CN116847851A - Antiviral compositions and devices and methods of use thereof - Google Patents

Antiviral compositions and devices and methods of use thereof Download PDF

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CN116847851A
CN116847851A CN202180092187.0A CN202180092187A CN116847851A CN 116847851 A CN116847851 A CN 116847851A CN 202180092187 A CN202180092187 A CN 202180092187A CN 116847851 A CN116847851 A CN 116847851A
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B·J·麦肯泰尔
B·S·巴尔
R·M·伯克
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Sintech
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Abstract

Antiviral compositions and devices, and methods of using the same to inactivate viruses in contact with the compositions and devices are described herein. The composition and/or device comprises silicon nitride at a concentration of 1wt.% to 15wt.% and the silicon nitride inactivates at least 85% of viruses in contact with the composition and/or device.

Description

Antiviral compositions and devices and methods of use thereof
Cross Reference to Related Applications
The present application claims priority from U.S. provisional application No. 63/143,370 filed on day 29 of 1 of 2021; the contents of the U.S. provisional application are incorporated by reference herein in their entirety.
Technical Field
The present disclosure relates to antiviral compositions, systems, methods, and devices. More specifically, the present disclosure relates to silicon nitride compositions, devices, and coatings for inactivation of viruses.
Background
There is a general need for safe and reliable inactivation or removal of viruses. There is a wide need to control pathogens that affect human health. There is a need for materials that not only have antiviral properties for human pharmacotherapy, but also for surface coatings and/or composites for various medical devices or instruments, examination tables, clothing, filters, masks, gloves, catheters, endoscopic instruments, and the like.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes a pandemic in COVID-19, remains viable and thus may be infectious on several materials. One strategy to prevent contaminant-mediated spread of covd-19 is to develop materials whose surface chemistry can spontaneously inactivate SARS-CoV-2.
Thus, there is a need for a safe and reliable method for inactivating and killing viruses that can be applied to medical devices, instruments, clothing, or other systems that may be in contact with the human body for prolonged periods of time.
Disclosure of Invention
Provided herein are embodiments of antiviral compositions comprising silicon nitride at a concentration of about 1wt.% to about 15wt.%, wherein the silicon nitride inactivates viruses that come into contact with the composition.
In some aspects, the virus may be contacted with the silicon nitride for a duration of at least 1 minute or at least 30 minutes. For example, the virus may be inactivated by at least 85% after contact with 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 alpha-Si 3 N 4 、β-Si 3 N 4 SiYAlON, beta-SiYAlON, siYON or SiAlON. The virus may be influenza A or SARS-CoV-2. In some aspects, the composition is a slurry, suspension A float, gel, spray, paint or toothpaste.
Further provided herein are embodiments of antiviral devices comprising silicon nitride at a concentration of about 1wt.% to about 15wt.%, wherein the silicon nitride inactivates viruses that are in contact with the composition.
In some aspects, the virus may be contacted with the silicon nitride for a duration of at least 1 minute or at least 30 minutes. For example, the virus may be inactivated by at least 85% after contact with 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 alpha-Si 3 N 4 、β-Si 3 N 4 SiYAlON, beta-SiYAlON, siYON or SiAlON. The virus may be influenza A or SARS-CoV-2. In some aspects, the apparatus may be a medical device, a medical instrument, an inspection station, a filter, a mask, a glove, a catheter, an endoscopic instrument, or a surface in daily contact. The device may be metallic, polymeric and/or ceramic, and the silicon nitride may be coated on or embedded in the surface of the device.
Also provided herein are embodiments of a method of preventing viral transmission, the method comprising: an antiviral device is contacted with a virus, wherein the device comprises silicon nitride at a concentration of about 1wt.% to about 15 wt.%.
In some aspects, the virus may be contacted with the silicon nitride for a duration of at least 1 minute or at least 30 minutes. For example, the virus may be inactivated by at least 85% after contact with 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 alpha-Si 3 N 4 、β-Si 3 N 4 SiYAlON, beta-SiYAlON, siYON or SiAlON. The virus may be influenza A or SARS-CoV-2.
Other aspects and iterations of the present invention are described more fully below.
Drawings
FIG. 1 is a schematic representation of influenza A virus.
Fig. 2A is an exposure to 0wt.%, 7.5wt.%, 15wt.%, and 30wt.% Si 3 N 4 Graphical representation of virus for up to 10 minutes.
FIG. 2B is a graph of the exposure to Si according to FIG. 2A for measurement 3 N 4 A graphical representation of the viability of virus-inoculated cells.
Fig. 3A is an exposure to 15wt.% Si 3 N 4 Graphical representation of viruses for 1 minute, 5 minutes, 10 minutes and 30 minutes.
FIG. 3B is a graph used to determine exposure of viruses to Si according to FIG. 3A 3 N 4 Graphical representation of the method of post-viability.
Fig. 4A is a graph according to fig. 2A exposed to 0wt.%, 7.5wt.%, 15wt.%, and 30wt.% Si 3 N 4 PFU/100 μl of influenza A for 10 min.
Fig. 4B is a graph of the exposure to 7.5wt.%, 15wt.% and 30wt.% Si according to fig. 2B 3 N 4 Graph of cell viability of influenza a vaccinated cells for up to 10 minutes.
FIG. 5 contains a process for treating a silicon substrate with Si that has been exposed to various concentrations 3 N 4 Is a photograph of cells inoculated at different ratios of virus to slurry.
Fig. 6A shows a fluorescence microscopy image of MDCK cells prior to inoculation.
Fig. 6B shows a fluorescence microscopy image of MDCK cells after inoculation with virus exposed to control.
FIG. 6C shows a process for exposure to 30wt.% Si 3 N 4 Fluorescent microscopy images of MDCK cells after virus inoculation of (c).
Fig. 7A is a room temperature exposure to 15wt.% Si 3 N 4 PFU/100 μl of influenza A up to 1, 5, 10 or 30 minutes.
FIG. 7B is a graph of Si exposed to 15wt.% at room temperature 3 N 4 Graph of cell viability of influenza a vaccinated cells for 1, 5, 10 or 30 minutes.
Fig. 8A is an exposure to 15wt.% Si at 4 deg.c 3 N 4 PFU/100 μl of influenza A up to 1, 5, 10 or 30 minutes.
FIG. 8B is a graph of Si exposure to 15wt.% at 4deg.C 3 N 4 For 1 min, 5 minGraph of cell viability of influenza a vaccinated cells at 10 min or 30 min.
Fig. 9A shows Raman spectra (Raman spectrum) of influenza a virus prior to inactivation.
Fig. 9B shows raman spectral changes of influenza a virus associated with chemical modification of RNA and hemagglutinin after inactivation after 1 minute of exposure.
FIG. 10 shows NH 3 Influenza a virus is inactivated by a basic transesterification mechanism.
FIG. 11 shows O-P-O stretching in a penta-coordinate phosphate group after inactivation.
Fig. 12A shows the vibration pattern of methionine in the hemagglutinin structure.
Fig. 12B shows the structural change of methionine in the presence of ammonia.
FIG. 13 shows the C-S stretch of methionine to homocysteine after inactivation.
