin Yang b

a

, Kelvin W. K. Yeung d, Haobo Pan e, Paul K. Chu c, Shuilin Wu a,b*

Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry-ofEducation Key Laboratory for the Green Preparation and Application of Functional Materials,

Hubei Key Laboratory of Polymer Materials, School of Materials Science & Engineering, Hubei University, Wuhan 430062, China b

c

School of Materials Science & Engineering, Tianjin University, Tianjin 300072, China

Department of Physics & Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong 999077, China

d

Department of Orthopaedics& Traumatology, Li KaShing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong 999077, China e

Center for Human Tissues and Organs Degeneration, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China * To whom correspondence should be addressed: E-mail: [email protected]; [email protected] (S.L. Wu)

KEYWORDS: Graphene oxide; Ag nanoparticle; Disinfection; Antimicrobial; Photodynamic; Implants

1 Environment ACS Paragon Plus


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ABSTRACT: Researchers have widely agreed that the broad spectrum antibacterial activity of silver nanoparticles (AgNPs) can be predominantly ascribed to the action of Ag+. This study marks the first report detailing the rapid and highly efficient synergistic bacteria killing of AgNPs, which is achieved by inspiring AgNPs’ strong photocatalytic capability to rapidly produce radical oxygen species using 660 nm visible light together with the innate antimicrobial ability of Ag+. These AgNPs were uniformly distributed into well-defined graphene oxide (GO) nanosheets through an in-situ reduction of Ag+ and subsequently wrapped with a thin layer of typeⅠcollagen. In vivo subcutaneous tests demonstrated that 20 minutes irradiation of 660 nm visible light could achieve a high antibacterial efficacy of 96.3% and 99.4% on the implant surface against Escherichia coli and Staphylococcus aureus, respectively. In addition, the collagen could reduce the coatings’ possible cytotoxicity. The results of this work can provide a highly effective and universal GO-based bioplatform for combination with inorganic antimicrobial NPs (i.e. AgNPs) with excellent photocatalytic properties, which can be utilized for facile and rapid in-situ disinfection as well as long-term prevention of bacterial infection through the synergistic bacteria-killing of both 660 nm light-inspired photodynamic action and their innate physical antimicrobial ability.


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1. INTRODUCTION Despite strict sterilization measures, infections caused by artificial implants have not been eliminated. Once protective bacterial biofilms form on the surface of implants, antibiotic therapy has been reported to have poor outcome.1,2 In addition, the abuse of antibiotics can induce bacterial resistance and a series of side effects.3 The coating strategy is proposed to prevent implant-related bacterial infections because functionalized coatings can endow the implant materials with highly effective self-antibacterial efficacy.4,5 As a broad-spectrum antimicrobial agent, silver nanoparticles (AgNPs) have been incorporated into coatings to enhance the antibacterial performance6,7 which can effectively resist a wide range of aerobic, anaerobic, Gram positive, and Gram negative fungi and viruses by attaching to the cell membrane to kill bacteria through the released Ag+.7-11 The micro-galvanic effect generated by AgNPs is favorable for the growth of osteoblasts.12 However, high concentrations of Ag+ can result in some cell toxicity,13,14 and meanwhile, the AgNPs’ size, shape, surface coating, and solution chemistry can affect the release of Ag+,15-19 which can also influence the surface plasmon resonance of AgNPs, and thus affecting the photodynamic antibacterial efficacy.20 To obtain high antibacterial activity and good cytocompatibility, the size and distribution of AgNPs in the coatings should be precisely controlled. Some recent studies have shown that graphene oxide (GO), the derivative of graphene with a single two-dimensional layer of sp2-bonded carbon atoms,21,22 is a promising bioplatform for scaffold substrates and drug or gene delivery carriers23-25 due to its high mechanical strength,



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large surface area, fast charge transfer,26,27 and excellent biocompatibility. Furthermore, some studies showed that GO could exhibit good antibacterial activity.28,29 In addition, GO has many oxygen-containing functional groups, which can be easily modified by other molecules to endow GO with specific biological functions.29-34 Hence, some recent studies tried to utilize GO as the carrier of AgNPs because this material is favorable for the adsorption and distribution of AgNPs.35 The antibacterial efficacy of GO/AgNPs composites is much higher than that of a single GO or AgNP, and the physical penetration of cell membranes by Ag+ is still believed to be the predominant bacteria-killing mechanism.36,37 However, most studies ignore AgNPs’ effective photocatalytic ability with the assistance of GO,38 which can promote the generation of reactive oxygen species (ROS) and thus possibly enhance antimicrobial efficiency.39 In addition, the cytocompatibility of Ag+ released from GO/AgNPs composites has not been addressed. As a nature material, collagen is the predominant component in the skin, tendon, and bone. It can be degraded into small molecules that can be absorbed by body. Hence, this material has no harm for the living organisms. In addition, it can be easily combined with GO through the reaction between carboxyl groups of GO and the amine groups of the collagen to effectively improve the biocompatibility of the composite coating and reduce the cytotoxicity.40 At the same time, GO/collagen composites can overcome the shortcomings of pure collagen, such as poor mechanics and rapid degradation.41 This study first reports a rapid and highly efficient in-situ disinfection route without cytotoxicity through integrating the innate antimicrobial ability of Ag+ and the photodynamic


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effects of AgNPs inspired by 660 nm visible light that is capable of penetrating tissues to some extent (Scheme 1).

