KR20230117410A - Nitride-based antipathogenic composition and device and method of use thereof - Google Patents

Abstract

Nitride selected from 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, silicon nitride, or combinations thereof Described herein are antipathogenic compositions and methods of using such compositions to inactivate viruses, bacteria and/or fungi.

Description

Nitride-based antipathogenic composition and device and method of use thereof

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to US Provisional Application No. 63/123,037, filed on December 9, 2020, which is incorporated herein by reference in its entirety.

Field

The present invention relates generally to antipathogenic compositions and devices, and in particular to systems and methods for nitride-based antipathogenic compositions and devices.

It is well known that surface spread of pathogenic organisms causes a number of diseases, disorders and deaths. In recent years, the discovery of new pathogens, such as the SARS-CoV-2 virus, has highlighted this problem. Methods to kill or inactivate surface-dwelling pathogens often include the use of cleaning agents, high heat or high pressure, or electromagnetic radiation. These methods can be costly, and in some cases present other risks to humans, such as exposure to toxic chemicals or intense ultraviolet light. Additionally, over time some pathogens develop resistance to conventional methods.

Accordingly, there is a need for methods and compositions useful for inactivating or killing pathogenic organisms.

Nitride-based compositions or devices for inactivating viruses, bacteria and/or fungi are provided herein. The nitride-based composition may be 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, silicon nitride in various forms, or these includes a combination of

Also provided herein are methods for inactivating pathogens by contacting viruses, bacteria and/or fungi with an antipathogenic nitride-based composition or device disclosed herein.

1A-1D are graphical representations showing inactivation of SARS-CoV-2 by nitride powders treated with 15 wt% Cu, AlN and Si ₃ N ₄ powders in aqueous medium for 1 and 10 minutes at room temperature; Control viruses were treated identically without the addition of powder. After centrifugation, the supernatant was subjected to TCID ₅₀ assay. Viral titers were determined using the Reed-Muench method. Percent reduction of TCID _50/50 μL (Figures 1A and 1B) and Figures 1C and 1D are shown for virus inactivation times of 1 minute and 10 minutes, respectively. Statistics according to unpaired two-tailed Student's t -test ( n=3 ) are shown in the inset.
2A-2D are graphic representations showing viral RNA that has undergone severe degradation after exposure to copper or nitride particles. In Figures 2a and 2b, viral suspensions were exposed to Cu, AlN and Si ₃ N ₄ powders for 1 minute, and viral RNAs in the supernatant and on particles were analyzed using viral N gene "set 1" and "set 2" primers, respectively. evaluated. Data collected from supernatant and pellet samples are presented relative to the amount of viral N gene RNA in the untreated suspension. In FIGS. 2C and 2D , RT-PCR test results after 10 min exposure of the supernatant to Cu, AlN and Si ₃ N ₄ powders for viral N gene “set 1” and “set 2” primers are shown, respectively. Statistics according to unpaired two-tailed Student's t-test (n=3) are shown in the inset.
3a-3e are images showing that Si ₃ N ₄ inhibits viral infection without affecting cell viability where Cu kills cells. VeroE6/TMPRSS2 cells were inoculated with unexposed virions (Fig. 3a) and 10 min UTE-exposed virions to Si ₃ N ₄ (Fig. 3b), AlN (Fig. 3c) and Cu (Fig. 3d). In Figure 3e, non-inoculated cells (labeled "mham-infected" cells) were also prepared and imaged for comparison. After fixation, cells were stained with anti-SARS coronavirus envelope antibody (red), phalloidin (green) to visualize F-actin, and DAPI (blue) to stain nuclei. Representative fluorescence micrographs of n=3 samples are shown.
Figure 4 is a graphical representation showing fluorescently labeled and unlabeled cells counted in fluorescence micrographs, % infected cells and % viable cells were calculated as: % infected cells = (stained with anti-SARS coronavirus coat antibody) number of stained cells) / (number of cells stained with DAPI) x 100; and % viable cells = (number of cells stained with phalloidin) / (number of cells stained with DAPI) x 100. Data are representative of n=3 samples. * and ** are p<0.05 and 0.01 respectively by unpaired two-tailed Student's t-test (n=3); ns = not important.
5A-5G are graphical representations of Raman spectra for: (a) uninfected cells (FIG. 5A) (ie, not exposed to virions) and (b) Si ₃ N ₄ (FIG. 5B), ( c) cells infected with SARS-CoV-2 virions exposed to AlN (Fig. 5c) and (d) Cu (Fig. 5d) for 10 min; In Figure 5E, Raman spectra of cells infected with unexposed virions (negative control). In Figure 5f, a plot of the average intensity of the two tryptophan T1 and T2 bands (756 and 875 cm-1, respectively) as a function of the fraction of cells infected by virions exposed for 10 min without exposure to different particles (see label) ; In the inset, the structure of N'-formylkynurenine, an intermediate in tryptophan catabolism following the enzymatic IDO reaction. In Fig. 5g, a graphical representation shows three possible structures of tyrosine-based peptides that can justify the disappearance of ring vibrations in tyrosine (Ty2 band) upon chelation of Cu(II) ions.
FIG. 6 shows the chemical and charge similarity between the protonated amine group Si-NH ₃ ⁺ on the Si ₃ N ₄ surface and the N-terminal C-NH ₃ ⁺ of intracellular lysine (left panel); and interactions of SARS-CoV-2 virus with charged molecular species on the Si ₃ N ₄ surface (specifically, positively charged protonated amines) and the eluted species NH ₃ /NH ₄ ⁺ (central panel). it is a schematic The eluted N leaves 3+ charged vacancies on the solid surface (purple regions) and forms a stalk with negatively charged silanols. A three-step process leading to cleavage of the RNA backbone by eluted nitrogen species (i.e., deprotonation of the 2'-hydroxyl group, formation of transient pentaphosphates, and phosphodiester linkages in the RNA backbone by alkaline transesterification via hydrolysis). cleavage) is shown in the right panel. The similarity between the protonated amine and the N-terminus of the lysine could trigger a highly effective “competitive binding” mechanism for SARS-CoV-2 virion inactivation, whereas the eluted ammonia had a combined “catch and kill” effect. lethally degrades virion RNA.
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 and 48 hours.
FIG. 8 is a chart showing RT-qPCR genomic testing of Washington state variants of SARS-CoV-2 virus versus α- and β-silicon nitride powders at 15% wt/vol for 30 min exposure.
9A is a chart showing plaque assay test results of a 30 minute exposure at 15 wt%/volume of α- and β-silicon nitride powder versus the Washington state variant of SARS-CoV-2 virus.
9B is a chart showing plaque assay test results of a 30 minute exposure at 15% wt/vol of α- and β-silicon nitride powder versus the South African variant of SARS-CoV-2 virus.
9C is a chart showing plaque assay test results of a 30 minute exposure at 15% wt/vol of α- and β-silicon nitride powder versus the British variant of SARS-CoV-2 virus.
Corresponding reference characters indicate elements of the drawings. Headings used in the drawings do not limit the scope of the claims.

Various embodiments of the invention are discussed in detail below. While specific implementations are discussed, it should be understood that this is done for illustrative purposes only. Those skilled in the art will recognize that other components and configurations may be used without departing from the spirit and scope of the invention. Accordingly, the following description and drawings are illustrative and should not be construed as limiting. Numerous specific details are set forth in order to provide a thorough understanding of the present invention. In some instances, however, well-known or common details are not described in order to avoid obscuring the description. A reference to one or one embodiment in this disclosure may be a reference to the same embodiment or any embodiment; Such reference refers to at least one embodiment.

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 present 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. Also described are various features that may be exhibited by some embodiments and not exhibited by others.

As used herein, "consisting of", "having" and "comprising" are used in an open and non-limiting sense. The terms "which", "which" and "the" are understood to include the plural as well as the singular. Accordingly, the term "mixtures thereof" also relates to "mixtures thereof".

As used herein, "inactivate" or "inactivation" refers to virus inactivation that completely removes a virus or renders the virus non-infectious so that the virus stops contaminating a product or object.

