CN117295398A - Nitride-based antipathogenic compositions and devices and methods of use thereof - Google Patents

Abstract

Described herein are anti-pathogenic compositions comprising a 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 a combination thereof; and methods of inactivating viruses, bacteria, and/or fungi using the compositions.

Description

Nitride-based antipathogenic compositions and devices and methods of use thereof

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/123,037, filed on 12/9/2020, the entire contents of which are incorporated herein by reference.

Technical Field

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

Background

It is well known that surface transmission of pathogenic organisms can lead to a number of diseases, disabilities and deaths. In recent years, the discovery of new pathogens (e.g., SARS-CoV-2 virus) has highlighted this problem. Methods of killing or inactivating pathogens living on surfaces typically involve the use of cleaning products, high heat or pressure, or electromagnetic radiation. These methods can be costly and in some cases can present other hazards to humans, such as exposure to toxic chemicals or intense ultraviolet radiation. Furthermore, some pathogens exhibit resistance to existing methods over time.

Thus, there is a need for methods and compositions suitable for inactivating or killing pathogenic organisms.

Disclosure of Invention

Provided herein are nitride-based compositions or devices for inactivating viruses, bacteria, and/or fungi. The nitride-based composition comprises 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.

Also provided herein are methods of inactivating pathogens by contacting a virus, bacterium, and/or fungus with an antipathogenic nitride-based composition or device disclosed herein.

Drawings

Fig. 1A to 1D are graphical representations showing: at room temperature with a composition containing 15wt.% Cu, alN and Si ₃ N ₄ Treatment of the powders in aqueous medium for 1 min and 10 min of the nitride powders inactivated SARS-CoV-2, wherein the control virus was treated identically without any addition of powder. After centrifugation, the supernatant was subjected to TCID ₅₀ And (5) measuring. The Reed-Muench method was used to determine virus titers. TCID showing virus inactivation times of 1 min and 10 min, respectively ₅₀ 50 μl (FIGS. 1A and 1B) and a decrease (FIGS. 1C and 1D). Statistics are given in the inset according to the unpaired two-tailed Student's t-test (n=3).

Figures 2A to 2D are graphical representations showing viral RNAs that undergo severe degradation after exposure to copper or nitride particles. In FIGS. 2A and 2B, the virus suspension is exposed to Cu, alN and Si ₃ N ₄ Powder for 1 min and viral RNA in the supernatant and on the particles were assessed using viral N gene "set 1" and "set 2" primers, respectively. The data collected on the supernatant and pellet samples were compared to the amount of viral N gene RNA in untreated suspension. In FIGS. 2C and 2D, the exposure of the supernatant to Cu, alN and Si of the viral N gene "set 1" and "set 2" primers, respectively, is shown ₃ N ₄ Results of RT-PCR test after 10 minutes of powder. Statistical data are given in the inset according to the unpaired two-tailed schwann t-test (n=3).

FIGS. 3A to 3E are diagrams showing Si ₃ N ₄ Images of inhibiting viral infection without affecting cell viability, wherein Cu kills cells. With (FIG. 3A) unexposed virions and exposure to Si ₃ N ₄ (FIG. 3B), alN (FIG. 3C) and Cu (FIG. 3D) for 10 minutes UTE virions were inoculated with VeroE6/TMPRSS2 cells. In fig. 3E, uninoculated cells (labeled "pseudo-infected" cells) were also prepared and imaged for comparison. After fixation, cells were stained with anti-SARS coronavirus envelope antibody (red), F-actin was visualized with Phalloidin (Phalindin) (green), and nuclei were stained with DAPI (blue). Fluorescence micrographs representing n=3 samples are shown.

FIG. 4 is a graphical representation showing the counts of fluorescently labeled and unlabeled cells on a fluorescence micrograph, and the% infected cells and% viable cells are calculated as follows: infected cell% = (number of cells stained with anti-SARS coronavirus envelope antibody)/(number of cells stained with DAPI) x 100; and live cell% = (number of cells stained with phalloidin)/(number of cells stained with DAPI) x 100. Data represent n=3 samples. P <0.05 and 0.01 by unpaired bipartite schwann t-test (n=3), respectively; n.s. =non-salient.

Fig. 5A to 5G are graphical representations of the following Raman spectra (Raman spectra): (a) Uninfected cells (FIG. 5A) (i.e., not exposed to virions) and to (b) Si ₃ N ₄ (FIG. 5B), (C) AlN (FIG. 5C) and (D) Cu (FIG. 5D) for 10 minutes in SARS-CoV-2 virion infected cells; in fig. 5E, raman spectra of unexposed virion (negative control) infected cells are shown. In FIG. 5F, the average intensity of two tryptophan T1 and T2 bands (at 756 and 875cm-1, respectively) is plotted as a function of fraction of virosome infected cells that were not exposed and exposed to different particles for 10 minutes (see label); in the inset, the structure of N' -formyl kynurenine is shown, which is an intermediate in tryptophan catabolism upon enzymatic IDO reaction. In fig. 5G, the graphical representation shows three possible conformations of the tyrosine-based peptide, which can demonstrate the disappearance of the ring vibration in tyrosine (Ty 2 band) upon chelation of Cu (II) ions.