Scheme 1. The schematical illustration of cytocompatibility and synergistic bacteria-killing through the innate antimicrobial ability of Ag+ and the photodynamic effects of AgNPs in GO/AgNPs/collagen hybrid coating using 660 nm visible light.

As illustrated in Scheme 2, the preparation of GO/AgNPs/collagen hybrid coating was composed of three steps. Briefly, a GO/AgNO3 mixed solution was irradiated by UV-light to reduce Ag+ into AgNPs to obtain GO/AgNPs composite. This composite was then grafted onto the surface of polydopamine (PDA) modfied Ti. Before PDA modification, Ti plates were hydrothermally treated in KOH. Finally, collagen was grafted onto the surface through the immersion of the samples in a collagen solution. 5 Environment ACS Paragon Plus


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Scheme 2. The schematical illustration of the synthetic route of PDA/GO/AgNPs/Col-Ti

2. EXPERIMENTAL PROCEDURES 2.1. Preparation of GO and GO/AgNPs composites According to the modified Hummer’s method,42 GO powders was prepared from graphite flakes (Feng xian, Shanghai china) and then dispersed in deionized (DI) water with a concentration of 1.5 mg/mL under ultrasonic conditions. AgNO3 (0.051g) were added into 30 mL GO solution and fully mixed for 30 minutes. The mixture was placed in a vacuum environment at room temperature for 2 days to ensure the fully combination of Ag+ with GO through electrostatic attraction. Finally, the mixture was irradiated for 1 hour by UV-light


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(λ=278 nm) to reduce Ag+ into AgNPs that were uniformly distributed on the surface of GO nanosheets.

2.2. Preparation of GO/AgNPs/Collagen hybrid coating Biomedical Ti plates (Φ6 mm × 2.5 mm, Baosteel Group Corp, Shanghai China) were mechanically polished using SiC grinding papers of 240, 400, 800, and 1200 grits successively and then ultrasonically cleaned for 30 minutes with alcohol and DI water. Afterwards, these Ti plates were hydrothermally treated according to the procedures reported in our previous work.43 The hydrothermally treated Ti plates were immersed in 2 mg/mL of dopamine hydrochloride (DPA, Feng xian, Shanghai, China) Tris-HCL buffer solutions (pH 8.5) for 24 hours in a dark environment.44 The obtained samples were called PDA-Ti. After being washed by DI water and air dried, the samples were put into the mixed GO/AgNPs solution for 24 hours in a vacuum condition and then washed with DI water. The corresponding samples were named PDA/GO/AgNPs-Ti. These dried samples were subsequently immersed into the 2 mg/mL collagen 2% acetic acid solution for 24 hours. The obtained samples were labeled as PDA/GO/AgNPs/Col-Ti.

2.3. Characterization Transmission electron microscopy (TEM) images were obtained by a Tecnai G20 electron microscope (FEI, USA) operated at an accelerating voltage of 200 kV. The surface chemical



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compositions were detected by X-ray photoelectron spectroscopy (XPS) (ESCALAB 250Xi, Thermo Scientific, USA). The surface morphologies of samples were examined by scanning electron microscopy (SEM) (JSM-6510LV, JEOL, Japan). The surface functional groups were determined using Fourier transform infrared method (FTIR, NICOLET iS10).

2.4. ROS detection In order to evaluate the photocatalytic properties of coatings, the electron spin resonance (ESR) spectroscopy was employed to detect the content of the reactive oxygen species (ROS) produced by coatings using the 2,2,6,6-tetramethyl piperidyl as a capture agent. First, all the samples were set in a dark environment to avoid the effect of light. Next, these samples were placed into 96-well plates, and 100 µL of capture agent was added into each well. Subsequently, these samples were irradiated for 20 min by 660 nm visible light with a laser power of 180 mW (MRL-III-660Dnm-500mW-16090712, China). Finally, the content of ROS was detected by ESR spectroscopy.

2.5. Ag ions release test To obtain the release profile of Ag+, the three PDA/GO/AgNPs/Col-Ti and PDA/GO/AgNPsTi samples were immersed in 5 mL PBS (pH 7.4) at 37 oC, respectively. The solutions were taken out at intervals of 1, 2, 3, 5, 7, 10, 14, 18, 22, and 30 days, and the immersion solutions were refreshed each time. Ag+ release quantities were determined by inductive coupled plasma


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atomic emission spectrometry (ICP-AES, Optimal 8000, PE, USA).

