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Biological and Environmental Phenomena at the Interface

Gold Coating of Silver Nanoplates for Enhanced Dispersion Stability and Efficient Antimicrobial Activity against Intracellular Bacteria Hiroaki Ichimaru, Ayaka Harada, Soichiro Yoshimoto, Yuta Miyazawa, Daigou Mizoguchi, Kaung Kyaw, Katsuhiko Ono, Hiroyasu Tsutsuki, Tomohiro Sawa, and Takuro Niidome Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00540 • Publication Date (Web): 15 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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Gold Coating of Silver Nanoplates for Enhanced Dispersion Stability and Efficient Antimicrobial Activity against Intracellular Bacteria

Hiroaki Ichimaru,† Ayaka Harada,† Soichiro Yoshimoto,† Yuta Miyazawa,‡ Daigou Mizoguchi,‡ Kaung Kyaw†, §, Katsuhiko Ono,|| Hiroyasu Tsutsuki,|| Tomohiro Sawa,|| Takuro Niidome†,*

†

Faculty of Advanced Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japan ‡ Dai Nippon Toryo Co., Ltd. 1382-12, Shimoishigami, Otawara, Tochigi, 324-8516, Japan § Department of Chemical Engineering, Yangon Technological University, Gyogone, Insein P.O., Yangon 11-011, Myanmar || Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan

*corresponding author: Takuro Niidome ([email protected])

Keywords: silver nanoplates; gold coating; dispersion stability; intracellular bacteria; antimicrobial activity

1 ACS Paragon Plus Environment

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ABSTRACT Silver nanoparticles have antibacterial activity. However, the nanoparticles are unstable and easily form aggregates, which decreases their antibacterial activity. To improve the dispersion stability of silver nanoparticles in aqueous media, and to increase their effectiveness as antibacterial agents, we coated triangular plate-like silver nanoparticles (silver nanoplates, Ag NPLs) with one or two layers of gold atoms (Ag@Au1L NPLs and Ag@Au2L NPLs, respectively). These gold coatings improved the dispersion stability in aqueous media with high salt concentrations. Ag@Au1L NPLs showed stronger antibacterial activity on pathogenic bacteria than Ag NPLs and Ag@Au2L NPLs. Furthermore, the Ag@Au1L NPLs decreased the number of bacteria in RAW 264.7 cells. The Ag@Au1L NPLs displayed no cytotoxicity towards RAW 264.7 cells, and the Ag@Au1L NPLs could be used as an antibacterial agent for intracellular bacterial infections.

INTRODUCTION Bacterial infections and related diseases are becoming a serious problem around the world. Traditional antibiotics are not effective against some bacteria because of mutations and evolved functions have led to drug tolerance. Emerging multi-drug-resistant bacteria cause many serious infectious diseases and are life-threatening.1 Researchers have been developing new antimicrobial agents by targeting specific bacterial cell structures, enzymes, or metabolic pathways. To evade the biological defenses of the human body, intracellular bacteria are internalized into macrophages after infection.2–4 Mycobacterium tuberculosis is an intracellular pathogen that can cause serious pulmonary tuberculosis.5 To overcome these


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bacterial infections, novel antibacterial agents with different bactericidal mechanisms to conventional antibiotics are needed. Generally, nanoparticles are recognized as exogenous materials and can be effectively taken up by macrophages.6,7 Surface modification of nanoparticles is an attractive strategy to increase their accumulation in macrophages. Mannose-modified poly(lactic-co-glycolic acid) (PLGA) nanoparticles showed increased uptake by macrophages compared with unmodified nanoparticles.8 Compared with free vancomycin, vancomycin-encapsulated PLGA nanoparticles can decrease the number of methicillin-resistant Staphylococcus aureus (MRSA) inside infected J774A.1 macrophages.9 Silver nanoparticles have antibacterial activity against various species of bacteria.10,11 Their antibacterial effect is caused by release of silver ions from the nanoparticles.12–14 Silver ions enter bacterial cells and produce reactive oxygen species, which can damage the cell membrane, DNA, and proteins, and lead to cell death.15 In addition, nanoparticles kill bacteria by adsorbing on bacterial membranes and damaging the membrane structure.16 Silver nanoparticles also prevent biofilm formation by pathogenic bacteria.17 Despite these properties, the use of silver nanoparticles as antibacterial agents against infectious diseases is not common because silver nanoparticles are unstable and tend to form aggregates under physiological conditions in electrolytes such as inorganic salts and proteins. Aggregate formation decreases the release of silver ions from the nanoparticles, and reduces antibacterial activity at the site of infection. To improve the stability of nanoparticles and apply them as effective antibacterial materials, we developed triangular plate-like silver nanoparticles (silver nanoplates, Ag NPLs) coated with four layers of gold atoms.18 The four layers of gold atoms on Ag NPLs improved their dispersion stability, however, their antibacterial activity was decreased by


