WO2022125797A1 - Nitride based antipathogenic compositions and devices and methods of use thereof - Google Patents
Nitride based antipathogenic compositions and devices and methods of use thereof Download PDFInfo
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- WO2022125797A1 WO2022125797A1 PCT/US2021/062650 US2021062650W WO2022125797A1 WO 2022125797 A1 WO2022125797 A1 WO 2022125797A1 US 2021062650 W US2021062650 W US 2021062650W WO 2022125797 A1 WO2022125797 A1 WO 2022125797A1
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- nitride
- pathogen
- composition
- virus
- silicon nitride
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Definitions
- the present disclosure generally relates to antipathogenic compositions and devices, and in particular to systems and methods for nitride-based antipathogenic compositions and devices.
- nitride based compositions or devices for inactivating viruses, bacteria, and/or fungi comprise aluminum nitride, boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorus nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zirconium nitride, various forms of silicon nitride, or combinations thereof.
- methods for inactivating pathogens by contacting a virus, bacteria, and/or fungus with an antipathogenic nitride based composition or device disclosed herein.
- FIGS. 2A-2D are graphical representations that show viral RNA that has underwent severe degradation after exposure to copper or nitride particles.
- virus suspensions were exposed to Cu, AIN and SisN4 powders for 1 minute, and viral RNA in supernatants and on particles were evaluated using viral N gene “set 1” and “set 2” primers, respectively. Data collected on supernatants and pellet samples are given in comparison with the amount of viral N gene RNA in suspension that was left untreated.
- FIGS. 3A-3E are images showing SisN4 suppressed virus infection without affecting cell viability in which Cu killed the cells.
- VeroE6/TMPRSS2 cells were inoculated with (FIG. 3A) unexposed virions, and virions 10-minute UTE-exposed to Si 3 N 4 (FIG. 3B), AIN (FIG. 3C), and Cu (FIG. 3D).
- FIGS. 5A-5G are graphical representations of Raman spectra for: (a) uninfected cells (FIG. 5A) (i.e. , unexposed to virions), and cells infected with SARS- CoV-2 virions exposed for 10 minutes to (b) SisN4 (FIG. 5B), (c) AIN (FIG. 5C), and (d) Cu (FIG. 5D); in FIG. 5E, Raman spectrum of cells infected by unexposed virions (negative control).
- FIG. 5A uninfected cells
- FIG. 5B unexposed to virions
- 5F a plot of the average intensity of the two tryptophan T1 and T2 bands (at 756 and 875 cm-1 , respectively) as a function of fraction of infected cells by virions unexposed and exposed for 10-min to different particles (cf. labels); in the inset, the structure of N’-formylkynurenine, an intermediate in the catabolism of tryptophan upon enzymatic IDO reaction.
- FIG. 5G a graphical representation shows three possible conformations of tyrosine-based peptides that can justify the disappearance of ring vibrations in tyrosine (Ty2 band) upon chelation of Cu(ll) ions.
- FIG. 6 is a schematic model illustrating a chemical and electrical charge similarity between the protonated amine groups, Si-NHs + , at the surface of SisN4 and the N-terminal of lysine, C-NHs + in cells (left panel); and, the interaction of SARS- CoV-2 viruses with the charged molecular species at the surface of SisN4 (specifically, at protonated amines charging plus) and the eluted species NHs/NH4 + (central panel).
- the eluted N leaves 3+ charged vacancies on the solid surface (violet-colored sites), which stem together with negatively charged silanols.
- RNA backbone cleavage by the eluted nitrogen species namely, deprotonation of 2’- hydroxyl groups, formation of a transient pentaphosphate, and cleavage of the phosphodiester bond in the RNA backbone by alkaline transesterification through hydrolysis
- the similarity between protonated amine and N-terminal of lysine might trigger an extremely effective “competitive binding” mechanism for SARS-CoV-2 virion inactivation, while eluted ammonia fatally degrades the virion RNA in a combined “catch and kill” effect.
- FIG. 7 is a logarithmic comparison of average bacterial growth on PEEK, boron nitride, aluminum nitride, Shapal (a combination of boron nitride and aluminum nitride), and silicon nitride materials at 24 hours and 48 hours.
- FIG. 8 is a chart showing RT-qPCR genomic testing of the Washington State variant of the SARS-CoV-2 virus versus a- and [3-silicon nitride powders at 15 wt.%/vol for 30 min exposure.
- FIG. 9A is a chart showing plaque assay test results of the Washington State variant of the SARS-CoV-2 virus versus a- and [3-silicon nitride powders at 15 wt.%/vol for 30 min exposure.
- FIG. 9B is a chart showing plaque assay test results of the South African variant of the SARS-CoV-2 virus versus a- and [3-silicon nitride powders at 15 wt.%/vol for 30 min exposure.
- FIG. 9C is a chart showing plaque assay test results of the British variant of the SARS-CoV-2 virus versus a- and [3-silicon nitride powders at 15 wt.%/vol for 30 min exposure.
- references 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.
- various features are described which may be exhibited by some embodiments and not by others.
- 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.
- object examples 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.
- PPE personal protective equipment
- body covers head covers, shoe covers, face masks, eye protectors, face and eye protectors, and gloves.
- silicon nitride includes a-SisN4, p-SisN4, SiYAION, [3-SiYAION, SiYON, SiAION, or combinations thereof.
