Materials. Gold (III) chloride trihydrate, silver (I) nitrate, tannic acid, sodium citrate, hydroquinone, polyvinylpyrrolidone (PVP), phosphotungstic acid hydrate (PTA), L-cysteine, monosodium phosphate, disodium phosphate and aniline were purchased from Sigma-Aldrich (St. Louis, USA). Ethanol and N-Methyl-2-pyrrolidone (NMP) was obtained from Duksan Pure Chemicals Co., Ltd. (Ansan, South Korea). Dulbecco’s Modified Eagle Medium (DMEM), Dulbecco’s phosphate buffered saline (DPBS, pH 7.4), and fetal bovine serum (FBS) were purchased from Gibco Laboratories (Gaithersburg, USA). Ellman’s reagent (5,5’-dithiobis(2-nitrobenzoic acid), DTNB) was purchased from Thermo Scientific (Waltham, USA). The Ez-cytox cell viability assay kit was purchased from Daeil Lab Service (Seoul, Korea). The RNeasy® mini kit and the QIAamp® viral RNA mini kit were purchased from QIAGEN (Hilden, Germany). A/California/04/2009 (H1N1), A/canine/Korea/GCVP01/2007(H3N2) and A/wild bird feces/Korea/KU-VI135874/2012(H9N2) were prepared.
Synthesis of metallic nanoparticle. PoGNP was prepared following the surfactant-free emulsion method 29. Aniline monomer (0.182 mL, 2 mmol) was dispersed in 20 mL of deionized water. Then, 1 mL of gold (III) chloride solution (0.1 M) was added to construct a polyaniline (PANI)-Au hybrid nanoparticle under emulsification for 30 min with ultrasonication. The produced PANI-Au nanoparticle was washed 3× by centrifugation. PANI was then removed from the PANI-Au composite by dissolving the composite in NMP. Finally, purified PoGNP was redispersed in DPBS.
AgNP was synthesized following the citrate reduction method 35. Tannic acid (42 mg, 0.025 mmol) and sodium citrate (150 mg, 0.5 mmol) were dissolved in 100 mL of deionized water, and the solution was heated to 95 °C with vigorous stirring. Then 1 mL of silver (I) nitrate solution (25 mM) was quickly injected and reacted for 1 h at 95 °C.
Non-porous sGNP was obtained following the seed-mediated growth method 36. First, gold nanoparticle (GNP) seed was synthesized using the citrate reduction method. Briefly, 100 mL of sodium citrate solution (5 mM) was heated to 95 °C with vigorous stirring. Thereafter, 1 mL of gold (III) chloride solution (0.1 M) was injected into the sodium citrate solution. After 30 min, a wine-colored GNP seed solution was obtained. Then, 100 μL of gold (III) chloride solution (25 mM) was added to GNP seed solution (0.8 μM) to give a final volume of 10 mL. Subsequently, 22 μL of sodium citrate solution (30 mM) was injected into the seed solution mixture, following the addition of 100 μL of hydroquinone solution (30 mM). The mixture was kept at room temperature (RT) for 24 h. PVP solution (2 μg/mL) was added to prevent the aggregation of nanoparticles, followed by centrifugation at 10,000 rpm for 10 min.
Size distribution and zeta potential of each nanoparticle was collected by ELS-Z2000 (Otsuka electronics Co., Ltd., Osaka), and concentration of particles were analyzed through an inductively-coupled plasma optical emission spectrometer (ICP-OES, Perkin Elmer, Waltham).
Binding efficiency between nanoparticle and influenza virus. 300 μL aliquots of H3N2 virus suspension were exposed to 2 × 10-1 mg/mL to 6.25 × 10-3 mg/mL of nanoparticle (PoGNP and sGNP) solution and incubated at RT for 1 h. Subsequently, the incubated samples were centrifuged at 8000 rpm for 10 min to separate the virus-bound nanoparticles from the supernatant solutions. The supernatants were transferred to other test tubes while the precipitates were dispersed in DPBS. Thereafter, real-time RT-PCR analysis was conducted on the prepared solutions to determine the binding efficiency of the nanoparticles.
Cytotoxicity assay for antiviral activities. The water-soluble tetrazolium salt-1 (WST-1) assay was performed to determine the cytotoxicity of the nanoparticle-treated IAVs on MDCK cells using the Ez-cytox cell viability assay kit (Daeil Lab Service). Twenty-four hours prior to the infection experiment, 2.0 × 105 of Madin-Darby canine kidney (MDCK) cells were seeded into 96-well plates with 100 μL of 10% fetal bovine serum (FBS) containing DMEM.