2.6. Antibacterial tests The antibacterial ability was evaluated by surface plate, antibacterial ratio, and zones of inhibition (ZOIs). Two types of bacteria, E. coli and S. aureus, were used. All bacteria were cultured separately in the sterile Luria-Bertain (LB) media (containing 1 g bacto-tryptone, 0.5 g bacto-yeast extract and 1 g NaCl in 100 mL of deionized water) at 37 oC. During the antibacterial tests, all the samples, including Ti, DPA/GO-Ti, PDA/GO/AgNPs-Ti, and PDA/GO/AgNPs/ColTi, were sterilized with 75% alcohol and then exposed under ultraviolet radiation for 30 minutes. The detail procedure of the surface plate test is as following: all the samples (in a dark environment before experiments) were put into 96-well plates, separately, and 100 µL of bacterial liquid was added into each well. Each group is divided into three lines (irradiated 20 minutes with 660 nm visible light, culturing for 20 minutes in the dark and culturing for 1 day in the dark) and the bacterial liquid had been diluted with LB media to 1×105 colony-forming units (CFU) per milliliter. For the group of 660 nm visible light irradiaton for 20 minutes, the surface temperature was recorded by Thermal Imager. After the incubation time, 10 µL of bacterial liquid was taken out for dilution with LB media 100 times. Thereafter, 10 µL of diluted bacterial liquid was taken out to be evenly poured into each plate, which included 7 mL of solid LB agar. Afterwards, the number of colonies was counted by digital photo after culturing for 1 day at 37 o

C. By counting the number of colonies on the plate, the antibacterial rate can be calculated. The



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antibacterial ratio was calculated using Equation (1) (N is the number of colonies). Equation (1):

Bactericidal Ratio (%) = ( N control − N sample ) N control

For the three groups, the samples after co-culturing with the bacteria were taken out and washed three times with PBS (pH 7.4). Next, 200 µL 2.5% glutaraldehyde was employed to fix bacteria onto the surface, and the surface was then washed three times with PBS. Finally, these samples were dehydrated with 30%, 50%, 70%, 90%, and 100% alcohol for 15 minutes. After drying, the bacterial morphologies on the coatings were observed by SEM. As for ZOIs assay, the bacterial liquid was diluted with a melted LB agar plate to 1×103 CFU per milliliter, and the 10-mL bacterial suspension was added into each plate. After solidification, the different samples were placed onto the inverted culture dish. The culture time was 12 hours for E. coli and 24 hours for S. aureus at 37 oC, respectively. The inhibition zones were observed by a digital camera. In order to examine the inner structures of bacteria, the bacteria were cultured through the aforementioned process, fixed with 2.5% glutaraldehyde at room temperature, and washed three times with PBS. Afterwards, the bacteria were fixed by 1% osmium tetraoxide with PBS as the solvent for 2 hours and then washed three times with PBS. Next, the bacteria were dehydrated by 50%, 70%, 80%, 90%, 95%, and 100% alcohol for 15 minutes, successively. In the final process, the bacteria were embedded into an embedding medium (Google biological, Wuhan) and then were made a sheet of 60 nm using a diamond knife (daitome, Ultra 45°) and stained with uranylacetate. The inner structure of the bacteria was observed using TEM.


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2.7. In vitro tests The biological activity was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and cell morphology observation. MC3T3-E1 cells (the mouse calvarial cell line) were used in this work (Tongji hospital, Wuhan). In the experiment, all the samples were sterilized with 75% alcohol and then treated with 30 minutes of ultraviolet radiation. The MTT assay was used to confirm the cell viability. During the experiment, all types of sterilized samples were put into a 48-well plate, and 350 µL 1×104 cells/ml cells were added into the plates. After incubation for 1, 3, 7, and 21 days at 37 oC in a 5% CO2 incubator, a 350 µL MTT solution with a concentration of 0.5 g/L (dissolved MTT powder into pH 7.4 PBS solution) was dropped into each well after removing the stock solution, and culturing continued for 4 hours. Finally, the liquid was taken out to measure the OD490 or OD570 through a microplate reader. The cell morphologies were observed by inverted fluorescence microscopy (IX73, Olympus, Japan). Using the same procedures as those of the MTT experiment, all samples were cultured in a 96-well plate in a 5% CO2 incubator at 37 oC for 7 days. Then, samples were washed three times with PBS (pH 7.4) and immersed in 4% formaldehyde PBS solution for 10 minutes. Afterwards, the samples were rinsed with PBS three times (each time for 10 min) and then soaked with FITC (100 nM YiSen, Shanghai) for 30 minutes in the dark at room temperature. After being washed three times with PBS, the samples were subsequently dyed using DAPI (100



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nM YiSen, Shanghai) for 30 seconds in the dark at room temperature and also washed three times with PBS again. After drying, the bacterial morphologies on the samples were observed using an inverted fluorescence microscope.