Fig. 14A is an exposure to 15wt.% or 30wt.% Si 3 N 4 PFU/100 μl of feline calicivirus (Feline calicivirus) up to 1, 10, or 30 minutes.
Fig. 14B is a view of a 30wt.% Si exposure 3 N 4 Graph of cell viability of feline calicivirus vaccinated cells for 1, 10, 30, or 60 minutes.
Fig. 15A shows that H1 influenza a virus (nucleoprotein, NP) was stained red after 10 minutes of exposure to a slurry of 15wt.% silicon nitride and green after inoculation into a biogenic medium containing MDCK cells in the presence of filiform actin (F-actin).
Figure 15B shows NP-stained H1 influenza a virus from figure 15A.
Fig. 15C shows F-actin-stained MDCK cells from fig. 15A.
Fig. 16A shows that H1 influenza a virus (nucleoprotein, NP) was stained red without exposure to silicon nitride and green after inoculation into a biogenic medium containing MDCK cells in the presence of filiform actin (F-actin).
FIG. 16B shows NP-stained H1H1 influenza A virus from FIG. 16A.
Fig. 16C shows F-actin-stained MDCK cells from fig. 16A.
Fig. 17 shows a trimodal distribution of silicon nitride powder.
FIG. 18 shows the viability of MDCK cells as beta-Si 3 N 4 Concentration (wt.%/mL).
FIG. 19 shows that influenza A is exposed to Si 3 N 4 Direct comparison of viral titers before and after 30 minutes of powder.
FIG. 20 shows the viability of MDCK cells as alpha-Si 3 N 4 Concentration (wt.%/mL).
FIG. 21 shows exposure of influenza A to alpha-Si 3 N 4 Comparison of virus titers before and after 30 minutes of powder.
Fig. 22 shows the trimodal particle size distribution of the silicon nitride powder.
FIG. 23 is an overview of an antiviral test method.
Fig. 24A shows Vero cell viability measured 24 hours after exposure to silicon nitride at concentrations of 5wt.%/vol, 10wt.%/vol, 15wt.%/vol, or 20wt.%/vol (n=4) along with cell culture media of Wen Yoda m, 5m, and 10 m.
Fig. 24B shows Vero cell viability measured 48 hours after exposure to silicon nitride at concentrations of 5wt.%/vol, 10wt.%/vol, 15wt.%/vol, or 20wt.%/vol (n=4) along with cell culture media of Wen Yoda m, 5m, and 10 m.
FIG. 25A shows the titres expressed as PFU/mL of silicon nitride at concentrations of 5wt.%/vo1, 10wt.%/vol, 15wt.%/vo1 and 20wt.%/vol, wen Yoda m, 5m and 10m with SARS-CoV-2 virus diluted in cell culture medium.
FIG. 25B shows titers in% inhibition of silicon nitride at concentrations of 5wt.%/vol, 10wt.%/vol, 15wt.%/vol and 20wt.%/vol, as measured by% inhibition of SARS-CoV-2 virus diluted in cell culture medium, wen Yoda m, 5m and 10 m.
Detailed Description
Various embodiments of the present disclosure are discussed in detail below. While specific embodiments are discussed, it should be understood that this is done for illustrative purposes only. One skilled in the relevant art will recognize that other components and configurations may be used without departing from the spirit and scope of the disclosure. The following description and drawings are, accordingly, illustrative and should not be construed as limiting. Numerous specific details are described to provide a thorough understanding of the present disclosure. However, in some instances, well known or conventional details are not described in order to avoid obscuring the description.
Several definitions will now be presented that apply throughout this disclosure. Reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Furthermore, various features are described which may be exhibited by some embodiments and not by others.
As used herein, the terms "include," "have," and "include" are used in their open, non-limiting sense. The terms "a/an" and "the" are to be interpreted as covering a plurality as well as the singular. Thus, the term "mixture thereof (a mixture thereof)" also relates to "mixture thereof (mixtures therof)".
As used herein, "about" refers to a value, including an integer, fraction, percentage, etc., whether or not explicitly indicated. The term "about" generally refers to a range of values that would be considered equivalent to the recited value (e.g., having the same function or result), for example, ±0.5% to 1%, ±1% to 5% or ±5% to 10% of the recited value.
As used herein, the term "silicon nitride" includes α -Si 3 N 4 、β-Si 3 N 4 SiyalON, beta-SiYAlON, siYON, siAlON, or a combination thereof.
As used herein, "inactivation" or "inactivation" refers to viral inactivation, wherein the virus is prevented from contaminating the product or subject by completely removing or rendering the virus non-infectious.
As used herein, the term "device" or "component" includes materials, compositions, devices, surface coatings, and/or composites. In some examples, the instrument may comprise various medical devices or equipment, examination tables, clothing, filters, personal protective equipment such as masks and gloves, catheters, endoscopic instruments, surfaces with which viral persistent infections may promote the spread of disease, and the like. The device may be metallic, polymeric, and/or ceramic (e.g., silicon nitride and/or other ceramic materials).
As used herein, "contacting" means physically contacting or sufficiently close to a composition or device to be affected by the composition or device.
Within the context of the present disclosure and in the specific context of use of each term, the terms used in this specification generally have their ordinary meaning in the art. Alternative language and synonyms may be used for any one or more of the terms discussed herein, and no special significance should be made whether or not a term is specified or discussed herein. In some cases, synonyms for certain terms are provided. The recitation of one or more synonyms does not exclude the use of other synonyms. The examples used anywhere in this specification (including examples of any terms discussed herein) are illustrative only and are not intended to further limit the scope and meaning of the disclosure or any example terms. As such, the present disclosure is not limited to the various embodiments set forth in the present specification.
Additional features and advantages of the disclosure will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the principles disclosed herein. The features and advantages of the disclosure may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the principles set forth herein.
Provided herein are compositions comprising silicon nitride (Si 3 N 4 ) For use in antiviral devices, compositions and apparatus for inactivating viruses. Silicon nitride has unique surface chemistry that is biocompatible and provides many biomedical applications, including 1) osteogenesis, osteoinduction, osteoconduction, and bacteriostasis as is concurrent in spinal and dental implants; 2) Killing both gram positive and gram negative bacteria according to different mechanisms; 3) Inactivation of human and animal viruses, bacteria and fungi; and 4) the polymer or metal matrix composite, natural or man-made fibers, polymer or metal containing silicon nitride powder retains key silicon nitride bone restoration, bacteriostatic, antiviral and antifungal properties.
In an embodiment, the antiviral composition may comprise silicon nitride. For example, the antiviral composition may be a device comprising silicon nitride powder. In some embodiments, the antiviral device may be a monolithic component comprising up to 100% silicon nitride. Such an assembly may be fully dense, having no internal porosity, or such an assembly may be porous, having a porosity in the range of about 1% to about 80%. The unitary assembly may be used as a medical device or may be used in an apparatus where it may be desirable to inactivate viruses. In another embodiment, the antiviral composition may be incorporated into a device or into a coating to inactivate viruses on or within the device. In some embodiments, the antiviral composition may be a slurry comprising silicon nitride powder.
