Controlling the

size of Ag NPs is as important to antiviral activity as the composition of the Ag NPs. We previously demonstrated an Bafilomycin A1 mw environmentally friendly process for producing Ag NPs with a narrow size distribution [25]. This process uses only three materials: a silver-containing glass powder as an Ag+ supplier, glucose as a reducing agent for Ag+, and water as a solvent. The stabilizing agent for Ag NPs is caramel, which is generated from glucose during heating to reduce Ag+. In this work, Ag NPs synthesized by this process were used to make the Ag NP/Ch composites, since the size of the Ag NPs could be easily controlled without the use or production of hazardous materials. Ag NP/Ch composites were synthesized in aqueous media at room temperature by mixing a chitosan solution and an Ag NP GSK872 nmr suspension. The surface and internal structure of the synthesized Ag NP/Ch composites were observed by scanning and transmission electron microscopies, respectively. The effect of introducing a small amount of Ag NPs into the chitosan matrices and the effect

of the size of the Ag NPs were evaluated with respect to the antiviral activity of the composites. Methods Materials Ag NP suspensions were synthesized from silver-containing glass powder (BSP21, silver content 1 wt%, average grain size 10 μm, Kankyo Science, Kyoto, Japan) and glucose aqueous solution, as described previously [25]. Ag NPs used in this work were spherical; their characteristics are summarized in Table 1. Phosphate-buffered saline (PBS), methanol, Giemsa stain solution, find more and 5 M hydrochloric acid (HCl) and 5 M sodium next hydroxide (NaOH) aqueous solutions were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and used without further purification. Chitosan solution (10 mg/mL) was prepared by mixing 0.1 g chitosan (average molecular weight 54 kg/mol, deacetylation ratio 84%; Yaizu Suisankagaku Industry Co., Ltd., Shizuoka, Japan), 10 mL of PBS, and 100 μL of 5

M HCl; following complete dissolution of the chitosan, the solution was filter-sterilized by passage through a 0.2-μm filter. Bovine serum albumin (BSA) solution was prepared using BSA powder (Sigma-Aldrich Japan, Tokyo, Japan) and PBS, then filter-sterilized as above. Trypsin was obtained from Life Technologies Co., (Carlsbad, CA, USA). Dulbecco’s Modified Eagle Medium (DMEM, high glucose) was purchased from Sigma-Aldrich Japan (Tokyo, Japan). Table 1 Characteristics of Ag NPs Sample number Average diameter ± SD (nm) Concentration of Ag NP in suspension (μg/mL) SN35 3.5 ± 1.8 73 SN65 6.5 ± 1.8 62 SN129 12.9 ± 2.5 77 Synthesis of Ag NP/Ch composites Chitosan solution (100 μL, 10 mg/mL) was mixed with Ag NP solution (0.25 to 4.5 mL) and 40 μL 5 M NaOH at room temperature, followed by vigorous stirring to precipitate the Ag NP/Ch composite. The obtained Ag NP/Ch composite was centrifuged at 6,000 rpm for 10 min.