Two forms of Si₃N₄ powders were used in this study: alpha silicon nitride (α-Si₃N₄) and sintered beta silicon nitride (β-Si₃N₄) prepared as previously described (Simpson et al., 2023). α-Si₃N₄ that possessed a median particle size of 0.6843 μm and a specific surface area of 9.5091 +/− 0.0732 m²/g was characterized via scanning electron microscopy (SEM) (Figure 1). The SEM analysis reveals a variety of α-Si3N₄ particle morphologies. The β-Si₃N₄ had a median particle size of 0.8904 μm and a specific surface area of 9.1800 +/− 0.0963 m²/g. It was also characterized via SEM (Figure 2). The SEM analysis demonstrated the morphology of sintered Si₃N₄ particles, equiaxed grains for α-Si₃N_4, and the telltale elongated, hexagonal cross-section grains for β-Si₃N₄.

Due to the release of reactive nitrogen species when Si₃N₄ is exposed to an aqueous environment, we first tested the impact of Si₃N₄ on cell viability (Pezzotti, 2018). Vero cells were exposed to clarified supernatants (Figure 3A) from increasing concentrations of α-Si₃N₄ (AP⁴) and β-Si₃N₄ (AP²) for 48 h. Cell viability decreased at the highest powder concentration (15% (w/v)) for AP⁴ and AP² to approximately 80 and 60%, respectively (Figure 3C). Then, the powders were tested at 15% (w/v) for virucidal activity by incubating AP² or AP⁴ with SARS-CoV-2 for 30 min. After incubation, the powders were pelleted by centrifugation, and the infectious virus in the supernatant was assessed via plaque assay (Figure 3B). Both phases of Si₃N₄ reduced SARS-CoV-2 (Lineage A) viral titers, as determined via plaque assay in a time-dependent manner (Figure 3D). Exposure of SARS-CoV-2 to AP² resulted in a 0.65–1.23 log₁₀ fold change (77.7 to 94.15% inhibition) in viral titers. AP⁴ had a greater impact on viral titer reduction with a 2.7, 3.4, and 3.5 log₁₀ fold change (99.8, 99.96, and 99.97% inhibition) at 1, 5, and 10 min, respectively. The greatest decrease in infectious virus occurred when SARS-CoV-2 was exposed to AP⁴ for 30 min, resulting in viral titers falling below detectable limits. Collectively, these results demonstrate that AP⁴ has less cytotoxicity and increased virucidal activity against SARS-CoV-2 (Lineage A) compared to AP².

To further explore the cause for the reduction in viral titers, we sought to test whether the decrease in virus was attributed to the virucidal activity of the Si₃N₄ powders or if it was a result of protocol design, where the virus might become bound to the pellets and therefore not being detected in the sample supernatants during plaque assays. SARS-CoV-2 was exposed to Si₃N₄ (AP⁴ and AP²) at 15% (w/v) for 30 min, with the virus only processed in parallel to serve as a control (Figure 4A). Viral RNA was quantified from clarified supernatants and resuspended Si₃N₄ pellets by RT-qPCR. The supernatants were simultaneously used to quantify infectious virus as determined by plaque assay. AP² exposure reduced viral titers by approximately 1.1 log₁₀ fold change (92.1% inhibition), consistent with our previous finding, and AP⁴ exposure resulted in zero detectable plaques (Figure 4B). The same pattern of reduction was observed for viral RNA detected in the supernatant by RT-qPCR, with virus exposed to AP⁴ having the largest decrease (99.9% inhibition) in genomic copies per reaction (Figure 4C). Little to no viral RNA (<1 genomic copy for N gene) was detected in the pellets. These findings suggest that the decrease in the infectious virus is due to the antiviral properties of Si₃N₄ rather than SARS-CoV-2 being bound to the pellets.