As used herein, the term “object,” “apparatus,” or “component” includes materials, compositions, devices, surface coatings, and/or composites. In some embodiments, the device 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 facilitate the spread of disease, etc. can include The device may be metal, polymeric and/or ceramic (eg silicon nitride and/or other ceramic materials).

As used herein, “contact” means physically touching or in close enough proximity to a composition or device to be affected by the composition or device.

As used herein, “personal protective equipment” or “PPE” refers to any device, article, or appliance worn or used by a person to minimize exposure to pathogens or other harmful substances. Non-limiting examples of PPE include body coverings, head coverings, shoe covers, face masks, eye protection, face and eye protection, and gloves.

As used herein, the term “silicon nitride” includes α-Si ₃ N ₄ , β-Si ₃ N ₄ , SiYAlON, β-SiYAlON, SiYON, SiAlON, or combinations thereof.

As used herein, the term “component” includes nitride-based materials, compounds, implants, devices, and the like useful for antipathogenic purposes.

As used herein, the term “effective concentration” is defined as the concentration of a substance required to inactivate at least 90% of pathogens for at least 30 minutes. As a non-limiting example, an effective concentration of α-Si ₃ N ₄ can be a concentration that reduces viral activity by at least 1-log ₁₀ within 30 minutes.

The terms used herein generally have their ordinary meaning in the art, within the context of the disclosure, and in the specific context in which each term is used. Alternate languages and synonyms may be used for one or more of the terms discussed herein, and no special meaning should be attached to whether a term is elaborated upon or discussed herein. In some cases, synonyms for certain terms are provided. The description of one or more synonyms does not preclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and is not intended to further limit the scope and meaning of this disclosure or any illustrative terms. Likewise, the present disclosure is not limited to the various embodiments provided herein.

Additional features and advantages of the present invention will be set forth in the description that 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 present invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims. These and other features of the present disclosure will be more fully apparent from the following description and appended claims, or may be learned by practice of the principles described herein.

I. SARS-CoV-2 inactivation using silicon nitride and aluminum nitride

Provided herein are methods of 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 subsequently binds (ie captures) and then inactivates the virus (eg "catches").

Silicon nitride has a unique surface chemistry that is biocompatible and has 1) simultaneous osteogenic, osteoinductive, osteoconductive and bacteriostatic applications such as 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) polymer or metal matrix composites, natural or man-made fibers, polymers or metal containing silicon nitride powders offer a number of biomedical applications including maintaining key silicon nitride bone restoration, bacteriostatic, antiviral and antifungal properties.

Silicon nitride (Si ₃ N ₄ ) is a non-oxide ceramic compound that has been used in many industries since the 1950's. The Si ₃ N ₄ formulation is FDA approved for use as an intervertebral spinal spacer in cervical and lumbar fusion surgery with proven long-term safety, efficacy and biocompatibility. Clinical data for Si ₃ N ₄ implants compare favorably with other spinal biomaterials such as allografts, titanium and polyetheretherketone. An interesting finding is that the Si ₃ N ₄ implant has a lower incidence of bacterial infection (ie less than 0.006%) when compared to other implant materials (2.7% to 18%). This property reflects the complex surface biochemistry of Si ₃ N _{4 ,} which elutes trace amounts of nitrogen that are converted to bacteria-inhibiting ammonia, ammonium and other reactive nitrogen species (RNS). A recent investigation found that viral exposure to sintered Si ₃ N ₄ powder in aqueous suspension neutralized H1N1 (influenza A/Puerto Rico/8/1934), feline calicivirus and enterovirus (EV-A71). Based on these results, Si ₃ N ₄ can inactivate SARS-CoV-2.

Silicon nitride may be antipathogenic due to the release of nitrogen-containing species when in contact with aqueous media or biological fluids and tissues. The surface chemistry of silicon nitride can be expressed as:

Si ₃ N ₄ + 6H ₂ O → 3SiO ₂ + 4NH ₃

SiO ₂ + 2H ₂ O → Si(OH) ₄

Because surface silanols are relatively stable, they elute faster (within minutes) than silicon. In the case of viruses, it has been surprisingly discovered that silicon nitride can provide RNA cleavage via alkaline transesterification resulting in loss of genome integrity and viral inactivation. It can also reduce the activity of hemagglutinin. With a concomitant increase in pH, elution of ammonia inactivates viruses, bacteria and fungi. As the examples show, it has been surprisingly found that silicon nitride and aluminum nitride each inactivate SARS-CoV-2.

The use of copper (Cu), a historically recognized biocide, is limited due to its cytotoxicity. Unlike Cu, ceramic devices or utensils made of Si ₃ N ₄ are biocompatible and non-toxic to humans. An advantage of Si ₃ N ₄ is its material versatility; Therefore, Si ₃ N ₄ is It can be incorporated into polymers, bioactive glasses and other ceramics to create composites and coatings that retain the beneficial biocompatibility and antiviral properties of Si ₃ N ₄ .

The present disclosure describes SARS-CoV-2 as an aqueous suspension of Si ₃ N ₄ and aluminum nitride (AlN) particles with two controls (ie, a suspension of copper (Cu) particles (positive control) and SARS-CoV without antiviral agent). -2 Compare the effects of exposure to a sham suspension of virions (negative control). Copper (Cu) was chosen as a positive control because of its well-known ability to inactivate various microorganisms, including viruses. Aluminum nitride was included in the test because, like Si ₃ N ₄ , it is a nitrogen-based compound that elutes nitrogen due to surface hydrolysis in an aqueous solution and increases the pH accordingly. As similar antiviral and antibacterial phenomena are believed to work for all nitride-based compounds, AlN was used to provide additional insight into the antipathogenic mechanisms of nitrogen-containing inorganic substances.

Persistence of human coronaviruses on common materials (e.g. metal, plastic, paper and textiles) and touch surfaces (e.g. doorknobs, handles, handrails, tables and desktops) may contribute to hospital and social spread of the disease. there is. Warnes et al. found that, at room temperature with 30%–40% humidity, the pathogenic human coronavirus 229E (HuCoV-229E) can survive at least 5 days on various materials such as Teflon, polyvinylchloride, ceramic tile, glass, stainless steel, and silicone rubber. reported that they maintained infectivity in a lung cell model even after sustaining viability for These investigators also showed rapid HuCoV-229E inactivation (within minutes) on simulated fingertip contamination on Cu surfaces. Cu ion release and production of reactive oxygen species (ROS) were involved in viral inactivation; And increasing contact time with copper and brass surfaces increases non-specific fragmentation of viral RNA, indicating irreversible viral inactivation. Recently, Doremalen et al. demonstrated the surface stability of SARS-CoV-1 and SARS-CoV-2 viruses on plastic, cardboard, stainless steel, and even Cu surfaces for 4–72 hours after application. A breathable N95-grade mask can capture particles before they can be inhaled, but SARS-CoV-2 virus particles remain active in the mask filter for up to 7 days. Therefore, contact killing of viruses, such as those observed on Cu surfaces, is gaining new interest as a disease mitigation strategy.

Surprisingly, compounds capable of endogenous nitrogen release, such as Si ₃ N ₄ and AlN, can inactivate the SARS-CoV-2 virus at least as effectively as Cu. Without being limited by any one theory, multiple antiviral mechanisms may be at work, such as RNA fragmentation and, in the case of Cu and AlN, direct metal ion toxicity; However, while Cu and AlN supernatants show cell lysis, Si ₃ N ₄ may not cause metabolic changes. Raman spectra of VeroE6 cells exposed to Si ₃ N ₄ viral supernatant were similar to uninfected sham. These results indicate that Si ₃ N ₄ , Cu and AlN can all inactivate the SARS-CoV-2 virus, but Si ₃ N ₄ is the safest.

The antiviral effect may be related to viral RNA fragmentation by electrical attraction (including "competitive binding" to the envelope glycoprotein hemagglutinin in the case of influenza viruses) and reactive nitrogen species (RNS). This phenomenon is due to the slow and controlled elution of nitrogen from the surface of Si ₃ N ₄ forming ammonia (NH ₃ ) and ammonium (NH ₄ ⁺ ) moieties coupled with the release of free electrons and negatively charged silanols in aqueous solution. am.