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the coating. In this study, we evaluated the antibacterial activities of Ag NPLs coated with one or two layers of gold atoms (Ag@Au1L NPLs and Ag@Au2L NPLs, respectively) using colony forming unit assays. The gold coating on the Ag NPLs improved their dispersion stability in aqueous media with high salt concentrations, and the Ag@Au1L NPLs greatly decreased the number of bacteria present in RAW 264.7 cells. These results suggested that the Ag@Au1L NPLs could be used as an antibacterial material for intracellular bacteria.

EXPERIMENTAL SECTION Preparation of Ag NPLs and Ag@Au NPLs. Ag NPLs and Ag@Au NPLs were prepared following the established methods.19,20 Briefly, Ag NPLs were synthesized in a two-step process. First, a seed solution of silver nanoparticles was prepared by mixing 20 mL of 2.5 mM sodium citrate, 1 mL of 0.5 g/L polystyrene sulfonate (MW 70 000), and 1.2 mL of 10 mM NaBH4 in a conical flask, and adding 50 mL of 0.5 mM AgNO3 at a rate of 20 mL/min. This solution was stirred for 60 min at 30 °C. Next, 1 mL of the seed solution was mixed with 200 mL of distilled water and 4.5 mL of 10 mM ascorbic acid. Then, with continued stirring, 120 mL of 0.5 mM AgNO3 was added at a rate of 30 mL/min, and the mixture was stirred for another 4 min. After that, 20 mL of 25 mM sodium citrate was added. The prepared solution was kept at 30 °C for 100 h. The Ag@Au1L NPLs were synthesized by mixing 120 mL of the prepared solution of Ag NPLs with 9.1 mL of 0.125 mM polyvinyl pyrrolidone (MW 40 000) and 1.6 mL of 0.5 M ascorbic acid. Then, 9.6 mL of 0.014 mM chloroauric acid was added with stirring at a rate of 0.5 mL/min. This solution was kept at 30 °C for 24 h. The Ag@Au2L NPLs were synthesized by the same method, except the concentration of chloroauric acid was


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increased to 0.14 mM. Characteristics of the Ag NPLs and Ag@Au NPLs. The optical properties of Ag NPLs and Ag@Au NPLs were measured using an UV/visible/near IR spectrophotometer (V-670; Jasco, Tokyo, Japan). The shapes and sizes of the NPLs were observed by TEM (JEM-1400Plus; Jeol). The size distributions and zeta potentials of the NPLs were measured by a zetasizer (Malvern Zetasizer Nano ZS; Malvern Instruments Ltd, Malvern, UK). Silver ions released from each NPL were detected by ICP-OES (Thermo iCAP 7000 Series ICP Spectrometer; Thermo Fisher Scientific, MA). Preparation of samples for TEM. A 100-µL droplet of each NPL solution was placed on parafilm and a TEM grid coated with carbon film (ELS-C10, Okenshoji Co., Ltd.) was placed inside the droplet for 5 min. After incubation, the grid was vacuum dried at room temperature overnight. Evaluation of the dispersion stability in LB. First, each NPL solution was centrifuged (12 000 ´ g, 10 min, 25 °C), the supernatant was removed, and same volume of ultrapure water was added. An aliquot (200 µL) of the 2.0 × 10−4 M NPL solution and 800 µL of LB were added to a cuvette and mixed well. Immediately after mixing, the absorption spectrum was measured by the UV/visible/near IR spectrophotometer. Absorption spectra were also measured at 3 and 6 h after mixing. Antibacterial activity towards planktonic bacteria. S. Typhimurium (LT-2 strain) and P. aeruginosa (laboratory strain) were used to evaluate the antibacterial activities of the NPLs. The antibacterial activity of each NPL was evaluated using a colony forming unit assay, as described in a previous report.18 Briefly, a bacterial stock solution was plated on a LB agar plate and incubated overnight at 37 °C. A single colony was selected from the ager plate and added to 2 mL of LB. The bacteria were incubated overnight at 37 °C.


