Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
  • A61K8/25—Silicon; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
  • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
  • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95

Definitions

• the present disclosure relates to antiviral compositions, systems, methods, and devices. More specifically, the disclosure relates to silicon nitride compositions, devices, and coatings for the inactivation of viruses.
• SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
• SARS-CoV-2 which is responsible for the COVID-19 pandemic, remains viable and therefore potentially infectious on several materials.
• One strategy to discourage the fomite- mediated spread of COVID-19 is the development of materials whose surface chemistry can spontaneously inactivate SARS-CoV-2.
• an antiviral composition comprising silicon nitride at a concentration of about 1 wt.% to about 15 wt.%, where the silicon nitride inactivates a virus in contact with the composition.
• the virus may be in contact with the silicon nitride for a duration of at least 1 minute or at least 30 minutes. For example, the virus may be at least 85% inactivated after contact with the silicon nitride for at least 1 minute.
• the silicon nitride may be present at a concentration of less than or equal to 10 wt.%.
• the silicon nitride may be a-SisN4, p-SisN4, SiYAION, [3-SiYAION, SiYON, or SiAION.
• the virus may be Influenza A or SARS-CoV-2.
• the composition is a slurry, suspension, gel, spray, paint, or toothpaste.
• an antiviral apparatus comprising silicon nitride at a concentration of about 1 wt.% to about 15 wt.%, wherein the silicon nitride inactivates a virus in contact with the composition.
• the virus may be in contact with the silicon nitride for a duration of at least 1 minute or at least 30 minutes.
• the virus may be at least 85% inactivated after contact with the silicon nitride for at least 1 minute.
• the silicon nitride may be present at a concentration of less than or equal to 10 wt.%.
• the silicon nitride may be a-SisN4,
• the virus may be Influenza A or SARS-CoV-2.
• the apparatus may be a medical device, medical equipment, examination table, filters, masks, gloves, catheters, endoscopic instruments, or commonly-touched surfaces.
• the apparatus may be metallic, polymeric, and/or ceramic and the silicon nitride may be coated on or embedded in a surface of the apparatus.
• Also provided herein are embodiments of a method of preventing transmission of a virus comprising: contacting an antiviral apparatus with the virus, where the apparatus comprises silicon nitride at a concentration of about 1 wt.% to about 15 wt.%.
• the virus may be in contact with the silicon nitride for a duration of at least 1 minute or at least 30 minutes.
• the virus may be at least 85% inactivated after contact with the silicon nitride for at least 1 minute.
• the silicon nitride may be present at a concentration of less than or equal to 10 wt.%.
• the silicon nitride may be a-SisN4, p-SisN4, SiYAION, [3-SiYAION, SiYON, or SiAION.
• the virus may be Influenza A or SARS-CoV-2.
• FIG. 1 is an illustration of the Influenza A virus.
• FIG. 2A is an illustration of a virus exposed to 0 wt.%, 7.5 wt.%, 15 wt.%, and 30 wt.% SisN4 for 10 minutes.
• FIG. 2B is an illustration of methods used to determine viability of cells inoculated with a virus exposed to SisN4 according to FIG. 2A.
• FIG. 3A is an illustration of a virus exposed to 15 wt.% SisN4 for 1 , 5, 10, and 30 minutes.
• FIG. 3B is an illustration of methods used to determine viability of a virus after exposure to SisN4 according to FIG. 3A.
• FIG. 4A is a graph of PFU/100 pl for Influenza A exposed to 0 wt.%, 7.5 wt.%, 15 wt.%, and 30 wt.% SisN4 for 10 minutes according to FIG. 2A.
• FIG. 4B is a graph of cell survivability of cells inoculated with Influenza A exposed to 7.5 wt.%, 15 wt.%, and 30 wt.% SisN4 for 10 minutes according to FIG. 2B.
• FIG. 5 includes photographs of cells inoculated with different ratios of virus to slurry that had been exposed to various concentrations of SisN4.
• FIG. 6A shows a fluorescence microscopy image of MDCK cells before inoculation.
• FIG. 6B shows a fluorescence microscopy image of MDCK cells after inoculation with a virus exposed to the control.
• FIG. 6C shows a fluorescence microscopy image of MDCK cells after inoculation with a virus exposed to 30 wt.% SisN4.
• FIG. 7A is a graph of PFll/100 pl for Influenza A exposed to 15 wt.% SisN4 for 1 minute, 5 minutes, 10 minutes, or 30 minutes at room temperature.
• FIG. 7B is a graph of cell survivability of cells inoculated with Influenza A exposed to 15 wt.% SisN4 for 1 minute, 5 minutes, 10 minutes, or 30 minutes at room temperature.
• FIG. 8A is a graph of PFll/100 pl for Influenza A exposed to 15 wt.% SisN4 for 1 minute, 5 minutes, 10 minutes, or 30 minutes at 4°C.
• FIG. 8B is a graph of cell survivability of cells inoculated with Influenza A exposed to 15 wt.% SisN4 for 1 minute, 5 minutes, 10 minutes, or 30 minutes at 4°C.
• FIG. 9A shows the Raman spectrum of Influenza A virus before inactivation.