FIG. 6 shows Si ₃ N ₄ Protonated amine-based Si-NH at the surface ₃ ⁺ N-terminal C-NH to lysine in cells ₃ ⁺ Schematic models of chemical and charge similarity between (left panel); at Si ₃ N ₄ SARS-CoV-2 virus with charged molecular species at the surface (in particular at protonated positively charged amine) and eluted species NH ₃ /NH ₄ ⁺ Is shown (middle panel). The eluted N leaves 3+ charge vacancies (purple sites) on the solid surface that are created with the negatively charged silanol. The right panel shows a three-step process of cleavage of the RNA backbone by eluted nitrogen species (i.e., deprotonation of the 2' -hydroxyl, transient pentaphosphate formation, and cleavage of phosphodiester linkages in the RNA backbone by base transesterification by hydrolysis). Note that the similarity between protonated amine and lysine N-terminus may trigger an extremely efficient "competitive binding" mechanism for SARS-CoV-2 virion inactivation, while eluted ammonia is in the combined "capture andkilling "effects deadly degradation of virion RNA.

FIG. 7 is a log comparison of average bacterial growth over PEEK, boron nitride, aluminum nitride, shape (combination of boron nitride and aluminum nitride) and silicon nitride materials for 24 hours and 48 hours.

FIG. 8 is a graph showing a 30 minute exposure RT-qPCR genomic test of the Washington state variant of SARS-CoV-2 virus relative to 15 wt/vol% of alpha-silicon nitride and beta-silicon nitride powders.

FIG. 9A is a graph showing plaque assay test results for 30 minute exposures of Washington's variant of SARS-CoV-2 virus relative to 15 wt/vol% of alpha-silicon nitride and beta-silicon nitride powders.

FIG. 9B is a graph showing plaque assay test results for 30 minute exposures of the south Africa variant of SARS-CoV-2 virus relative to 15 wt/vol% of alpha-silicon nitride and beta-silicon nitride powders.

FIG. 9C is a graph showing plaque assay test results for 30 minute exposures of the British variant of SARS-CoV-2 virus relative to 15 wt/vol% of alpha-silicon nitride and beta-silicon nitride powders.

Corresponding reference numerals indicate corresponding parts throughout the several views of the drawings. The headings used in the figures do not limit the scope of the claims.

Detailed Description

Various embodiments of the present disclosure are discussed in detail below. While specific embodiments are discussed, it should be understood that this is done for illustrative purposes only. One skilled in the relevant art will recognize that other components and configurations may be used without departing from the spirit and scope of the disclosure. The following description and drawings are, accordingly, illustrative and should not be construed as limiting. Numerous specific details are described to provide a thorough understanding of the present disclosure. However, in some instances, well known or conventional details are not described in order to avoid obscuring the description. References to one or an embodiment in the present disclosure may refer to the same embodiment or to any embodiment; and such references mean 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 disclosure. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Furthermore, various features are described which may be exhibited by some embodiments and not by others.

As used herein, the terms "include," "have," and "include" are used in their open, non-limiting sense. The terms "a/an" and "the" are to be interpreted as covering a plurality as well as the singular. Thus, the term "mixture thereof (a mixture thereof)" also relates to "mixture thereof (mixtures therof)".

As used herein, "inactivation" or "inactivation" refers to viral inactivation, wherein the virus is prevented from contaminating the product or subject by completely removing or rendering the virus non-infectious.

As used herein, the terms "object," "apparatus," or "component" include materials, compositions, devices, surface coatings, and/or composites. In some examples, the apparatus may include various medical devices or equipment, examination tables, personal protective equipment such as clothing, filters, masks and gloves, catheters, endoscopic instruments, surfaces with which viral persistent infections may promote disease transmission, and the like. The device may be metallic, polymeric, and/or ceramic (e.g., silicon nitride and/or other ceramic materials).

As used herein, "contacting" means physically contacting or sufficiently close to a composition or device to be affected by the composition or device.

As used herein, "personal protective equipment" or "PPE" means any device, article, or apparatus that is worn or otherwise used by an individual to minimize contact with pathogens or other harmful substances. Non-limiting examples of PPE include body covers, head covers, shoe covers, face masks, goggles, face masks and goggles, and gloves.

As used herein, the term "silicon nitride" includes α -Si ₃ N ₄ 、β-Si ₃ N ₄ SiyalON, beta-SiYAlON, siYON, siAlON, or a combination thereof.