Regarding SARS-CoV-2 virus inactivation, two important aspects of the surface chemistry of Si ₃ N ₄ play a fundamental role: (i) the protonated amino groups Si-NH ₃ ⁺ and lysine on the Si ₃ N ₄ surface. similarities between the N-terminus of C-NH ₃ ⁺ on viruses; and (ii) elution of gaseous ammonia due to Si ₃ N ₄ hydrolysis. A schematic representation of the interaction between SARS-CoV-2 and the Si ₃ N ₄ surface is shown in FIG. 6 (middle panel). Similarity is depicted in the left panel of this figure. This triggers a very effective “competitive binding” approach to SARS-CoV-2 inactivation, which has come from several other successful examples, such as hepatitis B and influenza A. The potent antiviral effect of eluted (gaseous) NH ₃ is due to virion penetration and reaction with the RNA backbone. RNA undergoes alkaline transesterification through hydrolysis of phosphodiester bonds. RNA phosphodiester bond cleavage is schematically depicted in the right panel of FIG. 6 . The results of RT-PCR and fluorescence microscopy in this study suggest the contribution of both mechanisms to the inactivation of SARS-CoV-2, consistent with previous work. The TCID ₅₀ results shown in FIGS. 1A-1D and RT-PCR data in FIG. 2 for viral RNA harvested from the supernatant or Si ₃ N ₄ particles provide important information about this mechanism. >99% inactivation was achieved after exposure to Si ₃ N ₄ for 1 min (FIG. 1B), whereas RNA harvested from Si ₃ N ₄ particles (FIG. 2B) was essentially completely fragmented, whereas only partial for the supernatant. Only viral RNA fragmentation was observed (Figure 2a) was essentially completely fragmented. In the case of Cu, the opposite effect was found. This suggests that the inactivation mechanism of Si ₃ N ₄ has a series of events of “competitive binding” and ammonia poisoning, which is a kind of “catch and kill” scenario, as shown in the left panel of FIG. 6 . Complete RNA fragmentation upon 10 min exposure to Si ₃ N ₄ suggests that nitrogen elution is a key process triggering a series of reactions leading to viral inactivation (see right panel of FIG. 6 ).

In some embodiments, an object, article or composition comprising silicon nitride or aluminum nitride may act to continuously bind a virus (eg, SARS-Cov-2) and then inactivate the virus.

The antiviral effect of Si ₃ N ₄ may be similar to Cu. Cu is an essential trace element for human health and is an electron donor/acceptor for several key enzymes by altering the redox state between Cu ⁺ and Cu2 ⁺ , but these properties can also cause cell damage. Its use as an antiviral agent is limited due to allergic dermatitis, hypersensitivity and multi-organ dysfunction. In contrast, the safety of Si ₃ N ₄ as a permanently implanted material during spinal fusion surgery has been well established by experimental and clinical data. Thus, an object, article or composition comprising silicon nitride may be as effective at inactivating viruses as Cu without the negative effects of Cu.

Si ₃ N ₄ is well known for its function as an industrial material. Load-bearing Si ₃ N ₄ prosthetic hip bearings and spinal fusion implants were initially developed due to the superior strength and toughness of Si ₃ N ₄ . Subsequent studies have shown other properties of Si ₃ N ₄ to be favorable for orthopedic implant design, such as enhanced bone conductivity, bacteriostasis, improved radiolucency, lack of implant sedimentation and wear resistance. Thus, the surface chemistry, topography and hydrophilicity of Si ₃ N ₄ contribute to a dual effect (ie upregulation of osteogenic activity to promote spinal fusion while simultaneously preventing bacterial attachment and biofilm formation). Besides its proven track record as a bioimplant, an advantage of Si ₃ N ₄ is its manufacturing versatility. The sintered powder of Si ₃ N ₄ is incorporated into other materials such as polymers, other ceramics, bio-glass and metals to create a composite structure that retains the index bone forming and antibacterial properties of monolithic Si ₃ N ₄ . Three-dimensional layered deposition of Si ₃ N ₄ could enable the production of protective surfaces for medical applications that reduce formite-mediated transmission of microbial diseases. Incorporating Si ₃ N ₄ particles into fabrics of personal protective equipment such as face masks, protective gowns and surgical cloths can contribute to the safety of healthcare workers and patients.

Si ₃ N ₄ inactivates the SARS-CoV-2 virus within minutes of exposure. Without being bound by any one theory, the mechanism of action may be shared with other nitrogen-based compounds that represent trace surface disinfectants, such as aluminum nitride.

In some embodiments, the object used to bind and inactivate the SARS-CoV-2 virus is a device or appliance that may include a silicon nitride and/or aluminum nitride composition on at least a portion of the surface of the object. A silicon nitride or aluminum nitride coating may be applied to the surface of an object in powder form. In some embodiments, the silicon nitride or aluminum nitride powder may be filled, embedded or impregnated into at least a portion of the object. In some embodiments, a 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 μm, from about 300 nm to about 1.5 μm, or from about 0.6 μm to about 1.0 μm. In other embodiments, silicon nitride or aluminum nitride may be incorporated into the device. For example, the object may include silicon nitride and/or aluminum nitride powder within the body of the object. In one embodiment, the device may be made of silicon nitride. In other embodiments, the object may be made of aluminum nitride. In another embodiment, the object may include a slurry or suspension of aluminum nitride or silicon nitride particles.

In some embodiments, the object may further comprise other materials, including but not limited to paper, cardboard, fabric, plastic, ceramic, polymer, stainless steel, metal, or combinations thereof. Some non-limiting examples of objects include surgical gowns, surgical drapes, shoe covers, cubicle curtains, tubing, garments, gloves, safety glasses, masks including surgical masks and face shields, PPE, tables such as hospital examination and operating tables, Air such as chairs, bed frames, bed trays, desks, fixtures, cabinets, equipment racks, carts, handles, knobs, handrails, toys, water filters and face mask filters, respirator filters, air filtration filters and air ventilation filters or air conditioning filters May contain filters. In some embodiments, the filter may be within the filtering device of an anesthesia machine, ventilator, or CPAP machine so that the filter's antibacterial surface layer can trap pulmonary pathogens as air moves into and out of infected lungs. In various embodiments, an object may be a medical device or instrument. Non-limiting examples of medical devices or instruments include orthopedic implants, spinal implants, pedicle screws, dental implants, indwelling catheters, endotracheal tubes, colonoscopy endoscopes, and other similar devices.

In another embodiment, the object may be a composition comprising silicon nitride or aluminum nitride powder, including but not limited to a slurry, suspension, gel, spray, paint or toothpaste. For example, silicon nitride or aluminum nitride can be added to a slurry such as paint and then applied to a surface to provide an antibacterial, antifungal and antiviral surface. In another embodiment, silicon nitride or aluminum nitride can 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.

The silicon nitride or aluminum nitride coating may be present on the surface of the object at a concentration of 1% to 100% by weight. Silicon nitride and/or aluminum nitride may be coated or laminated to the object. In various embodiments, the coating is about 1%, 2%, 5%, 7.5%, 8.3%, 10%, 15%, 16.7%, 20%, 25%, or about 30% by weight silicon nitride powder or aluminum nitride powder. In some embodiments, the coating may include about 10% to about 20% by weight of silicon nitride or aluminum nitride. In at least one embodiment, the coating includes about 15 weight percent silicon nitride or aluminum nitride. In some embodiments, the silicon nitride or aluminum nitride may be embedded in (as a filler) or on the surface of the object at a concentration of about 1% to about 100% by weight. In various embodiments, the object is about 1%, 2%, 5%, 7.5%, 8.3%, 10%, 15%, 16.7%, 20%, 25%, 30% by weight. %, 33.3%, 35%, 40%, 50%, 60%, 70%, 80%, 90% to 100% silicon nitride or aluminum nitride. In some embodiments, the silicon nitride or aluminum nitride may be present on the surface of the object in a concentration of 10% to 20% by weight. In at least one embodiment, the silicon nitride or aluminum nitride may be present at a concentration of about 15% by weight on the surface of the object. In some aspects, the concentration of silicon nitride or aluminum nitride may vary depending on the substrate material of the object, such as paper, cardboard, textile, plastic, ceramic, polymer, 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 to the amount of silicon nitride and/or aluminum nitride that may be incorporated into the object (ie, permeation limit).

