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Hyaluronic acid-templated Ag Nanoparticles/Graphene Oxide Composites for Synergistic Therapy of Bacteria Infection Xiang Ran, Ye Du, Zhenzhen Wang, Huan Wang, Fang Pu, Jinsong Ren, and Xiaogang Qu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 24, 2017

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ACS Applied Materials & Interfaces

Hyaluronic acid-templated Ag Nanoparticles/Graphene Oxide Composites for Synergistic Therapy of Bacteria Infection Xiang Ran, †,‡ Ye Du, ﹟ Zhenzhen Wang, †,‡Huan Wang, †,‡Fang Pu, † Jinsong Ren,*,† and Xiaogang Qu*

†

Laboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences,Changchun, Jilin 130022, P.R. China ﹟ Department of Breast Surgery, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China ‡ University of Chinese Academy of Sciences, Beijing 100039, P.R. China

KEYWORDS. graphene , hyaluronic acid, silver nanoparticle, antibacteria, synergistic therapy

ABSTRACT. Developing methods of decreasing the harm to cell and increasing the antibacterial efficiency is becoming a potential topic to medical treatments. We demonstrated a hyaluronidase-triggered photothermal platform for killing bacteria based on AgNPs and graphene oxide (GO). The property of the HAase-triggered release provided excellent antibacterial activity against S. aureus. Upon illumination of NIR light, the GO-based nanomaterials locally raised the temperature, resulting in high mortality of bacterial. The HAasetriggered AgNPs releasing approach for antibacterial allows AgNPs to be protected by HA

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template without affecting mammalian cells. The nanocomposites provided antibacterial activity against S. aureus while showed low toxicity to mammal cells. In addition, the GO-HA-AgNPs are prepared for in vivo experiments and show excellent antibacterial property in wound disinfection model.

1. INTRODUCTION Antibiotics play critical roles in treating pathogenic infections caused by bacteria in the past decades, however, the use of antibiotics leads to the rise of drug resistance.1 Benefit from the rapid development of nanotechnology, nanomaterials have opened up a new way in infection treating research.2 Among many reported nano-antibacterial agents, silver/silver-containing compounds have been used as effective biocides to reduce infections through preventing bacteria infected on human skin and catheters.3-5 Since the silver nanopartilces (AgNPs) demonstrate excellent antimicrobial activity, they are regarded as a promising candidate for combating with bacteria.6-8 Traditional Ag-based therapy methods often employ the AgNPs independently, which makes the AgNPs easily aggregated and suffers from their low antibacterial properties. To solve these problems, the antibacterial applications of AgNP-based nanocomposites have also attracted considerable interests.9-13 However, The AgNPs often attach to mammal cells, which will generate reactive oxygen species (ROS) and the extra activities also result in the noticeable cytotoxicity to mammal cells.14, 15 Therefore, it is necessary to develop methods for decreasing the harm to cell and increasing the antibacterial activity of AgNPs Graphene is a robust nanomaterial which consists of a single layer of tightly packed carbon atoms. This material possesses a special two-dimensional (2D) structure16,

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, large specific


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surface area18, remarkable electrical conductivity19-21, excellent adsorptivity22-25, and high chemical stability26. The unique optical properties make Graphene to absorb light irradiation and to release it as heat.27-30 This photothermal feature has been widely used in biological researches such as cancer therapy.31, 32 Due to the property of damaging cell membranes and the oxidization capacity towards glutathione (GSH), Graphene-based materials show good antibacterial activity.33 Moreover, the photothermal capability of graphene also has been successfully employed in bacteria therapy. Recently, Wu et al. have applied magnetic nanoparticlesfunctionalized graphene as photothermal agent for effective capturing and killing bacteria.34 Compared with conventional antibiotic therapies, photothermal therapy is not be restricted by antibiotic-resistance of bacteria and could resolve this issue. It shows great promises for combining the advantage of graphene and AgNPs in antibacterial agent. In this work, we presented a photothermal nanocomposite of hyaluronic acid (HA)-templated AgNPs integrated with graphene oxide triggered by HAase with low cytotoxicity and high antibacterial activity (Scheme 1). HA was used to synthesize and stabilize the AgNPs in aqueous solution. Owing to the high biocompatibility and the negative potential of HA,35, 36 we expected the HA-AgNPs possess low cytotoxicity to the mammal cells, compared with traditional citratetemplated AgNPs. Since several bacteria secreted hyaluronidase (HAase),37, 38 the HA could be degraded by these bacteria and the AgNPs could be released to inactivate bacteria. The HAasetriggered AgNPs releasing approach allows AgNPs to be protected by HA template without affecting mammalian cells, and presents controlled release in the presence of bacteria. Furthermore, the HA-AgNPs were functionalized on graphene oxide to yield GO-HA-AgNPs, which possess excellent photothermal property. After the HA was degraded, the sheetlike GO could accumulate on the surface of bacteria via intensive physical interactions.10,