• FIG. 9B shows changes in the Raman spectrum of the Influenza A virus relevant to chemical modifications in RNA and hemagglutinin after inactivation after 1 minute of exposure.
• FIG. 10 shows NHs inactivates Influenza A virus by the mechanism of alkaline transesterification.
• FIG. 11 shows O-P-O stretching in pentacoordinate phosphate group after inactivation.
• FIG. 12A shows vibrational modes of methionine in the hemagglutinin structure.
• FIG. 12B shows methionine’s structural change in the presence of ammonia.
• FIG. 13 shows C-S stretching methionine to homocysteine after inactivation.
• FIG. 14A is a graph of PFU/100 pl for Feline calicivirus exposed to 15 wt.% or 30 wt.% SisN4 for 1 minute, 10 minutes, or 30 minutes.
• FIG. 14B is a graph of cell survivability of cells inoculated with Feline calicivirus exposed to 30 wt.% SisN4 for 1 minute, 10 minutes, 30 minutes, or 60 minutes.
• FIG. 15A shows the H1 H1 Influenza A virus (nucleoprotein, NP) stained red after 10 minutes of exposure to a slurry of 15 wt.% silicon nitride and after its inoculation into a biogenic medium containing MDCK cells stained green for the presence of filamentous actin (F-actin) proteins.
• NP nucleoprotein
• FIG. 15B shows the NP stained H1 H1 Influenza A virus from FIG. 15A
• FIG. 15C shows the F-actin stained MDCK cells from FIG. 15A.
• FIG. 16A shows the H1 H1 Influenza A virus (nucleoprotein, NP) stained red without exposure to silicon nitride and after its inoculation into a biogenic medium containing MDCK cells stained green for the presence of filamentous actin (F- actin) proteins.
• FIG. 16B shows the NP stained H1 H1 Influenza A virus from FIG. 16A
• FIG. 16C shows the F-actin stained MDCK cells from FIG. 16A.
• FIG. 17 shows a trimodal distribution of silicon nitride powder.
• FIG. 18 shows the viability of the MDCK cells as function of p-SisN4 concentration (wt.%/mL).
• FIG. 19 shows a direct comparison of the viral titers before and after exposure of Influenza A to the SisN4 powder for 30 minutes.
• FIG. 20 shows the viability of the MDCK cells as function of a-SisN4 concentration (wt.%/mL).
• FIG. 21 shows a comparison of the viral titers before and after exposure of Influenza A to the a-SisN4 powder for 30 minutes.
• FIG. 22 shows a trimodal particle size distribution of silicon nitride powder.
• FIG. 23 is an overview of the antiviral testing method.
• FIG. 25A shows titers of silicon nitride at concentrations of 5, 10, 15, and 20 wt.%/vol incubated with SARS-CoV-2 virus diluted in cell culture media for 1 , 5, and 10m expressed as PFU/mL.
• FIG. 25B shows titers of silicon nitride at concentrations of 5, 10, 15, and 20 wt.%/vol incubated with SARS-CoV-2 virus diluted in cell culture media for 1 , 5, and 10m expressed as % inhibition.
• “about” refers to numeric values, including whole numbers, fractions, percentages, etc., whether or not explicitly indicated.
• the term “about” generally refers to a range of numerical values, for instance, ⁇ 0.5-1 %, ⁇ 1-5% or ⁇ 5-10% of the recited value, that one would consider equivalent to the recited value, for example, having the same function or result.
• silicon nitride includes a-SisN4, p-SisN4, SiYAION, [3-SiYAION, SiYON, SiAION, or combinations thereof.
• inactivate or “inactivation” refers to viral inactivation in which the virus is stopped from contaminating the product or subject either by removing virus completely or rendering them non-infectious.
• apparatus or “component” as used herein include materials, compositions, devices, surface coatings, and/or composites.
• the apparatus may include various medical devices or equipment, examination tables, clothing, filters, personal protective equipment such as masks and gloves, catheters, endoscopic instruments, commonly-touched surfaces where viral persistence may encourage the spread of disease, and the like.
• the apparatus may be metallic, polymeric, and/or ceramic (ex. silicon nitride and/or other ceramic materials).
• contact means in physical contact or within close enough proximity to a composition or apparatus to be affected by the composition or apparatus.
• antiviral devices, compositions, and apparatuses that include silicon nitride (SisN4) for the inactivation of viruses.
• Silicon nitride possesses a unique surface chemistry which is biocompatible and provides a number of biomedical applications including 1 ) concurrent osteogenesis, osteoinduction, osteoconduction, and bacteriostasis, such as in spinal and dental implants; 2) killing of both gram-positive and gram-negative bacteria according to different mechanisms; 3) inactivation of human and animal viruses, bacteria, and fungi; and 4) polymer- or metalmatrix composites, natural or manmade fibers, polymers, or metals containing silicon nitride powder retain key silicon nitride bone restorative, bacteriostatic, antiviral, and antifungal properties.
• an antiviral composition may include silicon nitride.
• the antiviral composition may an apparatus that includes silicon nitride powder.
