Centrifuged composites were washed with 1 mL PBS, followed by centrifugation at 6,000 rpm for 10 min. The washing process was repeated A-1210477 datasheet twice. The washed Ag NP/Ch composite was suspended in 250 μL PBS and used in antiviral assays the same day. Synthesis of the Ag NP/Ch composites was carried out in a laminar flow cabinet to prevent biological contamination. Microscopy observations Scanning electron microscopy (SEM) specimens of the composites were prepared by casting 5
μL of a water dispersion of the Ag NP/Ch composite, followed by drying at room temperature. Captisol Osmium plasma coating was conducted to enhance the conductivity of the specimens. Dried samples were coated using a plasma multi-coater PMC-5000 (Meiwafosis Co., Ltd., Tokyo, Japan). SEM observation was performed using a JSM-6340F (JEOL, Tokyo, Japan) at 5 kV. Transmission electron microscopy (TEM) specimens of the Ag NPs and Ag NP composites were prepared by casting 5 μL of Ag NP solution or a water dispersion of the composite onto a carbon-coated AZD4547 solubility dmso copper microgrid. Excess solution was removed using filter paper, and the specimens were dried at room temperature. Further staining was not
carried out for any specimen. TEM observation was performed using a JEM-1010 (JEOL) at 80 kV. Assaying the antiviral activity of the Ag NP/Ch composites Human influenza A virus (A/PR/8/34 (H1N1)), obtained from Life Technologies Co., was used and assayed using the fifty-percent tissue culture infectious dose (TCID50) method. Viral suspension in PBS (250 μL, titer ca. 1,000 TCID50/mL) was added to 250 μL Ag NP/Ch composite suspension. The mixture was stirred vigorously for 5 s and then left at room temperature for 1 h to allow the virus and composite particles to interact. Then,
the mixture was centrifuged at 6,000 rpm for 10 min to remove the composite particles. The supernatant (50 μL) was subjected to two-fold serial Liothyronine Sodium dilution with PBS 11 times in a 96-well cell culture plate sown with Madin-Darby canine kidney (MDCK) cells. Eight duplicate dilution series were prepared and assayed for each Ag NP/Ch sample. Samples were incubated at 37°C and 5% CO2 for 1 h to allow viral infection of the MDCK cells. MDCK cells were maintained by adding 50 μL DMEM (with the addition of 0.4% of BSA and 5 ppm of trypsin) to each well immediately following infection and again 5 days post-infection. Seven days post-infection, the living cells were fixed with methanol and stained with 5% Giemsa stain solution. The TCID50 of the sample solution was calculated from the number of infected wells using the Reed-Muench method [26, 27]. The antiviral activity of the Ag NP/Ch composite was estimated as the TCID50 ratio of the Ag NP/Ch-treated supernatant to the control (untreated) viral suspension.