- component includes a nitride based material, a compound, an implant, a device, or similar, that is useful for antipathogenic purposes.
- the term “effective concentration” is defined as the concentration of a material required to inactivate at least 90% of a pathogen in at least 30 min.
- the effective concentration of a-SisN4 may be a concentration which results in at least a 1 -logi o reduction in the activity of a virus within 30 min.
- a method for inactivating the SARS-CoV-2 virus by contacting the virus with an object or composition comprising silicon nitride and/or aluminum nitride.
- the silicon nitride and/or aluminum nitride successively binds (i.e. captures) and then inactivates the virus (e.g. “catch and kill”).
- 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 metal-matrix composites, natural or manmade fibers, polymers, or metals containing silicon nitride powder retain key silicon nitride bone restorative, bacteriostatic, antiviral, and antifungal properties.
- Si 3 N4 Silicon nitride
- a formulation of Si 3 N4 is FDA-cleared for use as an intervertebral spinal spacer in cervical and lumbar spine fusion surgery, with proven long-term safety, efficacy, and biocompatibility.
- Clinical data for Si 3 N4 implants compare favorably with other spine biomaterials, such as allograft, titanium, and polyetheretherketone. A curious finding is that Si 3 N4 implants have a lower incidence of bacterial infection (i.e., less than 0.006%) when compared to other implant materials (2.7% to 18%).
- Si 3 N4 This property reflects the complex surface biochemistry of Si 3 N4 that elutes minute amounts of nitrogen, which is converted to ammonia, ammonium, and other reactive nitrogen species (RNS) that inhibit bacteria.
- RNS reactive nitrogen species
- 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:
- the present disclosure compares the effects of exposing SARS- CoV-2 to aqueous suspensions of SisN4 and aluminum nitride (AIN) particles and two controls, (i.e. , a suspension of copper (Cu) particles (positive control) and a sham suspension of SARS-CoV-2 virions without any antiviral agent (negative control)).
- Copper (Cu) was chosen as a positive control because of its well-known ability to inactivate a variety of microbes, including viruses.
- Aluminum nitride was included in the testing because, like SisN4, it is a nitrogen-based compound whose surface hydrolysis in aqueous solution leads to the elution of nitrogen, with an attendant increase in pH.
- AIN Since comparable antiviral and antibacterial phenomena are believed to be operative for all nitride-based compounds, AIN was used to provide additional insight into the antipathogenic mechanisms of nitrogen-containing inorganic materials.
- compounds capable of endogenous nitrogen-release can inactivate the SARS-CoV-2 virus at least as effectively as Cu.
- multiple antiviral mechanisms may be operative, such as RNA fragmentation, and in the case of Cu and AIN, direct metal ion toxicity; but while Cu and AIN supernatants demonstrated cellular lysis, SisN4 may provoke no metabolic alterations.
- the Raman spectrum of VeroE6 cells exposed to the SisN4 viral supernatant was like that of the uninfected sham.
- the antiviral effect may be related to the electrical attraction (including “competitive binding” to an envelope glycoprotein hemagglutinin in the case of influenza virus) and viral RNA fragmentation by reactive nitrogen species (RNS). These phenomena are due to the slow and controlled elution of nitrogen from Sisi’s surface which forms ammonia (NHs) and ammonium (NH4 + ) moieties coupled with the release of free electrons and negatively charged silanols in aqueous solution.
- electrical attraction including “competitive binding” to an envelope glycoprotein hemagglutinin in the case of influenza virus
- RNS reactive nitrogen species
- Sisi’s surface chemistry play fundamental roles: (i) the similarity between the protonated amino groups, Si-NHs + at the surface of SisN4 and the N-terminal of lysine, C-NHs + on the virus; and, (ii) the elution of gaseous ammonia due to SisN4 hydrolysis.
- FIG. 6 central panel
- the similarity is depicted in the left panel of this figure.
- an object, article, or composition comprising silicon nitride or aluminum nitride may be operable to successively bind a virus (e.g. SARS-Cov-2) and then inactivate the virus.
- a virus e.g. SARS-Cov-2
- the antiviral effectiveness of SisN4 may be comparable to Cu. While Cu is an essential trace element for human health and an electron donor/acceptor for several key enzymes by altering redox states between Cu + and Cu 2+ , these properties can also cause cellular damage. Its use as an antiviral agent is limited by allergic dermatitis, hypersensitivity, and multi-organ dysfunction. In contrast, the safety of SisN4 as a permanently-implanted material during spine fusion surgery is well established by experimental and clinical data. Therefore, an object, article, or composition comprising silicon nitride may be as effective at inactivating a virus as Cu without the negative effects of Cu.
- SisN4 is well-known for its capabilities as an industrial material. Load-bearing SisN4 prosthetic hip bearings and spinal fusion implants were initially developed because of the superior strength and toughness of SisN4. Later studies showed other properties of SisN4 that are favored in designing orthopaedic implants, such as enhanced osteoconductivity, bacteriostasis, improved radiolucency, lack of implant subsidence, and wear resistance. Therefore, Sisls ’s surface chemistry, topography, and hydrophilicity contribute to a dual effect (i.e. , upregulation of osteogenic activity to promote spinal fusion while simultaneously preventing bacterial adhesion and biofilm formation).
- an advantage of SisN4 is its versatility of manufacture.
