key: cord-0966717-mc8wol5v authors: Jeremiah, Sundararaj S.; Miyakawa, Kei; Morita, Takeshi; Yamaoka, Yutaro; Ryo, Akihide title: Potent antiviral effect of silver nanoparticles on SARS-CoV-2 date: 2020-09-11 journal: Biochem Biophys Res Commun DOI: 10.1016/j.bbrc.2020.09.018 sha: b14dd6b7ffae67bfd21bc718d0a9e2bcc6bcda5c doc_id: 966717 cord_uid: mc8wol5v The pandemic of COVID-19 is spreading unchecked due to the lack of effective antiviral measures. Silver nanoparticles (AgNP) have been studied to possess antiviral properties and are presumed to inhibit SARS-CoV-2. Due to the need for an effective agent against SARS-CoV-2, we evaluated the antiviral effect of AgNPs. We evaluated a plethora of AgNPs of different sizes and concentration and observed that particles of diameter around 10 nm were effective in inhibiting extracellular SARS-CoV-2 at concentrations ranging between 1 and 10 ppm while cytotoxic effect was observed at concentrations of 20 ppm and above. Luciferase-based pseudovirus entry assay revealed that AgNPs potently inhibited viral entry step via disrupting viral integrity. These results indicate that AgNPs are highly potent microbicides against SARS-CoV-2 but should be used with caution due to their cytotoxic effects and their potential to derange environmental ecosystems when improperly disposed. subsequent to Ag mediated cytotoxicity or viral infection could be rapidly quantified using Cell-Titer Glo [6] . 50 µL of CellTiter-Glo Substrate (Promega) was added to the cells and their viability was measured based on the luminescence intensities detected by GloMax Discover System (Promega) 10 minutes later. Viral RNA was extracted from culture supernatants using QIAamp viral RNA Mini Kit (Qiagen) and stored at -80℃ until further analysis. The extracted viral RNA was quantified using CFX96 Real-Time System (Bio-rad) with a TaqMan Fast virus 1-Step Master Mix (Thermo) using 5'-AAATTTTGGGGACCAGGAAC-3' and 5'-TGGCAGCTGTGTAGGTCAA-3' as the primer set and 5'-FAM-ATGTCGCGCATTGGCATGGA-BHQ-3' as probe [7] . cAg or AgNPs at desired concentration were added to VeroE6/TMPRSS2 or Calu-3 cell lines grown in 96-well white plates and were incubated at 37℃ for 48 or 96 hours respectively after which the cells were washed with PBS and the viability was quantified using the CellTiter-Glo assay. Virus pre-treatment assay J o u r n a l P r e -p r o o f 8 VeroE6/TMPRSS2 (non-human origin) and Calu-3 (human lung epithelial cell). cAg was serially diluted and added to the cells in 96 well plates and the cell viability was assessed after 48 hours using CellTiter-Glo assay. Ag was found to exhibit cytotoxicity at concentrations from 20 parts per million (ppm) onward in both VeroE6/TMPRSS2 ( Fig 1A) and Calu-3 cell lines (Fig 1B) . Since cAg contains particle sizes varying from 1 to 1000 nm, we used it as an initial screen to ascertain the effect of AgNPs on SARS-CoV-2. The multiplicity of 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. Viral suspension at a fixed MOI was treated with each serial dilution of cAg for one hour and then inoculated to VeroE6/TMPRSS2 and Calu-3 cells. Cell viability of infected VeroE6/TMPRSS2 was assessed after 48 hours to identify the proportion of cells killed by the virus and the viral load was quantified in Calu-3 cells using real time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) after 96 hours. cAg showed robust antiviral effect denoted by increased viability of infected VeroE6/TMPRSS2 cells at concentrations ranging between 1 to 10 ppm (Fig 2A) . In Calu-3 cells, significant viral load reduction was observed at similar cAg concentration range ( Fig 2B) . While metal ions are known inhibitors of PCR at high concentrations, we confirmed that at the effective concentration (2ppm), Ag did not inhibit amplification and is suitable to analyze viral RNA in Ag containing samples (Fig S1) [9] . Since 2 ppm was also 10-fold lower than the cytotoxic concentration, it was chosen as the desired concentration for further testing. Previous studies have documented the size dependent potency of AgNPs in viral inactivation, with ≤10nm particle size reported to have maximum antiviral effect [10] . Since cAg contains varying sizes of Ag particles, we predicted that the particles around 10nm size in cAg would have exerted J o u r n a l P r e -p r o o f the antiviral effect. To prove this, we employed the virus pre-treatment assay (VPrA) to test antiviral effect of AgNPs of different sizes ranging from 2-100nm on