• the antiviral apparatus may be a monolithic component comprising up to 100% silicon nitride. Such a component can be fully dense possessing no internal porosity, or it may be porous, having a porosity that ranges from about 1 % to about 80%.
• the monolithic component may be used as a medical device or may be used in an apparatus in which the inactivation of a virus may be desired.
• an antiviral composition may be incorporated within a device or in a coating to inactivate viruses on or within the device.
• the antiviral composition may be a slurry comprising silicon nitride powder.
• the antiviral composition may inactivate or decrease the transmission of human viruses.
• viruses that may be inactivated by the antipathogenic composition include coronaviruses (e.g. SARS- CoV-2), Influenza A, H1 N1 , enterovirus, and Feline calicivirus.
• coronaviruses e.g. SARS- CoV-2
• Influenza A e.g. Influenza A
• H1 N1 e.g. enterovirus
• Feline calicivirus e.g. calicivirus
• a silicon nitride composition may be effective in the inactivation of the Influenza A virus.
• a silicon nitride composition may be effective in the inactivation of the SARS- CoV-2 virus.
• Silicon nitride may be antipathogenic due to release of nitrogen containing species when in contact with an aqueous medium, or biologic fluids and tissues.
• the surface chemistry of silicon nitride may be shown as follows:
• silicon nitride may provide for RNA cleavage via alkaline transesterification which leads to loss in genome integrity and virus inactivation. This may also reduce the activity of hemagglutinin.
• each of silicon nitride inactivates a coronavirus and Influenza A.
• the antipathogenic composition may exhibit elution kinetics that show: (i) a slow but continuous elution of ammonia from the solid state rather than from the usual gas state; (ii) no damage or negative effect to mammalian cells; and (iii) an intelligent elution that increases with decreasing pH.
• Cu copper
• Si 3 N 4 are biocompatible and not toxic to the human body.
• An advantage of Si 3 N 4 is the versatility of the material; thus Si 3 N 4 may be incorporated into polymers, bioactive glasses, and even other ceramics to create composites and coatings that retain the favorable biocompatible and antiviral properties of Si 3 N 4 .
• An antiviral device or apparatus may include a silicon nitride composition on at least a portion of a surface of the device for antiviral, antibacterial, or antifungal action.
• an antiviral device may include a silicon nitride coating on at least a portion of a surface of the device. The silicon nitride coating may be applied to the surface of the device the device as a powder.
• the silicon nitride powder may be filled, imbedded, or impregnated in at least a portion of the device.
• the powder may be micrometric or nanometer in size.
• the average particle size may range from about 100 nm to about 5 pm, from about 300 nm to about 1 .5 pm, or from about 0.6 pm to about 1 .0 pm.
• the silicon nitride may be incorporated into the device.
• a device may incorporate silicon nitride powder within the body of the device.
• the device may be made of silicon nitride.
• the composition can comprise a slurry or suspension of nitride particles.
• the silicon nitride coating may be present on the surface of an apparatus or within the apparatus in a concentration of about 1 wt.% to about 100 wt%.
• the coating may include about 1 wt.%, 2 wt.%, 5 wt.%, 7.5 wt.%, 8.3 wt.%, 10 wt.%, 15 wt.%, 16.7 wt.%, 20 wt.%, 25 wt.%, 30 wt.%, 33.3 wt.%, 35 wt.%, or 40 wt.% silicon nitride powder.
• the coating may include about 10 wt.% to about 20 wt.% silicon nitride. In at least one example, the coating includes about 15 wt.% silicon nitride. In some embodiments, silicon nitride may be embedded in (as a filler) or on the surface of a device or apparatus in a concentration of about 1 wt.% to about 100 wt.%.
• a device or apparatus may include about 1 wt.%, 2 wt.%, 5 wt.%, 7.5 wt.%, 8.3 wt.%, 10 wt.%, 15 wt.%, 16.7 wt.%, 20 wt.%, 25 wt.%, 30 wt.%, 33.3 wt.%, 35 wt.%, 40 wt.%, 50 wt.%, 60 wt.%, 70 wt. %, 80 wt.%, 90 wt.%, to 100 wt.% silicon nitride.
• the silicon nitride may be on the surface of the apparatus at a concentration of about 10 wt.% to about 20 wt.%. In at least one example, the silicon nitride may be on the surface of the apparatus at a concentration of about 15 wt.% silicon nitride.
• the antiviral composition may be a monolithic component consisting of the silicon nitride.
• a component can be fully dense possessing no internal porosity, or it may be porous, having a porosity that ranges from about 1 % to about 80%.
• the monolithic component may be used as a medical device or may be used in an apparatus in which the inactivation of a virus may be desired.
• a device or apparatus that includes silicon nitride for antiviral properties may be a medical device.
• medical devices or apparatuses include orthopedic implants, spinal implants, pedicle screws, dental implants, in-dwelling catheters, endotracheal tubes, colonoscopy scopes, and other similar devices.
• silicon nitride may be incorporated within or applied as a coating to materials or apparatuses for antiviral properties such as polymers and fabrics, surgical gowns, tubing, clothing, air filters and water filters, masks, tables such as hospital exam and surgical tables, desks, fixtures, handles, knobs, toys, and filters such as air conditioner filters, or toothbrushes.