- Sintered powders of SisN4 have been incorporated into other materials, such as polymers, other ceramics, bioglass, and metals, to create composite structures that maintain the index osteogenic and antibacterial properties of monolithic SisN4.
- Three-dimensional additive deposition of SisN4 may enable the manufacture of protective surfaces in health care that reduce fomite-mediated transmission of microbial disease.
- Incorporation of SisN4 particles into the fabric of personal protective equipment, such as face masks, protective gowns, and surgical drapes could contribute to health workers as well as patient safety.
- SisN4 inactivates the SARS-CoV-2 virus in a matter of minutes following exposure. Without being limited to any one theory, the mechanism of action may be shared with other nitrogen-based compounds that express trace amounts of surface disinfectants, such as aluminum nitride.
- the object used to bind and inactivate the SARS-CoV-2 virus is a device or apparatus that may include a silicon nitride and/or aluminum nitride composition on at least a portion of a surface of the object.
- the silicon nitride or aluminum nitride coating may be applied to the surface of the object as a powder.
- the silicon nitride or aluminum nitride powder may be filled, embedded, or impregnated in at least a portion of the object.
- the powder may have particles in the micron, submicron or nanometer size range.
- 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 or aluminum nitride may be incorporated into the device.
- an object may incorporate a silicon nitride and/or aluminum nitride powder within the body of the object.
- the device may be made of silicon nitride.
- the object may be made of aluminum nitride.
- the object can comprise a slurry or suspension of aluminum nitride or silicon nitride particles.
- the object may further comprise other materials including, but not limited to, paper, cardboard, fabric, plastic, ceramic, polymers, stainless steel, metal, or a combination thereof.
- the object may include surgical gowns, surgical drapes, shoe covers, cubicle curtains, tubing, clothing, gloves, eye protectors, masks including surgical masks and face shields, PPE, tables such as hospital exam and surgical tables, chairs, bed frames, bed trays, desks, fixtures, cabinets, equipment racks, carts, handles, knobs, railings, toys, water filters, and air filters such as face mask filters, respirator filters, air filtration filters, and air ventilation filters, or air conditioner filters.
- 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.
- the object may be a medical device or apparatus.
- medical devices or apparatuses include orthopedic implants, spinal implants, pedicle screws, dental implants, in-dwelling catheters, endotracheal tubes, colonoscopy scopes, and other similar devices.
- the object may be a composition incorporating silicon nitride or aluminum nitride powder therein including, but not limited to slurries, suspensions, gels, sprays, paint, or toothpaste.
- silicon nitride or aluminum nitride to a slurry, such as paint, that is then applied to a surface may provide an antibacterial, antifungal, and antiviral surface.
- silicon nitride or aluminum 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.
- the silicon nitride or aluminum nitride coating may be present on the surface of the object in a concentration of about 1 wt.% to about 100 wt.%.
- the silicon nitride and/or aluminum nitride may be coated onto or layered into the object.
- 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.%, or about 30 wt.% silicon nitride powder or aluminum nitride powder.
- the coating may include about 10 wt.% to about 20 wt.% silicon nitride or aluminum nitride. In at least one example, the coating includes about 15 wt.% silicon nitride or aluminum nitride. In some embodiments, silicon nitride or aluminum nitride may be embedded in (as a filler) or on the surface of the object in a concentration of about 1 wt.% to about 100 wt.%.
- the object 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 or aluminum nitride.
- the silicon nitride or aluminum nitride may be on the surface of the object at a concentration of about 10 wt.% to about 20 wt.%. In at least one example, the silicon nitride or aluminum nitride may be on the surface of the object at a concentration of about 15 wt.%. In some aspects, the concentration of silicon nitride or aluminum nitride may depend on the substrate material of the object, such as paper, cardboard, fabric, plastic, ceramic, polymers, stainless steel, and/or metal. In some embodiments, the substrate material of the object may be a polymer and the polymer may have a practical limit (i.e. percolation limit) on the amount of silicon nitride and/or aluminum nitride that may be incorporated into the object.
- the substrate material of the object may be a polymer and the polymer may have a practical limit (i.e. percolation limit) on the amount of silicon nitride and/or aluminum nitrid
- the object may be a monolithic component consisting of the silicon nitride or aluminum nitride.
- Such an object may 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 object may be used as a medical device or may be used in an apparatus in which the inactivation of a virus may be desired.
- the object may contact the SARS-CoV-2 virus for a limited period of time.
- the object may be in contact with the SARS-CoV-2 virus for about 1 min to about 2 hours in order to inactivate the virus.
- the object may contact the SARS-CoV-2 virus for at least 30 seconds, 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 object may be permanently implanted in the patient.
- the object may be worn externally by a user.
- the object may be permanently implanted in the patient.
- the object may be a high contact surface.
- the object may be in continuous or sustained contact with a body fluid of a patient.
- the body fluid may be blood or gas (e.g., 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 object 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 object for at least 1 minute. In another example, the virus is at least 99% inactivated after contact with the object for at least 30 minutes. In yet another example, the virus is at least 99% inactivated after contact with the object for at least 1 minute.
- the article may comprise silicon nitride or aluminum nitride incorporated into the article or the silicon nitride or aluminum nitride may be coated onto the surface of the article.
