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Mussel-Inspired Synthesis of NIR-Responsive and Biocompatible Ag-Graphene 2D Nano-Agents for Versatile Bacterial Disinfections Xin Fan, Fan Yang, Chuanxiong Nie, Ye Yang, Haifeng Ji, Chao He, Chong Cheng, and Changsheng Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16283 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 15, 2017

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ACS Applied Materials & Interfaces

Mussel-Inspired Synthesis of NIR-Responsive and Biocompatible Ag-Graphene 2D Nano-Agents for Versatile Bacterial Disinfections Xin Fan,a Fan Yang,a Chuanxiong Nie,a,b* Ye Yang,a Haifeng Ji,a Chao He,a Chong Cheng,a,b and Changsheng Zhaoa,c*

a College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, 610065, China

b Institute für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, 14195, Berlin, Germany

c National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064, China

* Corresponding authors. Tel: +86-28-85400453, Fax: +86-28-85405402,

E-mail:(C. X. Nie) [email protected]; (C.S. Zhao) [email protected] or [email protected].

KEYWORDS: Antibacterial nano-agents, Biocompatible graphene nano-composites, NIRresponsive, Bacterial disinfections, Self-sterilizing coating

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ABSTRACT: Pathogenic bacterial infection has been becoming as a global threat towards people’s health, especially the massive usage of antibiotics due to the lack of antibacterial agents with less side-effects. Developing new nano-agents to fight pathogenic bacteria has provided enormous new possibilities in the treatment of bacterial infections, such as the graphene based 2D antibacterial nano-agents with different bacterial inhibition capabilities; however, musselinspired design of NIR-responsive and biocompatible Ag-graphene nano-agents possessed with efficient and versatile bacterial disinfection activities have rarely been reported. In this study, we developed a new kind of antibacterial nano-agent, dopamine conjugated polysaccharide sulfates anchored and protected Ag-graphene (Ag@G-SAS) nanocomposite to combat baterial infection and contamination in different application fields. The Ag@G-SAS owned robust antibacterial activity towards both Escherichia coli and Staphylococcus aureus; notably, the nano-agent can significantly inhibit Staphylococcus aureus infection on wounded pig skin without or with NIR laser. Beyond the wound disinfection, the 2D Ag@G-SAS can also serve as a good layer-bylayer (LbL) building block for the construction of self-sterilizing coating on biomedical devices. All the results verified that the LbL assembled Ag@G-SAS coating exhibited favorable bactericidal activity, extraordinary blood compatibilities, and good promotion ability for cell proliferation. Owing to the shielding effects of heparin-like polysaccharide sulfates, the Ag@GSAS nano-agent showed limited cytotoxicity towards mammalian cells. Combined all the advantages mentioned above, it is believed that the proposed Ag@G-SAS nano-agent and its LbL assembled coatings may have versatile application potentials to avoid bacterial


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contaminations in different fields, such as wounded skin, disinfection of biomedical implants and devices, and food packaging sterilization.

1 INTRODUCTION Infectious disease caused by pathogenic bacteria is one of the most troublesome health challenges worldwide, afflicting various fields including biomedicine,

1

water,

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and food

industries.3 For the bacteria, Staphylococcus aureus (S. aureus) is a main cause of skin infection, soft-tissue damage, respiratory, bone and endovascular disorders.4 Even worse, S. aureus has extraordinary capacity to attach to the surfaces of medical devices and further cause secondary harms to patients.5 For decades, antibiotics play important roles in treating bacterial infection, while the indiscriminate usage of antibiotics has brought horrible consequences, especially the rise of drug-resisting pathogens.6

With the development of modern chemistry, various kinds of nanomaterial-based antimicrobials have been developed.7-9 Silver nanoparticle (AgNP)-based antimicrobials are the most generally explored due to their broad antibacterial spectrum and superior antimicrobial ability.10 However, traditional Ag-based therapies usually employed AgNPs independently, whereas the AgNPs tended to aggregate into clusters, leading to decrease of their antimicrobial activity.11 To solve the problems, other nanomaterials were utilized as a carrier to load AgNPs evenly and thus achieving better antibacterial activity.12 Graphene oxide (GO) is a twodimensional nano-sheet with large surface area;13 and can enhance the antibacterial effects for



AgNPs because it is able to increase the colloid stability and prevent the aggregation of AgNPs.14 Moreover, GO is an efficient photo-thermal agent not only to ablate tumor cells in cancer therapy,15 but also to effectively kill bacteria.16 Combining the advantages mentioned above, Ag@GO nano-composites own strong antibacterial activity from multiple origins, which may be a good candidate for sterilization usages.