In some embodiments, the object may be a monolithic component composed of silicon nitride or aluminum nitride. Such objects may be completely dense with no internal porosity, or may be porous with a porosity ranging from about 1% to about 80%. The monolithic object may be used as a medical device or may be used in instruments where inactivation of viruses may be required.

In some embodiments, the object may come into contact with the SARS-CoV-2 virus for a limited amount of time. An object may come into contact with the SARS-CoV-2 virus for about 1 minute to about 2 hours to inactivate the virus. In various embodiments the object may be in contact with 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. In at least one embodiment, the object may be permanently implanted into a patient. In at least one embodiment, the object may be worn externally by a user. In another embodiment, the object may be permanently implanted into a patient. In another embodiment, the object may be a high contact surface. In a further embodiment, the object may be in continuous or continuous contact with the patient's bodily fluid. The bodily fluid may be blood or gas (eg, inhaled or exhaled gas).

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 after contact with an object for at least 1 minute, at least 5 minutes, or at least 30 minutes. % inactive, at least 95% inactive or at least 99% inactive. In at least one embodiment, the virus is at least 85% inactivated after contact with an object for at least 1 minute. In another embodiment, the virus is at least 99% inactivated after contact with the object for at least 30 minutes. In another embodiment, the virus is at least 99% inactivated after contact with an object for at least 1 minute.

Articles of personal protective equipment having antiviral and antimicrobial properties are also provided herein. The article may include silicon nitride or aluminum nitride incorporated into the article, or silicon nitride or aluminum nitride may be coated on the surface of the article.

The silicon nitride or aluminum nitride coating may be present on the surface of the article at a concentration of about 1% to about 100% by weight. In various embodiments, the coating is about 1%, 2%, 5%, 7.5%, 8.3%, 10%, 15%, 16.7%, 20%, 25%, or about 30% by weight of silicon nitride powder or aluminum nitride powder. In some embodiments, the coating may include about 10% to about 20% by weight of silicon nitride or aluminum nitride. In at least one embodiment, the coating includes about 15 weight percent 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 at a concentration of about 1% to about 100% by weight. In various embodiments, the object is about 1%, 2%, 5%, 7.5%, 8.3%, 10%, 15%, 16.7%, 20%, 25%, 30% by weight. %, 33.3%, 35%, 40%, 50%, 60%, 70%, 80%, 90% to 100% silicon nitride or aluminum nitride. In some embodiments, silicon nitride or aluminum nitride may be present on the surface of the article in a concentration of about 10% to about 20% by weight. In at least one embodiment, the silicon nitride or aluminum nitride may be present on the surface of the article in a concentration of about 15% by weight. In some aspects, the concentration of silicon nitride or aluminum nitride may vary depending on the substrate material of the object.

In some embodiments, the article is PPE. In some aspects, the article is a body cover, head cover, shoe cover, face mask, face and eye protection, or glove. In some aspects, the article may be operable to inactivate the SARS-CoV-2 virus when the article contacts the virus.

II. Nitride-based antipathogenic composition and device and method of use thereof

Provided herein are antipathogenic compositions and devices comprising nitrides for inactivation of viruses, bacteria and fungi. As used herein, a nitride is a nitrogen compound in which nitrogen has a nominal oxidation state of -3 to +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 these includes a combination of Nitrides can have a high intrinsic nitrogen content. For example, silicon nitride (Si ₃ N ₄ ) contains about 40% nitrogen by weight, boron nitride (BN) contains about 56% nitrogen by weight, and aluminum nitride (AlN) contains 34% nitrogen by weight. and titanium nitride (TiN) contains about 22% nitrogen by weight.

In general, nitrides can be antipathogenic due to the release of nitrogen-containing species when in contact with aqueous media or biological fluids and tissues. For example, the surface hydrolysis chemistry of silicon nitride can be expressed as:

Si ₃ N ₄ + 6H ₂ O → 3SiO ₂ + 4NH ₃

SiO ₂ + 2H ₂ O → Si(OH) ₄

Similarly, as a second example, the surface hydrolysis chemistry of boron nitride can be expressed as:

BN + 3H ₂ O → B ₂ O ₃ + 2NH ₃

Also, in the presence of water, ammonia and ammonium exist in pH dependent equilibrium as follows:

NH₄ ⁺ (aq) + OH^- (aq)NH ₃ + H ₂ O

NH ₄ ⁺ (aq) + OH ^- (aq)

Elution of ammonia, protonation to ammonium and hydroxide ions and concomitant pH increase inactivate viruses, bacteria and fungi. Similar to silicon nitride, other nitride materials may exhibit antipathogenic properties due to surface hydrolysis of nitrogen and consequent formation of ammonia and ammonium. Previously, only β-silicon nitride was believed to exhibit antipathogenic properties. Surprisingly, however, other nitride materials were found to exhibit similar surface chemistries. This result was unexpected given the fact that all nitrides are essentially water insoluble and their hydrolysis was previously considered insufficient to produce antipathogenic properties.

In one embodiment, the antipathogenic nitride-based composition may exhibit elution kinetics exhibiting: (i) slow but continuous elution of ammonia from the solid state rather than the conventional gas phase; (ii) no damage or adverse effects on eukaryotic cells; and (iii) intelligent elution that increases with decreasing pH.

The antipathogenic composition and device disclosed herein is one of aluminum nitride, boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorus nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, and zirconium nitride. may contain more than In some embodiments, the compositions and devices may further include silicon nitride, such as α-silicon nitride. In other embodiments, the nitride may be α-silicon nitride. In some embodiments, the nitride may be aluminum nitride. In another embodiment, the antipathogenic composition and device may include a nitride mixture, such as a mixture of AlN and BN. Surprisingly, it has been found that these compositions are capable of inactivating viruses.

Nitride-based compositions can be powders, particulates, slurries, suspensions, coatings, films, and/or composites. In some embodiments, the composition may include micrometric or nanometric particles of nitride. The average particle size of the nitride may range from about 100 nm to about 5 μm, from about 300 nm to about 1.5 μm, or from about 0.6 μm to about 1.0 μm. In other embodiments, the composition may include a slurry or suspension of nitride particles.

In some embodiments, the antipathogenic composition may be a monolithic component composed of nitrides. Such components may be completely dense with no internal porosity, or may be porous with a porosity ranging from about 1% to about 80%. The monolithic component may be used as a medical device or may be used in instruments where inactivation of viruses, bacteria and/or fungi may be required.

In another embodiment, the antipathogenic nitride-based composition can be incorporated into a device or into a coating on a device surface to inactivate viruses, bacteria and fungi. At least a portion of the surface of the device may be coated with a coating comprising a nitride-based composition. Non-limiting examples of suitable devices include orthopedic implants, spinal implants, pedicle screws, dental implants, indwelling catheters, endotracheal tubes, endoscopes for colonoscopy, and other similar devices. The device or mechanism may be metal, polymeric and/or ceramic.

In some embodiments, nitrides are used in polymers and fabrics, surgical gowns, gloves, tubing, garments, air and water filters (eg, filtration devices in anesthesia machines, respirators, or CPAP machines), masks, hospital examinations and surgeries. It can be applied as a coating or incorporated into materials or appliances for antipathogenic properties such as tables, desks, fixtures, handles, knobs, toys and filters such as air conditioner filters or toothbrushes.

The nitride-based coating may be applied as a powder to the surface of the device. In some embodiments, nitride powder may be embedded or impregnated into at least a portion of the device. In some embodiments, powders may be micrometric or nanometer sized. In other embodiments, silicon nitride may be incorporated into the device. For example, the device may contain nitride powder within the device body.

In some embodiments, 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 buffered saline. In other embodiments, the nitride-based composition may be a suspension or emulsion of a nitride powder and a suitable vehicle for topical application (eg, a cream, gel, or lotion). The nitride powder may be present in the composition in a concentration of about 0.1% by volume to about 20% by volume. In various embodiments, the slurry may comprise about 0.1%, 0.5%, 1%, 1.5%, 2%, 5%, 10%, 15%, or 20% silicon nitride by volume. there is. In some embodiments, the concentration of nitride may be effective to inactivate pathogens.