extracellular viruses. Virus was treated with 2ppm solution of AgNPs of different sizes for one hour and the virus-AgNP mixture was added to VeroE6/TMPRSS2 and Calu-3 cells. The cell viability was quantified by CellTiter-Glo assay in VeroE6/TMPRSS2 and the viral copies in supernatant were quantified by RT-qPCR in Calu-3 cells. Antiviral effect was noted with AgNPs of size ranging from 2-15nm (Fig 2C,D) . AgNP 2 showed cytotoxicity at 2ppm while other sizes did not ( Fig S2) . Hence we chose PVP-AgNP 10 for further testing. Since we observed excellent antiviral activity in VPrA at 1 hour, we wanted to know the minimum contact time required by Ag for viral inhibition. Time course study performed based on VPrA with PVP-AgNP 10 revealed significant inhibition beyond 30 minutes of contact ( Fig S3) . We next performed the VPrA, cell post-treatment assay (CPoA) and the cell pre-treatment assay (CPrA) on SARS-CoV-2 using PVP-AgNP 10 in VeroE6/TMPRSS2 to observe the effect of Ag on extracellular and intracellular viruses ( Fig 3A) . VPrA showed effective inhibition of extracellular free virions characterized by both the reduction of cell death and also a steep fall in the viral load to negligible levels ( Fig 3B,C) . We further performed the CPoA to detect the ability of Ag to suppress virus in already infected cells. In this experimental design, VeroE6/TMPRSS2 cells were allowed to be infected with SARS-CoV-2 (MOI 0.05) for2 hours after which the extracellular viruses were washed and then the infected cells were treated with 2ppm of PVP-AgNP 10 . We observed significant protection of infected cells and suppression of viral load with PVP-AgNP 10 (Fig 3B,C) . Additionally we performed the CPrA to assess the ability of silver pre-treated cells to resist viral infection. VeroE6/TMPRSS2 cells were incubated with 2 ppm PVP-AgNP 10 for 3 hours after which the cells were washed to remove unbound silver followed by infection with SARS-CoV-2 (MOI 0.05). At the end of 48 hours, the virus was found only to be partially inhibited ( Fig 3C) . We confirmed the size dependent antiviral effect of PVP-AgNP 10 using the immunofluorescence analysis carried on VPrA experimental model; SARS-CoV-2 infection was effectively averted by AgNP 10 and not by AgNP 100 (Fig 4A) . The plaque assay revealed that silver achieved complete inhibition of 0.05 MOI which is one log 10 fold more than virus control. Partial inhibition was observed with higher viral loads starting from 0.5 MOI (Fig 4B) . To assess the role of AgNPs in viral entry, we performed the luciferase based pseudovirus entry assay. PVP-AgNP 10 potently inhibited pseudoviral entry characterized by significant reduction of luciferase activity similar to that of the neutralizing antibody used as control (Fig 4C) . Ag is long known for its antimicrobial effect and the antiviral property of AgNPs is being extensively researched with renewed interest in the recent past [1] . The exact mechanism by which AgNPs exert its killing effect on viruses is still obscure. However, it has been consistently observed that AgNPs interact with the structural proteins on the surface of extracellular viruses to inhibit infection in the early phase, by either preventing viral attachment or entry, or by damaging the surface proteins to affect the structural integrity of virions [11, 12] . In the current study, we have AgNPs have been shown to preferentially bind to viral surface proteins rich in sulfhydryl groups and cleave the disulfide bonds to destabilize the protein, thereby affecting viral infectivity [11, 13] . Studies on HIV have shown that AgNPs associate to the disulfide bonds that are in close proximity to the CD4 binding domain of the gp120 surface protein [11] . Hati and Bhattacharyya have demonstrated the importance disulfide bonds in binding of SARS-CoV-2 spike protein with the angiotensin converting enzyme-2 (ACE2) receptor and the disruption of which lead to impaired viral binding to the receptor [14] . Considering the mechanism of action of AgNPs shown by other authors, it can be presumed that AgNPs exert their antiviral effect on SARS-CoV-2 by disrupting the disulfide bonds on the spike protein and ACE2 receptors. Further studies are being conducted to find the antiviral mechanism of AgNPs on SARS-CoV-2 and elucidate it in detail subsequently. AgNPs have also been claimed to possess intracellular antiviral action by interacting with viral nucleic acids [15] . We observed a partial antiviral effect in CPrA, as