However, bare Ag@GO always showed strong cytotoxicity towards mammal cells because of the generation of reactive oxygen species (ROS)

17-18

and strong interaction between GO and

phospholipids.19-20 Thus, it is quite necessary to develop a new method for decreasing the damage to cells without weakening the antibacterial activity of Ag@GO nano-composites. Polymer shields have been reported to have potential to improve cell compatibility for Agcontaining materials.21-22 Agarwal et al., have proved that the Ag-impregnated polymeric films constructed by poly(acrylic acid) (PAA) and poly(allylamine hydrochloride) (PAH) showed no toxicity to mammalian fibroblast cells.23 Hsu et al. found that polyurethane shielded AgNPs showed no significant cytotoxicity towards fibroblasts and endothelial cells.24 Chaudhari et al. have also demonstrated that PEG coated Ag@CNT composites had no toxicity against eukaryocyte under the concentration which was enough to inhibit bacterial growth.25 These recent developed novel polymer shielded nano-agents had provided enormous new possibilities to fight pathogenic bacterial infection; however, rationally design of polymer shielded biocompatible Ag-graphene nano-agents possessed with efficient and versatile bacterial


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disinfection activities has rarely been reported.

In this study, we successfully develop a new type of antibacterial nano-agent by using dopamine conjugated polysaccharide sulfates to anchor and protect Ag-graphene, namely Ag@G-SAS. Sodium alginate sulfate is widely acknowledged as a heparin-like polymer with good anticoagulant activity. In our previous study, we demonstrated that the sodium alginate sulfate owned better blood compatibility and more excellent cytocompatibility than original sodium alginate.26 Therefore, in this study, we choose sodium alginate sulfate to modify our nanocomposites in order to achieve better hemocompatibility and cytocompatibility. The antibacterial property, disinfectant efficacy and cytotoxicity are systematically investigated. Moreover, the NIR-responsive effect of the Ag@G-SAS is utilized to further enhance its antimicrobial ability and allows more effective inhibition of both E. coli and S. aureus at low concentrations of nano-agent. It is verified that the Ag@G-SAS has great potential to serve as a disinfection agent for wounded skin owning to its robust antibacterial property, quick-acting disinfectant efficacy and limited cytotoxicity. Beyond the wound disinfection, the 2D Ag@GSAS can also serve as a good layer-by-layer (LbL) building block for the construction of selfsterilizing coating on biomedical devices. To achieve this, quaternized chitosan (QCS) coated graphene nano-sheet, namely G-QCS, is also obtained through mussel-inspired approach and then a self-sterilizing coating is constructed using the Ag@G-SAS and G-QCS for LbL assembly. Likewise, the coating exhibits strong antibacterial activity and favorable



biocompatibility at both cell and blood levels. The proposed Ag@G-SAS nano-sheets and their LbL self-assembly coatings with favorable biocompatibility show promising future in food packaging sterilization, medical disinfection, wound dressing and surface modification of biomedical devices.

2 EXPERIMENTAL SECTION Materials and methods are shown in Supporting Information. 3 RESULTS AND DISCUSSION 3. 1 Preparation and Characterization of Ag@G-SAS


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Figure 1. (A) Preparation of the Ag@G-SAS nano-composite and the TEM images (scale bar: 100 nm.). TEM images of other nano-composites mentioned in this study are shown in Figure S1, Supporting Information. (B) AFM image for Ag@G-SAS on freshly cleaved mica. The AFM image for pristine GO nano-sheets is shown in Figure S2, Supporting Information. The average height difference between the two arrows was about 10.0 nm, representing the average size of Ag nanoparticles. (C) XPS wide scan for pristine GO and Ag@G-SAS. (D) XPS Ag 3d spectra for pristine GO and Ag@G-SAS. (E) Element analysis for GO and Ag@G-SAS, which was estimated from XPS wide spectra.

In this study, a facile mussel-inspired adhesion approach was used to prepare the Ag@G-SAS. Catecholic groups are the major groups for mussel adhesion since they can form irreversible covalent bonds onto surfaces.27-28 Herein, a catecholic polymer, dopamine grafted sodium alginate sulfates (DA-SAS), was firstly synthesized through carbodiimide chemistry, as reported in our previous works.26,29 SAS is a negatively charged heparin-like polymer with excellent cell compatibility and blood compatibility. The 1H NMR spectra for DA-SAS are shown in Figure S3, Supporting Information. The grafting yield for DA was about 19.50% according to the 1H NMR spectra. Then, Ag@G-SAS was synthesized as illustrated in Figure 1A. In brief, Ag+ ions were firstly loaded in GO nano-sheets and then the GO-Ag+ were then reduced to Ag@G-SAS by DA-SAS.30-31 In this system, the positively charged Ag+ ions are firstly attracted by the negative charged carboxyl groups on GO surface. While the driving force of SAS assembly is


