Nano silver – zanamivir is capable of destroying drug-resistant H1N1 influenza A virus

As one of the most effective drugs for the treatment of influenza virus infections, the clinical application of zanamivir is limited in the presence of resistant influenza viruses. It is important to produce new drugs to combat the infection of the flu virus. In recent years, nano silver (AgNPs) have gained wide attention in the area of ​​antiviral. In this study, we demonstrated the surface coating of AgNPs using zanamivir (ZNV) to produce a compound with antiviral properties. In summary, the modified AgNPs (Ag @ ZNV) of zanamivir inhibited the neuraminidase activity of the H1N1 virus. Furthermore, the cytological effect showed that Ag @ ZNV significantly counteracted the apoptosis caused by the H1N1 virus on MDCK cells, involving DNA fragmentation, chromosomal concentration and caspase activation. -3. Ag @ ZNV effectively reduces the accumulation of reactive oxygen species (ROS) caused by the H1N1 virus and activates both the p38 and p53 signaling pathways. Taken together, our research indicates that Ag @ ZNV is a promising new drug against the infection of the H1N1 flu virus.

(Copyright NanoCMM Technology)

1. INTRODUCE

Influenza viruses that pose a great threat to the community are spreading across the global health system. There are three types of influenza virus A, B and C, that cause illness in people. However, influenza A infections, belonging to H1N1, account for the majority of hospitalizations. Pandemic H1N1 flu broke out in 2009 in Mexico and the United States, spreading rapidly among people and leading to more than 280,000 deaths worldwide, developing into the first human pandemic in 40 years. A is classified according to two main types of surface proteins, hemagglutinin (HA) and neuraminidase (NA). NA plays an important role in viral replication. It cleans sialic acid from HA, NA and cell surfaces, and helps progeny viruses separate from host cells. Studies have demonstrated that 2009 A (H1N1) virus is the result of a series of reclassification events involving swine, poultry, and human strains of influenza A virus, with an originating NA gene. The A / H1N1 strain resembles a Eurasian pig resembling a bird. An NA inhibitor is a type of antiviral compound that fights influenza viruses, targeting the highly conserved NA enzyme site of glycoprotein. Zanamivir and oseltamivir are often used as inhibitors of NA binding in the active sac of NA and to disrupt enzyme reactions. They are currently approved options in the US for immediate intervention against influenza virus infection. However, resistance to NA inhibitors typically arises due to amino acid mutations in the active NA sites, either in framework radicals or in catalytic residues. NA-resistant influenza viruses can be rapidly selected in treated patients or sometimes emerge without a specific relationship with the treatments. New methods of treatment and prevention against influenza virus have attracted great attention of scientists.

Based on special physical and chemical properties, nanomaterials have become new materials in medicine, food industry and environmental technology. Among them, silver nanoparticles (AgNP) have attracted wide attention in the biomedical field. In addition to its antibacterial, antifungal and anticancer activities, AgNPs are being explored as antiviral agents in recent years. Cumulative data have reported that AgNPs suppressed replication of respiratory syncytial virus, dengue 2 virus, rift valley fever virus, yellow bean mosaic virus, herpes simplex virus, parainfluenza virus. 3 and influenza virus H3N2. Therefore, we are looking to verify that zanamivir (Ag @ ZNV) modified AgNPs have outstanding activity to limit H1N1 virus infection. Reactive oxygen species (ROS) play an essential role in the pathophysiology of our bodies. Viral infection affects the redox balance, causing overproduction of ROS and leading to host cell apoptosis. Previous research has described that nano silver AgNPs stimulate ROS production to kill cancer cells, but little is known about the relationship and mechanism between two viral infections. In this study, we are aiming to explore how the modified zanamivir silver nanoparticles counteract MDCK apoptosis caused by the H1N1 influenza virus.