We expanded antiviral testing to include additional SARS-CoV-2 lineages and MERS-CoV to evaluate the pan-coronavirus efficacy of Si₃N₄. A reduction in infectious virus was observed across the four SARS-CoV-2 lineages exposed to both AP² and AP⁴. Again, AP² was less effective at reducing viral titers than AP⁴ but had the greatest impact on SARS-CoV-2 B.1.617.1 with a 2.22 log₁₀ fold change (99.4% inhibition) (Figure 5A). All viral titers for SARS-CoV-2 lineages fell below detectable limits when exposed to AP⁴ for 30 min. The same trend in viral reduction was observed for MERS-CoV when incubated with Si₃N₄, with a 2.04 log₁₀ fold change (99.1% inhibition) by AP² and plaques below detectable limits by AP⁴ as compared to the control (Figure 5B). AP⁴ had the greatest pan-coronavirus virucidal activity, outperforming AP².

Our overall scientific objective is to implement Si₃N₄-treated personal protective equipment (PPE) as a viral intervention in areas with high-exposure risk (e.g., hospitals). To that end, two types of α-Si₃N₄-infiltrated fabric were produced and studied. One fabric was infiltrated with AP⁴ (α-Si₃N₄) at a heavy load via an aqueous slurry as previously described (Simpson et al., 2023). The second fabric was infiltrated with AP⁴ (α-Si₃N₄) using a dry, alternating electric current method as described in the Methods section. SEM analysis of the dry-infiltrated spunbond polypropylene (SBPP) infiltrated with α-Si₃N₄-showed fine Si₃N₄ particles evenly distributed onto individual polypropylene fibers (Figure 6). Both slurry-infiltrated heavy Si₃N₄-loaded fabric and dry-infiltrated fabric were tested for antiviral activity against SARS-CoV-2 (Lineage A) at 10- and 30 min exposure, with testing done in accordance to ISO 18184 test method (Figure 7A). Slurry-infiltrated heavy Si₃N₄-loaded fabric was compared to untreated fabric (Figure 7B). To establish a proof of concept for our fabric testing, a virus only control (VOC) (virus in media) was included to determine if equivalent viral titers from the untreated fabric could be obtained. Slurry-infiltrated heavy Si₃N₄-load fabric exhibited a 1.28 log₁₀ fold change (94.1% inhibition) at 10 min, and a 1.45 log₁₀ fold change (96.1% inhibition) at 30 min compared to the untreated fabric. Additionally, no statistically significant differences were observed in viral titers for the untreated fabric and VOC, validating the controls within our experimental design.

We next compared the antiviral activity between the slurry-infiltrated heavy Si₃N₄-loaded fabric and the dry-infiltrated fabric. The dry-infiltrated fabric exhibited a 1.48–1.52 log₁₀ fold change (96.7–97% inhibition) in viral titers (PFU/mL) when incubated with SARS-CoV-2 for 30 min and 10 min (Figure 7C). The reduction in virus observed for dry-infiltrated fabric was comparable to the positive control, slurry-infiltrated heavy Si₃N₄-loaded fabric.

African green monkey kidney epithelial (Vero) cells (ATCC, CCL-81) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (VWR, 10128-208) supplemented with 10% fetal bovine serum (FBS) (VWR, 97068-085), 1% L-glutamine (VWR, 45000-676), and 1% penicillin/streptomycin (VWR, 45000-652). Vero E6 cells (ATCC, CRL-1586) were maintained in Eagle’s Minimum Essential Medium with L-glutamine (EMEM) (VWR, 10128-608) supplemented with 10% FBS and 1% penicillin/streptomycin. Vero E6-TMPRSS2-T2A-ACE2 (TMPRSS2) cells (BEI Resources, NR-54970) were maintained in DMEM supplemented with 10% FBS, 2% L-glutamine, and 10 μg per mL puromycin (Gibco, A1113803). All cells were maintained at 37 °C in a 5% CO₂ environment.