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NIR


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irradiation could emphasize and focus on the composites to release heat. The heat will dissipate into the surroundings, and temperature will rise and inhibit the bacteria growth and cause the death of bacteria, thus resulting in a synergistic therapy.27 The cytotoxicity and antibacterial efficiency were tested by cell experiment and bacteria experiment. 2. MATERIALS AND METHODS 2.1 Materials. AgNO3, NaBH4 and hyaluronic-acid (HA) were purchased from Aladdin (Shanghai, China). Graphite powder, potassium persulfate (K2S2O8),potassium permanganate (KMnO4), 30% hydrogen peroxide (H2O2), hydrochloric acid (HCl) were all purchased from. Beijing Chemical Company (Beijing, China). N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and adipic acid dihydrazide (ADH) were obtained from Sigma-Aldrich (St. Louis, USA). Milli-Q water (Millipore, USA) was used throughout the experimental process. 2.2 Synthesis of HA-ADH. 100 mg of HA dissolved in 30 mL of MES buffer (pH 6.0). After adding DMSO–DI water (1: 1, 1 mL) containing EDC (192 mg) and NHS (232 mg) to this solution, the pH of the solution was adjusted to 6.0 and the solution was stirred overnight. To finish the reaction, the pH was adjusted to 7.0 with a 30-fold molar excess of ADH added. The product was transferred to the dialysis tubing (3500 Da) and dialyzed exhaustively against 100mM NaCl for 2 days and DI water for 1 more day. Spongy-like HA-ADH was obtained by freeze-drying. 2.3 Synthesis of HA-AgNPs and cit-AgNPs. A mix solution (200 mL) of 0.25 mM of AgNO3 and 100mg of HA-ADH or 0.25 mM of trisodium citrate was stirred for 15 min. NaBH4 (6 mL,


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10 mM) was added dropwisely to the solution. The reaction was stirred for 30 min, and a yellow colloidal silver solution was obtained. Then HA-AgNPs and cit-AgNPs was stored at 4 °C. 2.4 Synthesis of GO-HA-AgNPs. 10 mg of fractionated GO sheets were dispersed in to 50 mL of MES buff (pH 6.0) and sonicated for 30 min. 48 mg of EDC and 58 mg of NHS were added to the solution. EDC and NHS were added again into the solution after sonication for 30 min,. The mixture was stirring for 12 h at 4 °C, and adjusting the pH to 7.0. Then HA-AgNPs was added to the system. After 12 h, the reactant mixture was dialyzed (10000 Da) against DI water for 3 days. 2.5 HAase Treatment. 200 µg of GO-HA-AgNPs was dispersed in 4 mL of PBS (10 mM, pH 7.4) buff. Different volumes (0 µL, 1 µL, 2 µL, 5 µL, 10 µL, 20 µL) of HAase (0.1 mg/mL) were added into the solution. The released AgNPs were collected by centrifugation at 12000 rpm for 10 min after being stirred for 30 min. The absorbance at 410 nm was used to determine the amount of released AgNPs. 2.6 Bacteria Culture. Cultures of S. aureus bacterial strains on the solid Luria-Bertani (LB) agar plate were transferred to 20 mL of liquid LB culture medium, The resultant bacteria grown at 37°C for 12 h and were isolated by 3500 rpm centrifugation . Next, the bacterial cells were rinsed with PBS buffer and designed bacterial concentrations were mearsured by OD600. 2.7 Bacteria Viability Test. Two groups of test bacterial suspensions (105–106 CFU/mL) were incubated with different concentration of GO-HA-AgNPs for 10 min. One group of the bacteria was then irradiated with NIR light for 2 min. The resultant bacterial suspensions were cultured in Petri dishes containing LB agar medium at 37 °C, overnight. The CFU of the bacteria was then estimated.