- the silicon nitride or aluminum nitride coating may be present on the surface of the article 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.%, or about 30 wt.% silicon nitride powder or aluminum nitride powder.
- the coating may include about 10 wt.% to about 20 wt.% silicon nitride or aluminum nitride. In at least one example, the coating includes about 15 wt.% silicon nitride or aluminum nitride. In some embodiments, silicon nitride or aluminum nitride may be embedded in (as a filler) or on the surface of the article in a concentration of about 1 wt.% to about 100 wt.%.
- the object 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 or aluminum nitride.
- the silicon nitride or aluminum nitride may be on the surface of the article at a concentration of about 10 wt.% to about 20 wt.%. In at least one example, the silicon nitride or aluminum nitride may be on the surface of the article at a concentration of about 15 wt.%. In some aspects, the concentration of silicon nitride or aluminum nitride may depend on the substrate material of the object.
- the article is PPE.
- the article is a body cover, a head cover, a shoe cover, a face mask, a face and eye protector, or gloves.
- the article is operable to inactivate a SARS-CoV- 2 virus when the article contacts the virus.
- compositions and devices that include a nitride for the inactivation of viruses, bacteria, and fungi.
- a nitride is a compound of nitrogen where nitrogen has a nominal oxidation state of between -3 and +5.
- Non-limiting examples of suitable nitrides include silicon nitride, aluminum nitride, boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorus nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zirconium nitride, or combinations thereof.
- Nitrides can have high intrinsic nitrogen content.
- silicon nitride (Si 3 N 4 ) contains about 40 wt.% nitrogen
- boron nitride (BN) has about 56 wt.% nitrogen
- aluminum nitride (AIN) has 34 wt.% nitrogen
- titanium nitride (TiN) has about 22 wt.% nitrogen.
- Nitrides in general may be antipathogenic due to release of nitrogen containing species when in contact with an aqueous medium, or biologic fluids and tissues.
- the surface hydrolysis chemistry of silicon nitride may be shown as follows:
- the antipathogenic nitride based 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 eukaryotic cells; and (iii) an intelligent elution that increases with decreasing pH.
- the antipathogenic compositions and devices disclosed herein can comprise one or more of aluminum nitride, boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, and zirconium nitride.
- the composition and devices can further comprise silicon nitride, such as a-silicon nitride.
- the nitride may be a-silicon nitride. In some embodiments, the nitride may be aluminum nitride. In still other embodiments, the antipathogenic compositions and devices may include nitride mixtures, such as a mixture of AIN and BN. It was surprisingly found that these compositions were capable of inactivating viruses.
- the nitride based compositions can be powders, particulates, slurries, suspensions, coatings, films, and/or composites.
- the compositions can comprise micrometer or nanometer particles of the nitride.
- the average particle size of the nitride 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 composition can comprise a slurry or suspension of nitride particles.
- the antipathogenic composition may be a monolithic component consisting of the 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, bacteria, and/or fungi may be desired.
- the antipathogenic nitride based composition may be incorporated within a device or within a coating on the surface of the device to inactivate viruses, bacteria, and fungi. At least a portion of the surface of the device maybe coated with coating comprising the nitride based composition.
- suitable devices include orthopedic implants, spinal implants, pedicle screws, dental implants, in-dwelling catheters, endotracheal tubes, colonoscopy scopes, and other similar devices.
- the device or apparatus may be metallic, polymeric, and/or ceramic.
- the nitride may be incorporated within or applied as a coating to materials or apparatuses for antipathogenic properties such as polymers and fabrics, surgical gowns, gloves, tubing, clothing, air and water filters (e.g. filtration devices of anesthesia machines, ventilators, or CPAP machines), masks, tables such as hospital exam and surgical tables, desks, fixtures, handles, knobs, toys, and filters such as air conditioner filters, or toothbrushes.
- materials or apparatuses for antipathogenic properties such as polymers and fabrics, surgical gowns, gloves, tubing, clothing, air and water filters (e.g. filtration devices of anesthesia machines, ventilators, or CPAP machines), masks, tables such as hospital exam and surgical tables, desks, fixtures, handles, knobs, toys, and filters such as air conditioner filters, or toothbrushes.
- air and water filters e.g. filtration devices of anesthesia machines, ventilators, or CPAP machines
- masks e.g. filtration devices of anesthesia machines, ventilators, or
- the nitride based coating may be applied to the surface of the device as a powder.
- the nitride powder may be imbedded or impregnated in at least a portion of the device.
- the powder may be micrometric or nanometer in size.
- the silicon nitride may be incorporated into the device.
- a device may incorporate the nitride powder within the body of the device.
- the antipathogenic nitride based composition may be a slurry of nitride powder in an aqueous solution.
- the aqueous medium may be water, saline, buffered saline, or phosphate buffer saline.
- the nitride base composition may be a suspension or emulsion of nitride powder and a suitable vehicle (e.g., cream, gel, or lotion) for topical application.
- the nitride powder may be present in the composition in a concentration of about 0.1 vol.% to about 20 vol.%.
- the slurry may include about 0.1 vol.%, 0.5 vol.%, 1 vol.%, 1.5 vol.%, 2 vol.%, 5 vol.%, 10 vol.%, 15 vol.%, or 20 vol.% silicon nitride.
- the concentration of the nitride may be effective to inactivate the pathogen.
- the nitride-based coating may be present on the surface of a device or within the device 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.% nitride powder.