In other embodiments, the nitride-based coating may be present on the surface of or within the device at a concentration of about 1% to about 100% by weight. In various embodiments, the coating is about 1%, 2%, 5%, 7.5%, 8.3%, 10%, 15%, 16.7%, 20%, 25%, 30% by weight. %, 33.3%, 35%, or 40% nitride powder. In at least one embodiment, the coating includes about 15 weight percent nitride. In some embodiments, the nitride may be present in or on the surface of the device or appliance in a concentration of about 1% to about 100% by weight. In various embodiments, the device or appliance may contain about 1%, 2%, 5%, 7.5%, 8.3%, 10%, 15%, 16.7%, 20%, 25%, 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 nitride may be included. there is. In some embodiments, the concentration of nitride may be effective to inactivate pathogens.

In some embodiments, the anti-pathogenic nitride-based composition or device is capable of inactivating or reducing the spread of viruses, bacteria and/or fungi. Viruses, bacteria and fungi can infect mammalian cells, animal cells and/or plant cells. Non-limiting examples of viruses that can be inactivated by the antipathogenic nitride-based composition include coronaviruses (eg, SARS-CoV-2), rhinoviruses, influenza viruses (A, B, C, D) and feline calicis. contains a virus Anti-pathogenic nitride-based compositions or devices are capable of killing both gram-positive and gram-negative bacteria. Examples of soluble fungi include downy mildew, powdery mildew, Botrytis rot, Fusarium rot, rust, Rhizoctonia rot, Sclerotinia rot, Sclerotium (Sclerotium) Including but not limited to those that cause rot, or other agricultural diseases.

In some embodiments, the pathogen may be on or in the surface of a human, animal or plant. In other embodiments, the pathogen may be on the surface of or inside an inanimate object.

Further provided herein is a method of inactivating a pathogen by contacting a virus, bacteria, and/or fungus with an antipathogenic nitride-based composition disclosed herein. In one embodiment, a method may include coating a device or appliance with a nitride-based composition and contacting the coated appliance with a virus, bacteria, or fungus. In another embodiment, the method may include contacting a virus, bacterium 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.

Also provided herein are methods for treating or preventing pathogens at a specific location in a human patient. For example, the pathogen may be a virus, bacterium or fungus. The method can include contacting a patient with a device, appliance, or composition comprising a nitride-based composition. The device, apparatus or composition may include from about 1% to about 100% nitride by weight. In some embodiments, a device or appliance may include from about 1% to about 100% nitride by weight on a surface of the device or appliance. In one embodiment, the device or mechanism may be a monolithic nitride-based ceramic. In other embodiments, the device or appliance may include a nitride coating such as a nitride powder coating. In other embodiments, the device or mechanism may incorporate nitride into the device body. For example, the nitride powder may be incorporated or impregnated into the device or body of the device using methods known in the art.

In some embodiments, the device or instrument may be in contact 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 embodiment, the device or appliance may be permanently implanted into a patient. In at least one embodiment, the device or appliance may be worn externally by a user.

Exemplary Embodiments

Embodiment 1: Effective concentration of a nitride selected from 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 A method for inactivating a pathogen comprising contacting a composition comprising a pathogen with a pathogen, wherein the effective concentration of the nitride inactivates the pathogen.

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 nitride is from about 0.1% to about 20% by volume.

Embodiment 7: The method of embodiment 1, wherein said composition comprises a powder of said nitride.

Embodiment 8: The method of embodiment 7, wherein the composition is coated over at least a portion of a surface of the device and/or incorporated into the device.

Embodiment 9: The method of embodiment 8, wherein the effective concentration of nitride is from 1% to 100% by weight.

Embodiment 10: The method of embodiment 8 or 9, wherein the device is an orthopedic implant, a spinal implant, a pedicle screw, a dental implant, an indwelling catheter, an endotracheal tube, a colonoscopy endoscope, a surgical gown, a mask, filter or tubing.

Embodiment 11: The method of any one of embodiments 1 to 10, wherein the pathogen is a virus, bacteria or 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 or inside a human, animal or plant.

Embodiment 14: The method of any one of embodiments 1 to 12, wherein the pathogen is on the surface of an inanimate object.

Embodiment 15: A nitride-based composition for inactivating pathogens comprising a slurry of nitride particles in an aqueous medium, wherein the nitride is present in a concentration of from about 0.1% to about 20% by volume, wherein the concentration is effective to inactivate pathogens. That is, a nitride-based composition.

Embodiment 16: The nitride-based composition of Example 15, wherein the nitride is boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorus nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, nitride A nitride-based composition selected from zirconium, or combinations thereof.

Embodiment 17: The nitride-based composition of Example 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, bacteria or 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 or inside a human, animal or plant.

Embodiment 23: The nitride-based composition of any one of embodiments 15 to 21, wherein the pathogen is on the surface of an inanimate object.

Embodiment 24: A nitride-based device for pathogen inactivation comprising a nitride powder coated on at least a portion of a surface of the device and/or incorporated into the device, wherein the nitride is present in a concentration of about 10% to about 30% by weight; wherein the concentration is effective to inactivate pathogens.

Embodiment 25: The nitride-based device of embodiment 24, wherein the nitride is boron nitride, chromium nitride, cerium nitride, hafnium nitride, lanthanum nitride, phosphorus nitride, sulfur nitride, tantalum nitride, titanium nitride, vanadium nitride, yttrium nitride, nitride A nitride-based device selected from zirconium, or combinations thereof.

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, bacteria, or 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 or inside a human, animal or plant.

Embodiment 32: The nitride-based device of any one of embodiments 24 to 30, wherein the pathogen is on the surface of an inanimate object.

Embodiment 33: A method for inactivating a pathogen comprising contacting a pathogen with a slurry comprising an effective concentration of α-silicon nitride, wherein the effective concentration of nitride inactivates the pathogen, and wherein the effective concentration of α-silicon nitride in the slurry is about 15% w/v.

Embodiment 34: The method of embodiment 33, wherein the slurry comprises 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 a nitride selected from combinations thereof.

Embodiment 35: A method for inactivating a pathogen comprising contacting a pathogen with a slurry comprising an effective concentration of aluminum nitride, wherein the effective concentration of 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 comprises 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 a nitride selected from combinations thereof.

Example

Example 1: Rapid inactivation of SARS-CoV-2 by silicon nitride or aluminum nitride

In this study, the SARS-CoV-2 virus was isolated from a suspension of silicon nitride (Si ₃ N ₄ ) particles, aluminum nitride (AlN) particles and two controls (i.e., copper (Cu) particles (positive control) and a suspension without antiviral agent ( The effect of exposure to an aqueous suspension of the negative control)) was compared. Cu was chosen as a positive control because of its well-known ability to inactivate various microorganisms, including viruses.

Preparation of test materials

Si ₃ N ₄ , Cu and AlN powders were purchased from commercial sources. Si ₃ N ₄ powder (nominal composition of 90 wt % Si ₃ N ₄ , 6 wt % Y ₂ O ₃ and 4 wt % Al ₂ O ₃ ) was obtained by aqueous mixing of the inorganic components and spray drying followed by sintering of the spray dried granules. (~1700°C for ~3 hours), hot isostatic pressing (~1600°C, 2 hours, N ₂ at 140 MPa), aqueous based milling and freeze drying. The average particle diameter of the obtained powder was 0.8±1.0 μm. As-received Cu powder (USP grade 99.5% purity) granules were milled to achieve particle sizes comparable to Si ₃ N ₄ . The average particle size of AlN powder was 1.2±0.6 μm, similar to that of Si ₃ N ₄ .

Preparation of Mammalian and Viral Cells

VeroE6/TMPRSS2 mammalian cells were used for viral assays. Cells were cultured in Dulbecco's Modified Eagle Minimum Essential Nutrient Medium (DMEM) supplemented with G418 disulfate (1 mg/ml), penicillin (100 units/mL), streptomycin (100 μg/mL), and 5% fetal bovine serum. Eagle's minimum essential medium) and maintained at 37°C in 5% CO/95% in a humidified atmosphere. SARS-CoV-2 virus stocks were propagated for 2 days at 37°C using VeroE6/TMPRSS2 cells. Viral titers were analyzed as the median tissue culture infectious dose (TCID ₅₀ ).