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2.8 Antibacterial Experiments. The as-prepared 500 µL bacteria solution (98 cfu/mL) was mixed with different concentrations of GO-HA-AgNPs for 10 min. Then, the solution was cultured for 24 h. Then the number of the bacteria colonies was observed. Control experiments were performed in parallel with GO-HA, cit-AgNPs and without GO-HA-AgNPs 2.9 HeLa Cell Viability. Methyl thiazolyl tetrazolium (MTT) assays were used to test the cellular viability. HeLa cells were seeded in 96-well assay plates at a density of 5000 cells/well. Cells were further incubated with MnO2 nanosheets or MCDM at the indicated concentrations for 24h. 10 µL of MTT solution (BBI) was added to each well of the microtiter plate. After that, the cells was incubated for 4 h. Then100 µL of DMSO was added to lyse the cells. Bio-Rad model-680 microplate reader was used to determine the absorbance of formazan at 490 nm. The results were expressed as the mean values of three measurements. 2.10 Wounds model. To evaluate the antibacterial effect of GO-HA-AgNPs in vivo, the wounds model was built. The four groups of 12 male Kunming mice with wound (three mice per group) were divided into control, NIR, GO-HA-AgNPs and GO-HA-AgNPs+NIR groups. The mice in four different groups with different band-aids on their wound were observed. The band-aids were changed with 24 h interval after taking the photograph. The NIR and GO-HA-AgNPs+NIR groups was irradiated by NIR (1.0 W/cm2) for 2 min after changing the band-aids. After 3 days, all mice were sacrificed, and the tissues of wounds were harvested, and the number of bacteria was quantified at each day of the therapeutic process. Aliquots of diluted homogenized intestinal tissues were placed on agar. The grown colonies were counted for analysis. All animal procedures were in accord with the guidelines of the Institutional Animal Care and Use Committee.


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3. RESULTS AND DISCUSSION 3.1 The Characteristics of GO-HA-AgNPs. The HA-templated AgNPs were synthesized based on a method published by Fernig.39 GO was synthesized from natural graphite powder using a modified Hummers method.40 The HA-AgNPs were subsequently integrated with GO according to a procedure reported by Char.41 Transmission electron microscope (TEM) image of the water-soluble HA-AgNPs exhibited the particles were 20 nm in diameter (Figure S1). Figure 1a and 1b showed the GO sheet alone and the AgNPs-dispersed GO sheet, which demonstrated the structure of GO-HA-AgNPs. Figure 2a showed a clear TEM images of GO-HA-AgNPs at higher magnifications. The presence of Ag, HA and GO was proved by element mapping by using energy-dispersive X-ray spectroscopy (Figure 2b). Atomic force microscope (AFM) images suggested the HA-AgNPs was functionalized on the GO sheet with the height from 1 nm to 50 nm (Figure 1c and 1d). As shown in Figure 3a and 3b, The structural features of GO-HAAgNPs composites have also been thoroughly investigated using FTIR spectroscopy. A new peak of HA–GO conjugates appeared at 1640 cm-1 corresponding to the carbonyl-amide vibration. UV-Vis spectra demonstrated that these GO-HA-AgNPs nanocomposites displayed an absorption peak at 410 nm and the characteristic absorption peak of GO at 230 nm (Figure 3c). According to the spectroscopic data and the thermogravimetric analysis (TGA) traces (Figure 3d), the approximate amount of GO sheets in HA–GO conjugates was estimated to be about 15%. Furthermore, zeta potential of GO and GO-HA-AgNPs was examined, showing that the surface was negatively charged (-8.3 mV and -12.7 mV, respectively).