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mainly generated from the catecholic structures of DA-SAS. Catecholic groups can adhere onto GO surface and then form cross-linked viscous polymer layers on GO surface mediated by oxidative Ag+, as well as weak alkaline environment. The morphologies of GO and Ag@G-SAS were visualized by TEM and AFM. As shown in Figure 1A, a flat-shape thin sheet, which was the typical structure of GO, was observed. For Ag@G-SAS, the AgNPs were anchored on GO sheets evenly under the coverage of transparent polymer layers. The average diameter for AgNPs was about 15 nm, according to the TEM image. Similar structures were also visualized by AFM. Moreover, the AFM images in Figure S2, Supporting Information, clearly revealed the monolayered structure for the GO nano-sheets, for which the thickness was only about 1.0 nm.32 For the Ag@G-SAS, as shown in Figure 1B, nanodots on graphene sheet with height about 10 nm were noticed, which should be the anchored AgNPs. The thickness for graphene sheet was also increased due to the adhesion of SAS.

The chemical components for pristine GO and Ag@G-SAS were investigated by XPS spectra, which are shown in Figure 1(C, D, E). The XPS data for G-SAS are shown in Figure S4, Supporting Information. As shown in Figure 1C, no peak of N, S and Ag was probed from GO; while the peaks of N, S and Ag were probed from Ag@G-SAS, manifesting that the polysaccharide and AgNPs were successfully anchored onto the surface of GO nano-sheets. The Ag 3d spectra that shown in Figure 1D also indicated the existence of the AgNPs. The Ag



contents of Ag@G-SAS was 0.25 At. % (approx. 1.9 wt. %), which was calculated from XPS wide scan.

3. 2 Bacterial Killing Properties of Ag@G-SAS

Figure 2. (A) Photographs of E. coli colonies grew on MH agar plates after incubation with Ag@G-SAS at the concentration of 3,15 and 75 µg mL-1. (B, C) Bacterial killing ratios for the samples at the concentration of 3, 15 and 75µg mL-1 against E. coli and S. aureus, respectively. (D, E) Real-time OD600 values of the samples at 15µg mL-1 against E. coli and S. aureus,


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respectively. (F) NIR-responsive heating images under laser irradiation (808 nm, 3 W cm-2, 7 min). The concentrations for the samples are fixed to 75 µg mL-1. (G) Real-time NIR-responsive heating curve of Ag@G-SAS and the bacterial killing percent. The concentration for Ag@GSAS was fixed to 3 µg mL-1. Values are expressed as mean ±SD, n=4.

The bacterial killing ratios of Ag@G-SAS dispersions against both E. coli and S. aureus were firstly studied by plate counting method, as shown in Figure 2(A, B, C). The pristine GO dispersion exhibited slight bactericidal properties against both E. coli and S. aureus, which might be due to the sharp edges of GO nano-sheets and strong interaction between lipid bilayers and GO nano-sheets, both of which would cause the damage of cell membranes and lead to the death of microbial cells.

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The bacterial killing ratio of G-SAS was lower than the pristine GO since

the polymer layers can weaken the “nano-knife” effect of GO and also the interaction between GO and phospholipids. After the loading of AgNPs, the nanomaterials significantly increased the bacterial killing activity, majorities of bacteria were inactivated at 15µg mL-1. To investigate the bacterial inhibition property in more details, real-time optical density at 600 nm (OD600 values) curves were measured as shown in Figure 2D and Figure 2E. For the control, the OD600 values of E. coli and S. aureus grew rapidly from 0.070 and 0.018 to 1.219 and 0.924 in 24 h, respectively. The pristine GO and G-SAS did not exhibit the significant inhibition towards bacteria. For Ag@G-SAS, the bacterial growth was obviously inhibited because of the remarkable antibacterial property of AgNPs.10, 34