In some embodiments, the antiviral composition can inactivate or reduce the transmission of human viruses. Non-limiting examples of viruses that can be inactivated by the anti-pathogenic composition include coronaviruses (e.g., SARS-CoV-2, influenza A, H1N1, enteroviruses, and feline calicivirus). For example, a silicon nitride composition may be effective in inactivating influenza a virus. In another example, the silicon nitride composition can be effective to inactivate SARS-CoV-2 virus.
Silicon nitride may be anti-pathogenic in that it releases nitrogen-containing species when contacted with aqueous media or biological fluids and tissues. The surface chemistry of silicon nitride can be shown as follows:
Si 3 N 4 +6H 2 O→3SiO 2 +4NH 3
SiO 2 +2H 2 O→Si(OH) 4
nitrogen elutes faster (within minutes) than silicon because surface silanol is relatively stable. For viruses, it has surprisingly been found that silicon nitride can provide RNA cleavage by basic transesterification, which results in loss of genome integrity and viral inactivation. This may also reduce the activity of hemagglutinin. Ammonia elution and the consequent increase in pH inactivate viruses, bacteria and fungi. As shown in the examples, it was surprisingly found that each of the silicon nitrides inactivated coronaviruses and influenza a.
In one embodiment, the anti-pathogenic composition may exhibit elution kinetics that show: (i) Slowly but continuously eluting ammonia from a solid state rather than from a generally gaseous state; (ii) no harm or negative effect on cells; and (iii) elution increases intelligently with decreasing pH.
The use of copper (Cu), a historically recognized virucide, is limited by its cytotoxicity. Contrary to Cu, from Si 3 N 4 The ceramic devices or apparatus are biocompatible and non-toxic to humans. Si (Si) 3 N 4 Has the advantages of multifunction of the material; thus Si is 3 N 4 Can be incorporated into polymers, bioactive glasses, and even other ceramics to produce Si-retaining materials 3 N 4 Is a complex and coating of advantageous biocompatible and antiviral properties.
An antiviral device or apparatus may comprise a silicon nitride composition on at least a portion of the surface of the device for antiviral, antibacterial or antifungal action. In embodiments, the antiviral device may comprise a silicon nitride coating on at least a portion of the surface of the device. The silicon nitride coating may be applied as a powder to the surface of the device. In some examples, the silicon nitride powder may be filled, embedded, or impregnated in at least a portion of the device. In some embodiments, the powder may be micro-scale or nano-scale in size. The average particle size may be in the range of about 100nm to about 5 μm, about 300nm to about 1.5 μm, or about 0.6 μm to about 1.0 μm. In other embodiments, silicon nitride may be incorporated into the device. For example, the device may incorporate silicon nitride powder into the body of the device. In one embodiment, the device may be made of silicon nitride. In another embodiment, the composition may comprise a slurry or suspension of nitrogen particles.
The silicon nitride coating may be present on the surface of the device or within the device at a concentration of about 1wt.% to about 100 wt.%. In various embodiments, the coating may comprise about 1wt.%, 2wt.%, 5wt.%, 7.5wt.%, 8.3wt.%, 10wt.%, 15wt.%, 16.7wt.%, 20wt.%, 25wt.%, 30wt.%, 33.3wt.%, 35wt.%, or 40wt.% silicon nitride powder. In some examples, the coating may comprise about 10wt.% to about 20wt.% silicon nitride. In at least one example, the coating comprises about 15wt.% silicon nitride. In some embodiments, the silicon nitride may be embedded in (as a filter) or on the surface of a device or apparatus at a concentration of about 1wt.% to about 100 wt.%. In various embodiments, a device or apparatus may comprise about 1wt.%, 2wt.%, 5wt.%, 7.5wt.%, 8.3wt.%, 10wt.%, 15wt.%, 16.7wt.%, 20wt.%, 25wt.%, 30wt.%, 33.3wt.%, 35wt.%, 40wt.%, 50wt.%, 60wt.%, 70wt.%, 80wt.%, 90wt.% to 100wt.% silicon nitride. In some examples, the silicon nitride may be located on the surface of the device at a concentration of about 10wt.% to about 20 wt.%. In at least one example, the silicon nitride may be located on the surface of the device at a concentration of about 15wt.% silicon nitride.
In some embodiments, the antiviral composition may be a monolithic component composed of silicon nitride. Such an assembly may be fully dense, having no internal porosity, or such an assembly may be porous, having a porosity in the range of about 1% to about 80%. The unitary assembly may be used as a medical device or may be used in an apparatus where it may be desirable to inactivate viruses.
In various embodiments, the device or apparatus comprising silicon nitride for antiviral properties may be a medical device. Non-limiting examples of medical devices or instruments include orthopedic implants, spinal implants, pedicle screws, dental implants, indwelling catheters, endotracheal tubes, colonoscopy, and other similar devices.
In some embodiments, the silicon nitride may be incorporated into or applied as a coating to the following materials or devices for antiviral properties: such as polymers and fabrics, surgical gowns, tubing, clothing, air and water filters, masks, tables such as hospital examination and surgical tables, desks, fixtures, handles, knobs, toys, and filters such as air conditioning filters or toothbrushes. In some examples, the filter may be within a filtering device of an anesthesia machine, ventilator, or CPAP machine, such that an antimicrobial surface layer in the filter may trap lung pathogens as air enters and exits the infected lung.
In other embodiments, the silicon nitride powder may be incorporated into a composition including, but not limited to, a slurry, suspension, gel, spray, paint, or toothpaste. For example, adding silicon nitride to a slurry (e.g., paint) and then applying it to a surface can provide an antibacterial, antifungal, and antiviral surface. In other embodiments, silicon nitride may be mixed with water along with any suitable dispersants and slurry stabilizers and then applied by spraying the slurry onto various surfaces. An example of a dispersant is Dolapix a88.
In some embodiments, the silicon nitride may be included in the antiviral composition at a concentration of about 5wt.% to about 20 wt.%. In at least one example, the composition may comprise about 15wt.% silicon nitride. Alternatively, in some embodiments, the silicon nitride is included in the antiviral composition at a concentration of about 5wt.% to about 20 wt.%. In at least one example, the composition may comprise about 15wt.% silicon nitride. In one 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 a concentration of about 0.1wt.% up to about 70 wt.%. In some embodiments, the silicon nitride powder may be present in the slurry at a concentration of about 0.1wt.% to about 55 wt.%. In other embodiments, the silicon nitride may be incorporated into the organic suspension, gel, spray, and/or coating at a concentration of about 0.1wt.% to about 70wt.% or about 0.1wt.% to up to about 55 wt.%. In various embodiments, the slurry, organic suspension, gel, spray, and/or coating may comprise about 0.1wt.%, 0.5wt.%, 1wt.%, 1.5wt.%, 2wt.%, 5wt.%, 10wt.%, 15wt.%, 20wt.%, 25wt.%, 30wt.%, 35wt.%, 40wt.%, 45wt.%, 50wt.%, or 55wt.% silicon nitride.
