Silver nanoparticles has a strong antiviral effect on COVID-19
The COVID-19 pandemic is spreading unchecked due to the lack of effective antiviral measures. Silver nanoparticles (AgNPs) have been studied for their antiviral properties and are believed to inhibit SARS-CoV-2. Due to the need for an effective agent against SARS-CoV-2, we evaluated the antiviral effects of AgNPs. We evaluated numerous AgNPs of different sizes and concentrations and observed that particles with a diameter of about 10 nm were effective in inhibiting extracellular SARS-CoV-2 at concentrations between 1 and 10 nm. ppm while cytotoxic effects were observed at concentrations of 20 ppm or more. Luciferase-based pseudoviral entry assay showed that AgNPs strongly inhibit viral entry step through disruption of viral integrity.
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1. Introduction
The elemental metal, Silver (Ag) has a broad spectrum antibacterial effect against various bacteria, fungi and viruses. Due to their versatility, silver nanoparticles (AgNPs) have now been found as a biocide for biological surfaces in various forms such as wound dressings, medical devices, deodorant sprays and fabric. Several studies have demonstrated the potent antiviral effects of AgNPs against various human pathogenic viruses such as respiratory syncytial virus (RSV), influenza virus, influenza virus, and influenza virus. Norovirus, hepatitis B virus (HBV) and human immunodeficiency virus (HIV) [ 1]. In addition to these viruses, since Ag has been shown to kill SARS-CoV, we hypothesized the strong ability of nanosilver to inhibit SARS-CoV-2 [ 2 , 3] . To date, no studies have directly demonstrated the effect of AgNPs on SARS-CoV-2. We tested colloidal silver nanoparticles (cAg), simple elemental Ag nanoparticles of different diameters (AgNP n ) and limited polyvinylpyrrolidone 10 nm silver nanoparticles (PVP – AgNP 10 ) against against SARS-CoV-2 to find out the most effective Ag size and concentration that can inhibit SARS-CoV-2. We propose that AgNPs can be used on inanimate and abiotic surfaces to effectively control the ongoing COVID-19 pandemic while taking care not to overdo it.
VeroE6/TMPRSS2 (VeroE6 cells stably expressing transmembrane serine protease TMPRSS2, JCRB #1819) and Calu-3 cell line were cultured in DMEM containing 10% FBS [ 4 ]. SARS-CoV-2 (JPN/TY/WK-521) was obtained from NIID, JAPAN, stored in special samples at -80°C and handled at biosafety level 3 (BSL3). While performing SARS-CoV-2 infection studies, DMEM containing 2% FBS was used.
2.2. Plaque test
Plaque assay was performed SARS-CoV-2 on VeroE6/TMPRSS2 and Calu-3 cell lines as described elsewhere with minor modifications [ 5 ]. 96-well plates were seeded with 5 × 10 4 cells per well in 10% FBS/DMEM and left to grow overnight. Viral solutions were diluted in 10-fold serial dilutions in 2% FBS/DMEM. The supernatant was removed and replaced with virus diluent in the correspondingly marked wells and incubated at 37 °C for 96 h, after which the cells were fixed with 4% formalin and stained with 0.25% crystal violet to visualize the patches on a white background. Mean tissue culture infection dose (TCID50) and multiple infection (MOI) were calculated from quadruple trials.
2.3. Silver Formula
PVP-AgNP 10 at a stock concentration of 20 ppm (Cat No: 795925) and cAg (Cat No: 85131) were obtained from Sigma. AgNPs have different sizes; AgNP 2 (Cat No: US7150), AgNP 15 (Cat No: US7091), AgNP 50 , AgNP 80 , and AgNP 100 (US1038W) were purchased from US Research Nanomaterials, Inc. concentration is prepared by dilution in sterile distilled water.
2.4. Cell viability test
The CellTiter-Glo (Promega) cell viability assay is a luminescence-based assay that quantitatively detects living cells based on adenosine triphosphate (ATP) levels. Cell death due to Ag-mediated cytotoxicity or viral infection can be rapidly quantified using Cell-Titer Glo [ 6 ]. 50 μl CellTiter-Glo Substrate (Promega) was added to the cells and their viability was measured against the luminescence intensity detected by the GloMax Discovery System (Promega) 10 min later.