SARS-CoV-2 isolates hCoV-19/USA-WA1/2020 (Lineage A) (NR-52281), hCoV-19/USA/CA-Stanford-15_S02/2021 (Lineage B.1.617.1; Kappa Variant) (NR-55486), hCoV-19/England/204820464/2020 (Lineage B.1.1.7; Alpha Variant) (NR-54000), hCoV-19/South Africa/KRISP-EC-K005321/2020 (Lineage B.1.351; Beta Variant) (NR-54008), and MERS-CoV (NR-44260) were obtained from BEI Resources. SARS-CoV-2 and MERS-CoV were propagated in Vero E6 cells. SARS-CoV-2 Lineage A was additionally propagated in TMPRSS2 cells when used for fabric antiviral testing as described below. Viral propagation was carried out in the respective media for each cell type with low FBS (2%) and harvested based on the presence of cytopathic effect (CPE) (20–72 hpi). Supernatants were centrifuged (at 21,801 x g for 10 min) to pellet cellular debris and filtered via a 0.2 μm filter (VWR, Cat. #28144-050). The stock virus was titrated according to the plaque assay described below.

AP⁴ and AP² powders were prepared as described previously (Simpson et al., 2023).

Vero cells were plated at 2 × 10⁴ cells/well in a 96-well plate (Corning, 3,903). Silicon nitride powder at 5, 10, and 15% (w/v) was added to one mL of complete DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. Samples were briefly vortexed prior to 30 min incubation at room temperature with rotation. Samples were then centrifuged at 554 x g at room temperature for 2 min, and the supernatants clarified through filtration with a 0.2 μm filter (VWR, Cat. #28144-050). The clarified supernatants (200 μL/well) were added to Vero cells (2 × 10⁴ cells/well, 96-well plate) (Corning, 3,903) and incubated for 48 h at 37 °C, 5% CO₂. Untreated cells (complete DMEM) served as the 100% viability control. CellTiter Glo (Promega, Cat #G7570) was used to measure ATP production as a determinant of cell viability.

SARS-CoV-2 isolates or MERS-CoV were diluted in complete DMEM supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin to a concentration of 2×10⁵ PFU/mL. One mL of virus was added to tubes containing Si₃N₄ at 15% (w/v) or without Si₃N₄ (virus only) to serve as the control. Samples were briefly vortexed and then incubated at room temperature for up to 30 min with rotation. Samples were then centrifuged at 554 x g at room temperature for 2 min and supernatants clarified through filtration with a 0.2 μm filter (VWR, Cat. #28144-050). Infectious virus was quantified by plaque assay.

Supernatants for powder antiviral testing were quantified via plaque assay using Vero cells for SARS-CoV-2 isolates (1.5 × 10⁵ cells/well, 12-well plate) and Vero E6 cells for MERS-CoV (2×10⁵ cells/well, 12-well plate). Samples from fabric antiviral testing were quantified using TMPRSS2 cells (2 × 10⁵ cells/well, 12-well plate). Clarified supernatants were serially diluted (1:10), and 200 μL per dilution was added to each well in the 12-well plate. Plates were incubated at 37 °C, 5% CO₂ for 1 h, rocking every 15 min to ensure adequate coverage of the cells. A 1:1 overlay of 1% agarose (Invitrogen, 16500-500) and 2x EMEM (VWR, Cat # 10128-758) supplemented with 5% FBS, 1% L-glutamine, 2% penicillin/streptomycin, 1% non-essential amino acids (VWR, Cat # 45000-700), and 1% sodium pyruvate (VWR, Cat # 45000-710) were added to the cells (1 mL/well). Plates were incubated at 37 °C, 5% CO₂ for 48 h or 72 h, for SARS-CoV-2 isolates and MERS-CoV, respectively. After incubation, cells were fixed with 10% formaldehyde and stained with 2% crystal violet in 20% ethanol for counting.

SARS-CoV-2 isolates hCoV-19/USA-WA1/2020 (Lineage A) were exposed to Si₃N₄ for 30 min, as written in the powder antiviral testing section. The clarified supernatants were divided for plaque assay, and 250 μL was then transferred to clean microcentrifuge tubes for RNA extraction. Residual media from the Si₃N₄ pellets were removed and discarded. Pellets were resuspended in 250 μL complete DMEM.