In some embodiments, the composition includes a toothpaste and the silicon nitride is in the form of a powder that directly replaces the silicon dioxide powder found in standard toothpastes. The silicon nitride powder may replace the silicon dioxide powder in the toothpaste at a concentration of about 1wt.% to about 30 wt.%. In some examples, silicon nitride may be present in the toothpaste at a concentration of about 1wt.%, 5wt.%, 10wt.%, 15wt.%, 20wt.%, silicon nitride not only acts as an antiviral and antibacterial agent in the toothpaste, but it may also act as a polishing agent similar to silica.
Also provided herein are methods of inactivating pathogens by contacting the virus with an antiviral composition comprising silicon nitride. In embodiments, the method may comprise coating a device or apparatus with silicon nitride and contacting the coated apparatus with a virus. The coating apparatus may comprise applying silicon nitride powder to a surface of the apparatus. In other embodiments, the silicon nitride powder may be filled, incorporated, or impregnated within a device or apparatus.
Without being limited by a particular theory, the antiviral composition may reduce viral effects and reduce hemagglutinin activity by basic transesterification. Surprisingly, it was found that silicon nitride powder (i) significantly reduces viral action by disrupting RNA internucleotide linkages by means of basic transesterification and (ii) significantly reduces hemagglutinin activity, thus disrupting host cell recognition by denaturing protein structures on the viral surface, resulting in viral inactivation, whether or not viral envelope is present.
In one embodiment, the anti-pathogenic composition may exhibit elution kinetics that show: (i) Slowly but continuously eluting ammonia from a solid state rather than from a generally gaseous state; (ii) no harm or negative effect on cells; and (iii) elution increases intelligently with decreasing pH. Furthermore, the inorganicity of silicon nitride may be more beneficial than the use of petrochemical or organometallic bactericides, virucides and fungicides that are known to damage mammalian cells or have residual effects in soil, plants and vegetables or fruits.
Also provided herein is a method of treating or preventing a pathogen at a location in a human patient. For example, the pathogen may be a virus. The method may comprise contacting the patient with an apparatus, device or composition comprising silicon nitride. Without being limited to any one theory, silicon nitride inactivates viruses (e.g., coronaviruses such as SARS-CoV-2 or influenza A virus). The apparatus, device, or composition may comprise about 1wt.% to about 100wt.% silicon nitride. In some examples, the device or apparatus may comprise about 1wt.% to about 100wt.% silicon nitride on a surface of the device or apparatus. In an embodiment, the device or apparatus may be a monolithic silicon nitride ceramic. In another embodiment, the device or apparatus may comprise a silicon nitride coating, such as a silicon nitride powder coating. In another embodiment, the device or apparatus may incorporate silicon nitride into the body of the device. For example, silicon nitride powder may be incorporated or impregnated into the body of the device or apparatus using methods known in the art.
In some embodiments, the composition or device 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. In at least one example, the apparatus or device may be permanently implanted in the patient. In at least one example, the apparatus or device may be externally worn by a user. In another example, the device may be a high contact surface. In further examples, the device may be in continuous or continuous contact with the body fluid of the patient. The body fluid may be blood or gas (inhaled or exhaled).
In some embodiments, 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 contacting with the silicon nitride in the composition or device for at least 1 minute, at least 5 minutes, or at least 30 minutes. In at least one example, the virus is at least 85% inactivated after contact with the silicon nitride in the composition or device for at least 1 minute. In another example, the virus is at least 99% inactivated after contact with the silicon nitride in the composition or device for at least 30 minutes.
Examples
Example 1: effect of silicon nitride concentration on viral inactivation
To show the effect of silicon nitride concentration on viral inactivation, influenza a was exposed to various concentrations of Si 3 N 4 And (3) powder. To prepare silicon nitride, a weight of silicon nitride powder is mixed with pure distilled water. For example, 7.5g of silicon nitride is dispersed in 92.5g of pure distilled water. Viruses were added to this mixture at concentrations of 1:1, 1:10 and 1:100 viruses/mixture, respectively. These mixtures were then incubated at 4℃for 10 minutes with gentle agitation. Influenza a was exposed to 0wt.%, 7.5wt.%, 15wt.% and 30wt.% Si at 4 ℃ 3 N 4 For 10 minutes as shown in fig. 2A. The mixture was then filtered to remove the silicon nitride powder.
Then for Si 3 N 4 Effectiveness in inactivating influenza a Martin-darbea's kidney (Madin-Darby canine kidney) (MDCK) cells vaccinated with influenza a virus were observed. The remaining mixture was then inoculated into Petri dishes containing live MDCK cells in the biogenic medium. The number of viable MDCK cells was then counted using a staining method after 3 days of exposure. According to FIG. 2B, by exposure to Si 3 N 4 3 days after the influenza a inoculation of MDCK cells, the viability of the cells was determined.
Fig. 4A is an exposure to 0wt.%, 7.5wt.%, 15wt.%, and 30wt.% Si 3 N 4 PFU/100 μl of influenza A for 10 min. Fig. 4B is with exposure to 7.5wt.%, 15wt.% and 30wt.% Si 3 N 4 For 10 minutesCell viability of influenza a vaccinated cells.
Example 2: effect of exposure time and temperature on viral inactivation
To show the effect of silicon nitride on viral inactivation, influenza a was exposed to a fixed concentration of Si at various times and temperatures 3 N 4 Powder (15 wt.%). The mixture was then incubated at room temperature and 4 ℃ for 1 to 30 minutes with gentle agitation. For example, influenza a is exposed to 15wt.% Si at room temperature or 4 °c 3 N 4 For 1 minute, 5 minutes, 10 minutes, or 30 minutes, as shown in fig. 3A. Then for Si 3 N 4 The effectiveness in inactivating influenza a was observed on martin-darbeol kidney (MDCK) cells vaccinated with influenza a virus. According to FIG. 3B, by exposure to Si 3 N 4 3 days after inoculation of MDCK cells with influenza a virus, the viability of the cells was determined.
Fig. 7A is a room temperature exposure to 15wt.% Si 3 N 4 PFU/100 μl of influenza A up to 1, 5, 10 or 30 minutes. FIG. 7B is a graph of Si exposed to 15wt.% at room temperature 3 N 4 Graph of cell viability of influenza a vaccinated MDCK cells for 1, 5, 10 or 30 minutes.
Fig. 8A is an exposure to 15wt.% Si at 4 deg.c 3 N 4 PFU/100 μl of influenza A up to 1, 5, 10 or 30 minutes. FIG. 8B is a graph of Si exposure to 15wt.% at 4deg.C 3 N 4 Graph of MDCK cell viability for influenza a vaccinated up to 1, 5, 10 or 30 minutes.
Example 3: effect of silicon nitride on H1H1 influenza A inactivation
To show the effect of silicon nitride on the inactivation of virus, influenza a was exposed to a slurry of 15wt.% silicon nitride for 10 minutes.
Figures 15A-15C show that H1 influenza a virus (a/polis/8/1934H 1N1 (PR 8)) was stained red (nucleoprotein, NP) after inoculation into a medium of biological origin containing MDCK cells that was stained green in the presence of filiform actin (F-actin) present in all eukaryotic cells. Figures 16A-16C show the effect of viruses on MDCK cells in the absence of silicon nitride.