2.5. RT-qPCR
Viral RNA was extracted from the culture using the QIAamp virus RNA Mini Kit (Qiagen) and stored at -80 °C until further analysis. Extracted viral RNA was quantified using the CFX96 Real-Time System (Bio-rad) with TaqMan Fast virus 1-Step Master Mix (Thermo) using 5′-AAATTTTGGGGACCAGGAAC-3 and 5′-TGGCAGCTGTGTAGGTCAA-3 as primer set and 5′-FAM-ATGTCGCGCATTGGCATGGA-BHQ-3 as probes [ 7 ].
2.6. Study of cytotoxicity
cAg or AgNPs at desired concentrations were added to the VeroE6/TMPRSS2 or Calu-3 cell lines cultured in a 96-well blank plate and incubated at 37 °C for 48 or 96 h respectively, after which the cells were washed with PBS and viability was quantified by the CellTiter-Glo assay.
3. Interaction study of nano silver and SARS-CoV-2
3.1. Virus pretreatment test
Virus at an MOI of 0.05 (for VeroE6/TMPRSS2) or 0.5 (for Calu-3) in DMEM containing 2% FBS was incubated with 2 ppm AgNP solution for 1 h at 37°C. Virus mixture -AgNP was added to VeroE6/TMPRSS2 or Calu-3 cells, respectively, in 96 well plates and incubated for 48 h or 96 h at 37 °C, respectively. Cell viability was quantified by CellTiter-assay Glo and viral copies in the supernatant were quantified by RT-qPCR.
3.2. Cell test after treatment
VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 (MOI = 0.05) and incubated for 2 h at 37 °C. Wells were washed with PBS to remove extracellular virus and coated with DMEM containing 2% FBS with 2 ppm PVP-AgNP 10 and incubated for 48 h at 37°C. Cell viability was quantified by the CellTiter-Glo assay and viral copies in supernatant were quantified by RT-qPCR .
3.3. Cell Pretreatment Test
VeroE6/TMPRSS2 cells were treated with 2 ppm PVP-AgNP 10 and incubated for 3 h at 37°C. The wells were then washed with PBS to remove free AgNPs in the medium and coated with DMEM containing 2% FBS with SARS-CoV-2 (MOI = 0.05) and incubated for 48 h at 37°C. Cell viability was quantified by the CellTiter-Glo assay and viral copies in the supernatant were determined. quantified by RT-qPCR.
3.4. Immunofluorescence
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X-100, and then blocked with Blocking One (Nacalai) at room temperature for 15 min. Cells were incubated with a polyclonal antibody against the SARS-CoV-2 Nucleocapsid Protein antibody (1:100 dilution, Novus NB100-56576) at room temperature for 1 h. After incubation, cells were stained with Alexa-labelled anti-rabbit antibody 568 (1:1000 dilution, Thermo) for 1 h at room temperature. Nuclei were stained with ProLong Gold Antifade Mountant with DAPI (Thermo). Images were taken with a BZ-9000 fluorescence microscope (Keyence)..
3.5. Pseudovirus Penetration Test
The lentiviral pseudotype was generated by transient infection of HEK293 cells with pNL4-3. Luc.RE− and pSARS2-Spike-FLAG at a 1:1 ratio. Pseudovirus-containing cultures were collected 48 h after transfection and filtered through a 0.45 μm Millex-HV filter (Merck). For the input assay, VeroE6/TMPRSS2 cells were seeded in a washed 96-well plate and inoculated with 100 μl of media containing pseudovirus with or without PVP-AgNP 10 . Neutralizing antibodies were used as a control for invasion inhibition. 48 h after inoculation, cells were washed and supplemented with 40 μl of Shiny Substrate (Promega). Luciferase activity was measured with the GloMax Discovery System (Promega).
4.1. Độc tính tế bào, liều lượng hiệu quả và thời gian tiếp xúc
AgNPs exhibit cytotoxicity to mammalian cells by inducing reactive oxygen species (ROS) [ 8 ]. We wanted to evaluate the concentration-dependent cytotoxicity expressed by Ag on VeroE6/TMPRSS2 (non-human) and Calu-3 (human lung epithelial cells) cell lines. The diluted cAg was monitored and added to cells in 96 well plates and cell viability was assessed after 48 h using the CellTiter-Glo assay. Ag was found to exhibit cytotoxicity at concentrations from 20 parts per million (ppm) onwards in both VeroE6/TMPRSS2 (Figure 1 A) and the Calu-3 cell line (Figure 1B).
Cytotoxicity of colloidal silver on 1A mammalian cells: Cytotoxicity expressed by serial concentrations of colloidal silver on VeroE6/TMPRSS2 cells. 1B : Cytotoxicity expressed by serial colloidal silver concentrations on Calu-3 cells.