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After incubation, the bacterial culture was diluted 102 fold using fresh LB, and then incubated for 4 h at 37 °C. After 4 h, the bacterial culture was diluted 104 fold with fresh LB. An aliquot (200 µL including 1.1× 108 and 1.9× 107 for S. Typhimurium and P. aeruginosa, respectively) of the diluted bacterial culture was added to a 96-well plate, followed by 50-µL aliquots of the NPLs. S. Typhimurium and P. aeruginosa were exposed to each NPL at concentrations of 2.5 × 10−4 M and 2.5 × 10−6 M, respectively, for 6 h at 37 °C. After incubation, 100 µL of each mixture was serially diluted 10-fold with PBS, and then 100 µL of each diluted solution was plated onto a separate LB agar plate. The plates were incubated overnight at 37 °C. After incubation, the number of colonies was counted. The experiment was performed in triplicate and data are expressed as mean ± standard error (n = 3) TEM observation of S. Typhimurium with Ag@Au1L NPLs. An aliquot (500 µL) of the Ag@Au1L NPLs was centrifuged (12 000 × g ,10 min, 25 °C), and then the supernatant was removed and same volume of ultrapure water was added back in. The NPL solution (200 µL, 2.0 ×10−4 M) was mixed with the bacterial culture and the mixture was incubated for 1 h. Then, the TEM grid was dipped in the bacterial culture. Finally, 0.1 % sodium phosphotungstate was used for staining the bacteria, and the TEM grid was dried for 2 d at room temperature. When the TEM grid was completely dry, the sample was observed by TEM. Quantification of silver ions released from NPLs in PBS. An aliquot (3 mL) of NPLs (silver atom concentration 2.0 × 10−4 M) was centrifuged (12 000 × g ,10 min, 25 °C), the supernatant was removed, and 3 mL of PBS was added. This solution was allowed to stand for 3 d at room temperature. The solution was centrifuged and silver ions contained in the supernatant were quantified by ICP-OES. The experiment was performed


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in triplicate and data are expressed as mean ± standard error (n = 3). Antibacterial activity against intracellular bacteria. Evaluation of antibacterial activity against intracellular bacteria was performed following previous reports.9 Antibacterial activity of the NPLs against intracellular bacteria was evaluated by the gentamicin protection assay to determine bacterial survival within macrophages. Briefly, RAW264.7 cells dispersed in DMEM containing 10 % FBS were seeded into a 96-well plate at 5.0 × 104 cells per well. The plates were incubated overnight and then infected with S. Typhimurium with a multiplicity of infection of one. After incubation for 1 h, planktonic bacteria were gently washed out using PBS and incubated with fresh DMEM containing 10 % FBS and 100 µg/mL gentamycin to kill any extracellular bacteria. After 2 h, the RAW 264.7 cells were washed with PBS, and 100 µL of 2.4 × 10−4 M NPLs in DMEM containing 20 µg/mL gentamycin was added to each well. After 6 h of incubation, the cells were gently washed with PBS and lysed by adding 200 µL of sodium deoxycholate (0.5% in PBS). Lysates were serially diluted and 100 µL of each solution was plated onto a LB agar plate. The number of viable bacteria was determined by colony forming units assay. The experiment was performed in triplicate and the data are expressed as mean ± standard error (n = 3) Evaluation of cytotoxicity on RAW 264.7 cells. The cytotoxicity of the NPLs were examined using a CCK-8 assay kit (Cell counting Kit-8; DOJINDO LABORATORIES, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, RAW 264.7 cells dispersed in DMEM containing 10 % FBS were seeded into 96-well plates at 1.5 × 104 cells per well and incubated overnight. Aliquots (100 µL) of each NPL (2.0 × 10−4 M dispersed in DMEM) were added to the cell culture. After incubation for 6 h, 10 µL of CCK-8 solution was added to the cell culture, followed by further incubation for 4 h. The


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absorbance at 450 nm was measured by a microplate reader (680; Bio-Rad, Hercules, CA). The experiment was performed in triplicate and the data are expressed as mean ± standard error (n = 3)