GO is an outstanding NIR-absorbing photothermal agent that has drawn widespread applications in biomedical field.35-36 Upon the NIR-laser irradiation, GO is able to convert optical energy into heat fast, leading to the thermal ablation of cells.37 Herein, the NIRresponsive property of the Ag@G-SAS dispersion was evaluated, as shown in Figure 2F. For MHB, nearly no temperature change was observed, which is shown in Figure S5, Supporting Information. Rapid temperature rises were observed for both Ag@G-SAS and G-SAS; their photo-thermal efficienty was even better than pristine GO. This might because that the GO was reduced by catecholic moieties during the synthesis process of Ag@G-SAS, and the NIR absorption of reduced GO (rGO) was higher than non-reduced GO.38 To explore the photothermally enhanced antibacterial property of Ag@G-SAS, the minimal inhibition concentrations (MIC) and the bacterial killing activity for the samples before-and-after NIR laser irradiation were measured. For Ag@G-SAS, the MIC for E. coli and S. aureus were 15 µg mL-1 and 10 µg mL-1, respectively. After a 7-min irradiation, the MIC of Ag@G-SAS against E. coli and S. aureus decreased to 10 µg mL-1 and 7.5 µg mL-1, respectively. As shown in Figure 2G, it was noticed that the bacterial viability decreased with increasing the irradiation time and only 18.2 ± 0.6 % S. aureus left after a 7-min irradiation. The results indicated that the presence of NIR could further enhance the antibacterial activity of Ag@G-SAS, which allows urgent bacteria killing usage. The NIR-responsive antibacterial ability of nano-agent can provide a more effective protection towards wounds and stop bacterial infection in time. But unfortunately, the


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NIR induced heating may cause mammalian cells death. Thus, the NIR-responsive antibacterial ability is suggested used only as an auxiliary means of emergency.

3.3 Skin Disinfection Test of Ag@G-SAS

Figure 3. (A) The procedures of S. aureus infection on pig-skin and wound disinfection by spraying Ag@G-SAS. (B) Photographs of bacterial contamination procedure for sterilizing pigskin. (C) Photographs of disinfection reagents treated wound after bacterial contamination. (D) Photographs for morphologies of infected wound and uninfected wound, respectively. (E, F) Typical SEM images of infected wound and uninfected wound, respectively. Scale bar: 10 µm.



S. aureus infection is considered to be a major causes of hospital-acquired infections because of its high lethality and sturdy resistance to antibiotics.4 Herein, Ag@G-SAS was found to have potential usage as a disinfection reagent, because it could effectively restrain bacterial proliferation at low dosage. In this study, we investigated the disinfection effect of Ag@G-SAS using an in vitro wounded pig-skin model. The main purpose of building this antibacterial infection model is to investigate whether the Ag@G-SAS can effective defense bacterial invasion when the skin wound is actually exposed to considerable numbers of bacteria. The pigskin is far better than normal agar plate here, since it has the similar structures and compositions to that of human skin tissues. More specifically, the pig-skin owns a three-dimensional fibrous structure and both of its physical structure and physiological environment are more similar to organisms than agar plate. Thus, it is more suitable and more convincing than agar as an antibacterial infection model. Therefore, all the experiments on pig-skin tissues are of guiding significance to the further clinical use of the human body. The detailed procedures of the tests are illustrated in Figure 3A. In short, the pig skin was firstly wounded and infected by S. aureus, and then the Ag@G-SAS was sprayed into the wounds. The pictures of wounds were obtained by digital camera (Figure 3(B, C, D)). As control in each experiment, the uninfected wound (shown in Figure S6, Supporting Information) and the infected wound without treatment were both prepared. As shown in Figure 3D, the wound infection with a lot of yellow spots and mucus was clearly observed in wounds without disinfection. After 4 days incubation, the Ag@G-SAS disinfectant wound had no visible yellow spot, indicating that bacterial infection was restrained


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effectively by Ag@G-SAS. The micro morphologies of skins were also visualized by SEM observations. As shown in Figure 3E and Figure 3F, for the infected skins, large quantities of agglomerated bacteria embedded on the surface of skin tissue and a thick bacterial biofilm was formed. While for the disinfectant wound, bacterial adhesion was found to be dispersive and the amount of adhered bacteria was much less than that of the infected skin, which indicated that the bacterial growth was successfully inhibited. Similar phenomenon could be observed by the fluorescence images as shown in Figure S7, Supporting Information. We also studied the NIR-responsive disinfection property of the Ag@G-SAS. As shown in Figure S8, Supporting Information, for the treated sample, almost no live bacteria could be found on wounded area. The results revealed that the Ag@G-SAS could vigorously kill large quantities of bacteria within 3 minutes in the presence of NIR laser. This kind of antibacterial reagent may have promising future in various fields due to its robust antibacterial activity and efficient NIR-responsive bactericidal efficacy. And the live/dead bacteria staining assay was also performed as shown in Figure S7, Supporting Information. For the infected wound, massive live bacteria were visualized. As for the Ag@GSAS treated wound, only a few live bacteria were found, which indicated that the strong bactericidal effect could be achieved by simple spraying Ag@G-SAS to the wounds. 3. 4 Layer-by-layer Assembly to Fabricate a Self-sterilizing Coating



In our study, the Ag@G-SAS was found to have excellent antibacterial activity and it exhibited flat 2D structure, which is favorable for surface coatings fabrication. Herein, we fabricated a self-sterilizing coating via the LbL assembly of Ag@G-SAS and its positively charged counterpart, quaternized chitosan (QCS) coated GO (G-QCS). The zeta potentials of assembly dispersions were measured as shown in Figure S9, Supporting Information, verifying the opposite charged modified GO dispersions were successfully obtained.