As cAg contains particle sizes varying from 1 to 1000 nm, we used it as an initial screen to determine the influence of AgNPs on SARS-CoV-2. The degree of multiple infection (MOI) of SARS-CoV-2 was calculated by independent experiments and was found to be 0.05 and 0.5 for VeroE6/TMPRSS2 and Calu-3, respectively. Virus suspensions at fixed MOI were treated with each serial dilution of cAg for 1 h and then inoculated into VeroE6/TMPRSS2 and Calu-3 cells. Figure 2 A). In Calu-3 cells, a significantly reduced viral load was observed at a similar cAg concentration range (Figure 2B). While metal ions are known to be PCR inhibitors at high concentrations, we confirm that at effective concentrations (2 ppm), Ag does not inhibit amplification and is suitable for analysis of viral RNA in samples containing Ag ( Figure S1 ) [ 9 ]. Since 2 ppm was 10 times lower than the cytotoxic concentration, it was chosen as the desired concentration for further testing.
The antiviral efficacy was concentration and dose dependent of the naked Silver nanoparticles on SARS-CoV-2. 2A: Colloidal silver saves VeroE6/TMPRSS2 cells from SARS-CoV-2-mediated cell death in a concentration-dependent manner. Error bars obtained from triple testing. p value ≤ 0.005 (∗∗∗). 2B: Concentration-dependent inhibition of SARS-CoV-2 replication in Calu-3 cells by colloidal silver. Error bars obtained from triple testing. value p ≤ 0.001 (∗∗∗). 2C : Silver nanoparticles exhibit a size-dependent antiviral effect on SARS-CoV-2 in Vero/TMPRSS2 cells. Error bars obtained from triple testing. value p ≤ 0.005 (∗∗∗). 2D: Size-dependent viral inhibition of SARS-CoV-2 by Silver nanoparticles in Calu-3 cells. Error bars obtained from triple testing. value p ≤ 0.001 (∗∗∗).
Previous studies have documented a size-dependent efficacy of AgNPs in virus inactivation, with particle sizes ≤10 nm reported to have maximal antiviral effects [ 10]. As cAg contains different sizes of Ag particles, we predicted that particles with a size of about 10 nm in cAg would exert antiviral effects. To demonstrate this, we used a virus pretreatment assay (VPrA) to examine the antiviral effects of AgNPs of different sizes from 2 to 100 nm on extracellular viruses. Viruses were treated with 2 ppm silver nanoparticle solution of different sizes for 1 h and the virus-AgNP mixture was added to VeroE6/TMPRSS2 and Calu-3 cells. Cell viability was quantified by CellTiter-Glo assay in VeroE6/TMPRSS2 and viral transcripts in supernatant were quantified by RT-qPCR in Calu-3 cells. Antiviral effects were observed with AgNPs ranging in size from 2 to 15 nm (Figures 2C and D). AgNP 2 showed cytotoxicity at 2 ppm while the other sizes did not ( Figure S2 ). Therefore, we choose PVP-AgNP 10 for further testing. Since we observed excellent antiviral activity in VPrA at 1 h, we wanted to know the minimum exposure time that Ag needed to inhibit the virus. The VPrA-based time course study with PVP-AgNP 10 showed significant inhibition after 30 min of exposure ( Figure S3 ).
4.2. Silver inhibits extracellular viruses by blocking viral entry
Next, we performed VPrA, cell post-treatment assay (CPoA) and cell pretreatment assay (CPrA) on SARS-CoV-2 using PVP-AgNP 10 in VeroE6/TMPRSS2 to observe observed the effects of Ag on extracellular and intracellular viruses (Figure 3 A). VPrA showed effective inhibition of extracellular free virions, characterized by both a reduction in cell death and also a reduction in viral load to negligible levels (Figures 3B and C). We further performed CPoA to detect Ag’s ability to suppress viruses in infected cells. In this experimental design, VeroE6/TMPRSS2 cells were allowed to be infected with SARS-CoV-2 (MOI 0.05) for 2 h after which the extracellular viruses were washed and then the infected cells were washed. treated with 2 ppm PVP-AgNP 10 . We observed significant protection of infected cells and suppression of viral load with PVP-AgNP 10 (Figures 3B and C). In addition, we performed CPrA to evaluate the ability of silver pretreated cells to resist viral infection. VeroE6/TMPRSS2 cells were incubated with 2 ppm PVP-AgNP 10 for 3 h, then the cells were washed to remove unbound silver, followed by SARS-CoV-2 infection (MOI 0.05). At the end of 48 h, the virus was only partially inhibited (Figure 3C).