RESULTS AND DISCUSSION Characterization of Ag@Au NPLs. Optical properties of the Ag NPLs, Ag@Au1L NPLs,

and

Ag@Au2L NPLs were measured

with an

UV/visible/near IR

spectrophotometer. The NPLs showed characteristic absorption spectra between 600–800 nm (Figure 1A). The shapes of the NPLs were studied using transmission electron microscopy (TEM). The results (Figure 1B) showed that the Ag@Au1L NPLs were uniformly dispersed and shaped like disks or triangles. Among 295 particles, 55.3 % were triangular and 44.7 % were disks or another shape. The Ag NPLs were the same shape as the Ag@Au1L NPLs (Figure S1). The size distributions of the Ag NPLs, Ag@Au1L NPLs, and Ag@Au2L NPLs were measured by dynamic light scattering. There were multiple distributions, which is typical of anisotropic nanoparticles that can have several modes of Brownian motion. In the dynamic light scattering measurements, the gold coating on the Ag NPLs did not affect their distributions (Figure 1C). For the Ag NPLs, some aggregated Ag NPLs were observed at 1 000–10 000 nm. Measurement of the zeta potentials of the NPLs showed the surface charges of all the NPLs were negative because of dispersing agents, such as sodium citrate and ascorbic acid (Figure S2). To examine the dispersion stabilities of the NPLs, Ag NPLs, Ag@Au1L NPLs, and Ag@Au2L NPLs, they were mixed in Luria Broth (LB) and the absorption spectra were measured with an UV/visible/near IR spectrophotometer. The absorption for the Ag NPLs disappeared immediately after incubation, indicating that the Ag NPLs formed aggregates


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after mixing with the LB (Figure 1D). By contrast, the absorption spectra for the Ag@Au1L NPLs and Ag@Au2L NPLs remained stable for 6 h after incubation in the LB (Figure 1E and F). No further change was observed after incubation for 24 h (data not shown). In particular, the Ag@Au2L NPLs showed no change in the absorption spectrum. Therefore, the gold coating on the Ag NPLs dramatically improved the dispersion stability.

Figure 1. Characterization of the Ag NPLs, Ag@Au1L NPLs, and Ag@Au2L NPLs. (A) Absorption spectra of the Ag NPLs (black line), Ag@Au1L NPLs (red line), and Ag@Au2L NPLs (blue line). (B) Transmission electron microscope (TEM) image of the Ag@Au1L NPLs. (C) Size distributions of NPLs dispersed in water. Spectral changes for the Ag NPLs (D), Ag@Au1L NPLs (E), and Ag@Au2L NPLs (F) in Luria Broth (LB). The black lines show absorption spectra recorded immediately after mixing. The red and


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blue lines show absorption spectra recorded after incubation for 3 h and 6 h, respectively.

Antibacterial activity against planktonic bacteria. To examine the effect of the gold coating on the Ag NPLs on antibacterial activity against pathogenic bacteria, we used a colony forming unit assay to measure the antibacterial activity against Salmonella enterica serovar Typhimurium (LT-2 strain) and Pseudomonas aeruginosa (laboratory strain). S. Typhimurium and P. aeruginosa were exposed to NPLs at concentrations of 2.5 × 10−4 M and 2.5 × 10−6 M, respectively, for 6 h. Against both pathogenic bacteria, Ag@Au1L NPLs showed the strongest antibacterial activity among the NPLs (Figure 2). For the Ag@Au2L NPLs, antibacterial activity was not observed against the bacteria, which indicated that the thick gold coating (two layers of gold atoms) on the Ag NPLs masked the antibacterial effect of the Ag NPLs. For the strong antibacterial activity of Ag@Au1L NPLs, we hypothesized that there would be some defects in the gold layer, and silver atoms could be released as silver ions through these defects. The high dispersion stability of Ag@Au1L NPLs compared with the Ag NPLs also contributed to the strong antibacterial activity.


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Figure 2. Antibacterial activities of NPLs against pathogenic bacteria (A) S. Typhimurium and (B) P. aeruginosa. S. Typhimurium and P. aeruginosa were exposed to NPLs at concentrations of 2.5 × 10−4 M and 2.5 × 10−6 M, respectively, for 6 h. Data are expressed as mean ± standard error (n = 3). *p < 0.05.