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Figure 4. (A) Construction of a self-sterilizing coating through immersed LbL self-assembly method and SEM image of PES-(Ag@G-SAS)3/G-SAS. Scale bar: 25 µm. The red arrows indicated the GO nano-sheets. (B, C) Inhibitory zones for the samples against E. coli and S. aureus, respectively. The white regions represent PES-(Ag@G-SAS)3/G-SAS and the red regions represent PES-(Ag@G-SAS)3. (D) OD600 values for the bacterial solutions (106 CFU mL1

) incubating with samples for 12h. (E) Bacterial killing ratios for the AgNPs containing samples,

which were calculated from agar plate count results. Values are expressed as mean ±SD, n=4. (F) SEM images that revealed the bacterial adhesion on the different membrane surfaces within 12 hours. The SEM images of other samples are shown in Figure S10, Supporting Information.

The coating was prepared by an immersive LbL-assembly method, which was illustrated in Figure 4A. In this study, PES membrane was chosen as the model substrate of coating because of its versatile applicability in biomedical field, like hemodialysis and tissue engineering scaffold, where the bacterial infections often occured thus leading to serious consequences.39-40 In short, the coating was fabricated by immersing substrates into Ag@G-SAS or G-SAS and GQCS alternatively. The sample contained 3 bilayers of (G-SAS/G-QCS) was named as PES-(GSAS)3; and the sample contained 3 bilayers of (G-QCS/Ag@G-SAS) was named as PES(Ag@G-SAS)3. Recent reports have demonstrated that exposing AgNPs to cells would generate large quantities of reactive oxygen species (ROS) and result in cell death.41-42 Therefore, to obtain a more cell compatible coating, an extra bilayer of (G-QCS/G-SAS) was coated outside



the PES-(Ag@G-SAS)3 to avoid the direct contacting between the sliver-containing layers and cells, namely PES-(Ag@G-SAS)3/G-SAS. Moreover, heparin-like macromolecules had remarkable biocompatibilities and their negative charges surfaces would also have potential to inhibit thrombosis and promote cells proliferation.43-44 Thus, in this study, the heparin-like SAS was applied as terminal layer on purpose to achieve better hemocompatibility and cytocompatibility.

The morphologies of the coating and pristine PES membrane were observed by SEM, as shown in Figure 4A and Figure S11, Supporting Information, respectively. Pristine PES membrane had a smooth and flat surface. While after coating 3 bilayers of (G-QCS/Ag@G-SAS) and 1 bilayer of (G-QCS/G-SAS), the surface exhibited the characteristic 3D porous structure, as shown in Figure 4A. As shown in Figure S12A, Supporting Information, the surface zeta potential of pristine PES substrate was about -15.4 ± 3.2 mV. After coating with PEI, Ag@GSAS and G-QCS, the zeta potential was fluctuated between -25.5 ± 2.3 mV ~ 45.1 ± 2.1 mV with the formation of new assembled layers, indicating that each assembly layer was successfully seated. Afterwards, XPS spectra were performed for chemical structure study, as shown in Figure S12B to Figure S12F, Supporting Information. As shown in Figure S12B to Figure S12F, Supporting Information, it was clearly noticed that the G-QCS and Ag@G-SAS had been assembled on the surface of PES successfully from the XPS spectra. The water contact angle results are shown in Figure S12G and Figure S12H, Supporting Information, indicating that the


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coating can improve the wettability of the membrane, which is beneficial for the antifouling property of the surface. Detailed discussions about XPS spectra and WCA data are shown in Supporting Information.

3. 5 Antibacterial Activity

As shown in Figure 4B to Figure 4F, the antibacterial activities of the coated-membranes were investigated through inhibition zones, OD600 values, bacterial killing ratios and bacteria adhesion observations. As shown in Figure S13, Supporting Information, no obvious inhibition zones for the pristine PES, PES-PEI and PES-(G-SAS)3 were observed. While, as for Ag@GSAS decorated membranes, the inhibition zones against E. coli and S. aureus were clearly noticed, as shown in Figure 4B and Figure 4C, respectively. The sizes of the inhibition zones towards E. coli were 4.2 mm for PES-(Ag@G-SAS)3 and 4.5 mm for PES-(Ag@G-SAS)3/GSAS. For S. aureus, the inhibition zones were 3.3 mm for PES-(Ag@G-SAS)3 and 3.2 mm for PES-(Ag@G-SAS)3/G-SAS. To evaluate the antibacterial property of coating in solution, OD600 value was monitored, as shown in Figure 4D. Vast bacterial growth was found on the surface of both the control and the pristine PES membrane. For the AgNPs contained sample, the bacterial growth was significantly suppressed; and the bacterial killing ratios were further calculated to verify the bactericidal activities of AgNPs contained samples. As shown in Figure 4E, the pristine PES had no obvious bacterial killing abilities. While after 3-bilayer coatings of (GQCS/Ag@G-SAS), the bacterial killing ratio against E. coli and S. aureus significantly increased