Example 2:

virus analysis

15% by weight (15 wt.%) of Si ₃ N ₄ , Cu and AlN powders were separately dispersed in 1 mL of PBS(-) and then virus suspension (2×10 ⁵ TCID ₅₀ in 20 μL) was added. Due to the high density of Cu powder, the volume fraction was approximately 1/3 of Si ₃ N ₄ . Mixing was performed for 1 and 10 minutes with slow manual rotation at 4°C. After exposure, the powder was pelleted by centrifugation (2400 rpm for 2 min) followed by filtration through 0.1 μm media. Supernatants were collected and subjected to TCID ₅₀ assay, real-time RT-PCR testing and fluorescence imaging. Experiments were performed in triplicate, including sham supernatants without antiviral powder. In 96-well plates, confluent monolayers of VeroE6/TMPRSS2 cells were inoculated with 50 μL/well of each virus suspension in 10-fold serial dilutions using 0.5% FBS DMEM (i.e. maintenance medium). Viral adsorption for 1 hour at 37°C was performed with a tilt every 10 minutes. Then, 50 μL/well of maintenance medium was added. Plates were incubated for 4 days at 37°C, 5% CO2 / 95% humidified atmosphere. The cytopathic effect (CPE) of the infected cells was observed by phase contrast microscopy. Cells were then fixed by adding 10 μL/well of glutaraldehyde and staining with 0.5% crystal violet. TCID ₅₀ was calculated according to the Reed-Muench method.

Viral RNA analysis

After exposure to the powder, 140 μL of the supernatant was used for viral RNA extraction. RNA was also extracted from the surface of the centrifuged and filtered powder. RNA purification was performed using the QIAamp Viral RNA Mini kit. A 16 μL aliquot of isolated RNA was reverse transcribed using ReverTra Ace® qPCR RT Master Mix. Quantitative real-time PCR was performed using Step-One Plus Real-Time PCR system primers/probes for two specific virus N gene sets. Each 20 μL reaction mixture contained 4 μL of cDNA, 8.8 pmol of each primer, 2.4 pmol of probe and 10 μL GoTaq Probe qPCR master mix. The amplification protocol consisted of 50 cycles of denaturation at 95 °C for 3 sec and annealing at 60 °C for 20 sec and extension.

Immunochemical Fluorescence Analysis

Vero E6/TMPRSS2 cells were inoculated with 200 μL of viral supernatant. After viral adsorption for 1 hour at 37° C., cells were incubated for 7 hours with maintenance medium in a CO2 incubator. For detection of infected cells, these cells were washed with TBS (20 mM Tris-HCl pH 7.5, 150 mM NaCl) and fixed with 4% PFA for 10 min at room temperature (RT) followed by 0.1% Triton in TBS for 5 min at RT. The membrane was permeabilized with X. Cells were blocked with 2% skim milk in TBS for 60 minutes at room temperature and stained with anti-SARS coronavirus enveloped (rabbit) antibody (dilution = 1:100) for 60 minutes at room temperature. After washing with buffer, cells were incubated with Alexa 594 goat anti-rabbit IgG (H+L) (1:500) and Alexa 488 phalloidin (1:50) for 60 minutes at room temperature in the dark. ProLong™ Diamond Antifade Mountant with DAPI was used as the mounting medium. Staining was observed with a fluorescence microscope BZX710. Total cell and infected cell counts were obtained using a Keyence BZ-X analyzer.

Raman spectroscopy

Vero E6/TMPRSS2 cells were infected at the free site with 200 μL of each virus suspension. After viral adsorption for 1 hour at 37° C., infected cells were incubated with maintenance medium in a CO ₂ incubator for 4 hours and fixed with 4% paraformaldehyde for 10 minutes at room temperature. After washing twice with distilled water, the infected cells were air dried and analyzed in situ using a Raman microprobe spectrometer. Raman spectra were collected using a high-sensitivity spectrometer with a 20× optical lens. It operated in microscopy mode using two-dimensional confocal imaging. A holographic notch filter in the optical circuit was used to efficiently achieve a spectral resolution of 1.5 cm-1 with a 532 nm excitation source operating at 10 mW. Raman emission was monitored using a single monochromator coupled to an air-cooled charge-coupled device (CCD) detector 1024 × 256 pixels). Acquisition time was fixed at 10 seconds. Thirty spectra were collected and averaged for each analysis time point. Raman spectra were deconvolved into Gaussian-Lorentzian subbands using commercially available software.

statistical analysis

Student's t-test determined statistical significance for n=3 and a p-value of 0.01 using Prism software.

Example 3:

Median tissue culture infectious dose

The TCID ₅₀ assay results for 15 wt % Si ₃ N ₄ , Cu and AlN powders are shown in FIGS. 1A-1D. Inactivation times of 1 min and 10 min are shown in FIGS. 1A and 1B as well as FIGS. 1C and 1D, respectively. Compared to the negative control, all three powders were effective in inactivating SARS-CoV-2 virions (>99%) during both exposure times.

RNA gene fragmentation

To investigate whether viral RNA was fragmented due to exposure to both supernatant and powder, RT-PCR tests were performed on the N gene set of viral RNA. Results are shown in FIGS. 2A and 2B as well as FIGS. 2C and 2D for 1 minute and 10 minute exposure, respectively. Again, compared to the negative control at 1 min exposure to the supernatant, almost complete fragmentation of RNA was observed for Cu while significant damage was caused by AlN and to a lesser extent by Si ₃ N ₄ . After 10 min exposure to the supernatant, significant cleavage of RNA was observed in all three materials. While Cu still showed the most fragmentation, Si ₃ N ₄ had a similar effect and AlN was essentially the same as the 1 min exposure condition. Viral RNA was virtually undetectable for all three materials based on RNA extracted from the pelleted powder at 1 min exposure (see Figures 2a and 2b). This result suggests that the reduction of viral RNA in the supernatant is due to direct degradation and not because RNA sticks to the powder.

immunofluorescence test

TCID ₅₀ assay and gene fragmentation results were confirmed using immunofluorescence imaging using an envelope antibody (red), phalloidin (green) to stain F-actin in living cells, and DAPI (blue) to stain cell nuclei. 3A-3D show VeroE6 inoculated with supernatants of (a) unexposed virions (ie, negative control) and 10 min exposed virions of (b) Si ₃ N ₄ , (c) AlN and (d) Cu. /shows a fluorescence micrograph representing the TMPRSS2 cell population. Figure 3E shows cells that were not inoculated with virus (labeled as "mock-infected" cells. Red spots in the negative control (Figure 3A) demonstrated that virions entered the metabolism of Vero6E cells and hijacked them. This is normal. Contrasted with sham-infected cells (Fig. 3e), which showed metabolic function.

Surprisingly, cells inoculated with supernatants of Si ₃ N ₄ and to a lesser extent AlN exhibited almost normal function with little infection. Conversely, cells inoculated with Cu supernatant were essentially dead (i.e., completely lacking F-actin, Fig. 3d), although based on the blue-red staining present in the nuclei, as virions appear to have hijacked some nuclei. Pre-survival may have been possible. This suggests that cell lysis is not only a result of viral infection but also due to the toxic effect of intracellular free Cu ions. Quantification of the colorimetric results from FIGS. 3A-3E is provided in FIG. 4 . These data demonstrate that about 35% of viable VeroE6 cells from the negative control were infected with the virions, whereas only 2% and 8% of cells inoculated with supernatants from Si ₃ N ₄ and AlN were infected, respectively. Quantitative evaluation of cells inoculated with Cu supernatant could not be evaluated due to premature death.