Example 4: evaluation of virucidal Activity of silicon nitride against influenza A Virus in MDCK cells
This study was designed to examine beta-silicon nitride (beta-Si) at the 30 minute incubation time point and a concentration of 15 wt./vol 3 N 4 ) Antiviral ability of the powder against influenza a virus. 15wt.% suspension was prepared in 1.5mL of virus diluted in DMEM without any additives.
Plaque assay methods were utilized. To adequately quantify plaque assays, the viability of madaidbis canine kidney cells (MDCK) as a result of exposure to varying concentrations of Si 3 N 4 A function of incubation period ranging from 30 minutes to 72 hours was evaluated. The results demonstrate that under preselected conditions, si 3 N 4 Is completely virucidal against influenza a virus, with a reduction in viral load of > 99.98%. The viability of MDCK cells was found to be time and dose dependent. For Si up to 15 wt.%/wt.% 3 N 4 At concentrations, substantially no loss of viability was observed. The change in survival at 15wt.% concentrations (i.e. survival of 83.3%, 59.7% and 44.0% respectively) was noted only at 24 hours, 48 hours and 72 hours.
Si used in this study 3 N 4 The nominal composition of the powder is 90wt.% alpha-Si 3 N 4 6wt.% yttria (Y) 2 O 3 ) And 4wt.% alumina (Al 2 O 3 ). It is prepared by mixing inorganic components with water and spray drying, then sintering the spray dried granules (about 1700 ℃ C. For about 3 hours), hot isostatic pressing (under N 2 About 1600 c, 2h,140 mpa), water-based comminution and freeze-drying. The resulting powder had a trimodal distribution with an average particle size of 0.8.+ -. 1.0 μm, as shown in FIG. 17. By Y 2 O 3 And Al 2 O 3 Doped Si 3 N 4 Helping to densify the ceramic and transform it from the alpha phase to the beta phase in the sintered device. The mechanism of densification is through dissolution of the alpha phase and subsequent precipitation of beta phase grains Is promoted by the formation of transient intergranular liquid that solidifies in the cooling device. Thus, beta-Si 3 N 4 Is formed from about 10wt.% of an inter-crystalline glassy phase (IGP) and 90wt.% of crystalline beta-Si 3 N 4 Composite material composed of crystal grains.
Three consecutive assays were performed in this study: (1) MDCK viability testing; (2) Influenza a supernatant titration test with and without centrifugation and filtration; (3) 15wt.%/wt Si was used 3 N 4 Virus titration was performed as a virus inhibitor with a latency of 30m.
In FIG. 18, the viability of MDCK cells as β -Si is shown 3 N 4 Concentration (wt.%/mL). Starting from 15wt.%, serial dilutions were performed to reach 0.047wt.%. At lower concentrations, cell viability was typically > 80% for all time points up to 72 hours. It should also be noted that for all concentrations except 15wt.%, cell viability generally increases with exposure time. At 15wt.% and 30 minutes exposure, cell viability was about 94.5%.
Twenty four hours after the determination of MDCK cell viability, MDCK cells were plated in 6-well plates at 1 x 10 6 The density of individual cells/wells was plated in 2mL volume of minimum essential medium of Dunaliella (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). On the day of the assay, a silicon nitride containing 15wt.% was used at 1X 10 4 Triplicate samples of virus diluted in DMEM without additives PFU/mL were incubated at room temperature with shaking for 30 minutes. After incubation, the samples were centrifuged at 4 ℃ and 12,000rpm for two minutes and further filtered through a 0.2 micron polyvinylidene fluoride (PVDF) filter. The samples were then pressed 1:5 serial dilutions and 7 concentrations were added to cells washed 2 times with Du's Phosphate Buffered Saline (DPBS) in triplicate at a volume of 400. Mu.L. The samples were incubated at 37℃for 1 hour with shaking every 15 to 20 minutes. After incubation, 2mL of plaque assay medium was added to the wells and the culture was incubated at 35 ℃/5% co 2 Lower Wen Yoda hours. After incubation, cells were stained with crystal violet and plaques were counted visually.
On the day of staining, the plagued medium was removed and the monolayer cells were washed twice with DPBS. Cells were then fixed with 70% ethanol for 10 min at room temperature. Ethanol was removed and 0.3% crystal violet solution was added to each well for 10 minutes at room temperature. After incubation, crystal violet was removed and the monolayer was washed twice with DPBS to remove residual crystal violet. The monolayers were air-dried overnight before plaque counting.
The virucidal test was performed at a concentration of 15 wt./vol and at 30 min. The process steps of centrifugation and filtration only reduce viral load by about 0.25log 10 . In view of this result, the virus is then not exposed to Si and is not exposed to Si 3 N 4 Subsequent titrations were performed up to 30 minutes. Based on ISO 21702 (measured for antiviral activity of plastics and other nonporous surfaces), si is not used 3 N 4 Is a priori selected to be 44 x 10 3 pfu/ml. On exposure to Si 3 N 4 After 30 minutes, no plaque formed on MDCK cells. Si (Si) 3 N 4 Is considered to be 100% effective in inactivating influenza a virus. The exposure to Si is provided in FIG. 19 3 N 4 Direct comparison of virus titers before and after 30m of powder. The data clearly demonstrate exposure to Si 3 N 4 Subsequent viral load reduction > 3.5log 10 (i.e., > 99.98%).
In summary, these tests demonstrate that Si is added at concentrations below 15wt.%/vol or for periods of time less than or equal to 30 minutes 3 N 4 Exposure to MDCK cells did not have adverse viability effects. At 15wt.%/vol Si 3 N 4 Under antiviral test conditions of 4.4X10) 3 Exposure to pfu/ml viral load for 30 min, si 3 N 4 Essentially 100% of the exposed virions are inactivated. Under these conditions, si is found 3 N 4 Is virucidal against influenza a virus.
Example 5: alpha-Si 3 N 4 Effects of powders on MDCK cells and influenza A
After exposure for 30 minutes, 24 hours, 48 hours and 72 hours, the alpha-Si was first evaluated 3 N 4 Toxicity of the powder to MDCK cells. In the supplement of 2%A 15 wt% (wt%) suspension was prepared in 1.5mL of Dulcitol Modified Eagle's Medium (DMEM) of Fetal Bovine Serum (FBS).
Twenty-four hours prior to adding the sample to the cells, a-Si prepared as described above was added 3 N 4 The powder suspension was incubated at room temperature for 30 minutes by shaking. After incubation, the suspension was centrifuged at 12,000rpm for two minutes at 4 ℃. The supernatant was further filtered through a 0.2 micron polyvinylidene fluoride (PVDF) filter and then serially diluted in 1/2 log increments. Six (6) concentrations were added to the pre-plated cells in triplicate in a volume of 200 μl. The plates were incubated for 30 min, 24 hr, 48 hr and 72 hr, at which time tetrazolium dye XTT (2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl) -5- [ (phenylamino) carbonyl was used]-2H-tetrazolium hydroxide) was evaluated for cytotoxicity as described below.