to 97.85 ± 0.52% and 93.58 ± 0.12%, respectively. The bacterial killing ability of PES-(Ag@GSAS)3/G-SAS was similar to that of the PES-(Ag@G-SAS)3. The AgNPs contained samples showed remarkable antibacterial activity due to the strong bactericidal nature of AgNPs. Li et al. noticed that AgNPs exhibited robust antibacterial properties to E. coli through destroying the structure of bacterial cell membranes and suppressing the activities of some enzymes of bacteria, which eventually caused bacteria death even the dosage of AgNPs was very low.45 Agarwal et.al found that the AgNPs contained polymeric multilayers showed remarkable antibacterial activities to both Gram-negative and Gram-positive bacteria without causing cytotoxity.46

SEM was utilized to observe the bacterial adhesion on the coatings. Figure 4F shows the bacteria adhesive patterns of both E. coli (rod-like shape) and S. aureus (sphere-like shape) on pristine PES and the coatings. It was visualized that large amounts of aggregated bacteria adhered to the surface of pristine PES. However, after coating, the adhesion of the bacteria was greatly inhibited and nearly no E. coli and S. aureus was observed.


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Figure 5. (A, B, C) Typical fluorescence images for E. coli adhered PES-(G-SAS)3, PES(Ag@G-SAS)3 and PES-(Ag@G-SAS)3/G-SAS, respectively. Scale bar: 10 µm. The live cells are stained green, while the dead cells are stained red. The concentrations of bacterial dispersion that introduced to A-C are fixed to 106 CFU/mL and the co-culture time is 12h. (D, E) Typical fluorescence images for E. coli adhered PES-(Ag@G-SAS)3/G-SAS before-and-after NIR-laser irradiation (808 nm, 3W cm-2, 5 minutes), respectively. And the inserted images show the coating surface temperature before-and-after NIR-laser irradiation. The concentrations of bacterial dispersion that introduced to D-E are fixed to 108 CFU/mL and the co-culture time is 6h. (F) Numbers of the adhered bacteria on the surfaces of A-E that mentioned above, which were estimated from the fluorescence images. Values are expressed as mean ±SD, n=4.



Furthermore, the fluorescence pictures of E. coli adhered coatings with double staining of live/dead bacteria are acquired and shown in Figure 5(A, B, C), while the other fluorescence pictures are shown in Figure S14, Supporting Information. The live/dead adhesion bacteria numbers were calculated from the fluorescence pictures, as shown in Figure 5F. It was noticed that large quantities of bacteria adhered on PES surface and only a few dead bacteria could be found. However, after coating 3 bilayers of (G-QCS/G-SAS), more than a half bacteria were dead due to the bactericidal ability of QCS. After the decoration with AgNPs, the adhered bacteria number significantly decreased; and for the PES-(Ag@G-SAS)3/G-SAS, almost no bacteria were found on the surface, which indicated that the self-sterilizing coating was successfully achieved. Moreover, the PES-(Ag@G-SAS)3/G-SAS was taken to investigate the long-lasting antibacterial activities. We recycled the coated-membranes that have been utilized for bacterial killing ratios and then re-measured their antibacterial efficiencies; data are shown in Figure S15, Supporting Information. It was found that even had been used for 3 times, the coated-membranes remained robust bacterial inhibition ability towards both E. coli and S. aureus. The samples that immersed in PBS for several weeks were applied to measure the bacterial killing ratios. As shown in Figure S15B in Supporting Information, the samples still maintained clear bacterial killing efficacy after being corroded by PBS for 28 days.