2. Experiment

2.1 Materials

The Madin Darby dog ​​kidney cell line (MDCK) used to study flu was purchased from the American Type Culture Collection (CCL-34TM). Influenza A / Hubei / 74/2009 (H1N1) was donated by the Wuhan Institute of Viruses, Chinese Academy of Sciences. Dulbecco’s cow serum (FBS) and modified eagle medium (DMEM) from Gibco were used for cell culture. l -1-Tosylamido-2-phenylethyl chloromethyl ketone (TPCK), zanamivir, vitamin C (VC), silver nitrate (AgNO 3), thiazolyl blue tetrazolium bromide (MTT), 4 ′, 6-diamidino-2-phenyindole (DAPI ), coumarin-6,2 ′, 7′-dichlorofluorescein diacetate (DCF-DA) and propidium iodide (PI) were all purchased from Sigma. The Lyso Tracker dark red molecular probe was obtained from Invitrogen. The deoxynucleotidyl transferase-mediated apoptosis test kit (TUNEL), neuraminidase detector, BCA protein test kit and caspase-3 activity test kit were acquired from the Beyotime Institute of Biotechnology. (Shanghai, China). p38, p-p38, p53, p-p53, PARP, caspase-3 and β-actin monoclonal antibodies are provided from Cell Signaling Technology. Milli-Q water for all experiments in this study was collected from milipore water purifiers.

2.2 Synthesis of nano silver AgNPs and Ag @ ZNV

AgNPs are prepared as (Li et al.): In a short time, 0.1 ml of freshly prepared 400 μg ml 1 VC solution is added drop by drop to 4 ml of new 400 μg ml 1 AgNO3 solution, Then stir from continuously for 30 minutes at room temperature. Then, 32 μl zanamivir 100 nM was added. Excess VC, AgNO 3 and zanamivir are removed by dialysis in 24 hours. Then, the concentration of Ag @ ZNV was detected by ICP-AES (inductive combined plasma atomic emission spectroscopy). Ag @ ZNV nanoparticle solution was ultrasonic in a water bath before going through a pore size filter of 0.22 μm. The final concentrations of nano silver AgNPs were 2.5 μg ml −1 and 0.8 nM zanamivir and the sample was stored at 4 ° C.

2.3 Characteristics of Ag @ ZNV

The morphology of Ag @ ZNV nanoparticles was characterized by transmission electron microscopy (TEM). Following this procedure, the samples are prepared by dispersing the powder particles onto a porous carbon film on a copper mesh. The micro-images were collected using Hitachi (H-7650) for the TEM operating at an accelerating voltage of 80 kV. Disperse X-rays (EDX) were performed using the EX-250 (Horiba) system to analyze the elemental composition of Ag @ ZNV. The size distribution and zeta potential of Ag @ ZNV were determined by Malvern Zetasizer Nano ZS particle analyzer.

MDCK cells were maintained in DMEM medium with 10% FBS, 100 U ml -1 penicillin and 50 U ml -1 streptomycin at 37 ° C in an incubator of 5% CO 2. H1N1 virus infection has been performed and 50% of the dose of tissue culture virus infection (TCID50) is counted as (Amatore et al.). Briefly, cells were seeded in 96-well culture plates at a density of 4 × 10 4 cells per well for 24 hours. The cells were then washed twice with phosphate buffered saline (PBS) and infected with H1N1 in DMEM without FBS. For the 50% tissue culture infectious dose (TCID50) test, the virus is diluted in 10 times a gradient of 10-1 to 10-10 in this step. After adsorption for 2 hours at 37 ° C, the supernatant was removed and cells cultured with DMEM containing 2% FBS and 2 μg ml -1 TPCK trypsin were treated after two washing. to remove the virus from getting stuck. Cell effect (CPE) was observed and TCID 50 was calculated using Reed-Muench method. All H1N1 viruses used in this study were at 100 TCID 50 ml.

2.5 Determination of cell viability

The cytotoxicity of Ag @ ZNV nanoparticles was determined by MTT assay. Briefly, MDCK cells were planted in 96-well culture plates and infected with H1N1. The cells were then treated to the indicated concentration of zanamivir with or without AgNPs. After 72 hours, 20 μl of 5 mg ml -1 MTT solution was added to each well and incubated at 37 ° C. The well solution was drawn out after 5 hours and DMSO was added 150 μl per well. The absorbance at 570 nm was recorded and the viability of MDCK cells for Ag @ ZNV was measured with a microfiber spectrophotometer.