Silver nanoparticles effectively inhibited extracellular SARS-CoV-2. 3A : Schematic representation of the virus pretreatment test (top panel), the cell posttreatment test (center panel) and the cell pretreatment test (lower panel). 3B : Performance of PVP-coated 10 nm Silver nanoparticles in three study designs involved in saving cells from SARS-CoV-2 infection. Error bars obtained from triple testing. value p ≤ 0.005 (∗∗∗). 2C : Performance of PVP-coated 10 nm Silver nanoparticles in three study designs regarding the reduction of SARS-CoV-2 replication. Error bars obtained from triple testing. value p ≤ 0.001 (∗∗∗).
We confirmed the size-dependent antiviral effect of PVP-AgNP 10 using immunofluorescence analysis performed on the experimental model VPrA; SARS-CoV-2 infection was effectively prevented by AgNP 10 but not AgNP 100 (Figure 4 A). Plaque test showed that silver achieved complete inhibition of 0.05 MOI, one log 10 fold higher than virus control. Partial inhibition was observed with higher viral loads starting at 0.5 MOI (Figure 4B). To evaluate the role of AgNPs in viral entry, we performed a luciferase-based pseudoviral entry assay. PVP-AgNP 10 strong inhibition of pseudovirus entry was characterized by a significant decrease in luciferase activity similar to that of the neutralizing antibody used as a control (Figure 4C)
Characterization of PVP-coated 10 nm Silver nanoparticles in SARS-CoV-2 infection. 4A : Immunofluorescence imaging comparing the effects of 10 nm and 100 nm Silver nanoparticles against SARS-CoV-2 infection in VeroE6/TMPRSS2 cells. Cell nucleus (blue) and the SARS-CoV-2 nucleocapsid protein in the cytoplasm (red). NC – Negative control. 4B : PVP-coated 10 nm silver nanoparticles protect VeroE6/TMPRSS2 cells from SARS-CoV-2 infection-mediated cell death. Crystal violet staining shows partial protection with visible patches (red arrowheads) and full protection without plaques (black arrowheads). 4C: Pseudovirus Penetration Test. PVP-coated 10 nm silver nanoparticles inhibited pseudovirus entry in VeroE6/TMPRSS2 cells. NC – Negative control, nAb – Neutralizing antibody. (For an explanation of the references to color in this figure legend, the reader is advised to refer to the Web version of this article.)
5. Discuss
Ag is long known for its anti-bacterial effects, and the antiviral properties of AgNPs are being studied extensively with renewed interest in the recent past [ 1 ]. The exact mechanism by which AgNPs exert their antiviral effects remains unclear. However, it has been consistently observed that AgNPs interact with structural proteins on the surface of extracellular viruses to inhibit early infection, by preventing binding or entry of the virus. virus, or by damaging surface proteins to affect the structural integrity of the virions [ 11 , 12]. In the present study, we obtained similar findings in VPrA where AgNPs effectively inhibit extracellular SARS-CoV-2 to protect target cells from infection, and the pseudovirus entry assay showed that AgNPs interfere with viral entry.
AgNPs have been shown to preferentially bind to sulfhydryl-rich viral surface proteins and cleave disulfide bonds to destabilize the protein, thereby affecting viral infectivity [ 11 , 13 ] . HIV studies have shown that AgNPs bind to disulfide bonds close to the CD4-binding region of the gp120 surface protein [ 11 ]. Hati and Bhattacharyya demonstrated the importance of disulfide binding in binding of the SARS-CoV-2 mutant protein to the angiotensin-converting enzyme-2 (ACE2) receptor and its disruption resulted in impaired binding of the SARS-CoV-2 mutant protein. virus with the [ 14] receptor. Considering the mechanism of action of AgNPs indicated by other authors, it can be assumed that AgNPs exert their antiviral effects against SARS-CoV-2 by breaking disulfide bonds on mutant proteins and proteins. ACE2 receptor. Further studies are underway to find the antiviral mechanism of AgNPs on SARS-CoV-2 and elucidate it in detail thereafter.