• the filters may be within filtration devices of anesthesia machines, ventilators, or CPAP machines such that an antimicrobial surface layer in the filter can trap pulmonary pathogens, as air moves in and out of infected lungs.
• silicon nitride powder may be incorporated into compositions including, but not limited to slurries, suspensions, gels, sprays, paint, or toothpaste.
• a slurry such as paint
• silicon nitride may be mixed with water along with any appropriate dispersants and slurry stabilization agents, and thereafter applied by spraying the slurry onto various surfaces.
• An example dispersant is Dolapix A88.
• silicon nitride may be included in an antiviral composition at a concentration of about 5 wt.% to about 20 wt.%. In at least one example, the composition may include about 15 wt.% silicon nitride. Alternatively, in some embodiments, silicon nitride may be included in an antiviral composition at a concentration of about 5 wt.% to about 20 wt.%. In at least one example, the composition may include about 15 wt.% silicon nitride. In an example, the antiviral composition may be a slurry of silicon nitride powder and water.
• Silicon nitride may be combined with water to form an aqueous slurry at concentrations of about 0.1 wt.% up to about 70 wt.%.
• the silicon nitride powder may be present in the slurry in a concentration of about 0.1 wt.% to about 55 wt.%.
• silicon nitride may be incorporated within organic suspensions, gels, sprays, and/or paints at concentrations of about 0.1 wt.% up to about 70 wt.% or about 0.1 wt.% up to about 55 wt.%.
• the slurry, organic suspension, gel, spray, and/or paint may include about 0.1 wt.%, 0.5 wt.%, 1 wt.%, 1.5 wt.%, 2 wt.%, 5 wt.%, 10 wt.%, 15 wt.%, 20 wt.%, 25 wt.%, 30 wt.%, 35 wt.%, 40 wt.%, 45 wt.%, 50 wt.%, or 55 wt.% silicon nitride.
• the composition comprises a toothpaste and the silicon nitride is in the form of a powder that is directly substituted for silicon dioxide powder found in standard toothpaste.
• the silicon nitride powder may be substituted for silicon dioxide powder in toothpaste at concentrations of about 1 wt.% to about 30 wt.%.
• silicon nitride may be present within a toothpaste at a concentration of about 1 wt.%, 5 wt.%, 10 wt.%, 15 wt.%, 20 wt.%,
• the silicon nitride not only serves to be an antiviral and antibacterial agent in the toothpaste, but it also may serve as a polishing agent similar to silicon dioxide.
• the method may include coating a device or apparatus with silicon nitride and contacting the coated apparatus with the virus.
• Coating the apparatus may include applying a silicon nitride powder to a surface of the apparatus.
• the silicon nitride powder may be filled, incorporated, or impregnated within the device or apparatus.
• the antiviral composition may decrease viral action by alkaline transesterification and reduce the activity of hemagglutinin. It was surprisingly found that silicon nitride powder (i) remarkably decreases viral action by alkaline transesterification through the breakage of RNA internucleotide linkages and (ii) markedly reduced the activity of hemagglutinin thus disrupting host cell recognition by denaturing protein structures on viral surfaces leading to the inactivation of viruses regardless of the presence of a viral envelope.
• the antipathogenic composition may exhibit elution kinetics that show: (i) a slow but continuous elution of ammonia from the solid state rather than from the usual gas state; (ii) no damage or negative effect to mammalian cells; and (iii) an intelligent elution which increases with decreasing pH.
• the inorganic nature of silicon nitride may be more beneficial than the use of petrochemical or organometallic bactericides, virucides, and fungicides which are known to harm mammalian cells or have residual effects in soil, on plants, and in vegetables or fruit.
• the pathogen may be a virus.
• the method may include contacting the patient with a device, apparatus, or composition comprising silicon nitride.
• the silicon nitride inactivates the virus (for example, a coronavirus, such as SARS-CoV-2, or Influenza A).
• the device, apparatus, or composition may include about 1 wt.% to about 100 wt.% silicon nitride.
• the device or apparatus may include about 1 wt.% to about 100 wt.% silicon nitride on the surface of the device or apparatus.
• the device or apparatus may be a monolithic silicon nitride ceramic.
• the device or apparatus may include a silicon nitride coating, such as a silicon nitride powder coating.
• the device or apparatus may incorporate silicon nitride into the body of the device.
• silicon nitride powder may be incorporated or impregnated into the body of the device or apparatus using methods known in the art.
• the composition or apparatus may be contacted with the patient or user for at least 1 minute, at least 5 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, or at least 1 day.
• the device or apparatus may be permanently implanted in the patient.
• the device or apparatus may be worn externally by a user.
• the apparatus may be a high contact surface.
• the apparatus may be in continuous or sustained contact with a body fluid of a patient.
• the body fluid may be blood or gas (inhalation or exhalation gas).
• the virus is at least 70% inactivated, at least 75% inactivated, at least 80% inactivated, at least 85% inactivated, at least 90% inactivated, at least 95% inactivated, or at least 99% inactivated after contact with the silicon nitride in the composition or apparatus for at least 1 minute, at least 5 minutes, or at least 30 minutes.