2.8. In vivo tests The model used were specific pathogen-free Wistar male mice of 180 g that were obtained from Google biological (Wuhan, China). The mice were cultured in quarantine for acclimatization and detection for 1 week to build a subcutaneous infection model. The mice were divided into two groups of 10 mice each group. One group was implanted with pure Ti samples while the other group was implanted with PDA/GO/AgNPs/Col-Ti samples. Two mice in each group were irradiated for 20 minutes with 660 nm visible light after implantation of samples and bacteria. Firstly, the mice received general anesthesia and disinfection for the right back skin. Then, a surgical incision was made to obtain subcutaneous tissue on the right, and the pure Ti and PDA/GO/AgNPs/Col-Ti samples were implanted. At the same time, 20 µL S. aureus of 1×106 CFU mL-1 were injected into the place of implants. Finally, the mice were sacrificed after culturing for 1, 4, 7, and 14 days. Four mice that had received 660 nm light irradiation were also sacrificed after culturing for 1 day to prove the synergistic antibacterial effect of photodynamic action and physical action. These implants were removed, and 5 µL tissue fluids were extracted to dilute 400 times with LB media. And 10 µL diluent was added evenly into each well with 7 mL LB agar inside. The numbers of colonies were counted after culturing for 1 day at 37 oC.


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The tissues contacting implant samples were extracted for each mouse and fixed in 4% paraformaldehyde for 1 day, and the tissues were dehydrated with 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, 100% alcohol for 1 hour, alcohol benzene for 5 to 10 minutes, xylol for 5 to 10 minutes, and wax for 1 hour. The paraffin was sectioned into 4 µm using a paraffin slicing machine (Leica instrument, RM2016, Shanghai China) after the tissue were embedded into paraffin by Histocentre (Junjie, JB-P5, Wuhan China). Next, the paraffin section was immersed in hematoxylin for 3 to 8 minutes after being dehydrated by xylol for 20 minutes, 100% alcohol for 10 minutes, 95% alcohol for 5 minutes, 80% alcohol for 5 minutes, and 70% alcohol for 5 minutes. The section was then immersed in eosin for 3 minutes after being washed with DI water. The paraffin section was observed using a microscope (NIKON ECLIPSE CI, Japan) after being dehydrated with 95% alcohol for 5 minutes, 100% alcohol for 5 minutes, and xylol for 5 minutes.

3. RESULTS AND DISCUSSIONS

3.1. Characterization of GO and GO/Ag composites. As shown in Figure 1a, the as-prepared GO nanosheets exhibit a thin and wizened form. The XPS survey scan detects a strong signal of O1s and C1s (Figure S1a). The narrow scan of C1s (Figure S1b) shows that there are four main peaks at 288.8 (O=C-OH), 287.7 (C=O), 286.7 (CO), and 284.9 eV (C=C/C-C). As shown in Figure S1c, the narrow scan of O1s, there are two peaks located at 533.2 (C-OH) and 531.9 eV (O=C-OH). FTIR spectra (Figure S1d) displays that



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the absorption peaks appear at 3450, 1720, 1630, and 1050 cm-1, and the corresponding functional groups are O-H, C=O, C=C, and C-O. These results confirm that GO nanosheets were successfully fabricated.45,46 As shown in Figure 1b, small black nano dots are homogeneously distributed on the synthesized GO nanosheets, and the average size of each nano dot is about 6 nm. The selected area electron diffraction (SAED) pattern displays that these uniformly distributed dots are simple substance particles, confirming the successful reduction of Ag+ into metallic Ag, which can be further proven with an XRD pattern (Figure 1c). The uniform distribution of AgNPs was ascribed to myriads of negatively charged oxygen functional groups in GO nanosheets, which absorbed Ag+ in the solution through electrostatic incorporation (illustrated in Scheme 2). As shown in Figure 1c, the as-prepared GO has a typical peak at 2θ ≈ 10.2o, which belongs to the (002) crystal diffraction peak of GO.47 Compared with GO spectra, the typical peaks of GO/AgNPs composites are located at 2θ ≈ 38.1o, 44.3o, 64.4o, and 77.3o, which correspond to the (111), (200), (220), and (311) diffractions of metallic Ag.48,49 Due to the incorporation of AgNPs, the structure of the GO was damaged, resulting in the disappearance of the GO’s typical peak.


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Figure 1. Structure analysis of GO and GO/AgNPs composites. TEM images of: (a) GO, and (b) GO/AgNPs composites; (c) X-ray diffraction (XRD) pattern of GO and GO/AgNPs composites.

3.2. Characterization of PDA/GO/AgNPs/Col-Ti. The hydrothermal pretreatment induced the formation of interconnected reticular



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nanostructures on the surface, which can increase the superficial area and draw out many hydroxy (-OH) groups on the surface,43 favoring the grafting of more PDA in the next step.49 As shown in Figure 2a, for the PDA-Ti sample, the PDA grafting did not evidently change the surface morphologies, but did introduce numerous -NH2 groups that are helpful for further grafting of GO/AgNPs composites through the reactions between -NH2 and -COOH or -OH of GO, which can be further confirmed by XPS and FTIR measurements. After grafting with GO/AgNPs, the surface of PDA/GO/AgNPs-Ti became smooth. The EDS results showed that Ag was homogenously distributed on the surface; these results are in good agreement with the results shown in Figure 1b. The further grafting of collagen of the PDA/GO/AgNPs/Col-Ti sample weakens the Ag signal. As shown in Figure 2b, XPS survey spectra obtained from PDA-Ti samples contains O, Ti, N, and C elements, and the signal of C1s is much stronger than that of O1s. After grafting with GO/AgNPs, a stronger Ag signal can be detected, and the signal of O1s increases while C1s decreases because the GO includes many oxygen-containing functional groups. The further covering of collagen weakens the signal of Ag and increases the intensity of C1s, O1s, and N1s due to the large amount of C, O, and N in collagen. The disappearance of Ti2p can be ascribed to the thicker collagen film. The high-resolution Ag3d spectra obtained from both PDA/GO/AgNPs-Ti (Figure S2a) and PDA/GO/AgNPs/Col-Ti (Figure S2b) exhibit two peaks at 374.4 eV and 368.4 eV, indicating the metallic status of Ag in the two coatings.50 The variation of C1s spectra indicates different groups on the surface after different modifications (Figure S2c,