AgNPs are also thought to exert an intracellular antiviral effect by interacting with viral nucleic acids [ 15 ]. We observed a partial antiviral effect in CPrA, as there was some reduction in viral load in cells pretreated with PVP-AgNP 10 . Although the reason for this effect is currently unknown, it may be explained by destruction of disulfide bridges on the ACE2 receptor or by a true intracellular mechanism (by inhibiting the transmission of ACE inhibitors). serial viral infections of newly generated viruses from infected cells to uninfected cells). In addition, because Ag binds nonspecifically to proteins, their use as antivirals may also induce some cellular dysfunction. Further studies are needed to more accurately explain the overall effects of Ag in vivo.
Several studies have reiterated the size-dependent antiviral effect of AgNPs with particles with a diameter of 10 nm being the most effective [ 1 ]. This is attributed to the higher stability of the interaction with viral proteins achieved by 10 nm particles that larger particles are not capable of [ 11 ]. Consistent with this, we also observed anti-SARS-CoV-2 activity only with AgNPs with a diameter of 2 to 15 nm. Our immunofluorescence study confirmed the above phenomenon, as we observed that PVP-AgNP 10 completely inhibited SARS-CoV-2 but AgNP 100 did not.
AgNPs can be generated by a number of methods and can contain reducing and capping agents along with metal particles [ 16 ]. Coated or capped AgNPs are believed to be more beneficial than conventional AgNPs because the coating increases stability, reduces aggregation, and reduces cytotoxicity of AgNPs [ 17 ]. Among the coated AgNPs, PVP coated nanoparticles are widely studied for biological use. It was observed that the PVP coating of AgNPs did not interfere with their antiviral activity whereas other coatings had [ 18 ]. PVP-AgNP 10 has been shown to have excellent antiviral activity against enveloped viruses such as RSV and HIV [ 11 , 19 ]. This is the reason for choosing PVP-AgNP10 for the study, and we have demonstrated a strong antiviral effect of PVP-AgNP 10 against SARS-CoV-2.
The antiviral effect of AgNPs is also concentration dependent. Most studies have observed antiviral efficacy of AgNPs at concentrations between 10 and 100 ppm [ 1 ]. However, 0.5 ppm cAg has been shown to be effective in inhibiting Influenza virus and is the least reported concentration showing antiviral activity [ 20 ]. In the present study, we observed naked AgNPs to inhibit SARS-CoV-2 at concentrations between 1 and 10 ppm and become cytotoxic to mammalian cells of 20 ppm or more.
The cytotoxicity of nanosilver against mammalian cells depends on the cell type and also the type of AgNPs. Mehrbod et al. Cytotoxicity was observed in Madin-Darby Canine Kidney (MDCK) cells with naked cAg particles at concentrations higher than 0.5 ppm [20]. Bare AgNPs with NaBH4 reductant were found to induce apoptosis in colon adenocarcinoma cells at 11 ppm, while Citrate-stabilized naked silver nanoparticles were observed to exhibit cytotoxicity. at concentrations higher than 30 ppm [ 21 , 22 ]. In this regard, PVP-coated AgNPs have been shown to be the least cytotoxic with no demonstrable cytotoxicity even at 50 ppm in human alveolar basal epithelial cells [ 19]. . Smaller particles have a higher toxicity potential due to the larger surface area for interaction with the bound protein [ 23 ]. We observed this effect because AgNP 2 showed cytotoxicity even at 2 ppm whereas no larger particles were cytotoxic at this concentration. Therefore, caution should be exercised when using Ag on biological surfaces.
Various formulations of ingestible and inhaled Ag are being marketed as a cure for COVID-19, available for purchase over the counter. The cytotoxic potential of these formulations should be considered prior to individual use. In addition, Ag is a very broad spectrum bactericide. Illegal use of Ag can create an imbalance in the general microbiome leading to unintended consequences [ 24 ]. AgNPs can be used on various inanimate surfaces to combat the ongoing COVID-19 pandemic [ 3 ]. Ag-coated masks have been shown to be effective in inhibiting SARS-CoV-2 and can be effective when applied on air filters of air conditioners and medical equipment [ 25 ] . Nanosilver-incorporated polycotton fabric has been shown to inhibit SARS-CoV-2 [ 26]. Ag-based sanitizers and disinfectants are also being used to disinfect hands and inanimate surfaces respectively [ 27]. However, the effect of nanosilver on microbial life when released into the environment is still unknown [ 16 ]. Appropriate handling procedures should be developed for Ag-containing products to avoid causing untimely imbalance in the environmental microbiome upon disposal after use..