In this study, NIR was noticed to be able to enhance the antibacterial activity of Ag@G-SAS. Hence, the NIR-responsive antibacterial property of the coatings was further explored. However,


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when the concentration of bacterial dispersion that co-cultured with PES-(Ag@G-SAS)3/G-SAS was 106 CFU/mL, the bacterial activity was almost completely inhibited. In order to simulate the situation that the coatings were exposed to a large quantity of bacteria in short period, we adjusted the concentration of bacteria dispersion and decreased the co-culture time of membranes and bacteria; and in this case, the silver nanoparticles were not able to inhibit the growth of bacteria on the surface of coatings well and then the function of NIR laser could be further highlighted. As shown in Figure 5D and Figure 5E, large amounts of live bacteria were noticed in unirradiated membranes. However, only after a 5-min of irradiation, almost all visible bacteria were dead on membrane surfaces. The NIR-responsive effect could not only significantly improve antibacterial efficiency, but also broaden the antibacterial spectrum of coating.47 In this sense, the AgNPs and QCS that contained in coating play the roles of long-effective antibacterial composition, which can further prevent the repeated bacterial infection.

3.6 Biocompatibility 3.6.1 Cytotoxicity Test



Figure 6. (A) Cell viability of MC3T3-E1 cells determined from CCK-8 assay after exposing with different concentrations of GO and other modified GO dispersion for 24h. Values are expressed as mean ±SD, n=4. *p < 0.05 compared with control. (B) CCK-8 assay for the cell cultured membranes. Value are expressed as mean ±SD, n=4. *p < 0.05 compared with control. (C, D, E) Representative fluorescence images for the MC3T3-E1 adhered on membrane surfaces. Scale bar: 15 µm. (F) Protein absorbed amounts for the coated-membranes. (G) Hemolysis ratio


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for the coated-membranes. The cytotoxicity of GO dispersions is also a crucial factor, and it must be carefully evaluated before being used in biomedical area.48 In this study, CCK-8 assay was employed to study the cytotoxicity of GO dispersions towards MC3T3-E1 cells. Polystyrene plate was used as the control. The cytotoxicity of the pristine and modified GO was calculated, as shown in Figure 6A. For the pristine GO, the cell viability decreased with increasing the dosage of GO, and the cell viability remained at a low level (only 63.5 ± 1.3%) even though the GO dosage was as low as 3 µg mL-1. However, after the coverage by the biomacromolecules, the GO presented lower cytotoxicity, and all the cell viabilities were higher than 80% when the concentrations were below 15 µg mL-1, indicating that the polymer coating could suppress the cytotoxicity. CCK-8 assay was also used to evaluate the cytotoxicity of the coatings, and the results are shown in Figure 6B. It was found the absorbance of cell suspension that co-cultured with the coatedmembranes was higher than those of the control and pristine PES, which revealed that the MC3T3-E1 cells compatibility was increased after coating. It was also noticed that the cells cocultured with PES-(Ag@G-SAS)3/G-SAS exhibited higher viability than those co-cultured with PES-(Ag@G-SAS)3, which indicated higher cell compatibility could be achieved by coating an extra bilayer of (G-QCS/G-SAS). And this conclusion could also be further demonstrated by fluorescence images in Figure 6D. 3.6.2 Cell Adhesion The cell morphologies of the MC3T3-E1 cells cultured on the membrane surfaces were



visualized by fluorescence microscope observations with a double staining of cell nucleus (blue) and cytoskeleton (red).; data are shown in Figure 6(C, D, E). The MC3T3-E1 cells cultured on PES membrane showed no significant plasmodesmata and most of them remained their original shuttle-like shape. For the PES-(Ag@G-SAS)3, much more filopodia were visualized and the cells tended to spread with ruffling of peripheral cytoplasm. The cells cultured on PES-(Ag@GSAS)3/G-SAS exhibited more distinct regional aggregation and larger spreading area compared to the cells cultured on PES-(Ag@G-SAS)3 surfaces, which demonstrated that the extra bilayer of (G-QCS/G-SAS) might promote cell growth and proliferation. The good cell compatibility of PES-(Ag@G-SAS)3/G-SAS could be attributed to two factors: (1) the heparin-like layer was beneficial to cell growth because of their unique ability to immobilize cell growth factors;49 (2) the extra bilayer functioned as shielding barrier which could effectively cut off the direct contaction between cells and AgNPs, and thereby limiting the cytotoxicity of AgNPs.23, 49 3.6.3 Blood Compatibility Blood compatibility is an important factor to evaluate whether a biomaterial is applicable for blood contacting. Herein, we further investigated the blood compatibility of our coatedmembranes to make sure their applicability in blood contacting field; typical data are shown in Figure 6F and Figure 6G. In this work, protein adsorption was performed by measuring the invitro adsorbed amounts of bovine serum albumin (BSA) and bovine serum fibrinogen (BFG). It was noted that the protein adsorbed amounts were obviously suppressed for PES-(Ag@GSAS)3/G-SAS. The suppressed protein adsorption was attributed to the improved hydrophilicity