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S2d and S2e), which can be further proven by FTIR spectra (Figure S3). In the case of PDA-Ti, the absorption bands at 3410, 1640, and 650 cm-1 are assigned to -OH, -NH2, C=O, and -NH. When the PDA is grafted onto the surface of Ti plates, it will be positively charged. Since the oxygen-containing functional groups can make the GO nanosheets negatively charged, the GO sheets will be successfully grafted onto the surfac of Ti plates by electrostatic adsorption with PDA. Because the GO has the same functional groups with PDA in spite of nitrogen functional groups, the PDA/GO/AgNPs-Ti samples should have no absorption bands at 1340~1020 cm-1 for C-N. However, for AHT/PDA/GO/AgNPs/Col samples, N-H and C-N bonds can be obviously observed due to the covalent grafting of collagen through the reaction between -NH2 of collagen and -COOH or -OH of GO. The antibacterial properties of the samples rely on Ag content.51 The release profiles of accumulated Ag+ from both PDA/GO/AgNPs-Ti and PDA/GO/AgNP/Col-Ti samples are shown in Figure 2c. The two groups display a similar release trend to that of Ag+: a rapid release ratio at the initial stage within 7 days followed by stability. The accumulative release content of Ag from the two types of samples is almost same, but the initial release concentration of PDA/GO/AgNPs/Col-Ti is slightly higher than PDA/GO/AgNPs-Ti, and the release process can last for over 30 days. These results indicate that Ag ions in PDA/GO/AgNPs/Col-Ti can be easily released from hybrid coating compared with PDA/GO/AgNPs-Ti; this phenomenon can be attributed to the use of acetic acid during collagen grafting.51 A small amount of Ag was turned into Ag+ by acetic acid and then liable to release from the surface. The corresponding surface



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morphologies of PDA/GO/AgNPs-Ti and PDA/GO/AgNPs/Col-Ti after immersion in neutral PBS for 30 days are shown in Figure 2d. The surface morphologies of the two coatings have no significant change compared with Figure 2a. The XPS spectra obtained from these immersed samples can also show obvious signals of Ag, C, O, and N (Figure S4), which are almost the same as those before immersion (Figure 2b), indicating that as-prepared coatings are chemically stable and can maintain a long-term release.

Figure 2. Structure analysis of PDA/GO/AgNPs/Col-Ti. SEM images of: (a) PDA-Ti, PDA/GOTi, PDA/GO/AgNPs-Ti, PDA/GO/AgNPs/Col-Ti and the EDS images of PDA/GO/AgNPs-Ti, PDA/GO/AgNPs/Col-Ti. (b) XPS survey spectra of PDA-Ti, PDA/GO/AgNPs-Ti and


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PDA/GO/AgNPs/Col-Ti. (c) Ag+ release profiles obtained by immersion of three samples in 5 mL neutral PBS. (d) SEM images of PDA/GO/AgNPs-Ti and PDA/GO/AgNPs/Col-Ti after immersion for 30 days.

3.3. Antimicrobial activity Figure 3a and 3b presents the antibacterial activities of the samples against E. coli and S. aureus through a surface plate, and the number of colonies per surface plate visually depicts the antimicrobial ability. After irradiation for 20 minutes by 660 nm visible light, there are almost no bacteria on both PDA/GO/AgNPs-Ti and PDA/GO/AgNPs/Col-Ti samples, which reveals that the composite coating can exhibit rapid and highly effective photodynamic antibacterial capacity. To the contrary, the bacterial colonies on PDA/GO-Ti show little difference with the pure Ti samples. As shown in Figure S5, the corresponding antibacterial activity of PDA coating is very poor. Although PDA has strong near-infrared absorption and high photothermal conversion efficiency,52 the content of PDA in the hybrid coating is very small (Figure 2a), resulting in little photothermal effect. Meanwhile, the physical antimicrobial effect of PDA is depended upon its structure, smooth PDA has poor antibacterial effect.53 These results suggest that both PDA and GO in this work have little antibacterial activity. So the photodynamic antibacterial action is predominantly caused by AgNPs, which can be further proven by ESR spectra shown in Figure 3e. The photodynamic antibacterial ability of materials is determined by the production of ROS that can kill bacteria effectively.54,55 In addition, although 660 nm visible light can also inspire