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3.2 The Photothermal Effect of GO-HA-AgNPs. To investigate the photothermal effect of NIR light on the materials, several experiments were carried out under different conditions. Heat conversion efficiency of GO-HA-AgNPs and GO was measured under 808nm NIR irradiation (0.5 W/cm2 1.0 W/cm2 and 2.0 W/cm2) while a PBS buffer was used as a control. The GO-HAAgNPs exhibited significant temperature change from 25.5 ºC to 35.1 ºC, 49.7 ºC, and 58.4 ºC under different power densities of NIR irradiation, respectively. Compared with GO and control, GO-HA-AgNPs presented relatively high heat conversion efficiency (Figure S2). The higher heat conversion efficiency of the GO-HA-AgNPs might be benefited from the higher specific surface area of GO with higher dispersion after linked with HA. The laser power density of 1.0 W/cm2 was used to test the efficiency of different concentrations of GO-HA-AgNPs (Figure S3). The 100 µg/mL of GO-HA-AgNPs showed better photothermal capability than other concentrations. Thus, we expect the material to be utilized as photothermal agent for antibacterial treatment. 3.3 The Release Experiment of AgNPs with HAase Treatment. Before the in vitro therapy, we explored the release of AgNPs triggered by HAase. 50 µg/mL of GO-HA-AgNPs solution was treated with different amounts of HAase. After centrifugation, the released AgNPs in supernatant were measured by UV-vis spectrometer. As shown in Figure S4a, in the absence of HAase, few AgNPs was detected in the supernatant. Upon the addition of HAase, more AgNPs were released from GO-HA-AgNPs composites. On the other hand, the concentration of released AgNPs did not increase obviously under the NIR irradiation (Figure S4b). These indicated that the AgNPs exposed only under HAase stimulation. Additionally, Hela cells were used as a model to explore the cytotoxicity of these materials. The MTT results obtained by incubating Hela cells with HA-AgNPs, citrate-templated AgNPs (citrate-AgNPs) and HAase-treated HA-AgNPs under different experiment conditions were shown in Figure S5a. Most of the cells incubated with the


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materials survived after 4 hours. HA-AgNPs presented much less impact on the cell growth, compared with traditional citrate-AgNPs and HAase-treated HA-AgNPs. Then we examined whether illumination of these materials under NIR light could affect the growth of human cells. Figure S5b exhibited that more than 84% of the Hela cells survived although the NIR light was introduced to the GO-HA-AgNPs. These results presented that our materials owned low cytotoxicity and high biocompatibility without HAase and could be used as a safe photothermal agent. 3.4 The Synergistic Antibacterial Effect of GO-HA-AgNPs. The efficiency of antibacterial photothermal treatment of GO-HA was investigated in a still S. aureus solution. The optical density of 600 nm (OD600) indicated the density of bacteria in a medium.42 Therefore, the viabilities of bacteria were represented by OD600 of the suspension. The different concentrations of GO-HA were incubated with bacteria for 10 min followed by treated with and without NIR, respectively. The survival rates of bacteria treated without NIR were all above 90%, which indicated GO-HA did not produce significant bacteria damage under dark. In contrast, viabilities of bacteria dramatically decreased upon NIR laser irradiation (1.0 W/cm2). Since a temperature gradient was formed with the heat dissipated into the surroundings when the system was under NIR irradiation and local temperatures were high enough to kill the bacteria, the survival rates of bacteria decreased by 45% at the concentration of 100 µg/mL (Figure S6). In the control group, the bacteria survival rate in the absence of photothermal agents was both about 100% under NIR laser irradiation or under dark condition, implying insufficient antimicrobial effect to bacteria under only NIR irradiation. The antibacterial activity of GO-HA-AgNPs nanocomposites was further tested with S. aureus by the measurement of surviving cells under the treatment of GO-HA-AgNPs. As shown in