The pellets and clarified supernatants were then extracted for RNA, in which 750 μL TRIzol LS (Thermo Fisher Scientific, Cat. #10-296-028) was added and the samples vortexed and incubated at room temperature for 5 min. Chloroform (200 μL) (MP Biomedicals, Cat #194002) was added to the samples, vortexed, and incubated at room temperature for 5 min. Samples were briefly vortexed post-incubation and centrifuged at 13,845 x g at room temperature for 10 min. The upper aqueous phase was transferred to a fresh microcentrifuge tube and processed for RNA with the RNeasy Mini Kit (Qiagen, Cat. #74106) using an adapted protocol. RLT buffer (600 μL) was added to the samples and mixed, followed by the addition of absolute ethanol (700 μL), and the samples were mixed. Further sample processing was done in accordance with manufacture’s guidelines (Quick-Start Protocol RNeasy Mini Kit, Part 1). Samples were eluted in 35 μL molecular grade water (VWR, Cat. #95000-094).

Viral RNA was quantified via RT-qPCR using 5 μL of purified RNA with the RNA UltraSense One-Step RT-PCR System (Thermo Fisher Scientific, Cat. #11732927) and a primer-probe set for 2019-nCoV-N1 (IDT, Cat. #10006770), targeting the nucleocapsid (N) gene, on a QuantStudio 3 Real-Time PCR System (Applied Biosystems, Cat. #A28567). Thermal cycling conditions were adapted from the RNA UltraSense One-Step RT-PCR System as follows: 50 °C for 15 min, 95 °C for 2 min, and 40 cycles of 95 °C for 15 s and 55 °C for 30 s. Absolute quantification was determined based on standard curves generated from serial dilutions (1:10) of quantitative synthetic SARS-CoV-2 RNA (BEI, NR-52358) containing fragments from the ORF 1ab, Envelope (E), and Nucleocapsid (N) regions.

Spunbond polypropylene (SBPP) was pre-cleaned and pre-treated as previously described (Simpson et al., 2023). SBPP was infiltrated with α-Si₃N₄ and polybutylene succinate (PBS) using a commercial alternating electric current technology (D-Preg, Fibroline, Lyon, France). The α-Si₃N₄-PBS powder blend (50 wt.% α-Si₃N₄/50 wt.% PBS) was scattered on the SBPP surface, and an alternative electric field was applied to charge the powder, break up the powder agglomerates, and impregnate the non-woven textile. The α-Si₃N₄-impregnated SBPP was thermally treated with a heat press at 130 °C at 1 bar for 30 s to melt the PBS binder and allow the α-Si₃N₄ particles to adhere or embed into the substrate. SEM characterization was performed on the final treated fabric (Figure 6). Antiviral test swatches of α-Si₃N₄-impregnated SBPP were prepared by cutting 25 mm squares and ultrasonically welding three squares together. Four three-layer squares weighing approximately 0.40 ± 0.01 g each were used as one sample for antiviral testing in accordance with the standard ISO 18184 test method.

SARS-CoV-2 isolate hCoV-19/USA-WA1/2020 (Lineage A) was diluted in complete DMEM to a concentration of 1×10⁷ PFU/mL. Four squares of fabric constituted one sample. Virus was spotted five times onto each fabric square in 10 μL spots, totaling 200 μL of virus per sample. The four pieces of fabric were sandwiched together and placed in 50 mL conical tubes. Virus only (200 μL) control was added to an empty 50 mL conical tube for processing in parallel. Tubes were incubated at room temperature for 30 min. After incubation, 20 mL complete DMEM was added to the fabric. Samples were kept on ice the entire time after the addition of media. Tubes were vortexed 5 times (5 s per time). Ten mL of homogenate was transferred to a 15 mL conical tube. Tubes were centrifuged at 1,256 x g at 4 °C for 2 min and supernatants clarified through filtration with a 0.2 μm filter. Infectious virus was quantified by plaque assay.

Samples consisted of three biological replicates unless specified otherwise. Statistical analyzes were performed using GraphPad Prism Software Version 10.4.1. Tests used to determine statistical significance are described within the figure legends. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.