2.6 Intracellular localization of Ag @ ZNV

The intracellular localization of 6-coumarin Ag @ ZNV in MDCK cells detected by Lyso Tracker Red as a lysosome marker. Cells were cultured in 35 mm cell culture plates with 80 nM Lyso Tracker for 120 min. When 70% of the confluence is stained, 1 mg ml of -1 DAPI is added for 30 minutes. Then, 6-coumarin labeled Ag @ ZNV and Lyso Tracker Red were incubated at 37 ° C for different durations. Finally, the cells were washed with PBS three times and analyzed with a fluorescence microscope.

2.7 Test for neuraminidase inhibition

H1N1 was mixed with or without Ag @ ZNV for 2 hours before incubation at 37 ° C. Then, the neuraminidase (NA) activity of the influenza virus was determined using a head-measured NA detector. read fluorescent microflora with excitation wavelength 360 nm and emission wavelength 460 nm as (Wang et al.).

2.8 Flow cytology analysis

The effect of Ag @ ZNV on cell cycle distribution was measured by flow cytometry (Song et al.). H1N1-infected MDCK cells and treated with or without Ag @ ZNV were harvested and fixed with 70% ethanol at -20 ° C overnight. After PI staining, DNA content was quantified and cell cycle analysis by MultiCycle software. The cell ratios in the G0 / G1, S, and G2 / M phases are represented by DNA plots.

2.9 TUNEL and DAPI staining test

DNA fragmentation and nuclear concentration were examined by fluorescent staining with apoptosis TUNEL and DAPI detection kits such as (Li et al.), After fixation with 3.7% formaldehyde and impregnated with 0.1% Triton X-100 in PBS, MDCK cells were marked with a TUNEL reaction mixture for 60 min and with 1 μg ml -1 DAPI for 15 minutes at 37 ° C. Cells were observed under fluorescence microscope.

2.10 Activity Caspase-3

The caspase-3 activity was monitored as instructed by the caspase-3 activity assay, the cell protein was extracted after concentration determination using the BCA protein assay kit. Protein mixed with specific caspase-3 substrate Ac-DEVD-pNA was added to a 96-well plate at 37 ° C for 1 hour. The absorbance was recorded at 405 nm.

2.11 Measurement of reactive oxygen species (ROS) generation

ROS accumulation inhibited by Ag @ ZNV-treated MDCK cells after H1N1 infection was determined by cell staining with a DCF assay such as (Yang et al.). Cells were collected and incubated with 10 μM DCF-DA in PBS at 37 ° C for 30 minutes. ROS generation was determined by fluorescence intensity with excitation and emission wavelengths at 488 nm / 525 nm.

2.12 Western blot analysis

Expression of proteins relative to the signaling pathways identified by the Western blot is (Ren et al.). MDCK cells treated with or without Ag @ ZNV for 24 hours after H1N1 infection were lysed with RIPA buffer and total protein was obtained. Protein concentration was quantified using the BCA assay kit. The protein equivalent was then separated on sodium dodecyl sulfate (SDS) –polyacrylamide gel, then transferred to the PVDF membrane. After being intercepted in 5% skim milk, the films were incubated with specific primary antibodies and a secondary antibody bound to horseradish peroxidase (HRP). The bolts are developed with enhanced chemical luminescent reagent (ECL).

2.13 Statistical analysis

All data were expressed as mean ± SD. GraphPad Prism 5.0 software was used for data analysis. Data were analyzed using the bilateral Student t-test to assess the differences between the two groups or one-way analysis of variance (ANOVA) for multi-group comparison. The difference is considered statistically significant with P <0.05 (*) or P <0.01 (**).