• the virus is at least 85% inactivated after contact with the silicon nitride in the composition or apparatus for at least 1 minute.
• the virus is at least 99% inactivated after contact with the silicon nitride in the composition or apparatus for at least 30 minutes.
• Example 1 Effect of silicon nitride concentration on virus inactivation
• Influenza A was exposed to 0 wt.%, 7.5 wt.%, 15 wt.%, and 30 wt.% SisN4 for 10 minutes at 4°C, as illustrated in FIG. 2A. The mixtures were then filtered to remove the silicon nitride powder.
• Influenza A virus-inoculated Madin-Darby canine kidney (MDCK) cells were then observed for the effectiveness of SisN4 in inactivating the Influenza A.
• the remaining mixtures were then inoculated into Petri dishes containing living MDCK cells within a biogenic medium.
• the amount of living MDCK cells were subsequently counted using staining methods after 3 days exposure.
• the viability of MDCK cells was determined after inoculating the cells for 3 days with Influenza A exposed to SisN4 according to FIG. 2B.
• FIG. 4A is a graph of PFll/100 pl for Influenza A exposed to 0 wt.%, 7.5 wt.%, 15 wt.%, and 30 wt.% SisN4 for 10 minutes.
• FIG. 4B is a graph of cell survivability of cells inoculated with Influenza A exposed to 7.5 wt.%, 15 wt.%, and 30 wt.% SisN4 for 10 minutes.
• Influenza A was exposed to a fixed concentration of SisN4 powder (15 wt.%) for various times and temperatures. The mixture was then allowed to incubate under gentle agitation for 1-30 minutes at room temperature and at 4°C. For example, Influenza A was exposed to 15 wt.% SisN4 for 1 , 5, 10, or 30 minutes at room temperature or 4°C, as illustrated in FIG. 3A.
• Influenza A virus-inoculated Madin-Darby canine kidney (MDCK) cells were then observed for the effectiveness of SisN4 in inactivating the Influenza A. The viability of MDCK cells was determined after inoculating the cells for 3 days with Influenza A exposed to SisN4 according to FIG. 3B.
• FIG. 7A is a graph of PFll/100 pl for Influenza A exposed to 15 wt.% SisN4 for 1 minute, 5 minutes, 10 minutes, or 30 minutes at room temperature.
• FIG. 7B is a graph of cell survivability of MDCK cells inoculated with Influenza A exposed to 15 wt.% SisN4 for 1 minute, 5 minutes, 10 minutes, or 30 minutes at room temperature.
• FIG. 8A is a graph of PFll/100 pl for Influenza A exposed to 15 wt.% SisN4 for 1 minute, 5 minutes, 10 minutes, or 30 minutes at 4°C.
• FIG. 8B is a graph of MDCK cell survivability inoculated with Influenza A exposed to 15 wt.% SisN4 for 1 minute, 5 minutes, 10 minutes, or 30 minutes at 4°C.
• Example 3 Effect of silicon nitride on H1H1 Influenza A inactivation
• Influenza A was exposed to a slurry of 15 wt.% silicon nitride for 10 minutes.
• FIGS. 15A-15C show the H1 H1 Influenza A virus (A/Puerto Rico/8/1934 H1 N1 (PR8)) stained red (nucleoprotein, NP) after its inoculation into a biogenic medium containing MDCK cells stained green for the presence of filamentous actin (F-actin) proteins which are found in all eukaryotic cells.
• FIGS. 16A-16C shows the effect of the virus on the MDCK cells without the presence of silicon nitride.
• Example 4 Evaluation of Influenza A viricidal activity by silicon nitride in MDCK cells [0093] This study was designed to examine the antiviral capabilities of beta-silicon nitride (
• a plaque assay methodology was utilized. To adequately quantify the plaque assay, the viability of Madin Darby Canine Kidney Cells (MDCK) were assessed as a function of exposure to various concentrations of SisN4 for incubation periods ranging from 30 minutes to 72 hours. The results demonstrated that SisN4 was completely viricidal to Influenza A with a reduction of > 99.98% in viral load at the preselected conditions. The viability of the MDCK cells was found to be time- and dosedependent. Essentially no loss in viability was observed for SisN4 concentrations up to 15 wt.%/wt. Changes in viability were only noted for the 15 wt.% concentration at 24, 48, and 72 hours (/.e., 83.3%, 59.7%, and 44.0% viable, respectively).
• MDCK Madin Darby Canine Kidney Cells
• the SisN4 powder used in this study had a nominal composition of 90 wt.% a-SisN4, 6 wt.% yttria (Y2O3), and 4 wt.% alumina (AI2O3). It was prepared by aqueous mixing and spray-drying of the inorganic constituents, followed by sintering of the spray-dried granules ( ⁇ 1700°C for ⁇ 3 h), hot-isostatic pressing ( ⁇ 1600°C, 2 h, 140 MPa in N2 ), aqueous-based comminution, and freeze-drying. The resulting powder had a trimodal distribution with an average particle size of 0.8 ⁇ 1 .0 pm as shown in FIG. 17.