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the photothermal effect of samples to some extent (Figure S6), the temperature can only reach up to 42℃, exhibiting little influence on bacteria within 20 min, which can be proven by the surface plate results shown in Figure S6. The bacterial colonies on the light irradiated Ti samples were almost the same as the one on the same batch cultured for 20 minutes in the dark, indicating that pure Ti samples have no antibacterial activity after irradiation under 660 nm visible light. In addition, the corresponding ROS yield is also very low due to the poor photocatalytic effect of thin TiO2 and titanate film on Ti plates.56 However, the PDA/GO-Ti sample can exhibit slightly higher antibacterial activity to some extent under the same condition. This is because the coated GO sheets not only have photocatalytic action,57 but also can enhance the photocatalytic action of the former slightly, resulting in a bit higher antibacterial efficacy.58 The photodynamic effect of AgNPs originates from the fact that the photons during light irradiation can make the surface plasmon resonance of AgNPs to produce free electrons,38,59 which can lead to a large amount of ROS,11 and thus kill bacteria. Generally, a large amount of pure AgNPs are easily agglomerated to weaken the antibacterial efficiency.60 However, once AgNPs bind to GO through electrostatic adsorption, the agglomeration of AgNPs can be eliminated because GO nanosheets with a large surface area and a myriad of negatively charged groups can favor the homogeneous dispersion of AgNPs. Meanwhile, the high conductivity of GO nanosheets can enhance the photocatalytic performance of AgNPs.38,39 This is because AgNPs have been reported to show an absorption peak at 631 nm,61 indicating that 660 nm


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visible light can inspire the AgNPs to release electrons. These excited electrons can be rapidly transferred to the surface of GO sheets where ROS are generated to enhance the photocatalytic activity of the GO/AgNPs system.38 However, after culturing for 20 minutes in the dark, the number of bacteria on all types of samples is almost the same, indicating no antibacterial efficacy of these samples within a short time in a completely dark environment. It can be concluded that the physical action of AgNPs has almost no effect on bacteria within a short time while the rapid and highly efficient photodynamic action of AgNPs can be achieved by irradiation of 660 nm visible light. After culturing for 1 day in the dark, the samples without AgNPs, both Ti and PDA/GO-Ti, have many obvious colonies. However, AgNPs-containing samples display no colonies, indicating that enhanced antibacterial activity resulted from the incorporation of AgNPs and the physical action of released Ag+ on bacteria.51 Obviously, the physical action needs a relatively long time, and this result is in accordance with the zones of inhibition (ZOIs) tests shown in Figure S7. The AgNPs-containing samples can kill the bacteria to form ZOIs through the diffusion of silver ions. These released Ag ions can not only induce the destruction of the cell membrane but also destroy the protein and DNA within the bacteria by infiltration into the cell, resulting in the death of bacterial cells.39 In addition, PDA/GO/AgNPs-Ti samples exhibit less colonies than PDA/GO/AgNPs/Col-Ti samples, suggesting that the grafting of collagen can weaken the cytotoxicity of AgNPs because collagen has excellent biocompatibility and can possibly have a chelating reaction with released Ag+.14,40 These results are in good agreement with antibacterial



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ratio measurements (Figure 3c and 3d). In fact, even without light irradiation, PDA/GO/AgNPsTi exhibited antibacterial efficacy of over 99.8% and 99.6% against E. coli and S. aureus, respectively, while PDA/GO/AgNPs/Col-Ti showed an efficiency of over 98.6% and 96.7% against E. coli and S. aureus, respectively. Essentially, the antibacterial ability of AgNPs against E. coli is more effective than that against S. aureus.14 As a whole, both PDA/GO/AgNPs-Ti and PDA/GO/AgNPs/Col-Ti have excellent physical antibacterial properties after contacting bacteria for a long period due to the physical actions of Ag+. The number of bacterial colonies for PDA/GO/AgNPs/Col-Ti is more than that of PDA/GO/AgNPs-Ti. This phenomenon can be ascribed to two reasons. One is that the covering of the thin collagen layer can slightly weaken the light intensity to AgNPs and also block the electronic transfer, thus reducing the yield level of ROS, which can be proven by ESR spectra (Figure 3e). And it can also prevent the diffusion of Ag+ by chelating reaction, reducing the possible cytotoxicity caused by Ag+. On the other hand, collagen can also be conducive to the cell survival due to its innate biocompatibility.40


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Figure 3. In vitro antibacterial assay. The surface plate of (a) S. aureus, and (b) E. coli. The experiments are carried out with three groups: irradiation 20 minutes with 660 nm light, in the dark 20 minutes, and in the dark 24 h. The histogram of antibacterial efficacy of (c) photodynamic action, and (d) physical action. (e) The ERS spectra detected the yield level of ROS is AHT/PDA/GO/AgNPs-Ti > AHT/PDA/GO/AgNPs/Col-Ti >> AHT/PDA/GO-Ti > pure Ti.