- the coating includes about 15 wt.% of the nitride.
- nitride may be present in 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.%, 60 wt.%, 70 wt.%, 80 wt.%, 90 wt.%, to 100 wt.% of the nitride.
- the concentration of the nitride may be effective to inactivate the pathogen.
- the antipathogenic nitride based composition or device may inactivate or decrease the transmission of viruses, bacteria, and/or fungi.
- the viruses, bacteria, and fungi may infect mammalian cells, animal cells, and/or plant cells.
- viruses that may be inactivated by the antipathogenic nitride based compositions include coronaviruses (e.g., SARS-CoV-2), rhinoviruses, influenza viruses (A, B, C, D), and Feline calicivirus.
- the antipathogenic nitride based compositions or devices may kill both gram-positive and gram-negative bacteria.
- fungi examples include, without limit, those that cause downy mildew, powdery mildew, Botrytis rot, Fusarium rot, rust, Rhizoctonia rot, Sclerotinia rot, Sclerotium rot, or other agriculturally relevant diseases.
- the pathogen may be on a surface of or within a human, an animal, or a plant. In other embodiments, the pathogen may be on a surface of or within an inanimate object.
- the method may include coating a device or apparatus with the nitride based composition and contacting the coated apparatus with the virus, bacterium, or fungus.
- the method may include contacting a virus, bacteria, and/or fungus with a composition comprising a nitride based composition.
- the composition may be a slurry comprising nitride powder or particles.
- the composition may be a suspension or emulsion comprising the nitride powder.
- the pathogen may be a virus, bacterium, or fungus.
- the method may include contacting the patient with a device, apparatus, or composition comprising nitride based composition.
- the device, apparatus, or composition may include about 1 wt.% to about 100 wt.% of the nitride.
- the device or apparatus may include about 1 wt.% to about 100 wt.% of the nitride on the surface of the device or apparatus.
- the device or apparatus may be a monolithic nitride based ceramic.
- the device or apparatus may include a nitride coating, such as a nitride powder coating.
- the device or apparatus may incorporate the nitride into the body of the device.
- the nitride powder may be incorporated or impregnated into the body of the device or apparatus using methods known in the art.
- the device 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.
- Embodiment 1 A method for inactivating a pathogen comprising contacting the pathogen with a composition comprising an effective concentration of a nitride chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zirconium nitride, or a combination thereof, wherein the effective concentration of the nitride inactivates the pathogen.
- a nitride chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum n
- Embodiment 2 The method of embodiment 1 , wherein the composition further comprises silicon nitride.
- Embodiment 3 The method of embodiment 1 , wherein the composition further comprises aluminum nitride.
- Embodiment 4 The method of embodiment 1 , wherein the nitride is boron nitride.
- Embodiment 5 The method of embodiment 1 , wherein the composition comprises a slurry of nitride particles in an aqueous medium.
- Embodiment 6 The method of embodiment 5, wherein the effective concentration of the nitride is about 0.1 vol.% to about 20 vol.%.
- Embodiment 7 The method of embodiment 1 , wherein the composition comprises a powder of the nitride.
- Embodiment 8 The method of embodiment 7, wherein the composition is coated over at least part of a surface of a device and/or is incorporated into the device.
- Embodiment 9 The method of embodiment 8, wherein the effective concentration of the nitride is about 1 wt.% to about 100 wt.%.
- Embodiment 10 The method of embodiments 8 or 9, wherein the device is an orthopedic implant, a spinal implant, a pedicle screw, a dental implant, an in-dwelling catheter, an endotracheal tube, a colonoscopy scope, a surgical gown, a mask, a filter, or tubing.
- Embodiment 11 The method of any one of embodiments 1 to 10, wherein the pathogen is a virus, a bacteria, or a fungus.
- Embodiment 12 The method of embodiment 11 , wherein the virus is a coronavirus.
- Embodiment 13 The method of any one of embodiments 1 to 12, wherein the pathogen is on a surface or within a human, animal, or plant.
- Embodiment 14 The method of any one of embodiments 1 to 12, wherein the pathogen is on a surface of an inanimate object.
- Embodiment 15 A nitride-based composition for inactivating a pathogen, the composition comprising: a slurry of nitride particles in an aqueous medium, wherein the nitride is present at a concentration of about 0.1 vol.% to about 20 vol.%, and wherein the concentration is effective to inactivate the pathogen.
- Embodiment 16 The nitride-based composition of embodiment 15, wherein the nitride is chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zirconium nitride, or a combinations thereof.
- the nitride is chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zi
- Embodiment 17 The nitride-based composition of embodiment 16, wherein the composition further comprises silicon nitride.
- Embodiment 18 The nitride-based composition of embodiment 16, wherein the composition further comprises aluminum nitride.
- Embodiment 19 The nitride-based composition of embodiment 16, wherein the nitride is boron nitride.
- Embodiment 20 The nitride-based composition of embodiment 15, wherein the pathogen is a virus, a bacteria, or a fungus.
- Embodiment 21 The nitride-based composition of embodiment 20, wherein the virus is a coronavirus.
- Embodiment 22 The nitride-based composition of any one of embodiments 15 to 21 , wherein the pathogen is on a surface or within a human, animal, or plant.
- Embodiment 23 The nitride-based composition of any one of embodiments 15 to 21 , wherein the pathogen is on a surface of an inanimate object.