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Figure S7, at concentrations from 5-100 µg/mL, GO-HA-AgNPs significantly decreased survival rates of S. aureus cells in a dose-dependent manner. Since the HA could be degraded by bacteria to expose the AgNPs, the GO-HA-AgNPs nanocomposites showed strong antibacterial effect. the viabilities of S. aureus reduced to 79% and 39% when the concentrations of agents were 5 ug/mL and 100 µg/mL, respectively. Meanwhile, the control group and cit-AgNPs instead of GO-HA-AgNPs were tested in the experiment, respectively. At a concentration of 100 µg/mL, the cit-AgNPs only reduced the viabilities of S. aureus by 40%. Fewer bacteria were killed after treatment with GO-Polyacrylic acid(PAA)-AgNPs. The negative charge of GO-PAA-AgNPs which prevented the composites from interacting with bacteria might be responsible for its lack of bacteria therapy efficiency. The data indicated that GO-HA-AgNPs exhibited better antibacterial efficiency as compared to AgNPs. Therefore, the GO-HA-AgNPs composites showed high antibacterial activity. We then integrated the advantages of antibacterial activity of AgNPs and photothermal therapy of the GO-HA-AgNPs to achieve a synergistic effect for bacteria therapy. To this end, bacterial strains were treated with GO-HA-AgNPs, GO-HA and cit-AgNPs under NIR irradiation (1.0 W/cm2) conditions, respectively. The survival rate of bacteria was still about 100%, since the NIR alone was harmless to the bacteria in the absence of antibacterial agents (Figure 4a). In contrast, the viabilities of S. aureus treated with GO-HA or cit-AgNPs decreased upon the NIR laser irradiation. Although cit-AgNPs were not able to converse NIR into heat, it still showed antibacterial activity. On the other hand, GO-HA-AgNPs demonstrated better antibacterial efficiency towards bacteria compared with GO-HA or cit-AgNPs owning to both the heat conversion ability of GO and the antibacterial activity of AgNPs. We also compared the bacteria survival rate in the presence of GO-HA-AgNPs with or without NIR irradiation. As shown in


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Figure S8, the viabilities of S. aureus decreased to 23% and 50% after treated by GO-HA-AgNPs under NIR irradiation and under dark condition, respectively. As a comparison, Escherichia coli (E. coli) was also treated with GO-HA-AgNPs under NIR irradiation. Since the E. coli did not secrete HAase, the survival rate reduced by only 32% (Figure S9). The above results indicated that GO-HA-AgNPs presented a synergistic antibacterial effect with HAase triggering. The scanning electron microscopy was also used to supporting the results. The surfaces of bacteria were smooth, which indicated that the cells were healthy without materials treating (Figure 4b). However, the cells were damaged seriously after treated with GO-HA-AgNPs, which showed the antimicrobial activity of the nanocomposites (Figure 4c). 3.5 The bactericidal effect of GO-HA-AgNPs. The bactericidal effect of GO-HA-AgNPs nanocomposites against S. aureus then tested in the nutrient solution. The bacteria were incubated with different concentrations of GO-HA-AgNPs and the bactericidal kinetics of bacteria was measured. The bacterial growth was also monitored by OD600. The bacteria were grown to an OD600 of 0.05 followed by various concentrations of GO-HA-AgNPs nanocomposites added in with NIR irradiation (1.0 W/cm2, 2 min). After 8 h, the bacteria in control medium grew into the stationary phase, whereas 10 µg/mL GO-HA-AgNPs showed a significant bactericidal effect against S. aureus (Figure 5a). After we increased the concentration of GO-HA-AgNPs, the bactericidal effect against bacteria became more significant., We then measured colony-forming units (CFUs) of surviving cells under the treatment of GO-HA-AgNPs to further investigate the antibacterial activity of GO-HA-AgNPs. The S. aureus was first exposed to different concentrations of GO-HA-AgNPs with NIR irradiation for 2 min. Then the CFUs of the surviving S. aureus were measured (Figure 5b). As showed in Figure 5c, control sample presented no obvious antibacterial activity. However, the colonies treated with GO-HA