The TC50 value of the test material is obtained by measuring the degree of 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 solutions were prepared daily in DMEM without additives as 1mg/mL stock. Phenazine Methosulfate (PMS) solutions were prepared in Du's Phosphate Buffered Saline (DPBS) at a concentration of 0.15mg/mL and stored in the dark at-20 ℃. The XTT/PMS stock was prepared immediately prior to use, with 40 μl of PMS added per mL of XTT solution. Fifty μl (504) of XTT/PMS was added to each well of the plate, and the plate was incubated at 37 ℃ for 4 hours. The 4 hour incubation was empirically determined to be within the linear response range of XTT dye reduction, with the number of cells per assay specified. The plates were sealed and inverted several times to mix the soluble formazan product and read at 450nm (650 nm reference wavelength) using a molecular device spectromax Plus 384 96-well plate spectrophotometer.
MDCK cells use 6 concentrations of α -Si ranging from 15wt.% to 0.047wt.% 3 N 4 Powder treatment was carried out for 30 minutes, 24 hours, 48 hours and 72 hours. In FIG. 20, the viability of MDCK cells as α -Si is shown 3 N 4 Concentration (wt.%/mL). After 30 minutes of exposure, the viability of the cells treated with all concentrations was greater than 90%, butCells treated with 4.7wt.% and 15wt.% had 89% and 83% viability, respectively. Cell viability was still higher than 92% at 24 hours with each concentration. In the cells treated with 1.5wt.%, 4.7wt.% and 15wt.%, viability at 48 hours was reduced to below 90% (89.1%, 88.7% and 74.0%, respectively), but only cells treated with 15wt.% were less than 90% (87.5%) at 72 hours.
Then 15wt.% of alpha-Si is evaluated 3 N 4 Virucidal activity of the powder against influenza A virus strain A/PR/8/34 in MDCK cells. 15wt.% suspension was prepared in 1.5mL of virus diluted in DMEM without any additives.
Twenty-four hours prior to addition of virus and sample to cells, MDCK cells were plated in 6-well plates at 1 x 10 6 The density of individual cells/wells was plated in 2mL volume of minimum essential medium of Dunaliella (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). On the day of the assay, will contain 15wt.% of alpha-Si 3 N 4 1×10 of (C) 4 Triplicate samples of virus diluted in DMEM without additives PFU/mL were incubated at room temperature with shaking for 30 minutes. After incubation, the samples were centrifuged at 4 ℃ and 12,000rpm for two minutes and further filtered through a 0.2 micron polyvinylidene fluoride (PVDF) filter. Samples were then serially diluted 1:5 and 7 concentrations were added to cells washed 2 times with Du's Phosphate Buffered Saline (DPBS) in triplicate in a volume of 400 mL. The samples were incubated at 37℃for 1 hour with shaking every 15 to 20 minutes. After incubation, 2mL of plaque assay medium was added to the wells and the culture was incubated at 35℃C/5% CO 2 Lower Wen Yoda hours. After incubation, cells were stained with crystal violet and plaques were counted visually.
On the day of staining, the spotting medium was removed and the monolayer cells were washed twice with DPBS. Cells were then fixed with 70% ethanol for 10 min at room temperature. Ethanol was removed and 0.3% crystal violet solution was added to each well for 10 minutes at room temperature. After incubation, crystal violet was removed and the monolayer was washed twice with DPBS to remove residual crystal violet. The monolayers were air-dried overnight before plaque counting.
15wt.% alpha-Si was evaluated 3 N 4 Virucidal activity of the powder against influenza A virus strain A/PR8/34 in MDCK cells. Target virus titer 1×10 4 PFU/mL, and the actual individual replica is 3.1X10 3 、3.8×10 3 And 4.7X10 3 PFU/mL, resulting in an average titer (and standard deviation) of 3.9X10 3 ±0.8×10 3 PFU/mL. This actual titer is within twice the target PFU/mL. Through alpha-Si 3 N 4 The powder-treated sample had one well with a single plaque resulting in a PFU/mL of 4.1.
log reduction was 2.98 and calculated using the following equation: log of 10 (a/B), wherein a is an untreated virus and B is a treated virus. The percentage reduction was 99.89% and was calculated using the following equation: (a-B) x100/a, wherein a is an untreated virus and B is a treated virus. FIG. 21 provides for the exposure to α -Si 3 N 4 Comparison of virus titers before and after 30m of powder. Thus, 15wt.% of α -Si 3 N 4 The powder was virucidal to influenza a virus strain a/PR/8/34 after 30 minutes of exposure.
Example 6: two forms of Si 3 N 4 Virucidal Activity of the powder against influenza A Virus in MDCK cells
Preparation of 5wt.% and 10wt.% of alpha-Si in 1.5mL of virus diluted in DMEM without any additives 3 N 4 And beta-Si 3 N 4 Powder suspension.
Twenty-four hours prior to addition of virus and sample to cells, MDCK cells were plated in 6-well plates at 1 x 10 6 The density of individual cells/wells was plated in 2mL volume of minimum essential medium of Dunaliella (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). On the day of the assay, will contain 10wt.% and 5wt.% of alpha-Si 3 N 4 And beta-Si 3 N 4 Powder 1X 10 4 Triplicate samples of virus diluted in DMEM without additives PFU/mL were incubated at room temperature with shaking for 30 minutes. After incubation, the samples were centrifuged at 12,000rpm at 4℃for two minutes and passed through 0.2 μm for aggregationThe vinylidene fluoride (PVDF) filter was further filtered. The samples were then pressed 1:5 serial dilutions and 7 concentrations were added to cells washed 2 times with Du's Phosphate Buffered Saline (DPBS) in triplicate at a volume of 400. Mu.L. The samples were incubated at 37℃for 1 hour with shaking every 15 to 20 minutes. After incubation, 2mL of plaque assay medium was added to the wells and the culture was incubated at 35 ℃/5% co 2 Lower Wen Yoda hours. After incubation, cells were stained with crystal violet and plaques were counted visually.
On the day of staining, the plagued medium was removed and the monolayer cells were washed twice with DPBS. Cells were then fixed with 70% ethanol for 10 min at room temperature. Ethanol was removed and 0.3% crystal violet solution was added to each well for 10 minutes at room temperature. After incubation, crystal violet was removed and the monolayer was washed twice with DPBS to remove residual crystal violet. The monolayers were air-dried overnight before plaque counting.
5wt.% and 10wt.% of alpha-Si were evaluated 3 N 4 And beta-Si 3 N 4 Virucidal activity of the powder against influenza A virus strain AIPR8/34 in MDCK cells. This was done in four separate experiments. Target virus titer 1×10 4 PFU/mL。
In the first experiment, the untreated virus samples were repeated 5.3X10 individually 3 、5.9×10 3 And 4.1X10 3 PFU/mL, resulting in an average titer (and standard deviation) of 5.1X10 3 ±0.9×10 3 PFU/mL. With 5wt.% and 10wt.% beta-Si 3 N 4 Treatment of the virus for 10 minutes resulted in a PFU/mL of < 21 with 10wt.% of the virus and a month.PFU/mL of 21 with 5wt.% of the virus (1 plaque formed). In this sample, the log reduction was 2.4 and calculated using the following equation: log10 (NB), where a is untreated virus and B is treated virus. The percentage reduction was 99.5% and was calculated using the following equation: (a-B) x100/a, wherein a is an untreated virus and B is a treated virus.