- Embodiment 24 A nitride-based device for inactivating a pathogen, the device comprising: a powder of a nitride coated over at least part of a surface of the device and/or is incorporated into the device, wherein the nitride is present at a concentration of about 10 wt.% to about 30 wt.%, and wherein the concentration is effective to inactivate the pathogen.
- Embodiment 25 The nitride-based device of embodiment 24, wherein the nitride is chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zirconium nitride, or a combinations thereof.
- the nitride is chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zi
- Embodiment 26 The nitride-based device of embodiment 25, wherein the device further comprises silicon nitride.
- Embodiment 27 The nitride-based device of embodiment 25, wherein the device further comprises aluminum nitride.
- Embodiment 28 The nitride-based device of embodiment 25, wherein the nitride is boron nitride.
- Embodiment 29 The nitride-based device of embodiment 24, wherein the pathogen is a virus, a bacteria, or a fungus.
- Embodiment 30 The nitride-based device of embodiment 29, wherein the virus is a coronavirus.
- Embodiment 31 The nitride-based device of any one of embodiments 24 to 30, wherein the pathogen is on a surface or within a human, animal, or plant.
- Embodiment 32 The nitride-based device of any one of embodiments 24 to 30, wherein the pathogen is on a surface of an inanimate object.
- Embodiment 33 A method for inactivating a pathogen comprising contacting the pathogen with a slurry comprising an effective concentration of a-silicon nitride, wherein the effective concentration of the nitride inactivates the pathogen, and wherein the effective concentration of a-silicon nitride in the slurry is about 15% w/v.
- Embodiment 34 The method of embodiment 33, wherein the slurry further comprises a nitride chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zirconium nitride, or a combination thereof.
- a nitride chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zi
- Embodiment 35 A method for inactivating a pathogen comprising contacting the pathogen with a slurry comprising an effective concentration of a aluminum nitride, wherein the effective concentration of the nitride inactivates the pathogen, and wherein the effective concentration of aluminum nitride in the slurry is about 15% w/v.
- Embodiment 36 The method of embodiment 35, wherein the slurry further comprises a nitride chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zirconium nitride, or a combination thereof.
- a nitride chosen from boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorous nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, zi
- Example 1 Rapid Inactivation of SARS-CoV-2 by Silicon Nitride or Aluminum Nitride
- the present study compared the effects of exposing the SARS- CoV-2 virus to aqueous suspensions of silicon nitride (SisN4) particles, aluminum nitride (AIN) particles, and two controls, (i.e. , a suspension of copper (Cu) particles (positive control) and no antiviral agent (negative control)).
- SiN4 silicon nitride
- AIN aluminum nitride
- Cu copper
- Cu no antiviral agent
- SisN4, Cu, and AIN powders were acquired from commercial sources.
- SisN4 powder (nominal composition of 90 wt.% SisN4, 6 wt.% Y2O3, and 4 wt.% 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 an average particle size of 0.8 ⁇ 1 .0 pm.
- As-received Cu powder (USP grade 99.5% purity) granules were comminuted to achieve a particle size comparable to the SisN4.
- AIN powder had an average particle size of 1 .2 ⁇ 0.6 pm as-received, which was comparable to SisN4.
- VeroE6/TMPRSS2 mammalian cells were used in the viral assays. Cells were grown in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) supplemented with G418 disulfate (1 mg/ml), penicillin (100 units/mL), streptomycin (100 pg/mL), 5% fetal bovine serum, and maintained at 37°C in a 5% CO2 195% in a humidified atmosphere. The SARS-CoV-2 viral stock was propagated using VeroE6/TMPRSS2 cells at 37°C for 2 days. Viral titers were assayed by a median tissue culture infectious dose (TCIDso).
- TCIDso median tissue culture infectious dose
- Vero E6/TMPRSS2 cells on cover glass were inoculated with 200 pL of virus supernatant. After viral adsorption at 37°C for 1 hour, the cells were incubated with the maintenance medium in a CO2 incubator for 7 hour. For the detection of infected cells, these cells were washed with TBS (20 mM Tris-HCI pH 7.5, 150 mM NaCI) and fixed with 4% PFAfor 10 min at room temperature (RT) followed by membrane permeabilization with 0.1 % Triton X in TBS for 5 minutes at RT.
- TBS 20 mM Tris-HCI pH 7.5, 150 mM NaCI
- Vero E6/TMPRSS2 cells were infected with 200 pL of each virus suspension onto glass sites. After viral adsorption at 37 °C for 1 hours, the infected cells were incubated with the maintenance medium in a CO2 incubator for 4 hours and fixed with 4% paraformaldehyde for 10 minutes at RT. After washing with distilled water twice, infected cells were air-dried and in situ analyzed using a Raman microprobe spectrometer. Raman spectra were collected using a highly sensitive spectroscope with a 20x optical lens. It operated in microscopic measurement mode with confocal imaging in two dimensions.
- a holographic notch filter within the optical circuit was used to efficiently achieve a spectral resolution of 1.5 cm-1 via a 532 nm excitation source operating at 10 mW.
- Raman emissions were monitored using a single monochromator connected to an air-cooled charge-coupled device (CCD) detector 1024 x 256 pixels). The acquisition time was fixed at 10 seconds. Thirty spectra were collected and averaged for each analysis time-point. Raman spectra were deconvoluted into Gaussian-Lorentzian sub-bands using commercially available software.
