Antibacterial Activity of Partially Oxidized Ag/Au Nanoparticles against the Oral Pathogen Porphyromonas gingivalis W83

Advances in nanotechnology provide opportunities for the prevention and treatment of periodontal disease. While physicochemical properties of Ag containing nanoparticles (NPs) are known to influence the magnitude of their toxicity, it is thought that nanosilver can be made less toxic to eukaryotes by passivation of the NPs with a benign metal. Moreover, the addition of other noble metals to silver nanoparticles, in the alloy formulation, is known to alter the silver dissolution behavior. Thus, we synthesized glutathione capped Ag/Au alloy bimetallic nanoparticles (NPs) via the galvanic replacement reaction between maltose coated Ag NPs and chloroauric acid (HAuCl4) in 5% aqueous triblock F127 copolymer solution. We then compared the antibacterial activity of the Ag/Au NPs to pure Ag NPs on Porphyromonas gingivalis W83, a key pathogen in the development of periodontal disease. Only partially oxidized glutathione capped Ag and Ag/Au (Au:Ag≈0.2) NPs inhibited the planktonic growth of P. gingivalis W83. This effect was enhanced in the presence of hydrogen peroxide, which simulates the oxidative stress environment in the periodontal pocket during chronic inflammation.


A. Material Characterization
Instrumentation. UV-Vis spectra of nanomaterials were recorded using a Varian Cary 300 spectrophotometer equipped with a temperature controller. All UV-Vis measurements were made using a quartz cell with a 1 cm path length at 25 o C.
A Thermo NNS Energy-dispersive X-ray (EDX) analyzer attached to a Vega LSH scanning electron microscope was used to determine the composition of the Ag/Au bimetallic nanoparticles (NPs). EDX measurements were performed on at least five different regions on two different samples.
Atomic force microscopy (AFM) images were generated with a multimode 8 scanning probe microscope (Bruker, Santa Barbra CA) in the peak force tapping (k = 0.4 Nm -1 , f = 70 kHz) mode as previously described [1,2]. Briefly, NPs were prepared for AFM measurements by removing excess reagents (samples were centrifuged at 10,000 × g for 15 min and suspended in water twice) followed by dropping 50 L of solution onto a freshly prepared parafilm. A silanized (0.1% w/v) 3-Aminopropyltriethoxysilane (APTES) 18 mm mica disk (Ted Pella, Redding CA) was then applied onto the sample facedown to obtain spreading for at least 10 minutes. The mica disk was then rinsed with ethanol and water prior to imaging. To prepare P. gingivalis W83 for imaging, the bacteria were grown to early exponential phase before being exposed to glutathione capped Ag/Au NPs (OD = 1) for 10 min or 5 hours. The bacteria (1 mL) were then washed by centrifugation at 2,500 × g for 5 min and suspended in 10 mM phosphatebuffered saline (1 mL final volume). The resuspended bacteria (100 μL) were further diluted to 1 mL in 10 mM phosphate-buffered saline. The resulting bacterial suspension (40 μL) was dried onto a silanated mica disk under nitrogen. Samples were rinsed well with water and allowed to dry prior to same day imaging. All microscopy (TEM and AFM) measurements were processed using the Gwyddion analysis tool (downloadable from http://gwyddion.net).
Digital transmission electron microscopy was carried out on a Philips Tecnai 12 instrument operating at 80 kV fitted with a Gatan camera. Samples were prepared for electron microscopy measurements by removing excess reagents (samples were centrifuged at 10,000 × g for 15 min and resuspended in water twice) followed by dropping 5 to 10 L of solution onto a silanated 200 mesh carbon-coated Cu grid (Ted Pella, Redding CA). Samples were allowed to air dry.
Fourier transform infrared spectroscopy (FTIR) was carried out using a Jasco FTIR 4100 with a deuterated triglyceride sulfate detector. In order to obtain the FTIR spectra a drop of the nanoparticle solution was placed on the surface of an attenuated total reflectance germanium crystal and dried under nitrogen. Spectra were obtained at 4 cm −1 resolution and 64 scans were obtained for statistical averaging.
Dynamic light scattering (DLS) was used to evaluate the colloidal stability of glutathione capped and uncapped Ag/Au alloy NPs. A Nicomp™ 380 XLS Zeta Potential/Particle Sizer (PSS Nicomp, USA) equipped with a He-Ne laser wavelength 638 nm and a power output of 60 mW was used for all DLS experiments. Briefly, 240 μL of glutathione capped or uncapped Ag/Au NPs (OD = 1) were suspended to a final volume of 3 mL in 10mM phosphate buffered saline (pH 7.5). Volume weighted DLS was used to evaluate nanoparticle aggregation immediately after dilution as well as 1, 2, 4, 6, 8, and 24 hours after dilution. All data were collected at 25 °C and at a scattering angle of 168.6° with a square acrylic cuvette (3 mL volume). Refractive index of water and viscosity were assumed to be 1.33 and 8.9×10 −4 Ns m −2 , respectively.    gingivalis W83 growth at 12 and 24 hours. P. gingivalis W83 was exposed to water (control), uncapped Ag/Au NPs, or glutathione capped Ag/Au NPs. The absorbance at 600 nm was measured at for 12 and 24 h after incubation at 37 °C under anaerobic conditions to assess bacterial growth. Glutathione capping was done using method I.

B. Supplemental Figures
Error bars are standard deviation.