Raman spectroscopy

Raman spectroscopy examined VeroE6 cells exposed to various supernatants to evaluate biochemical cellular changes due to infection and ion (i.e., Cu and Al) toxicity. 5A-5G show (a) uninfected VeroE6/TMPRSS2 cells, and (b) Frequency range for cells inoculated with supernatant containing virions exposed for 10 min to Si ₃ N ₄ , (c) AlN, (d) Cu (positive control) and (e) no antiviral compound (negative control). It shows a Raman spectrum of 700-900 cm-1. Of fundamental importance are the vibrational bands of ring respiration and the H-scissor ring of the indole ring of tryptophan (at 756 and 875 cm-1, respectively, denoted T1 and T2). Tryptophan plays an important role in protein synthesis and creation of molecules for various immune functions. Its stereoisomer serves to fix proteins in the cell membrane, and its catabolite has an immunosuppressive function. The catabolism of tryptophan is induced by viral infection. This occurs through the enzymatic activity of indoleamine-2,3-dioxygenase (IDO), which protects host cells from an overreactive immune response. IDO reduces tryptophan to kynurenine and then to N'-formyl-kynurenine. Increased IDO activity depletes tryptophan. Consequently, the intensities of the tryptophan bands (T1 and T2) are indicative of these biochemical changes. Except for the Cu-treated samples, the data presented in Figure 5f show an exponential decrease in the bound tryptophan band that correlates with the fraction of infected cells. (The chemical structure of N'-formyl-kynurenine is shown in the inset for clarity.) Copper's anomalies provide further evidence of its toxicity. VeroE6 cells consumed tryptophan to reduce Cu ²⁺ and stabilize it to Cu ⁺ .

Raman signals by ring-stretching vibrations of adenine, cytosine, guanine, and thymine were found at 725, 795, 680, and 748 cm-1, and are indicated as A, Cy1, G, and Th in Figs. 5a-5e, respectively. This band was preserved after virus exposure. However, for cells infected with Cu-exposed virions, there were abnormalities for lines representing tyrosines at 642 and 832 cm −1 labeled Ty1 and Ty2, respectively. The ring-respiration band of tyrosine, Ty2, was very weak compared to other samples (see Figs. 5d and 5b). Conversely, the C–C binding-related Ty1 signal remained strong. This suggests that the aromatic ring of tyrosine chelated the Cu ion. This explains why only the tyrosine ring-respiration mode decreased while the C-C signal remained unchanged. Three possible Cu(II) chelate forms on tyrosine are shown in Figure 5g.

For VeroE6 cells exposed to AlN-treated virions (Fig. 5c), the tryptophan T1 and T2 bands were preserved, but 615 and ∼615 due to ring bending of DNA cytosines (labeled Cy2 and Cy3, respectively, in Figs. 5a-5e). The band almost disappeared at 700 cm-1. Their loss is due to progressive internucleosome DNA cleavage or complex formation, both of which are associated with toxicity. Loss of cytosine signaling translates to a toxic effect by Al ions, although much less significant than copper. Al ₃₊ interacts with the carbonyl O and/or N ring donor at the nucleotide base and binds selectively by chelation to the backbone of the PO2 group and/or to the guanine N-7 site of ^the GC base pair.

Unlike exposure of VeroE6 cells to Cu and AlN supernatants resulted in moderate to severe toxicity, Si ₃ N ₄ did not induce modification of tryptophan, tyrosine and cytosine. The shape of the spectrum for the Si ₃ N ₄ viral supernatant was almost identical to that of the uninfected sham suspension (see Figs. 5a and 5b).

Example 4: Biofilm analysis of aluminum nitride and boron nitride materials

This study was conducted to compare Staphylococcus epidermidis biofilm growth on biomaterial variations of silicon nitride and other ceramic materials with PEEK as a positive control. Ceramic materials under observation include as-fired silicon nitride (AFSN), aluminum nitride (AlN), two grades of boron nitride (BN: AX05BN and PCBN1000) and Shapal (a mixture of AlN and BN).

Three replicates were selected for each sample material at the 24 and 48 hour time points for basic statistical analysis. After washing with alcohol under ultrasonic agitation for 5 minutes. Rinse with deionized water using ultrasonic agitation for 5 min. Sterilize the samples by UV-C light exposure for 30 min on each side. Samples were prepared by resting the samples for at least 60 minutes prior to inoculation.

Bacterial medium was prepared by mixing 7% glucose, 1X PBS and 10% human plasma, which was then inoculated with a small amount of Staphylococcus epidermidis. Initial absorbance values were taken using a spectrophotometer. The medium was then placed in a shaking incubator at 37° C. and 175 rpm for 6 hours and allowed to grow until the bacteria reached an absorbance value of 0.05 AU (corresponding to 10 ⁵ cells/mL per growth curve generated previously).

Each sterile sample was placed in a well plate and 7 mL of liquid culture was added. The well plate was placed in a shaking incubator at 37° C. and 125 rpm for 24 hours. At t = 24 hours the liquid culture was removed and 7 mL of fresh medium was added. The well plate was returned to the shaking incubator for an additional 24 hours.

Samples were removed at appropriate time points (24 or 48 hours) and rinsed with 5 mL of 1X PBS in a fresh well plate in a shaking incubator at 125 rpm for 2 minutes. Each sample was immersed in fresh 1X PBS and rinsed. Samples were placed in 10 mL 1X PBS in a 50 mL centrifuge tube and vortexed vigorously for 2 minutes.

Surface area measurements were obtained using disc diameter, width and weight. The number of colonies on each petrifilm was counted, recorded and compared.

The biofilm solution of each sample was serially diluted in 1X PBS to be 1x, 1/10x, 1/100x, 1/1,000x and 1/10,000x the concentration of the original sample. Each dilution was plated on petrifilm to allow the lowest calculable dilution to be used in data comparisons for each sample. After growing for 24 hours in a culture environment at 37°C, the bacteria formed colonies called colony forming units (CFUs). After obtaining the number of CFUs, the number was multiplied by the appropriate dilution factor to calculate the CFU per sample. Total CFU is divided by surface area to accurately compare samples of different sizes, so the final data reported are in units of CFU/mm ² . Statistical analysis was performed and graphed. Table 1 and Figure 7 show the average for each material at both 24 and 48 hours. Statistical analysis was performed by applying a heteroskedastic two-tailed t-test with 95% confidence intervals for all samples.

Table 1

Bacterial growth of PEEK at 24 hours was 10-fold greater than calcined β -Si ₃ N ₄ (AFSN), AlN and Shapal, all with p<0.05 _. Statistical differences were found between all materials except PEEK and AX05BN. AX05BN was also found to have a statistically significant difference in biofilm growth when compared to all other materials. Biofilm growth comparisons between AlN, Shapal, AFSN and PCBN1000 were not significantly different overall. It can be concluded that AlN, Shapal, PCBN1000 and AFSN contain the most effective antibacterial properties at the 24 hour time point.

At 48 hours, the serial dilution and plating procedure described in [0096] was performed on the remaining samples. Biofilm growth on PEEK was consistent with the results found at 24 h, as the CFU count remained at least an order of magnitude higher than either ceramic material. Representative p-values for this comparison did not reflect visual differences due to one PEEK sample outlier. Due to the expected biological variability, this outlier does not change the conclusions drawn from the overall results. Significant differences were found for both AX05BN and PCBN1000 compared to AFSN, Shapal and AlN. Compared to AX05BN, the p-values for AFSN, AlN, and Shapal were p=0.032, p=0.031, and p=0.033, respectively. The 48 hour results are consistent with the trend from 24 hours. All materials showed a consistent increase in biofilm growth over time.

As expected at both the 24 and 48 hour time points, Staphylococcus epidermidis were found to form much denser biofilms on PEEK than either ceramic variant. AX05BN did not perform as efficiently as the other ceramic materials evaluated in this experiment, as the resulting CFU count was not significantly different from PEEK. Shapal, AlN and AFSN showed similar biofilm growth, and Staphylococcus epidermidis Emphasizes ability to resist surface attachment and colonization. Although their bacterial resistance was fairly similar, AlN outperformed PEEK, AX05BN, PCBN1000, Shapal and AFSN in resistance to biofilm formation at both the 24 and 48 hour time point assessments. Shapal and AFSN closely followed AlN in antibacterial properties.

These results support previous conclusions that ceramic materials significantly outperform PEEK in biofilm resistance. Comparisons within ceramic samples show that AX05BN is much less effective at resisting bacterial attachment than other ceramic compositions. Although there was no significant difference between Shapal, AlN and AFSN, AlN showed slightly less biofilm formation at both time points.