In order to evaluate the antiviral activity of the nanoparticles, various nanoparticle concentrations (3.125 × 10-2 mg/mL to 2 × 10-1 mg/mL of PoGNP, sGNP, and AgNP) with two-fold serial dilutions and 300 μL aliquots of IAVs (H1N1, H3N2, and H9N2) were prepared. 75 μL of each nanoparticle solution was added to the virus aliquots, for a final nanoparticle concentration of 6.25 × 10-3 mg/mL to 2 × 10-1 mg/mL. Samples were incubated at RT for 10 min and 60 min, with vortex mixing every 10 min. In the meantime, the MDCK cells were washed 3x with 100 μL of PBS. Then 100 μL aliquots of nanoparticle-treated IAV samples were added to each well and incubated for an additional 1 h. The nanoparticle-treated IAVs were then removed, cells were rinsed once with PBS, and then incubated for an additional 72 h with 200 μL of fresh DMEM.
To determine cell viability, 20 μL of Ez-cytox was added to the incubated cells and their optical density (OD) at 450 nm was measured after 2 h using the UV-Vis SpectraMax 190 Microplate Reader (Molecular Devices, San Jose, USA).
For intracellular viral RNA quantification, a 24 h MDCK cell incubation period was followed by the RNeasy® mini kit protocol (QIAGEN) to prepare samples for real-time RT-PCR analysis.
Transmission electron microscopy (TEM) analysis. 30 μL of the nanoparticle and virus suspension mixtures, as previously described in Section 2.4, were incubated at RT for 10 and 60 min and then transferred by pipette to carbon-coated copper TEM grids (400 mesh). After 10 min at RT, the liquid was blotted with filter paper and a droplet of 3 wt% PTA solution was loaded onto the grid for negative staining. The excess PTA solution was blotted with filter paper after 10–40 sec, followed by washing 2x with a droplet of deionized water, and the grid was dried for 4 h prior to analysis. TEM images were obtained with a JEM-1011 transmission electron microscope (JEOL Ltd., Tokyo, Japan).
Real-time RT-PCR. A/canine/Korea/GCVP01/2007(H3N2) virus was serially diluted 10-fold (108.25–102.25 EID50/mL). Viral genomic RNA from each dilution was extracted using the QIAamp® viral RNA mini kit (QIAGEN), according to the manufacturer's instructions. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed to quantify the viral load in the samples using the QuantiTect Probe RT-PCR Kit (QIAGEN) and the LightCycler 96 system (Roche, Basel, Switzerland). The 50 μL final reaction volume contained 0.4 μM of matrix (M) gene-specific primer (forward: GACCRATCCTGTCACCTCTGAC; reverse: AGGGC ATTYTGGACAAAKC GTCTA) and 0.2 μM of specific probe (FAM-TGCAGTCCTCGCTCACTGGGCACG-BHQ-1). The thermal cycling conditions were: reverse transcription at 50 °C for 30 min, initial denaturation at 95 °C for 5 min, followed by 40 cycles of [94 °C for 15 s, 60 °C for 60 s] according to the manufacturer's protocol (Roche). The primers targeted the region of the M gene highly conserved across all IAV strains, as formulated by the CDC (Biosearch Technologies, Inc. http://www. who.int/csr/resources/publications/swineflu/ CDCrealtimeRTPCRprotocol_20090428.pdf). A standard curve was generated with EID values and copy numbers. The amount of viral RNA in nanoparticle-treated samples (in logEID50/mL) was calculated from the standard curve generated by real-time RT-PCR data using canine H3N2 virus (Fig. S4).
Ellman’s assay. 10 mM of DTNB in 10% ethanol solution and 0.1 M sodium phosphate buffer pH 7.4 was prepared. Then 50 μL of 0.2 mg/mL PoGNP, sGNP and AgNP aliquot was mixed with 50 μL of H3N2 virus and left for 60 min at RT. 100 μL of 1 mM DTNB solution was set as a negative control for the signal, and 100 μL of H3N2 virus suspension reacted with 10 mM TCEP for 60 min was prepared as a positive control. Subsequently, 20 μL of 10 mM DTNB solution was added to the mixture. Centrifugation of reactant was followed to remove nanoparticles in the mixture. Next, absorbance at 412 nm (A412) was observed using the UV-Vis SpectraMax 190 Microplate Reader (Molecular Devices, San Jose, USA).