The test material was spunbond nonwoven polypropylene fabric containing ~ 3% cuprous-oxide microparticles (MedCu Technologies, Ltd. Figure 1), hereafter referred as the Test Fabric.
The cuprous oxide particles were characterized by x-ray diffraction (XRD) using an x-ray diffractometer (Y-2000) with Cu Kα radiation (λ = 1.5418 Å). A scan efficiency of 0.1°·S-1 was applied to record the powder patterns in the range of 0° ≤ 2θ ≤ 60°. Three peaks at 2θ = 29.61°, 36.49° and 42.37° were indexed to (110), (111), and (200) planes of the cubic phase with lattice constant α = 0.4266 nm, which is in accordance with the spectrum for Cu2O in JCPDS–International Centre for Diffraction Data (PDF, Powder Diffraction File, No. 05–0667, 1996). Impregnation of cuprous oxide microparticles in the polypropylene fibers was achieved by adding the cuprous oxide microparticles to the polypropylene during the master batch preparation stage.8 The concentration of the cuprous oxide microparticles in the fabric was based on previous studies that demonstrated that this concentration endows the fabric with wide spectrum biocidal properties (e.g. ref8,13). As a negative test control material, the same spunbond polypropylene fabric without cuprous-oxide was used, hereafter referred as Control Fabric.
After being UV sterilization for 1 hour, 20 mm by 20 mm pieces of Control and Test Fabrics were aseptically placed together in separate vial containers (final weight of 0.20 ± 0.005 g per tested sample), hereafter referred to as Control or Test Fabric specimens, respectively.
As the virus source, -80°C cryopreserved SARS-CoV-2 clinical isolate was used. The stock virus titer was determined by infecting Vero-E6 (CRL-1586™; American Type Culture Collection) plated cells grown in Gibco phosphate-free, high glucose DMEM media (catalog number: 11971025) containing 10% fetal bovine serum (FBS) and determining the plaque forming units (PFU)/ml.
The determination of the direct contact virus inactivation of the test items was performed as follows according to the ISO Standard 18184:2019 -Textiles — Determination of antiviral activity of textile products: 100 µl of the virus stock suspension were placed onto each test specimen, making sure all the liquid was thoroughly absorbed. The vial containers were then tightly closed and incubated at of 25°C for 5, 30, or 60 min (triplicate Control or Test Fabric specimens per time point). After the respective incubation time, 10 ml of Earle's Balanced Salt Solution (EBSS, Gibco) were added to each container; the containers were closed and agitated for 60 seconds by vortexing. The virus suspensions were collected. From each of the recovered eluent suspensions after vortexing, 2-times sequential dilutions were prepared using phosphate free DMEM media.
For potential deactivation of the virus by possible eluting elements from the Control or Test Fabric specimens (i.e. not by direct contact inactivation), the following control was performed: 10 ml of EBSS were added to 3 Control Test specimens and to 3 Test Fabric specimens placed in the vial containers. The containers were then closed, vortexed for 60 s, and 5 ml of the solution from each vial container were transferred to new tubes. 50 µl of virus suspension were then added to each tube. The tubes were kept at 25°C in the CO2 incubator for 30 min.
The infectivity of the virus recovered from the Control or Test Fabric specimens were then determined by conducting a plaque assay using Vero-E6 cells plated in 96 well culture plates 12–24 hours in advance, and grown to 80–90% confluency. Briefly, the cells were washed once with PBS and then 100 µl of sequential dilutions the above-described suspensions were added to each well. The cells inoculated with the virus were incubated for 1 hour at 37°C in 5% CO2 incubator, with gentle shaking every 15 minutes. After 1 hour of absorption, 100 µl of a semi-solid maintenance solution preparation (1% methylcellulose in DMEM) were added to each well. The plates were incubated for 3 days at 37°C in 5% CO2 incubator. The number of plaques formed was monitored every day. After 72 hours of incubation, when the formation of plaques was obvious, 50 µl of 2% paraformaldehyde were added per well in order to fix the cells. The supernatants were discarded after 30 minutes, and 30 µl of crystal violet were added per well. After 2 minutes, the cells were washed 5 times with water. The number of plaques was determined by using an inverted microscope.
The plaque forming units (PFU) per ml were calculated using the following formula:
PFU/ml = number of plaques counted/(dilution factors x inoculation volume).
Each PFU/ml was converted to log 10 and the average of the Initial viral load (Log10) and Output viral load (Log10) were then determined.
The log10 Reduction was calculated in the following manner:
Log10 Reduction = Average Initial viral load (Log10) – Average Output viral load (Log10).
The percent reduction of the infectious titers was calculated as follows:
Percent reduction from Initial titer = 100-[(Mean final titer / Mean initial titer)*100]
A t-test was performed to compare the means of log reductions from direct contact inactivation as well as the total reduction for statistical significance between control and test fabrics. A p-value of less than 0.05 was considered a statistically significant difference.
The amount of copper eluting from the mask into human dermatomed skin was determined in an independent laboratory (Dermal Technology Laboratory Ltd., Med IC4, Keele University Science and Business Park, Keele, Staffordshire, UK) according to the OECD Test No. 428: Skin Absorption: In Vitro Method. Regular surgical face masks without copper were used as negative controls. The surgical face mask materials (triplicate samples) were applied to the surface of the skin in static diffusion cells and the skin was otherwise left unoccluded for an exposure period of 24 hours. Various skin compartments of a typical OECD 428 study14 were collected and analyzed for copper by Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES).
The amount of copper that eluted from the masks into the air under simulated breathing conditions was determined by an independent lab (Nelson Laboratories, Inc. 6280 S. Redwood RD., Salt Lake City, UT, USA). To simulate human inhalation, air was pulled under vacuum of 1 CFM through a capsule HEPA filter and a Concha Therm III® unit, which can heat and humidify air. The temperature of the test was maintained at 37 ± 2°C and > 90% relative humidity. In addition, a 4 ± 2% concentration of carbon dioxide was introduced into the air stream. The air was pulled through the test sample and releasable particles were drawn through a Pall® zefluor 3 µm PTFE membrane, with a particle collection efficiency of > 99.996% of 0.3 µm diameter. After 5 hours of exposure to simulated inhalation, the test was stopped and the collection membranes were removed and the amount of copper was determined by ICP-OES. The test was done in triplicates. Regular surgical masks without copper were used as negative controls. As a positive control, 0.1 grams of cuprous oxide powder were added to 100 ml of USP water and 1 ml of the mixture was inoculated onto a PTFE particle collection membrane and the solution was allowed to dry.
The filtration properties of the masks were determined according to EN 14683 (European standard for face masks) by an independent lab (TUV SUD Products Testing Co., Ltd. B-3/4, No. 1999 Du Hui Road, Minhang District, Shanghai, China), which included determination of Bacterial Filtration Efficiency (BFE), Differential Pressure and Synthetic Blood Penetration tests.