3 Results and discussion

3.1 Preparation and characterization of nano silver – zanamivir Ag @ ZNV

The morphology of silver nanoparticles AgNPs and Ag @ ZNV was firstly characterized by TEM. As shown in Figure 1A, Ag @ ZNV presents spherical and dispersed single particles with high uniformity. Besides, when the average particle size distribution in Figure 1C, Ag @ ZNV was effectively reduced from 3 nm to 2 nm compared with nano silver AgNPs. The small size of Ag @ ZNV contributes to a highly stable nanostructured which makes it easy to penetrate the cell membrane. In Figure 1B, the zeta potential of AgNPs alone is -15.2 mV and increases to -24.7 mV after zanamivir loading, which explains the higher stability of Ag @ ZNV. Furthermore, the size distribution of Ag @ ZNV in Figure 1D shows that the mean size of Ag @ ZNV ranges from 2 nm to 2.3 nm and remains stable for 30 days. Meanwhile, we also detected the size distribution of nano-silver AgNPs in Figure 1E. The size of AgNPs ranged from 2.0 nm to 3.0 nm and remained stable for 28 days. In Figure 1F, an elemental composition analysis using EDX shows the presence of strong signals from the Ag (59.5%) atoms, along with the C (23%), N (8) atomic signal. , 6%) and O (8,9%) which is from ZNV. No apparent peaks were observed for other elements or impurities. The presence of N and O atoms shows that ZNV has self-assembled on the surface of AgNPs. Based on EDX analysis results, representative chemical formula of Ag @ ZNV is calculated as (Ag 7 ZNV) n. Ag / ZNV nanoparticle ratios have been reported previously.

Figure 1 Characteristics of nano silver AgNPs and Ag @ ZNV. (A) TEM image of nano silver AgNPs and Ag @ ZNV (graduated in 2 nm divisions). (B) The Zeta potential of AgNPs and Ag @ ZNV. (C) Size distribution of nano silver AgNPs and Ag @ ZNV. (D) and (E) Time-based size distribution of Ag @ ZNV and AgNPs in aqueous solution using the nano-zs zetasizer particle analyzer. (F) Ag @ ZNV EDX analysis. Bars with different characters are statistically different at P <0.05 (*).

3.2 In vitro antiviral activity of nano silver – zanamivir Ag @ ZNV

The antiviral activity of nano silver AgNPs, ZNV and Ag @ ZNV was investigated by MTT assay. Cell viability of nano silver and ZNV is shown in Figures 2A and B, AgNPs and ZNV reduce cell viability in a dose-dependent manner. As shown in Figure 2C, the concentrations of Ag @ ZNV, AgNPs and ZNV are 2.5 μg ml −1, 2.5 μg ml −1 and 0.8 nM. The viability of MDCK cells infected with the H1N1 virus is 36.32%. Cells treated with zanamivir and nano silver AgNPs were 63.23% and 61.87%, respectively. However, Ag @ ZNV-treated cells reached 82.26%. Meanwhile, a synergy assessment has been done by previous reports. The data is explained by calculating the fractional inhibitory concentration index (FIC) as follows: FIC = (MIC drug A / MIC combination drug A) + (MIC combination drug B / MIC drug combination B). In this study, FIC is basically understood as follows: FIC below 0.5, synergy; FIC from 0.5 to 2.0, indifferent, FIC higher than 2.0, antagonistic. MIC drug One combination is the concentration found in Ag @ ZNV of Ag. Combination MIC drug B is the concentration present in Ag @ ZNV of ZNV. Particularly drugs MIC A and B correspond to AgNPs and “free” ZNV. The combined drug concentration detected by ICP, synergy was assessed by in vitro calculation The fractional inhibitory concentration index was as follows: Combination MIC A drug is the concentration in Ag @ ZNV of Ag (0 , 5 μg ml −1). Combination MIC drug B is the concentration present in Ag @ ZNV of ZNV (0.2 nM). Particularly, the MIC A drug corresponds to free AgNPs (2.5 μg ml -1). Particularly, the MIC B drug corresponds to free ZNV (0.8 nM). FIC = (MIC combination drug A / MIC drug combination A) + (MIC combination drug B / MIC drug combination B) = 0.5 μg ml −1 / 2.5 μg ml −1 + 0.2 nM / 0.8 nM = 0.45. FIC less than 0.5 indicates synergy. Results showed that Ag @ ZNV has enhanced antiviral activity. As shown in Figure 2D, the MDCK cells infected with H1N1 show cytoplasmic shrinkage, reduced number of cells and loss of cell-to-cell communication. Cell effects were reduced when treated with Ag @ ZNV. Cell viability and imaging showed that Ag @ ZNV effectively suppressed the replication of the H1N1 virus.