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The morphology and membrane integrity of bacterial cells on samples are shown in Figure 4. Both E. coli and S. aureus cells are almost intact on the surface of both Ti and PDA/GO-Ti. However, the membranes are shrinked or even cracked on the surface of both PDA/GO/AgNPsTi and PDA/GO/AgNPs/Col-Ti, which are marked by green arrows for E. coli and purple arrows for S. aureus, respectively (Figure 4a). Figure 4b shows the bacterial section images after culturing for 1 day in a dark environment. The bacterial membranes obtained from Ti samples are complete and thick while the bacterial membranes of both PDA/GO/AgNPs-Ti and PDA/GO/AgNPs/Col-Ti are incomplete. In addition, the cell membranes of two types of bacteria obtained from these AgNPs-containing samples had been melting. EDS spectra show the existence of the Ag element in the inside of the broken cells, indicating the penetration of Ag into the bacterial membrane. However, no Ag signal was detected in the inside of the bacteria obtained from Ti samples. After culturing for 1 day, the Ag ion can penetrate cells and form permeable pits, resulting in an osmotic collapse, the crackage of cell membrane, and a release of intracellular materials.49 As shown in Figure 4b, the broken bacterial cells exhibit a lot of white space in the inside of the cells. This may be from the DNA and proteins damaged by endosmotic Ag ions.39,56 These intracellular materials may be released from the cracked membrane.49 Consequently, the bacterial cell inside became blank. These results indicate that AgNPscontaining coatings can kill both E. coli and S. aureus by using physical penetration of Ag ions to damage the bacterial membranes and cell structures.9,10


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Figure 4. Bacterial morphologies and structures. (a) Surface morphologies of bacteria growth on the surface of samples. The bacterial membranes have shrinked or even cracked marked by green



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arrows and red arrows, respectively. (b) Bacterial section images observed by TEM and the EDS patterns.

3.4. In vitro cytocompatibility For clinical application, the implants should not only possess an antimicrobial capability but also have good biocompatibility. Some studies reported that Ag+ ions have cytotoxicity in high concentrations.51,62 Figure S8a shows cell viability and proliferation of MC3T3-E1 by MTT assay. In the case of PDA/GO/AgNPs/Col-Ti and PDA/GO/AgNPs-Ti, cell viability increases as the culturing time increases. In the early stage, the release rate of Ag+ is rapid: 0.17 µg/mL and 0.177 µg/mL for PDA/GO/AgNPs-Ti and PDA/GO/AgNPs/Col-Ti at 3 days, respectively (Figure 2c), which is lower than the first day, inducing a slightly higher local Ag+ concentration in the culture medium and consequently higher cytotoxicity, thus resulting in a lower cell viability and proliferation compared to the samples without AgNPs. As the culturing time increases to 7 days, the cell viability increases due to the lower Ag+ release rate (shown in Figure 2c). After culturing for 21 days, the cytotoxicity of Ag+ could be ignored for AgNPs-containing samples, as even the cell viability for PDA/GO/AgNPs/Col-Ti is higher than Ti because the Ag+ release rate (Figure 2c) is much lower, and collagen has excellent biocompatibility. These results are consistent with observations obtained using a fluorescence microscope (Figure S8b). After 7 days of culturing, osteoblasts can normally grow on the surface of both PDA/GO/AgNPs/Col-Ti and PDA/GO/AgNPs-Ti regardless of the reduced cell number


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unlike samples without AgNPs. Under the same condition, there are more cells on PDA/GO/AgNPs/Col-Ti than PDA/GO/AgNPs-Ti, indicating that collagen is beneficial for the growth of osteoblasts.

3.5. In vivo evaluation To evaluate the photodynamic and physical effects on antibacterial activity of surface modified samples in vivo, the subcutaneous infection model was built. After implantation for 1 day, in comparison with the untreated Ti group, the bacterial count from PDA/GO/AgNPs/Col-Ti group was quite small; in other words, the PDA/GO/AgNPs/Col-Ti samples demonstrated much more robust physical antibacterial activity. After irradiation for 20 minutes by 660 nm visible light, the group implanted with PDA/GO/AgNPs/Col-Ti showed no bacteria after culturing for 1 day. Under the same condition, the control group implanted with Ti had almost the same bacterial number as the one without light irradiation, indicating that the AgNPs in PDA/GO/AgNPs/Col coating have strong photocatalytic effects while photodynamic effects of Ti can be ignored, which complies with in vitro results shown in Figure 3 and 4. It also suggests that the photodynamic action of AgNPs-containing coatings combined with their physical effects can achieve much faster and higher antibacterial ability than the physical action alone. As the implantation time is increased to 4 days, there are no bacteria for PDA/GO/AgNPs/Col-Tiimplanted mice while the numbers of bacteria for the Ti-implanted mice significantly increase. Obviously, after 4 days, all bacteria have been killed by AgNPs in the PDA/GO/AgNPs/Col