In the second experiment, the untreated virus samples were repeated 7.5X10 individually 3 、7.2×10 3 And 5.0X10 3 PFU/mL, resulting in an average titer (and standard deviation) of 6.6X10 3 ±1.4×10 3 PFU/mL. With 5wt.% and 10wt.% beta-Si 3 N 4 The PFU/mL of both virus production treated for 5 minutes was < 21.
In the third experiment, 6.9X10 s alone were repeated 3 、7.8×10 3 And 5.0X10 3 PFU/mL, resulting in an average titer (and standard deviation) of 6.6X10 3 ±1.4x10 3 PFU/mL. With 5wt.% and 10wt.% of alpha-Si 3 N 4 The PFU/mL of both virus production treated for 10 minutes was < 21.
In the fourth experiment, 8.8X10 s alone were repeated 3 、1.0×10 4 And 7.5X10 3 PFU/mL, resulting in an average titer (and standard deviation) of 8.8X10 3 ±1.3×10 3 PFU/mL. With 5wt.% and 10wt.% of alpha-Si 3 N 4 The PFU/mL of both virus production treated for 5 minutes was < 21.
In each experiment, the actual titer determined for the untreated virus control was within twice the target PFU/mL. With 5wt.% and 10wt.% of alpha-Si 3 N 4 And beta-Si 3 N 4 Treatment of the virus for both 5 and 10 minutes with the powder resulted in PFU/mL < 1 (no plaque observed), but 5wt.% of beta-Si 3 N 4 The powder was treated for 10 minutes except for the sample which had one well with a single plaque, resulting in a PFU/mL of 21.
Example 7: silicon nitride in vitro inactivation of SARS-CoV-2
Preparation of an alpha-Si with nominal composition of 90wt.% by mixing with Water and spray drying an inorganic composition 3 N 4 6wt.% yttria (Y) 2 O 3 ) And 4wt.% alumina (Al 2 O 3 ) Is doped with Si 3 N 4 Powder (beta-Siyalon) and then sintered spray dried granules (about 1700 ℃ C., about 3 h), hot isostatic pressed (under N 2 About 1600 c, 2h,140 mpa), water-based comminution and freeze-drying. The resulting powder had a trimodal distribution with an average particle size of 0.8.+ -. 1.0 μm, as shown in FIG. 22. By Y 2 O 3 And Al 2 O 3 Doped Si 3 N 4 The ceramic is densified and transformed from the alpha phase to the beta phase during sintering. The mechanism of densification is through dissolution of the alpha phase and subsequent precipitation of beta phase grains, facilitated by the formation of transient inter-crystalline liquids that solidify in the cooling device. Thus, beta-Si 3 N 4 Is formed from about 10wt.% of an inter-crystalline glassy phase (IGP) and 90wt.% of crystalline beta-Si 3 N 4 Composite material composed of crystal grains.
Vero green african monkey kidney epithelial cells were chosen for this analysis because of their ability to support high levels of SARS-CoV-2 replication and their use in antiviral assays. These cells were cultured in DMEM supplemented with 10% fbs, 1% l-glutamine and 1% penicillin/streptomycin. The cells were maintained at 37℃and 5% CO 2 And (3) downwards. SARS-CoV-2 isolate USA-WA1/2020 is available from BEI Resources Inc. Vero cells were inoculated with SARS-CoV-2 (MOI 0.1) to produce a virus stock. Cell-free supernatant was collected 72 hours post infection and clarified by centrifugation at 10,000rpm for 10 minutes and filtered through a 0.2 μm filter. Stock viruses were titrated according to the plaque assay protocol detailed below.
Si is mixed with 3 N 4 The powder was suspended in 1mL DMEM growth medium in a microcentrifuge tube. The tube was vortexed for 30 seconds to ensure adequate contact, and then the tube was placed on a tube rotator mixer for 1, 5, or 10 minutes. At each time point, the samples were centrifuged and the supernatant was collected and filtered through a 0.2 μm filter. The clarified supernatant was added to the cells for 24 or 48 hours. Untreated cells were maintained aside as controls. Cells were tested at each time point using CellTiter Glo to measure ATP production to determine cell viability.
SARS-CoV-2 was diluted to 2X 10 in DMEM growth medium 4 PFU/mL concentration. Four mL of diluted virus were added to tubes containing 20%, 15%, 10% and 5% (w/v) silicon nitride. Parallel treatment of Si-free 3 N 4 As a control. The tube was vortexed for 30 seconds to ensure adequate contact, and then placed on a tube rotator mixer,for 1 minute, 5 minutes or 10 minutes, whereas the virus-only control was incubated for a maximum of 10 minutes. At each time point, the samples were centrifuged and the supernatant was collected and filtered through a 0.2 μm filter. The infectious virus remaining in the clarified supernatant was quantified by plaque assay. An overview of the antiviral testing method is provided in fig. 23. In step 1, SARS-CoV-2 virus is diluted in culture medium. In step 2, 4mL of diluted virus is added to a tube containing 20%, 15%, 10% or 5% (w/v) silicon nitride. In step 3, the tube was vortexed for 30s to ensure adequate contact, and then the tube was placed on a tube rotator mixer for 1m, 5m or 10m (only virus control will be incubated for up to 10 m). In step 4, at each time point, the samples were centrifuged and the supernatant was collected and filtered through a 0.2 μm filter. In step 5, the clarified supernatant is used to perform a plaque assay. Samples were serially diluted (10-fold) and added to fresh Vero for incubation for 1h, shaking every 15m, and then covered with agarose medium and incubated for 48h. After 48h incubation, cells were fixed with 10% fa and counted with crystal violet staining.
One day before plaque assay, vero cells were plated at 2X 10 5 Individual cells/well were seeded in 12-well plates. Clear supernatants from anti-virus assays were serially diluted (10-fold) and 200 μl was added to Vero cells at 37 ℃, 5% co 2 Incubate for 1 hour. The plates were shaken every 15 minutes to ensure adequate coverage and 0.6% agarose and 2 XEMEM supplemented with 5% FBS, 2% penicillin/streptomycin, 1% nonessential amino acids (VWR, catalog No. 45000-700), 1% sodium pyruvate and 1% L-glutamine were added to the cells at a 1:1 ratio at 1 hour, then at 37 ℃, 5% CO 2 Lower Wen Yoda hours. After incubation, cells were fixed with 10% formaldehyde and stained with 2% crystal violet in 20% ethanol for counting.