• Doping SisN4 with Y2O3 and AI2O3 is useful to densify the ceramic and convert it from its a- to [3-phase during sintering.
• the mechanism of densification is via dissolution of a- phase and subsequent precipitation of [3-phase grains facilitated by the formation of a transient intergranular liquid that solidifies during cooling.
• (3-SisN4 is therefore a composite composed of about 10 wt.% intergranular glass phase (IGP) and 90 wt.% crystalline (3-SisN4 grains.
• the viability of the MDCK cells is shown as function of [3- SisN4 concentration (wt.%/mL). Starting at 15 wt.%, serial dilutions were conducted to arrive at 0.047 wt.%. At the lower concentrations, cell viability was generally > 80% for all timepoints up to 72 h. Note also that cell viability generally increased with exposure time for all concentrations except 15 wt.%. At 15 wt.% and 30-minutes exposure the cell viability was ⁇ 94.5%.
• MDCK cells were plated in a 6-well plate at a density of 1 x 10 6 cells/well in a volume of 2 mL in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
• DMEM Dulbecco's Minimum Essential Medium
• FBS fetal bovine serum
• the samples were centrifuged for two minutes at 4°C and 12,000 rpm, and further filtered through a 0.2-micron polyvinylidene difluoride (PVDF) filter.
• PVDF polyvinylidene difluoride
• the samples were then serially diluted 1 :5 and 7 concentrations were added to cells that had been washed 2 times with Dulbecco's Phosphate Buffered Saline (DPBS) in triplicate in a volume of 400 pL.
• DPBS Dulbecco's Phosphate Buffered Saline
• the samples were incubated for 1 hour at 37°C with rocking every 15 to 20 minutes.
• 2 mL of the plaque assay media was added to the wells and the cultures were incubated for 48 hours at 35°C/5% CO2. After incubation, the cells were stained with crystal violet and the plaques were enumerated visually.
• the viricidal test was conducted at a concentration of 15 wt.%/vol and at 30 min. The process steps of centrifugation and filtration only reduced the viral load by about 0.25 log . Given this result, a subsequent titration was then conducted without and with the exposure of the virus to SisN4 for 30 minutes.
• the concentration for the titration without SisN4 was a priori selected to be 4.4 x 10 3 pfu/ml based on ISO 21702 (Measurement of antiviral activity on plastics and other non-porous surfaces). After 30 minutes of exposure to SisN4, no plaques formed on the MDCK cells. SisN4 was deemed to be 100% effective in inactivating Influenza A.
• FIG. 19 A direct comparison of the viral titers before and after exposure to the SisN4 powder for 30m is provided in FIG. 19.
• the data clearly demonstrate > 3.5log reduction in viral load after exposure to SisN4 (i.e., >99.98%).
• these tests demonstrated that exposure of SisN4 to MDCK cells had no adverse viability effects at concentrations less than 15 wt.%/vol or time periods of ⁇ 30 minutes.
• SisN4 At antiviral test conditions of 15 wt.%/vol SisN4 at 30 minutes exposure at a viral load of 4.4 x 10 3 pfu/ml, SisN4 inactivated essentially 100% of the exposed virions. SisN4 was found to be viricidal to Influenza A under these conditions.
• Example 5 Effect of a-Si'3N4 Powder on MDCK Cells and Influenza A
• a-SisN4 powder was first evaluated for toxicity to MDCK cells following exposure for 30 minutes, 24 hours, 48 hours and 72 hours.
• a 15 weight % (wt.%) suspension was prepared in 1.5 mL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2% fetal bovine serum (FBS).
• DMEM Dulbecco's Modified Eagle Medium
• FBS fetal bovine serum
• the a-SisN4 powder suspension prepared as described above was incubated for 30 minutes at room temperature with shaking. Following the incubation, the suspension was centrifuged for two minutes at 4°C at 12,000 rpm. The supernatant was further filtered through a 0.2-micron polyvinylidene difluoride (PVDF) filter and then serially diluted in 1 /2-logarithmic increments. Six (6) concentrations were added to the preplated cells in triplicate in a volume of 200 pL.
• PVDF polyvinylidene difluoride
• the plates were incubated for 30 minutes, 24, 48, and 72 hours at which time the cells were evaluated for cellular toxicity using the tetrazolium dye XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide), as described below.
• XTT 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide
• TC50 values for the test materials were derived by measuring the reduction of the tetrazolium dye XTT.
• XTT in metabolically active cells is metabolized by the mitochondrial enzyme NADPH oxidase to a soluble formazan product.
• XTT solution was prepared daily as a stock of 1 mg/mL in DMEM without additives.
• Phenazine methosulfate (PMS) solution was prepared at 0.15 mg/mL in Dulbecco's Phosphate Buffered Saline (DPBS) and stored in the dark at -20°C.
• DPBS Dulbecco's Phosphate Buffered Saline
• XTT/PMS stock was prepared immediately before use by adding 40 pL of PMS per mL of XTT solution.
• Fifty pL (504) of XTT/PMS was added to each well of the plate and the plate incubated for 4 hours at 37°C.