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coatings through physical actions while for the Ti group, and bacteria proliferate dramatically due to a lack of antibacterial activity. For Ti-implanted mice, as the implantation time is increased to 7 days and 14 days, the count of bacteria is gradually decreased due to the biological protection of a mouse’s innate immune system. The histological sections of the bacteria-infected wound tissues contacting with samples are shown in Figure 5. Once infected by bacteria, all the tissues for the two groups have neutrophils inflammatory cells (indicated by a black arrow) at 1 day, and the number of neutrophils inflammatory cells in the Ti group is far higher than that of the PDA/GO/AgNPs/Col-Ti group. In other words, the Ti group is infected significantly more seriously than the PDA/GO/AgNPs/Col-Ti group, which demonstrates the robust antibacterial activity of the PDA/GO/AgNPs/Col coating in vivo. After implantation for 4 and 7 days, the Ti group not only has a large number of inflammatory cells

but

also

presents

tissue

necrosis

(indicated

by

yellow

arrows)

while

the

PDA/GO/AgNPs/Col-Ti group has no tissue necrosis, and the connective tissue arrangement is relatively orderly (indicated by a red arrow). This confirms that the PDA/GO/AgNPs/Col coating not only possesses highly effective antibacterial activity but also has good biocompatibility. After implantation for 14 days, the PDA/GO/AgNPs/Col-Ti group displayed good tissue organizational structures while the tissues for the Ti group had just began recovery, and the capillaries in the newly formed tissues had started to expand (indicated by a green arrow).


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Figure 5. In vivo assay. Two groups: Pure Ti and PDA/GO/AgNPs/Col-Ti samples. Each group has been evaluated by two methods: the surface plate test and histological section. The mice cultured for 1 day was divided into two parts: one part was irradiated for 20 minutes using 660 nm light, another part was cultured without light irradiation. For the Histological section, the neutrophils inflammatory cells were indicated by black arrows, yellow arrows showed the tissue necrosis cells, red arrows indicated relatively orderliness connective tissues, and green arrows indicated newly formed tissues.



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For the PDA/GO/AgNPs/Col-Ti group, the group irradiated with 660 nm had few inflammatory cells than the group that had not undergone irradiation. However, there were no big differences before and after irradiation for the Ti samples. These results further confirm that the synergy between the photodynamic and physical effects of AgNPs in PDA/GO/AgNPs/Col coating can achieve more rapid and effective antibacterial activity; this result is in accordance with the results of the surface plate.

4. CONCLUSIONS

This work developed a novel hybrid coating composed of GO nanosheets, AgNPs, and collagen. For the first time, we investigated the synergistic effects of both photodynamic action and physical action of AgNPs on the antibacterial activity of this hybrid coating using 660 nm visible light irradiation. In vitro tests indicate that the antibacterial ability of AgNPs-containing coatings is determined by the yield level of ROS produced during light irradiation, and photodynamic action of GO/AgNPs coatings is more rapid and effective for killing bacteria than their physical action within a shor time. GO nanosheets could favor the uniform distribution of AgNPs, charge transfer, and suppression of electron-hole pairs, thus enhancing the photodynamic properties of AgNPs-contaning coatings. The in vivo subcutaneous model also reveals that the synergy between the photodynamic and physical effects of AgNPs in


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GO/AgNPs/Col hybrid coating can achieve rapid and highly effective bacteria-killing without side-effects through a short period of 660 nm visible light irradiation. In addition, collagen covering can improve the biocompatibility of this coating. This work provides a novel route to endow the implant materials with highly effective self-antibacterial activity as well as good biocompatibility through combining the physical bacteria-killing of AgNPs with their photodynamic action inspired by 660 nm visible light with some penetrating capacity, thus achieving the rapid in-situ disinfection of subcutaneous implants.

ASSOCIATED CONTENT

Supporting Information

This material is available free of charge via the Internet at http://pubs.acs.org. Structure analysis of GO and GO/AgNPs composites; the high-resolution spectra of Ag3d spectra; the FTIR spectra of PDA-Ti, PDA/GO/AgNPs-Ti and PDA/GO/AgNPs/Col-Ti; the XPS survey spectra of PDA/GO/AgNPs-Ti and PDA/GO/AgNPs/Col-Ti after immersion in neutral PBS for 30 days; the antibacterial activity of PDA-Ti irradiated 20 minutes with 660 nm visible light and cultured 24 h at dark; the temperature change curve of samples irradiated under 660 nm visible light and the surface plate of bacteria; the zones of inhibition of samples against E. coli and S. aureus; in vitro evaluation of cell viability and the fluorescence microscope observation.



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AUTHOUR INFORMATION

Corresponding Author * To whom correspondence should be addressed: E-mail: [email protected]; [email protected] (S.L. Wu)

ACKNOWLEDGEMENT

This work is financially supported by the National Natural Science Foundation of China, Nos. 51422102, and 51671081, and the National Key Research and Development Program of China No. 2016YFC1100600 (sub-project 2016YFC1100604), as well as well as Shenzhen Peacock Program (1108110035863317).

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The schematical illustration of cytocompatibility and synergistic bacteria-killing through the innate antimicrobial ability of Ag+ and the photodynamic effects of AgNPs in GO/AgNPs/collagen hybrid coating using 660 nm visible light. 26x14mm (600 x 600 DPI)

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Synergistic Bacteria Killing through Photodynamic and Physical

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