Preparation and characterization of nanoparticles
The powdered zinc oxide nanoparticles were purchased from US Research Nanomaterials (USA; Product Number: US3590). Nanoparticles were suspended in Dulbecco’s Modified Eagle’s medium (DMEM) (Invitrogen, USA) and the suspension were subjected to sonication to prevent agglomeration and make different concentrations. Oseltamivir purchased from Sigma-Aldrich (St. Louis, MO, USA) were dissolved in DMEM and used as a standard drug against influenza at different concentrations. Polyethylene glycol (PEG) 6000 was also purchased from Sigma-Aldrich. PEGylated ZnO-NPs were synthesized by mechanical method, as described in detail previously [16]. Inductively coupled plasma mass spectrometry (ICP-MS), X-ray diffraction analysis (XRD), Transmission Electron Microscopy (TEM), and Field Emission Scanning Electron Microscope (FE-SEM) were used for characterization of nanoparticles. Thermogravimetric analysis (TGA) was also performed to demonstrate the presence of PEG on the surface of ZnO nanoparticles [16].
Cell culture and virus propagation
Madin-Darby canine kidney (MDCK)-SIAT1 cells were a gift from the Razi Vaccine and Serum Research Institute (Karaj, Iran). The cells were grown at 37 °C in 5% CO2 in DMEM, supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, USA), 1 mM sodium pyruvate (Merck, Germany), 2 mM L-glutamine (Merck, Germany), and 100 U/ml penicillin and 100 μg/ml streptomycin sulfate (Sigma-Aldrich, USA).
Influenza A/Puerto Rico/8/34 (H1N1; PR8) was also obtained from the Razi Vaccine and Serum Research Institute, and propagated in MDCK-SIAT1 cells. For virus-stock preparation, MDCK-SIAT1 cell monolayer in 25-cm2 flask (SPL Life Science, South Korea) was washed three times with phosphate-buffered saline (PBS, Bio-Idea, Iran), and the cells were infected with the virus at a multiplicity of infection (MOI) of 0.1 for 1 h at 35 °C. Afterwards, the virus inoculum was removed and the cells were overlaid with infection medium containing serum-free DMEM, 2 μg/ml trypsin-TPCK (Merck, Germany), 25 mM HEPES buffer (Sigma-Aldrich, USA), and 0.14% of bovine serum albumin (BSA; Sigma-Aldrich, USA), and the flask was then incubated at 35 °C for an additional 48 h. The virus-containing supernatants were harvested at 48 h post infection, clarified by centrifugation at 2500 rpm for 10 min at 4°, and filtered by sterile syringe filter 0.22 μm (Millipore, Ireland). The virus was then aliquoted into sterile cryovials and stored frozen at − 80 °C until use. Virus was titrated using the tissue culture infectious dose 50 (TCID50) method according to the Reed and Muench formula [17], and was used for the next in vitro experiments at the titer of 100 TCID50/mL.
Determination of cell viability
The cytotoxicity of nanoparticles was assessed by MTT assay. Briefly, MDCK-SIAT1 cells at a density of 1 × 105 cells/mL were seeded in a flat-bottomed 96-well microtiter plate (SPL Life Science, South Korea), and were incubated for 24 h at 37 °C and 5% CO2. A range of concentrations from 25 to 225 μg/ml of nanoparticles was prepared using the cell culture medium and was added to the plate in triplicate. After 48 h, the treatments were removed, and 10 μL of MTT reagent and 100 μL RPMI (Bio-Idea, Iran) were added to each well and incubated for a further 4 h. The medium was then removed and 50 μL of DMSO solution was added to the wells. Finally, the plate was read at 550 nm by a microplate reader (Hiperion MPR 4+, Germany).
Assessment of antiviral activities
Virucidal activity
To evaluate direct effects of ZnO and ZnO-PEG nanoparticles on H1N1 influenza particles, equal volumes of the viral suspensions (100 TCID50/ml) and nanoparticles suspensions in non-toxic concentration ranges were mixed and incubated at 37 °C for 4 h in a humidified 5% CO2 atmosphere. The mixture (100 μL) was then added in triplicated wells of the confluent monolayer of MDCK-SIAT1 cells (2 × 104 cells/well) in a flat-bottomed 96-well microtiter plate and further incubated for 1 h at 35 °C. The virus control (infected but untreated) and cell control (uninfected untreated cells) were kept in each plate prepared throughout the experiment. After 1 h incubation, the mixture was discarded, and the cells were washed three times with PBS to remove non-absorbed viruses and overlaid with infection medium. The plate was then incubated for 48 h at 35 °C in a humidified 5% CO2 atmosphere.