• the 4-hour incubation has been empirically determined to be within the linear response range for XTT dye reduction with the indicated numbers of cells for each assay.
• the plates were sealed and inverted several times to mix the soluble formazan product and the plate was read at 450 nm (650 nm reference wavelength) with a Molecular Devices SpectraMax Plus 384 96 well plate format spectrophotometer.
• MDCK cells were treated with 6 concentrations of the a-SisN4 powder ranging from 15 wt.% to 0.047 wt. % for 30 minutes, 24 hours, 48 hours and 72 hours.
• the viability of the MDCK cells is shown as function of a-SisN4 concentration (wt.%/mL). Following 30 minutes of exposure cells treated with all concentrations had viability greater than 90% except for cells treated with 4.7 wt.% and 15 wt.% which had 89% and 83% viability, respectively. At 24 hours viability of cell treated with each concentration remained above 92%.
• a-SisN4 powder at 15 wt.% was then evaluated for virucidal activity against Influenza A strain A/PR/8/34 in MDCK cells.
• a 15 wt.% suspension was prepared in 1.5 mL of virus diluted in DMEM with no additives.
• MDCK cells were plated in a 6-well plate at a density of 1 x 10 6 cells/well in a volume of 2 mL in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
• DMEM Minimum Essential Medium
• FBS fetal bovine serum
• the samples were centrifuged for two minutes at 4°C and 12,000 rpm, and further filtered through a 0.2- micron polyvinylidene difluoride (PVDF) filter.
• PVDF polyvinylidene difluoride
• the samples were then serially diluted 1 :5 and 7 concentrations were added to cells that had been washed 2 times with Dulbecco’s Phosphate Buffered Saline (DPBS) in triplicate in a volume of 400 mL.
• DPBS Dulbecco’s Phosphate Buffered Saline
• the samples were incubated for 1 hour at 37°C with rocking every 15 to 20 minutes.
• 2 mL of the plaque assay media was added to the wells and the cultures were incubated for 48 hours at 35°C/5% CO2.
• the cells were stained with crystal violet and the plaques were enumerated visually.
• the plaquing media was removed, and the monolayers were washed two times with DPBS.
• the cells were then fixed with 70% ethanol for 10 minutes at room temperature.
• the ethanol was removed, and 0.3% crystal violet solution was added to each well for 10 minutes at room temperature.
• the crystal violet was removed, and the monolayers were washed two times with DPBS to remove residual crystal violet.
• the monolayers were air-dried overnight prior to counting the plaques.
• the virucidal activity of 15 wt.% a-SisN4 powder was evaluated against Influenza virus A strain A/PR8/34 in MDCK cells.
• the target virus titer was 1 x 10 4 PFU/mL and the actual individual replicates were 3.1 x 10 3 , 3.8 x 10 3 , and 4.7 x 10 3 PFU/mL yielding a mean titer (and standard deviation) of 3.9 x 10 3 ⁇ 0.8 x 10 3 PFU/mL. This actual titer is within two-fold of the targeted PFU/mL.
• the a-SisN4 powder treated samples had one well with a single plaque which resulted in a PFU/mL of 4.1 .
• the log reduction was 2.98 and was calculated using the following equation: log (A/B) where A is untreated virus and B is treated virus.
• the percent reduction was 99.89% and was calculated using the following equation: (A-B) x 100/A where A is untreated virus and B is treated virus.
• a comparison of the viral titers before and after exposure to the a-SisN4 powder for 30m is provided in FIG. 21. Therefore, the a-SisN4 powder at 15 wt.% was virucidal to influenza A virus strain A/PR/8/34 following a 30-m inute exposure.
• Example 6 Influenza A Virucidal Activity by two forms of Si'3N4 powder in MDCK Cells [0111] A 5 and 10 wt.% suspension of a-SisN4 and p-SisN4 powder was prepared in 1.5 mL of virus diluted in DMEM with no additives.
• MDCK cells were plated in a 6-well plate at a density of 1 x 10 6 cells/well in a volume of 2 mL in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
• DMEM Dulbecco's Minimum Essential Medium
• FBS fetal bovine serum
• the samples were centrifuged for two minutes at 4°C and 12,000 rpm, and further filtered through a 0.2-micron polyvinylidene difluoride (PVDF) filter.
• PVDF polyvinylidene difluoride
• the samples were then serially diluted 1 :5 and 7 concentrations were added to cells that had been washed 2 times with Dulbecco's Phosphate Buffered Saline (DPBS) in triplicate in a volume of 400 pL.
• DPBS Dulbecco's Phosphate Buffered Saline
• the samples were incubated for 1 hour at 37°C with rocking every 15 to 20 minutes.
• 2 mL of the plaque assay media was added to the wells and the cultures were incubated for 48 hours at 35°C/5% CO2. After incubation, the cells were stained with crystal violet and the plaques were enumerated visually.
• the virucidal activity of 5 and 10 wt.% of a-SisN4 and p-SisN4 powder was evaluated against Influenza virus A strain AIPR8/34 in MDCK cells. This was performed in four individual experiments.
• the target virus titer was 1 x 10 4 PFU/mL.