- TCID50 assay results for the 15 wt.% SisN4, Cu, and AIN powders are shown in FIGS. 1A-1D.
- Inactivation times of 1 and 10 minutes are shown in FIGS. 1A and 1B as well as FIGS. 1C and 1D, respectively.
- Relative to the negative control all three powders were effective in inactivating SARS-CoV-2 virions (>99%) for the two exposure times.
- RNA was fragmented from exposure to both the supernatants and powders RT-PCR tests were conducted on the N gene sets of the virus’ RNA. The results are shown in Fig. 2A and 2B as well as FIGS. 2C and 2D for 1 - and 10-minute exposures, respectively. Again, in comparison to the negative control at 1 minute of exposure to the supernatants, almost complete fragmentation of the RNA was observed for Cu while significant damage was caused by AIN and to a lesser extent by SisN4. After 10-minute exposure to the supernatants, substantial cleavage of the RNA was seen for all three materials.
- FIGS. 3A-3D show fluorescence micrographs representative of the VeroE6/TMPRSS2 cell populations that were inoculated with supernatants of (a) unexposed virions (i.e., negative control) and 10-minute-exposed virions of (b) SisN4, (c) AIN, and (d) Cu.
- FIG. 3E shows cells that were not inoculated with the virus (labeled as “sham-infected” cells. The red-colored spots in the negative control (FIG. 3A) demonstrated that the virions had entered and hijacked the Vero6E cells’ metabolism. This contrasts with the sham-infected cells (FIG. 3E) which showed normal metabolic function.
- FIGS. 5A-5G show Raman spectra in the frequency range OOGOO cm-1 for (a) uninfected VeroE6/TMPRSS2 cells, and cells inoculated with supernatants containing virions exposed for 10-minutes to (b) SisN4, (c) AIN, (d) Cu (positive control), and (e) no antiviral compounds (negative control).
- Tryptophan plays a vital role in protein synthesis and the generation of molecules for various immunological functions. Its stereoisomers serve to anchor proteins within the cell membrane and its catabolites possess immunosuppressive functions. The catabolism of tryptophan is triggered by a viral infection. This occurs via the enzymatic activity of indoleamine-2,3-dioxygenase (IDO) which protects the host cells from an over-reactive immune response.
- IDO indoleamine-2,3-dioxygenase
- IDO reduces tryptophan to kynurenine and then to N’- formyl-kynurenine.
- An increase in IDO activity depletes tryptophan. Consequently, the intensity of the tryptophan bands (T1 and T2) is an indicator of these biochemical changes.
- the data presented in FIG. 5F show an exponential decline in the combined tryptophan bands that correlates with the fraction of infected cells. (The chemical structure of N’-formyl-kynurenine is given in the inset for clarity.)
- the anomaly for copper provides further evidence of its toxicity.
- the VeroE6 cells consumed tryptophan to reduce Cu 2+ and stabilize it as Cu + .
- the Raman signals due to ring-stretching vibrations of adenine, cytosine, guanine, and thymine were found at 725, 795, 680, and 748 cm-1 , and are labeled as A, Cy1 , G, and Th, respectively, in FIGS. 5A-5E). These bands were preserved after virus exposure. However, there was an anomaly for lines representative of tyrosine at 642 and 832 cm-1 labeled as Ty1 and Ty2, respectively for cells infected with Cu-exposed virions. The ring-breathing band Ty2 of tyrosine was very weak compared to the other samples (cf. FIG. 5D with FIG. 5B).
- SisN4 invoked no modifications of tryptophan, tyrosine, and cytosine.
- the morphology of the spectrum for the SisN4 viral supernatant closely matched that of the uninfected sham suspension (cf. FIGS. 5A and 5B)
- a bacterial medium was prepared by combining 7% glucose, 1X PBS, and 10% human plasma, then inoculating it with a small aliquot of Staphylococcus epidermidis. An initial absorbance value was taken using a spectrophotometer. The medium was then placed in a shaking incubator at 37°C and 175 rpm for six hours where the bacteria was allowed to proliferate until an absorbance value of 0.05 AU was achieved (corresponding to 10 5 cells/mL per a previously generated growth curve).
- Each sterile sample was placed in a well plate and 7 mL of liquid culture was added.
- Samples were removed at appropriate time points (24 or 48 hours) and rinsed in 5 mL of 1X PBS in a fresh well plate in the shaking incubator for 2 minutes at 125 rpm. Each sample was dip-rinsed in fresh 1X PBS. Samples were placed in 10mL 1X PBS in a 50mL centrifuge tube and vortexed vigorously for 2 minutes.
- Each sample’s biofilm solution was serially diluted in 1X PBS, resulting in concentrations 1x, 1/1 Ox, 1/1 OOx, 1/1 ,000x, and 1/10,000x of the original sample.
- Each dilution was plated onto a Petrifilm to allow the lowest countable dilution to be used in data comparison for each sample.
- the bacteria formed colonies, referenced as colony forming units (CFU). Once obtaining the CFU count, the CFUs per sample were calculated by multiplying the count by the appropriate dilution factor.
- the [3-silicon nitride powder had a nominal composition of 90 wt.% p-SisN4, 6 wt.% yttira (Y2O3), and 4 wt.% alumina (AI2O3).