Example 5: Rapid inactivation of SARS-CoV-2 by alpha-silicon nitride and beta-silicon nitride

Two silicon nitride powders were prepared. The β-silicon nitride powder had a nominal composition of 90 wt% β-Si ₃ N _{4 ,} 6 wt% yttria (Y ₂ O ₃ ) and 4 wt % alumina (Al ₂ O ₃ ). The powder was prepared by aqueous mixing and spray drying of each inorganic component, followed by sintering the spray dried granules at about 1700° C. for about 3 hours. Next, the sintered granules were hot isostatically pressed at about 1600 DEG C for 2 hours at 140 MPa in a nitrogen atmosphere. Pressing was followed by aqueous based milling and freeze drying.

The α-silicon nitride powder was 98 wt % pure Si ₃ N ₄ with about 2 wt % SiO ₂ . It was prepared by heating commercially available high-purity α-silicon nitride in air at about 300° C. for about 1 hour and then cooling to room temperature.

To perform antiviral testing, the Washington state variant of SARS-CoV-2 virus was diluted to a concentration of 2 x 10 ⁴ virions/mL in DMEM growth medium. Then, 4 mL of the diluted virus solution was added to the tube containing 15 wt%/volume (w/v) α- or β-silicon nitride powder. A virus without Si ₃ N ₄ was treated at the same time as a control. The tube was vortexed for 30 seconds to ensure proper contact and then placed in a tube revolver for 30 minutes. Samples were centrifuged and the supernatant was collected and filtered through a 0.2 μm filter. RNA of infectious virus remaining in the clarified supernatant was isolated along with the pellet and quantified by RT-qPCR method.

The supernatant was also subjected to the plaque assay test method. The results presented in Figure 8 show that the genomic copy of the virus only control had a concentration of about 1 x 10 ⁶ /mL, while one of the β-silicon nitride powders had reduced virions to approximately 4.3 x 10 ⁴ /mL (95.9%). and two different lots of α-silicon nitride showed a virus reduction of approximately 1 x 10 ³ /mL (99.9%). Pellet samples from both α-silicon nitride in this same test series were found to be completely free of viable virions.

Plaque assay test results are provided in FIG. 9A. These data showed a reduction in viral activity for β-silicon nitride powders ranging from 1.25 to 3.5 log ₁₀ (~93% to 99.97%), while α-silicon nitride powders showed >4.5 log ₁₀ reduction (>99.997%). Changes in the surface hydrolysis of the β-silicon nitride powder are believed to have resulted in the range of observed results. However, a comparison of RT-qPCR and plaque assay methods showed that SARS-CoV-2 virions do not pellet with silicon nitride and their RNA structure is damaged when incubated with silicon nitride.

Plaque assay tests were performed on the effectiveness of α- and β-silicon nitride powders against the South African strain of SARS-CoV-2 virus using the same powders and methods described above. The results are shown in Figure 9b. α-silicon nitride proved to be very effective with >4.5 log ₁₀ reduction (99.9997%) in viruses and β-silicon nitride powder exhibited approximately 1-log ₁₀ reduction (˜90%).

Similarly, a plaque assay was performed on the effectiveness of α- and β-silicon nitride powders against the British strain of SARS-CoV-2 virus using the same materials and procedures as presented above. Results are shown in Figure 9c. α-silicon nitride powder reduced virus count by about 4- log ₁₀ (99.99%) and β- Silicon nitride powder reduced about 1-log ₁₀ (~90%).

Having described several embodiments, those skilled in the art will recognize that various modifications, alternative constructions and equivalents may be used without departing from the spirit of the invention. In addition, many well-known processes and elements have not been described in order to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the present invention.

Those skilled in the art will appreciate that the presently disclosed embodiments teach by way of example and not limitation. Therefore, matters contained in the above description or shown in the accompanying drawings are illustrative and should not be construed in a limiting sense. The following claims are intended to cover all general and specific features described herein, as well as all statements as to the scope of the present methods and systems that may be said to lie therebetween as a matter of language.

Claims (36)

A composition comprising an effective concentration of a nitride selected from 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 A method of inactivating a pathogen comprising contacting a pathogen with a pathogen, wherein the effective concentration of nitride inactivates the pathogen. The method of claim 1 , wherein the composition further comprises silicon nitride. The method of claim 1 , wherein the composition further comprises aluminum nitride. The method of claim 1, wherein the nitride is boron nitride. The method of claim 1 , wherein the composition comprises a slurry of nitride particles in an aqueous medium. 6. The method of claim 5, wherein the effective concentration of the nitride is from about 0.1% to about 20% by volume. The method of claim 1 , wherein the composition includes a powder of the nitride. 8. The method of claim 7, wherein the composition is coated over at least a portion of a device surface and/or incorporated into a device. The method of claim 8, wherein an effective concentration of the nitride is 1% to 100% by weight. 10. The device according to claim 8 or 9, wherein the device is an orthopedic implant, a spinal implant, a pedicle screw, a dental implant, an indwelling catheter, an endotracheal tube, a colonoscopy endoscope, a surgical gown, a mask, a filter or tubing. which is, how. The method according to any one of claims 1 to 10, wherein the pathogen is a virus, bacteria or fungus. 12. The method of claim 11, wherein the virus is a coronavirus. 13. The method according to any one of claims 1 to 12, wherein the pathogen is on or inside a human, animal or plant. 13. The method according to any one of claims 1 to 12, wherein the pathogen is on the surface of an inanimate object. A nitride-based composition for inactivating pathogens comprising a slurry of nitride particles in an aqueous medium, wherein the nitride is present in a concentration of from about 0.1% to about 20% by volume, wherein the concentration is effective to inactivate pathogens. Nitride-based composition. 16. The method of claim 15, wherein the nitride is from 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 a combination thereof. Which is selected, the nitride-based composition. 17. The nitride-based composition according to claim 16, wherein the composition further comprises silicon nitride. 17. The nitride-based composition according to claim 16, wherein the composition further comprises aluminum nitride. The nitride-based composition according to claim 16, wherein the nitride is boron nitride. The nitride-based composition according to claim 15, wherein the pathogen is a virus, bacteria or fungus. 21. The nitride-based composition according to claim 20, wherein the virus is a coronavirus. The nitride-based composition according to any one of claims 15 to 21, wherein the pathogen is present on or inside a human, animal or plant. 22. The nitride-based composition according to any one of claims 15 to 21, wherein the pathogen is on the surface of an inanimate object. A nitride-based device for pathogen inactivation comprising a nitride powder coated over and/or incorporated into at least a portion of a surface of the device, wherein the nitride is present in a concentration of about 10% to about 30% by weight, wherein the concentration is A nitride-based device effective for inactivating pathogens. 25. The method of claim 24, wherein the nitride is from 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 a combination thereof. A nitride-based device, which is selected. 26. The nitride-based device of claim 25, wherein the device further comprises silicon nitride. 26. The nitride-based device of claim 25, wherein the device further comprises aluminum nitride. 26. The nitride-based device of claim 25, wherein the nitride is boron nitride. 25. The nitride-based device of claim 24, wherein the pathogen is a virus, bacterium or fungus. 30. The nitride-based device of claim 29, wherein the virus is a coronavirus. 31. The nitride-based device according to any one of claims 24 to 30, wherein the pathogen is on or inside a human, animal or plant. 31. A nitride-based device according to any one of claims 24 to 30, wherein the pathogen is on the surface of an inanimate object. A method for inactivating a pathogen comprising contacting a pathogen with a slurry comprising an effective concentration of α-silicon nitride, wherein the effective concentration of nitride inactivates the pathogen, and wherein the effective concentration of α-silicon nitride in the slurry is about 15% w /v, which way. 34. The method of claim 33, wherein the slurry is from 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. further comprising a selected nitride. A method for inactivating a pathogen comprising contacting a pathogen with a slurry comprising an effective concentration of aluminum nitride, wherein the effective concentration of nitride inactivates the pathogen, wherein the effective concentration of aluminum nitride in the slurry is about 15% w/w which would be v, how. 36. The method of claim 35, wherein the slurry is from 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. further comprising a selected nitride.

Images