• the log reduction was 2.4 and was calculated using the following equation: log10(NB) where A is untreated virus and B is treated virus.
• the percent reduction was 99.5 % and was calculated using the following equation: (A-B) x 100/A where A is untreated virus and B is treated virus.
• a doped SisN4 powder ([3-SiYAION) with a nominal composition of 90 wt.% a-Si3N4, 6 wt.% yttria (Y2O3), and 4 wt.% alumina (AI2O3) was prepared by aqueous mixing and spray-drying of the inorganic constituents, followed by sintering of the spray-dried granules ( ⁇ 1700°C for ⁇ 3 h), hot-isostatic pressing ( ⁇ 1600°C, 2 h, 140 MPa in N2), aqueous-based comminution, and freeze-drying.
• the resulting powder had a trimodal distribution with an average particle size of 0.8 ⁇ 1 .0 pm as shown in FIG. 22.
• Doping SisN4 with Y2O3 and AI2O3 densified the ceramic and converted it from its a- to /3-phase during sintering. The mechanism of densification is via dissolution of a-phase and subsequent precipitation of /3-phase grains facilitated by the formation of a transient intergranular liquid that solidifies during cooling.
• /3-SisN4 is therefore a composite composed of about 10 wt.% intergranular glass phase (IGP) and 90 wt.% crystalline /3- SisN4 grains.
• Vero green African monkey kidney epithelial cells were chosen for this analysis due to their ability to support high levels of SARS-CoV-2 replication and their use in antiviral testing. These cells were cultured in DMEM supplemented with 10% FBS, 1 % L-glutamine, and 1 % penicillin/streptomycin. Cells were maintained at 37°C and 5% CO2. SARS-CoV-2 isolate USA-WA1/2020 was obtained from BEI Resources. Vero cells were inoculated with SARS-CoV-2 (MOI 0.1 ) to generate viral stocks. Cell-free supernatants were collected at 72 hours post-infection and clarified via centrifugation at 10,000 rpm for 10 minutes and filtered through a 0.2 pm filter. Stock virus was titered according to the plaque assay protocol detailed below.
• the SisN4 powder was suspended in 1 mL DMEM growth media in microcentrifuge tubes. Tubes were vortexed for 30 seconds to ensure adequate contact and then placed on a tube revolver for either 1 , 5, or 10 minutes. At each time point, the samples were centrifuged, and the supernatant was collected and filtered through a 0.2 pm filter. Clarified supernatants were added to cells for either 24 or 48 hours. Untreated cells were maintained alongside as controls. Cells were tested at each time point using CellTiter Gio, which measures ATP production, to determine cell viability.
• SARS-CoV-2 was diluted in DMEM growth media to a concentration of 2 x 10 4 PFU/mL.
• Four mL of the diluted virus was added to tubes containing silicon nitride at 20, 15, 10, and 5% (w/v).
• the virus without SisN4 was processed in parallel as a control. Tubes were vortexed for 30 seconds to ensure adequate contact and then placed on a tube revolver for either 1 , 5, or 10 minutes, while a virus only control was incubated for the maximum 10 minutes.
• the samples were centrifuged, and the supernatant was collected and filtered through a 0.2 pm filter. The remaining infectious virus in the clarified supernatant was quantitated by plaque assay.
• step l SARS-CoV-2 virus was diluted in media.
• 4 mL of diluted virus was added to tubes containing silicon nitride at 20, 15, 10, or 5% (w/v).
• step 3 tubes were vortexed for 30 s to ensure adequate contact and the placed on a tube revolver for either 1 m, 5 m, or 10 m (virus only control was incubated for the maximum 10 m).
• step 4 at each time point, the samples were centrifuged, and the supernatant was collected and filtered through a 0.2 pm filter.
• clarified supernatant was used to perform plaque assays.
• Vero cells were plated at 2 x 10 5 cells/well in a 12-well plate on the day before the plaque assay. Clarified supernatants from the antiviral testing were serially diluted (10-fold) and 200 pL was added to Vero cells which were incubated for 1 hour at 37°C, 5% CO2.
• SisN4 was resuspended in cell culture media at 5, 10, 15, and 20% (w/v). Samples were collected at 1 , 5, and 10 minutes and added to Vero cells. Vero cell viability was measured at 24 and 48 hours post-exposure (FIG. 24A and 24B). No significant decrease in cell viability was observed at either 24 or 48 hours post-exposure with 5%, 10%, or 15% silicon nitride. A small impact on cell viability ( ⁇ 10% decrease) was observed at 48 hours in cells exposed to 20% SisN4.
• SARS-CoV-2 virions were exposed to SisN4 at these concentrations for 1 , 5, or 10 minutes. Following SisN4 exposure, the infectious virus remaining in each solution was determined through plaque assay. At each timepoint, the samples were centrifuged, and the supernatant was collected and filtered through a 0.2um filter. The clarified supernatant was used to perform plaque assay in duplicate. Virus processed in parallel but only exposed to cell culture media contained 4.2 x 10 3 PFU/mL. SARS-CoV-2 titers were reduced when exposed to all concentrations of SisN4 tested (FIGS.

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