- the powders were each prepared by aqueous mixing and spray-drying of the inorganic constituents, followed by sintering the spray-dried granules at about 1700°C for about 3 hours. Next, the sintered granules were hot-isostatic pressed at about 1600°C for 2 hours at 140 MPa in nitrogen. The pressing was followed by aqueous based comminution and freeze-drying.
- the a-silicon nitride powder was 98 wt.% pure SisN4 with about 2 wt.% SiO2. It was prepared by heating commercially available high-purity a-silicon nitride at about 300°C for about 1 hour in air and then cooling it to room temperature.
- the Washington State variant of the SARS-CoV-2 virus was diluted in DMEM growth media to a concentration of 2 x 10 4 virions/mL.
- Four mL of the diluted virus solution were then added to tubes containing either a- or [3-silicon nitride powders at 15 wt.%/vol (w/v).
- the virus without SisN4 was processed in parallel as a control.
- the tubes were vortexed for 30 seconds to ensure adequate contact and then placed on a tube revolver for 30 minutes.
- the samples were centrifuged, and the supernatant collected and filtered through a 0.2 pm filter.
- the RNA in the remaining infectious virus within the clarified supernatant was isolated along with the pellets and quantified by RT-qPCR methods.
- the supernatants were also subjected to plaque assay test methods.
- the results, provided in FIG. 8, showed that the genomic copies of virus only control had a concentration of about 1 x 10 6 /mL, whereas one of the [3-silicon nitride powders demonstrated a reduction in virions to approximately 4.3 x 10 4 /mL (95.9%) and two different lots of a-silicon nitride showed viral reductions to approximately 1 x 10 3 /mL (99.9%).
- the pelleted samples from the two a-silicon nitrides were found to be completely free of live virions.
- the plaque assay test results are provided in FIG. 9A. These data showed a reduction in viral activity for the [3-silicon nitride powders that ranged from 1 .25 to 3.5 logi o ( ⁇ 93% to 99.97%) whereas the a-silicon nitride powders demonstrated > 4.5 logw reduction (>99.997%). It is believed that variations in the surface hydrolysis of the [3-silicon nitride powders led to the observed range of results. However, comparing the RT-qPCR and plaque assay methods suggests that the SARS-CoV-2 virions are not being pelleted with the silicon nitride and that their RNA structure is being damaged when incubated with silicon nitride.
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KR1020237022797A KR20230117410A (en) | 2020-12-09 | 2021-12-09 | Nitride-based antipathogenic composition and device and method of use thereof |
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US20100040655A1 (en) * | 2006-02-16 | 2010-02-18 | Queen Mary & Westfield College | Anti-viral Formulations Nanomaterials And Nanoparticles |
WO2015125367A1 (en) * | 2014-02-20 | 2015-08-27 | 昭和電工株式会社 | Antiviral composition, antiviral agent, photocatalyst and virus inactivation method |
US20200079651A1 (en) * | 2018-09-06 | 2020-03-12 | Sintx Technologies, Inc. | Antipathogenic devices and methods thereof |
WO2021211697A1 (en) * | 2020-04-14 | 2021-10-21 | Sintx Technologies, Inc. | Antipathogenic face mask |
WO2022005550A1 (en) * | 2020-06-29 | 2022-01-06 | Sintx Technologies, Inc. | Systems and methods for rapid inactivation of sars-cov-2 by silicon nitride and aluminum nitride |
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US20100040655A1 (en) * | 2006-02-16 | 2010-02-18 | Queen Mary & Westfield College | Anti-viral Formulations Nanomaterials And Nanoparticles |
WO2015125367A1 (en) * | 2014-02-20 | 2015-08-27 | 昭和電工株式会社 | Antiviral composition, antiviral agent, photocatalyst and virus inactivation method |
US20200079651A1 (en) * | 2018-09-06 | 2020-03-12 | Sintx Technologies, Inc. | Antipathogenic devices and methods thereof |
WO2021211697A1 (en) * | 2020-04-14 | 2021-10-21 | Sintx Technologies, Inc. | Antipathogenic face mask |
WO2022005550A1 (en) * | 2020-06-29 | 2022-01-06 | Sintx Technologies, Inc. | Systems and methods for rapid inactivation of sars-cov-2 by silicon nitride and aluminum nitride |
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LEHMAN CAITLIN W., FLUR RAFAELA, KEHN-HALL KYLENE, MCENTIRE BRYAN J., BAL B. SONNY, BOCK RYAN M.: "Silicon Nitride Inactivates SARS-CoV-2 in vitro", BIORXIV, 29 August 2020 (2020-08-29), XP055949615, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.08.29.271015v1> [retrieved on 20220808], DOI: 10.1101/2020.08.29.271015 * |
PEZZOTTI GIUSEPPE, OHGITANI ERIKO, SHIN-YA MASAHARU, ADACHI TETSUYA, MARIN ELIA, BOSCHETTO FRANCESCO, ZHU WENLIANG, MAZDA OSAM: "Rapid Inactivation of SARS-CoV-2 by Silicon Nitride, Copper, and Aluminum Nitride", BIORXIV, 20 June 2020 (2020-06-20), XP055895687, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.06.19.159970v1.full.pdf> [retrieved on 20220225], DOI: 10.1101/2020.06.19.159970 * |
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