Figure 2 Effect of Ag @ ZNV in H1N1-infected MDCK cells. (A – C) Antiviral activity of nano silver AgNPs, ZNV and Ag @ ZNV was measured by MTT assay. (D) Morphological changes of MDCK cells observed with phase contrast microscopy (magnification, × 40). The concentrations of Ag @ ZNV, AgNPs and ZNV were 2.5 μg ml −1, 2.5 μg ml −1 and 0.8 nM. Bars with different characters are statistically different at P <0.05 (*) or P <0.01 (**).

3.3 Intracellular localization of nano silver – zanamivir Ag @ ZNV

Intracellular production against nanoparticles has important implications for the transmission of drug-loaded nanosystems. In this study, the positioning of coumarin-6 Ag @ ZNV in MDCK cells was monitored by concomitant staining. As Figure 3 suggests, the yellow area covered with fluorescent green and red shows the localization of Ag @ ZNV and the lysosome. The binding accumulates on the MDCK cell membrane, after which Ag @ ZNV exits the lysosome and is released into the cytoplasm after 60 minutes. Gradually enhanced fluorescence showed time-dependent cell penetration over Ag @ ZNV’s antiviral activity.

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Figure 3 Intracellular trade of Ag @ ZNV in MDCK cells. Ag @ ZNV-treated cells were loaded with coumarin-6 for more than 120 minutes, and stained with Lyso Tracker for lysosomes and DAPI for the nucleus. The cells were observed under fluorescence microscope at different times (magnification, × 100).

3.4 Neuraminidase Activity

Neuraminidase (NA) activity was used to estimate the viral effect of infected cells. Zanamivir is commonly used as an NA inhibitor for clinical treatment against influenza virus infection. In this study, we compared the effects of nano silver AgNPs, zanamivir and Ag @ ZNV on the activity of H1N1 NA. Data in Figure 4 show that the H1N1 virus treated with Ag @ ZNV had lower NA activity (42.68%) compared to that treated with zanamivir (72.42%) or AgNPs (58.37%). , showed that these compounds were more effective in inhibiting the NA Activity of the H1N1 virus.

Figure 4 Inhibition of H1N1 NA activity by Ag @ ZNV. NA inhibition tests are performed by quantifying fluorescence using a micro-plate reader. Bars with different characters are statistically different at P <0.05 (*) or P <0.01 (**). The concentrations of Ag @ ZNV, AgNPs and ZNV were 2.5 μg ml −1, 2.5 μg ml −1 and 0.8 nM.

3.5 Inhibition of apoptosis of nano silver – zanamivir Ag @ ZNV

Flow cytometric analysis and TUNEL – DAPI assay were performed to evaluate the antiviral mechanism of Ag @ ZNV. As shown in Figure 5A, the G1 apoptotic cell population was significantly increased to 29.4% in the DNA plot. However, Ag @ ZNV significantly reduced it to 17.3%. DNA fragmentation is considered an important biochemical marker in cellular apoptosis. In Figure 5B, the MDCK cells infected with the H1N1 virus exhibit typical apoptotic traits with nuclear condensation and DNA fragmentation. However, DNA fragmentation and nuclear morphological changes caused by the H1N1 virus have been effectively countered when treated with Ag @ ZNV. These data indicate that MDCK cells are protected by Ag @ ZNV from apoptosis caused by the H1N1 virus.

igure 5 Ag @ ZNV inhibited apoptosis in H1N1-infected MDCK cells. (A) DNA content was quantified by clonal cytology of apoptosis and changes in cell cycle distribution. (B) DNA fragmentation and nuclear condensation reduced by Ag @ ZNV were detected by TUNEL-DAPI co-staining assay. MDCK cells were processed with or without Ag @ ZNV after infection with the H1N1 virus (magnification, × 100). The concentrations of Ag @ ZNV, AgNPs and ZNV were 2.5 μg ml −1, 2.5 μg ml −1 and 0.8 nM.