Mechanism of antimicrobial activity. The antibacterial activity of silver nanoparticles reported primarily arises from the release of silver ions from the nanoparticles.[12-14] Some reports have also suggested that the antibacterial effect is caused by the silver nanoparticles themselves, with silver nanoparticles adsorbing on bacterial membranes and disrupting the membrane regularity.[16] To examine the possibility of direct interaction of the particles on the bacteria, TEM images were recorded of bacteria incubated with Ag@Au1L NPLs for 1 h. The TEM image showed that the Ag@Au1L NPLs did not always adsorb on the bacterial membrane (Figure 3A, 11 ACS Paragon Plus Environment

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Figure S3). TEM images of bacteria after further incubation with Ag@Au1L NPLs revealed the destruction of bacteria at 3 h and 6 h; however, any specific accumulation of Ag@Au1L NPLs to the dead bacteria was not observed (Figure S3). Therefore, the strong antibacterial activity of the Ag@Au1L NPLs was not caused by direct interaction of Ag@Au1L NPLs with bacteria. To confirm the contribution of silver ions, release of silver ions from the NPLs was measured by inductively coupled plasma optical emission spectroscopy (ICP-OES). The amount of silver ions released from the Ag@Au1L NPLs after 3d incubation in PBS was about 2.4 times than that released from the Ag NPLs and 4.0 times than that released from the Ag@Au2L NPLs (Figure 3B). The amount of silver ions released at 6h, corresponding to the incubation time in the evaluation of the antibacterial activity, could not be detected because it was lower than the detection limit of ICP-OES; however, the faster release of the silver ions would contribute higher antibacterial activity of Ag@Au1L NPLs than Ag@Au2L NPLs. The higher release of silver ions from the Ag@Au1L NPLs suggested that the gold coating had some defects, which released silver ions effectively and facilitated antimicrobial activity. It was reported that chloride ions can affect the migration of gold atoms on the silver surface.21 The migration of the gold atom might affect the exposure of silver metals to the culture medium and release of silver ions. On the other hand, the Ag NPLs formed aggregates in LB medium containing high salt concentration as shown in Figure 1D. Gao et al. previously reported that silver nanoplates formed aggregates in PBS within 1 min, while gold-coated silver nanoplates with approximately four layers of Au atoms were stable in PBS for 48 days.19 Reduction of surface area of the particles as a result of the aggregates formation was disadvantageous to the release of silver ions. High dispersion stability and some defects in the gold layer would be the important keys for the efficient antimicrobial


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activity of the Ag@Au1L NPLs.

Figure 3. Mechanism of the antibacterial activity of Ag@Au1L NPLs. (A) TEM image of S. Typhimurium incubated with Ag@Au1L NPLs for 1 h. (B) Quantification of silver ions released from NPLs in phosphate buffered saline (PBS) at 3 d. Data are expressed as mean ± standard error (n = 3). *p < 0.05.

Antibacterial activity against intracellular bacteria. To investigate the potential of the NPLs as antibacterial drugs for intracellular bacterial infections, we evaluated their antibacterial activities against intracellular bacteria. Here, we used S. Typhimuriuminfected RAW 264.7 cells as a model. The infected RAW 264.7 cells were exposed to NPLs at a concentration of 2.4 × 10−4 M for 6 h. The Ag@Au1L NPLs decreased the


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number of bacteria (Figure 4), whereas the Ag NPLs and Ag@Au2L NPLs did not. With the Ag NPLs, a number of large aggregates of Ag NPLs were observed in the cell culture medium under optical microscopy (data not shown). This suggests that the large Ag NPLs are not internalized by the RAW264.7 cells, resulting in low antibacterial activity. With the Ag@Au1L NPLs and Ag@Au2L NPLs, no aggregation was observed in the cell cultures. Therefore, Ag@Au1L NPLs and Ag@Au2L NPLs would be more efficient than Ag NPLs, and the Ag@Au1L NPLs would release more silver ions in the infected RAW 264.7 cells.

Figure 4. Antibacterial activities of NPLs against intracellular bacteria. S. Typhimurium infected RAW 264.7 cells were exposed to NPLs at a concentration of 2.4 × 10−4 M for 6 h. After lysis of the RAW264.7 cells, we counted the number of bacteria that survived. Data are expressed as mean ± standard error (n = 3). *p < 0.05.