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and the negatively charged surface after the coating of G-SAS or Ag@G-SAS nanocomposites.50 Hemolysis is one of the important indicators to determine whether a new developed material has enough hemocompatibility for blood contacting application. Earlier reports have shown that both GO nano-sheets and AgNPs have high hemolysis ratio when directly exposed to red blood cells (RBCs).51, 52 Therefore, we evaluated the hemolysis ratio of the coated-membranes that contained both GO and AgNPs; the results are shown in Figure 6G. It was noted that all the samples exhibited low hemolysis ratio (below 5%). As was expected, the hemolysis ratio of PES-(Ag@G-SAS)3/G-SAS (0.608 ± 0.058%) was lower than that of PES(Ag@G-SAS)3 (2.221 ± 0.023%), which might attribute to the extra bilayer of G-QCS/G-SAS, limiting the direct contact between AgNPs and RBCs. And the SEM images of RBCs after incubating with the coated-membranes were also observed and shown in Figure S16, Supporting Information. The morphology of the RBCs incubated with the coated-membranes maintained well round shapes, which further indicated that the coated-membranes had favorable RBCs compatibility.

The enzyme linked immunosorbent assay(ELISA) and platelet adhesion were also carried out to evaluate whether the inflammatory response or unfavorable clotting effects would happen when the coated-membranes contacted with blood; data are shown in Figure S17 and Figure S18, Supporting Information. Briefly speaking, there was no inflammation response and platelet activation would be ocuured when the coated-membranes contacted with blood.



4 CONCLUSIONS In this study, we reported a new kind of antibacterial nano-composite, Ag@G-SAS, synthesized via a facile mussel inspired polysaccharide sulfate polymer for AgNPs anchoring and protection. The TEM and AFM images clearly showed that the nano-composite had 2D flat shape and the AgNPs were anchored on the surface of GO nano-sheet evenly. The antibacterial tests and the cytotoxicity measurement showed that the Ag@G-SAS presented the dose-dependent antibacterial property and cytotoxicity. When the concentration below 15 µg mL-1, the Ag@GSAS showed limited cytotoxicity towards MC3T3-E1 cells but robust antibacterial ability against both Gram positive S. aureus and Gram negative E. coli. Moreover, owing to the strong NIRlaser absorption of Ag@G-SAS, we further evaluated the bacterial killing activities of Ag@GSAS upon NIR-laser irradiation. The results showed that the presence of NIR-laser could further enhance the antibacterial activity of Ag@G-SAS, which allows bacterial killing at low dosage and urgent bacteria killing usage. Afterwards, we successfully fabricated the as-prepared Ag@G-SAS into a self-sterilizing coating on PES surface. The coating exhibited strong antibacterial activity and favorable biocompatibilities at both cell and blood levels. Beyond that, the bactericidal efficiency of the coating could be also further increased with the help of NIR laser. The proposed Ag@G-SAS nano-composites and their LbL self-assembly with favorable biocompatibilities show promising future in food packaging sterilization, medical disinfection, wound dressing and surface modification of biomedical devices.


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ASSOCIATED CONTENT Supporting Information Supporting Information including: materials and experimental methods used in this work; TEM images of G-SAS and G-QCS; AFM images of GO ; 1H NMR spectra for SAS and DA-SAS; XPS data of G-SAS; NIR-responsive heating images of MHB and the real-time temperature change curves of samples; Photographs and SEM image of control wound; Fluorescence images of

infected wound, Ag@G-SAS treated wound and infected wound before-and-after NIR irradiation; Zeta potentials of nano-composites; SEM image of pristine PES; XPS spectra and WCA data for samples; SEM images of bacterial adhesion on coated-membranes; Bacterial growth at the presence of PES, PES-PEI and PES-(G-SAS)3; Fluorescence images that revealed live/dead bacteria on samples; Images that revealed the long-term antibacterial activities of samples; SEM images of RBCs adhesion on samples; Enzyme linked immunosorbent assay of samples; SEM images that revealed platelet adhesion on membrane surfaces. AUTHOR INFORMATION Corresponding Authors: *E-mail: (C. X. Nie) [email protected];

*E-mail: (C.S. Zhao) [email protected] or [email protected].

Tel.: +86-28-85400453. Fax: +86-28-85405402.

Notes 29 Environment ACS Paragon Plus


The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was financially sponsored by the National Natural Science Foundation of China(Nos. 51433007, 51503125 and 51673125), the State Key Research Development Programme of China (2016YFC1103000 and 2016YFC1103001), and the Younth Science and Technology Innovation Team of Sichuan Province (Grant No. 2015TD0001). C. X. Nie acknowledges the support of China Scholarship Council. Dr. Cheng acknowledges the support of the DRS POINT Fellowship of Freie Universität Berlin, and an Alexander von Humboldt Fellowship. We should also thank our laboratory members for their generous help.

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