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and cit-AgNPs were partly inhibited. More importantly, after exposed under NIR irradiation for 2 min, GO-HA-AgNPs showed bactericidal effect against S. aureus colonies. 3.6 Wounds Healing Experiments In Vivo. To test the antibacterial effect of GO-HA-AgNPs in vivo, we utilized a model of mice with their back wounded. We divided the mice into 4 groups: treated with blank-band-aid, blank-band-aid+NIR irradiation, GO-HA-AgNPs-band-aid, and GO-HA-AgNPs-band-aid+NIR irradiation. After injury on the back, photos of the wounds of the mice from four different groups were taken at 24 h interval before we changed the band-aids (Figure 6). After 72 h of therapy, scabs were formed on the wounds of mices treated with GOHA-AgNPs+NIR irradiation. The wounds of mice in this group did not appear to have erthema. In contrast, different levels of erythema were observed from other 3 groups. After 72 h therapy, the tissues of wounds were harvest to determine the antibacterial effect of GO-HA-AgNPs. We counted the grown colonies after the bacteria were cultured overnight. As shown in Figure S10, the bacteria of GO-HA-AgNPs+NIR irradiation treated groups were 2 orders lower than the control and the NIR irradiation groups. In addition, the GO-HA-AgNPs group also showed antibacterial activity since the released AgNPs could inhibit the growth of bacteria. 4. CONCLUSIONS In conclusion, we have demonstrated a HAase-triggered photothermal platform for killing bacteria based on AgNPs and GO. Due to the protection of HA, the nanocomposites exhibited low toxicity to mammal cells. Furthermore, the property of the HAase-triggered release provided excellent antibacterial activity against S. aureus. Upon illumination of NIR light, the GO-based nanomaterials locally raised the temperature, resulting in high mortality of bacterial. These results indicated that the GO-HA-AgNPs could be used as new antimicrobial materials for


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synergistic therapy of bacteria without harming cell. Our strategy will open an avenue for clinical pathogenic bacteria diagnosis and treatment.


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Scheme 1. Schematic illustration of hyaluronic-acid-templated Ag nanoparticles/graphene oxide composites for synergistic therapy of bacteria.


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Figure 1. Transmission electron microscopy images of (a) GO and (b) GO-HA-AgNPs. Atomic Force Microscope images of (c) GO and (d) GO-HA-AgNPs. The height profile showed the height distribution on the line.


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Figure 2. (a) higher magnifications TEM images of GO-HA-AgNPs. (b) dark-field TEM image of GO-HA-AgNPs and corresponding energy-dispersive spectroscopic element mapping of C Kedge, N K-edge, O K-edge and Ag K-edge signals.


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Figure 3. FITR spectra of (a) GO, GO-COOH and (b) GO-HA. (c) The UV-Vis spectra of GOHA and GO-HA-AgNPs. (d) TGA traces of the GO and GO-HA.


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Figure 4. (a)Survival rate of S. aureus incubated with GO-HA-AgNPS, GO-HA and cit-AgNPs at concentrations under NIR irradiation for 2 min. SEM images of (b) S. aureus without materials treating and (c) with GO-HA-AgNPs under NIR irradiation for 2 min.


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Figure 5. (a) Survival rate of S. aureus

with various concentrations of GO-HA-AgNPs

nanocomposites under NIR irradiation. (b) Colony-forming units of surviving cells under the treatment of GO-HA-AgNPs. (c) Inhibition of colonies after treated with GO-HA, cit-AgNPs and GO-HA-AgNPs.


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Figure 6. Photographs of wound on the mice from the four groups at different times during the therapeutic process.

ASSOCIATED CONTENT Supporting Information. TEM images of HA-AgNPs, heat conversion curves of different concentration of materials, the release curve of AgNPs with HAase treatment, cytotoxicity data of materials, the survival rates of S. aureus treated with the materials under different conditions, and the viability of E.coli treated with GO-HA-AgNPs+NIR were included in the Supporting Information. The Supporting Information is available free of charge on the ACS.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Financial support was provided by National Basic Research Program of China (Grant Nos. 2012CB720602) and the National Natural Science Foundation of China (Grant Nos. 21210002, 21303182, 21431007, and 21533008)

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