Evaluation of the cytotoxicity towards RAW 264.7 cells. The cytotoxicity of NPLs towards RAW 264.7 cells was evaluated using a CCK-8 assay. RAW 264.7 cells were exposed to NPLs at a concentration of 2.0 × 10−4 M for 6 h. All the NPLs showed good biocompatibility with the RAW 264.7 cells (Figure 5). We confirmed that Ag@Au1L


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NPLs showed antibacterial activity against S. Typhimurium inside RAW 264.7 cells in the previous section. The low cytotoxicity of the Ag@Au1L NPLs combined with their antibacterial activity against intracellular bacteria could kill pathogenic bacteria without any cytotoxicity.

Figure 5. Cytotoxicity of NPLs. RAW 264.7 cells were exposed to each NPL at a concentration of 2.0 × 10−4 M for 6 h. Data are expressed as mean ± standard error (n = 3).

CONCLUSION To improve the dispersion stability of nanoparticles in aqueous media with high salt concentrations, we prepared gold-coated Ag NPLs with different thicknesses for the gold layer (Ag@Au1L NPLs and Ag@Au2L NPLs) and applied them as antibacterial agents. Among the Ag NPLs, the Ag@Au1L NPLs showed the strongest antibacterial activity because of their high dispersion stability in LB and the release of silver ions through defects in the gold layer. Furthermore, the Ag@Au1L NPLs could be internalized by RAW264.7 cells, where they killed bacteria within the cells. Therefore, we propose


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Ag@Au1L NPLs could be used as an antibacterial agent for intracellular bacterial infections and related diseases.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.xxxxxx. Characterization data (PDF)

AUTHOR INFORMATION ORCID Takuro Niidome: 0000-0002-8070-8708 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This study was supported by a Research Grant from the GSST Research Core and Group for Research B of Kumamoto University. The authors would like to acknowledge the Japan International Cooperation Agency (JICA) for its support of this project. We thank Gabrielle David, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

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Table of Contents/Abstract Graphic


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Figure 1. Characterization of the Ag NPLs, Ag@Au1L NPLs, and Ag@Au2L NPLs. (A) Absorption spectra of the Ag NPLs (black line), Ag@Au1L NPLs (red line), and Ag@Au2L NPLs (blue line). (B) Transmission electron microscope (TEM) image of the Ag@Au1L NPLs. (C) Size distributions of NPLs dispersed in water. Spectral changes for the Ag NPLs (D), Ag@Au1L NPLs (E), and Ag@Au2L NPLs (F) in Luria Broth (LB). The black lines show absorption spectra recorded immediately after mixing. The red and blue lines show absorption spectra recorded after incubation for 3 h and 6 h, respectively. 159x188mm (300 x 300 DPI)

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Figure 2. Antibacterial activities of NPLs against pathogenic bacteria (A) S. Typhimurium and (B) P. aeruginosa. S. Typhimurium and P. aeruginosa were exposed to NPLs at concentrations of 2.5 × 10−4 M and 2.5 × 10−6 M, respectively, for 6 h. Data are expressed as mean ± standard error (n = 3). *p < 0.05. 141x167mm (300 x 300 DPI)


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Figure 3. Mechanism of the antibacterial activity of Ag@Au1L NPLs. (A) TEM image of S. Typhimurium incubated with Ag@Au1L NPLs for 1 h. (B) Quantification of silver ions released from NPLs in phosphate buffered saline (PBS) at 3 d. Data are expressed as mean ± standard error (n = 3). *p < 0.05. 141x181mm (300 x 300 DPI)


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Figure 4. Antibacterial activities of NPLs against intracellular bacteria. S. Typhimurium infected RAW 264.7 cells were exposed to NPLs at a concentration of 2.4 × 10−4 M for 6 h. After lysis of the RAW264.7 cells, we counted the number of bacteria that survived. Data are expressed as mean ± standard error (n = 3). *p < 0.05. 129x77mm (300 x 300 DPI)


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Figure 5. Cytotoxicity of NPLs. RAW 264.7 cells were exposed to each NPL at a concentration of 2.0 × 10−4 M for 6 h. Data are expressed as mean ± standard error (n = 3). 77x46mm (300 x 300 DPI)


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Table of Contents/Abstract Graphic 64x61mm (300 x 300 DPI)


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Gold Coating of Silver Nanoplates for Enhanced ... - ACS Publications

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