Test Si 3 N 4 Impact on eukaryotic cell viability. Si (Si) 3 N 4 Resuspended at 5%, 10%, 15% and 20% (w/v) in cell culture medium. Samples were collected at 1, 5 and 10 minutes and added to Vero cells. 24 and after exposureVero cell viability was measured for 48 hours (fig. 24A and 24B). No significant decrease in cell viability was observed 24 or 48 hours after exposure to 5%, 10% or 15% silicon nitride. On exposure to 20% Si 3 N 4 A small effect on cell viability (about 10% drop) was observed at 48 hours. Interestingly, an increase in Vero cell viability of about 10% was observed at 48 hours in the 5% -10 min and 10% -10 min samples (fig. 24B), indicating Si 3 N 4 It is possible to stimulate cell growth or cell metabolism under these conditions. These data indicate Si 3 N 4 The impact on Vero cell health and viability is minimal and up to 20 wt./vol.
In view of 5%, 10%, 15% and 20% Si 3 N 4 Vero cells were non-toxic and thus tested for antiviral at these concentrations. SARS-CoV-2 virions are exposed to Si at these concentrations 3 N 4 For 1 minute, 5 minutes or 10 minutes. In Si 3 N 4 After exposure, residual infectious virus in each solution was determined by plaque assay. At each time point, the samples were centrifuged and the supernatant was collected and filtered through a 0.2um filter. Clear supernatants were used for plaque assays in duplicate. Parallel processing but only exposed to the content of 4.2×10 3 PFU/mL cell culture medium. When exposed to Si at all concentrations 3 N 4 When tested, SARS-CoV-2 titer was decreased (FIGS. 25A and 25B). This inhibition was dose dependent with SARS-CoV-2 exposure for 1 min, 5% Si 3 N 4 Reducing virus titer by about 0.8log 10 ，10％Si 3 N 4 Reduced by about 1.2log 10 ，15％Si 3 N 4 Reduced by 1.4log 10 And 20% Si 3 N 4 Reduced by 1.7log 10 (FIG. 25A). Similar results were observed with samples of 5 minutes and 10 minutes. This reduction in viral titer corresponds to a reduction in 5% Si 3 N 4 Lower 85% viral inhibition at 10% Si 3 N 4 93% at 15% Si 3 N 4 Lower 96%, at 20% Si 3 N 4 Lower 98% viral inhibition (fig. 25B). Longer higher Si 3 N 4 The concentration will increase the inhibition-producing at 20% Si 3 N 4 And 99.6% viral inhibition at 10 min exposure (figure 25B). These data indicate Si 3 N 4 Has strong antiviral effect on SARS-CoV-2.
Surprisingly, it was found that at 5% Si 3 N 4 Exposure to the solution for one minute resulted in 85% inactivation of SARS-CoV-2, while Vero cell viability was barely affected even after 48 hours of exposure to the same material at 20% concentration.
While several embodiments have been described, it will be understood by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the invention. In other instances, well known processes and elements have not been described in detail in order to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the invention.
Those skilled in the art will appreciate that the presently disclosed embodiments are taught by way of example and not limitation. Accordingly, what is included in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all of the generic and specific features described herein as well as all statements of the scope of the inventive methods and systems which, as a matter of language, might be said to fall therebetween.

Claims (30)

1. An antiviral composition comprising silicon nitride at a concentration of 1wt.% to 15wt.%, wherein the silicon nitride inactivates at least 85% of viruses in contact with the composition for at least 1 minute.
2. The antiviral composition of claim 1, wherein the virus is contacted with the silicon nitride for a duration of at least 5 minutes.
3. The antiviral composition of claim 1, wherein the virus is contacted with the silicon nitride for a duration of at least 30 minutes.
4. The antiviral composition of claim 1, wherein the silicon nitride is present at a concentration of less than or equal to about 10 wt.%.
5. The antiviral composition of claim 1, wherein the silicon nitride comprises a-Si 3 N 4 、β-Si 3 N 4 SiYAlON, beta-SiYAlON, siYON or SiAlON.
6. The antiviral composition of claim 1, wherein the virus is influenza a, enterovirus, or SARS-CoV-2.
7. The antiviral composition of claim 1, wherein the virus is at least 99% inactivated after contact with the silicon nitride for at least 30 minutes.
8. The antiviral composition of claim 1, wherein the composition comprises a slurry, suspension, gel, spray, paint, or toothpaste.
9. The antiviral composition of claim 8, wherein the composition comprises a toothpaste and the silicon nitride is in the form of a powder that directly replaces the silica powder found in standard toothpastes.
10. An antiviral device comprising silicon nitride, wherein the silicon nitride inactivates at least 85% of viruses in contact with the device for at least 1 minute.
11. The antiviral device of claim 10, wherein the virus is in contact with the silicon nitride for a duration of at least 5 minutes.
12. The antiviral device of claim 10, wherein the virus is in contact with the silicon nitride for a duration of at least 30 minutes.
13. The antiviral apparatus of claim 10, wherein the antiviral apparatus is a monolithic silicon nitride device comprising up to 100wt.% silicon nitride.
14. The antiviral apparatus of claim 10, wherein the silicon nitride is present at a concentration of 1wt.% to about 15 wt.%.
15. The antiviral apparatus of claim 14, wherein the silicon nitride is present at a concentration of less than or equal to about 10 wt.%.
16. The antiviral apparatus of claim 10, wherein the silicon nitride comprises a-Si 3 N 4 、β-Si 3 N 4 SiYAlON, beta-SiYAlON, siYON or SiAlON.
17. The antiviral device of claim 10, wherein the virus is influenza a, enterovirus, or SARS-CoV-2.
18. The antiviral device of claim 10, wherein the virus is at least 99% inactivated after contact with the silicon nitride for at least 30 minutes.
19. The antiviral apparatus according to claim 10, wherein the antiviral apparatus is a medical device, a medical instrument, an examination table, a filter, a mask, a glove, a catheter, an endoscopic instrument, or a surface in daily contact.
20. The antiviral device of claim 10, wherein the device comprises a substrate having a metal composition, a polymer composition, and/or a ceramic composition, and the silicon nitride is coated on or embedded in a surface of the substrate.
21. A method of preventing the transmission of a virus, the method comprising:
contacting the antiviral device with the virus for at least 1 minute,
wherein the antiviral device comprises silicon nitride at a concentration of 1wt.% to 100wt.%, and
wherein the silicon nitride inactivates at least 85% of viruses in contact with the antiviral device.
22. The method of claim 21, wherein the silicon nitride inactivates the virus.
23. The method of claim 21, wherein the virus is contacted with the silicon nitride for a duration of at least 5 minutes.
24. The method of claim 21, wherein the virus is contacted with the silicon nitride for a duration of at least 30 minutes.
25. The method of claim 21, wherein the antiviral apparatus is a monolithic silicon nitride device comprising up to 100wt.% silicon nitride.
26. The method of claim 21, wherein the silicon nitride is present at a concentration of 1wt.% to about 15 wt.%.
27. The method of claim 26, wherein the silicon nitride is present at a concentration of less than or equal to 10 wt.%.
28. The method of claim 21, wherein the silicon nitride comprises a-Si 3 N 4 、β-Si 3 N 4 SiYAlON, beta-SiYAlON, siYON or SiAlON.
29. The method of claim 21, wherein the virus is influenza a, enterovirus, or SARS-CoV-2.
30. The method of claim 21, wherein the virus is at least 99% inactivated after contact with the silicon nitride for at least 30 minutes.
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