there was some amount of reduction in the viral load in cells pre-treated with PVP-AgNP 10 . While the reason for this effect is not known at present, it is possibly explained to be either due to the destruction of disulfide bridges on ACE2 receptor or due to a true intracellular mechanism (there by inhibiting serial viral infection of newly produced virus from infected cells to uninfected cells). Also, since Ag binds nonspecifically to proteins, their use as antiviral agents might also cause some cellular dysfunction. Further studies are required to more precisely explain the holistic effect of Ag in vivo. Several studies have reiterated the size dependent antiviral effect of AgNPs with particles around 10nm diameter being most effective [1] . This has been attributed the higher stability of interaction J o u r n a l P r e -p r o o f to the viral protein achieved by 10 nm particles which is not capable by larger particles [11] . Consistent with this, we also observed anti-SARS-CoV-2 activity only with AgNPs of diameters ranging from 2-15 nm. Our immunofluorescence study corroborated 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 several methods and can contain reducing agents and capping agents along with the metal particles [16] . Coated or capped AgNPs are found to be more advantageous than plain AgNPs as coating increases stability, decreases agglomeration and reduces cytotoxicity of AgNPs [17] . Among the coated AgNPs, PVP capped nanoparticles are widely studied for biological use. It has been observed that PVP coating of AgNPs does not hinder their antiviral activity while other coating agents do [18] . PVP-AgNP 10 has been demonstrated to possess excellent antiviral activity against enveloped viruses such as RSV and HIV [11, 19] . This was the rationale to select PVP-AgNP 10 for the study and we have demonstrated the robust antiviral effect of PVP-AgNP 10 against SARS-CoV-2. Antiviral effect of AgNPs is also concentration dependent. Most studies have observed the antiviral efficacy of AgNPs at concentrations ranging between 10 to 100 ppm [1] . However, 0.5 ppm cAg has been shown effective in inhibiting Influenza virus and is the least concentration that has been reported to show antiviral activity [20] . In the current study, we observed naked AgNPs to inhibit SARS-CoV-2 at concentrations ranging between 1 to 10 ppm and become cytotoxic to mammalian cells from 20 ppm and above. Cytotoxicity of AgNPs to mammalian cells depends on the cell type and also the type of AgNPs. cAg particles at concentrations higher than 0.5 ppm [20] . Naked AgNPs with NaBH 4 reducing agent J o u r n a l P r e -p r o o f were found to induce apoptosis in colon adenocarcinoma cells at 11 ppm, while Citrate-stabilized naked AgNPs have been observed to exhibit cytotoxicity at concentrations higher than 30 ppm [21, 22] . In this regard, PVP coated AgNPs have been demonstrated 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 toxic potential due to the greater surface area of interaction with the bound protein [23] . We observed this effect as AgNP 2 showed cytotoxicity even at 2 ppm while none of the bigger particles were cytotoxic at this concentration. Therefore, care should be exercised when Ag is used on biological surfaces. Various ingestible and inhalable formulations of Ag are being marketed as cure for COVID-19, which available to purchase over the counter. The cytotoxic potential of these formulations should be considered before personal use. Also, Ag is a very broad spectrum microbicide. Illicit use of Ag might create an imbalance in the commensal microbiota leading to unforeseen consequences [24] . AgNPs can be used on a variety of inanimate surfaces to combat the ongoing COVID-19 pandemic [3] . Ag coated masks have been found to be effective in inhibiting SARS-CoV-2 and could potentially be effective when applied on the air filters of air conditioners and medical devices [25] . AgNP incorporated polycotton fabrics have been proven to inhibit SARS-CoV-2 [26] . Ag based sanitizers and disinfectants are also being used for disinfection of hands and inanimate surfaces respectively [27] . However, the effect of AgNPs on influencing the microbial life when released in the environment is unknown [16] . A proper disposal protocol should be developed for Ag containing products to avoid causing untoward imbalances in the environmental microbial pattern when discarded after use. 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