3.6 Inhibition of caspase-3 activation by nano silver – zanamivir Ag @ ZNV

Caspase-3 plays an important role in apoptosis, as it is responsible for protein breakdown of many important proteins, such as poly-ADP-ribose polymerase (PARP). To investigate the activation of caspase-3 and PARP in the inhibition of H1N1 virus by Ag @ ZNV, we performed a fluorometric and Western blot measurement assay. As revealed in Figure 6A, the caspase-3 activity of the MDCK cells infected with the H1N1 virus reached 326%, while the Ag @ ZNV group decreased significantly to 189%. In Figure 6B, the expression levels of caspase-3 and PARP in Ag @ ZNV-treated cells were significantly reduced compared to the untreated gain-regulating cell group following H1N1 infection. The results demonstrate that Ag @ ZNV counteracts the activity of the H1N1 virus through inhibition of caspase-3-mediated cell death.

Figure 6 Inhibition of PARP and caspase-3 by Ag @ ZNV in H1N1-infected MDCK cells. (A) Cell was treated with Ag @ ZNV and caspase-3 activity was detected. (B) Expression of PARP and caspase-3 levels by Western blot, β-actin is used as load control. Bars with different characters are statistically different at P <0.01 (**). The concentrations of Ag @ ZNV, AgNPs and ZNV were 2.5 μg ml −1, 2.5 μg ml −1 and 0.8 nM.

3.7 Resistance to ROS generation of nano silver – zanamivir Ag @ ZNV

Including influenza viruses, infection with certain viruses develops concurrently with the production of excess reactive oxygen species (ROS) involved in cell apoptosis. To find out if Ag @ ZNV nanoparticles limit ROS-mediated apoptosis caused by the H1N1 virus or not. We monitored intracellular ROS levels by examining the fluorescence intensity of dichlorofluorescein (DCF). As shown in Figure 7A, the ROS generation of MDCK cells increased significantly after infection with the H1N1 virus without treatment. Zanamivir or nano silver AgNPs alone inhibited generation slightly when Ag @ ZNV was significantly reduced. From the image in Figure 7B, we observed a strong fluorescence intensity of DCF in MDCK cells infected with the H1N1 virus. Fluorescence in cells treated with zanamivir alone or nano silver AgNPs became weak and dark after Ag @ ZNV treatment. These results showed that Ag @ ZNV inhibited ROS generation caused by H1N1 virus infection.

Figure 7 Restriction of ROS generation by Ag @ ZNV in H1N1-infected MDCK cells. (A) The level of ROS was detected by the intensity of fluorescence DCF. (B) Cells were incubated with 10 μM DCFH-DA for 30 minutes then treated with Ag @ ZNV (magnification, × 100). Bars with different characters are statistically different at P <0.01 (**). The concentrations of Ag @ ZNV, AgNPs and ZNV were 2.5 μg ml −1, 2.5 μg ml −1 and 0.8 nM.

3.8 ROS-mediated signal path

Overproduction of ROS induces DNA damage and further leads to cellular apoptosis through regulation of signaling pathways such as p38 and p53. Due to inhibition of ROS generation caused by H1N1 virus, we further examined the expression of proteins after Ag @ ZNV treatment by Western blot method. As shown in Figure 8B, H1N1 infection leads to the regulation of expression of p38, p-p38, p53, and p-p53. In contrast, they were partially regulated when the cells were treated with Ag @ ZNV. These results reflect that the ROS-mediated pathways of p38 MAPK and p53 signaling inhibition of Ag @ ZNV-induced apoptosis.