Pre-exposure antiviral activity
The confluent monolayer of MDCK-SIAT1 cells (2 × 104 cells/well) in a flat-bottomed 96-well microtiter plate were pre-incubated with different concentrations of ZnO and ZnO-PEG nanoparticles in non-toxic concentration ranges in triplicates for 3 h at 37 °C. The media containing nanoparticles was discarded from the wells, and the cells were washed three times with PBS and then incubated for 1 h at 35 °C with 100 TCID50/mL virus. Afterwards, the virus inocula were removed from the wells, and the cells were washed three times with PBS, and were then overlaid with infection medium. The plate was further incubated for 48 h at 35 °C in a humidified 5% CO2 atmosphere. The virus and cell controls were kept as described above.
Cell co-treatment assay
The co-treatment assay was performed to evaluate the functions of nanoparticles in inhibiting viral binding. MDCK-SIAT1 cells were grown in a flat-bottomed 96-well microtiter plate at the density of 2 × 104 cells/well. The media was removed from all wells and 100 μL of nanoparticles suspensions at their non-toxic concentrations and 100 μL of 100 TCID50/mL viral suspensions were added simultaneously to the cells in triplicated and incubated for 1 h at 35 °C. The virus and cell controls were also included in this assay. Following 1 h incubation, the solution on the cells was discarded and the cells were washed three times with PBS, and were overlaid with infection medium. The plate was incubated for an additional 48 h at 35 °C with 5% CO2.
Post-exposure antiviral activity
The confluent monolayer of MDCK-SIAT1 cells (2 × 104 cells/well) in all wells of a flat-bottomed 96-well microtiter plate were incubated with 100 μL of 100 TCID50/mL H1N1 virus suspensions for 1 h at 35 °C in a humidified 5% CO2 incubator. The virus inocula were then discarded from the wells, and the cells were washed three times with PBS for removing unattached viruses. Different non-cytotoxic concentrations of ZnO and ZnO-PEG nanoparticles suspended in infection medium were then added in triplicate to the wells and the plate further incubated for 48 h at 35 °C in a humidified 5% CO2 atmosphere. The virus control (virus + DMEM) and the cell control (uninfected cells in DMEM) were also included in this experiment. This assay was also carried out for oseltamivir and soluble polyethylene glycol.
At the indicated time of all above experiments (at 48 h), the supernatant of each well was harvested and was subjected to TCID50 and quantitative Real-Time PCR assays to determine the amount of total progeny virus.
Quantitative Real-Time PCR assay
To determine influenza viral load, a quantitative system using Real-Time PCR assay was carried out. Total RNA was extracted from the supernatants using the AccuPrep® Viral RNA Extraction Kit (Bioneer, South Korea), based on manufacturer’s protocol. The extracted RNA was then subjected to reverse transcription using the cDNA Synthesis Kit (Roche Diagnostics, Germany), according to manufacturer’s recommendations. Finally, quantitative Real-Time PCR was performed using the Genesig® Advanced kit (PrimerDesign Ltd., United Kingdom), according to manufacturer’s instructions. The kit contains primers and probe designed for detection of all influenza A subtypes. The assay was performed using the Rotor-Gene Q instrument (Qiagen, Germany) under the following conditions: 5 min activation of Taq DNA polymerase at 95 °C, followed by 40 cycles of 10s at 95 °C and 60s at 60 °C.
Indirect immunofluorescence assay (IFA)
MDCK-SIAT1 cells (5.0 × 104) were seeded on sterile glass coverslips (Nunc, Denmark) in a 24-well plate and grown until 80–90% confluence. The media was discarded and the cells were incubated with 200 μL of 100 TCID50/mL H1N1 virus suspensions for 1 h at 35 °C in a humidified 5% CO2 incubator. The virus inocula were then discarded from the wells, and the cells were washed three times with PBS. The maximum non-cytotoxic concentrations of nanoparticles (75 and 200 μg/ml of ZnO-NPs and ZnO-PEG-NPs, respectively) suspended in serum-free DMEM supplemented with trypsin-TPCK, HEPES buffer, and BSA were then added to the wells and the plate was incubated at 35 °C with 5% CO2. The virus and cell controls were also included in this experiment. After 24 h, the cells were fixed with cold acetone (4 °C) for 20 min, and the fixed cells were overlaid with anti-influenza A monoclonal antibody (Chemicon-Millipore, USA), followed by incubation at 37 °C for 45 min. In the next step, the cells were washed three time with PBS, and were then overlaid with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human antibody (Dako, Germany), followed by incubation at 37 °C for 30 min. Afterwards, the cells were washed three time with PBS and coverslips were mounted in slides with glycerol buffer. Ultimately, the cells were visualized under the Olympus BH2-RFCA fluorescence microscope (Tokyo, Japan).
Statistical analysis
Values represent the mean of three independent experiments. The results were tabulated, and differences between means were statistically analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. All analyses were carried out using the GraphPad Prism software, version 7.0 (GraphPad Software, USA), and P values less than 0.05 were taken as statistically significant.