As used herein, the term "component" includes nitride-based materials, compounds, implants, devices, or the like suitable for anti-pathogenic purposes.

As used herein, the term "effective concentration" is defined as the concentration of material required to inactivate at least 90% of pathogens in at least 30 minutes. As a non-limiting example, α -Si ₃ N _{4 (4)} The effective concentration can be at least 1-log reduction in viral activity within 30 minutes ₁₀ Is a concentration of (3).

Within the context of the present disclosure and in the specific context of use of each term, the terms used in this specification generally have their ordinary meaning in the art. Alternative language and synonyms may be used for any one or more of the terms discussed herein, and no special significance should be made whether or not a term is specified or discussed herein. In some cases, synonyms for certain terms are provided. The recitation of one or more synonyms does not exclude the use of other synonyms. The examples used anywhere in this specification (including examples of any terms discussed herein) are illustrative only and are not intended to further limit the scope and meaning of this disclosure or any example terms. As such, the present disclosure is not limited to the various embodiments set forth in the present specification.

Additional features and advantages of the disclosure will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the principles disclosed herein. The features and advantages of the disclosure may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the principles set forth herein.

I. Inactivation of SARS-CoV-2 Using silicon nitride and aluminum nitride

Provided herein is a method for inactivating SARS-CoV-2 virus by contacting the virus with a subject or composition comprising silicon nitride and/or aluminum nitride. The silicon nitride and/or aluminum nitride sequentially bind (i.e., capture) and then inactivate the virus (e.g., "capture and kill").

Silicon nitride has unique surface chemistry that is biocompatible and provides many biomedical applications including 1) osteogenesis, osteoinduction, osteoconduction, and bacteriostasis as is concurrent in spinal and dental implants; 2) Killing both gram positive and gram negative bacteria according to different mechanisms; 3) Inactivating human and animal viruses, bacteria, and fungi; and 4) the polymer or metal matrix composite, natural or synthetic fibers, polymer or metal-containing silicon nitride powder maintains key silicon nitride bone restoration, bacteriostasis, antiviral and antifungal properties.

Silicon nitride (Si) ₃ N ₄ ) Are non-oxide ceramic compounds that have been used in many industries since the 50 s of the 20 th century. Si (Si) ₃ N ₄ Is FDA approved for use as an intervertebral spinal spacer in cervical and lumbar fusion procedures, and has long-term safety, efficacy, and biocompatibility. Si (Si) ₃ N ₄ Clinical data for implants have advantages over other spinal biomaterials, such as allografts, titanium and polyetheretherketone. An interesting finding is that Si is compared to other implant materials (2.7% to 18%) ₃ N ₄ The bacterial infection rate of the implant was lower (i.e., less than 0.006%). This property reflects the trace amount of Si eluting nitrogen ₃ N ₄ Converts the nitrogen into ammonia, ammonium and other bacteria-inhibiting Reactive Nitrogen Species (RNS). Recent studies have also found that virus is exposed to sintered Si in aqueous suspension ₃ N ₄ The powder was neutralized by H1N1 (influenza A/Podoconcha (Puerto Rico)/8/1934), feline calicivirus (Feline calicivirus) and enterovirus (EV-A71). Based on these findings, si ₃ N ₄ It may be possible to inactivate SARS-CoV-2.

Silicon nitride may be anti-pathogenic in that it releases nitrogen-containing species when contacted with aqueous media or biological fluids and tissues. The surface chemistry of silicon nitride can be shown as follows:

Si ₃ N ₄ +6H ₂ O→3SiO ₂ +4NH ₃

SiO ₂ +2H ₂ O→Si(OH) ₄

Nitrogen elutes faster (within minutes) than silicon because surface silanol is relatively stable. For viruses, it has surprisingly been found that silicon nitride can provide RNA cleavage by alkali esterification, which results in loss of genome integrity and viral inactivation. This may also reduce the activity of hemagglutinin. Ammonia elution and the consequent increase in pH inactivate viruses, bacteria and fungi. As shown in the examples, it was surprisingly found that each of silicon nitride and aluminum nitride inactivated SARS-CoV-2.

The use of copper (Cu), a historically recognized virucide, is limited by its cytotoxicity. Contrary to Cu, from Si ₃ N ₄ The ceramic devices or instruments produced are biocompatible and non-toxic to humans. Si (Si) ₃ N ₄ Has the advantages of multifunction of the material; thus Si is ₃ N ₄ May be incorporated into polymers, bioactive glasses, and even other ceramics to produce Si-retaining materials ₃ N ₄ Is a complex and coating with advantageous biocompatibility and antiviral properties.

The present disclosure compares exposure of SARS-CoV-2 to Si ₃ N ₄ And the effect of an aqueous suspension of aluminium nitride (AlN) particles and two controls, namely a suspension of copper (Cu) particles (positive control) and a pseudo-suspension of SARS-CoV-2 virions without any antiviral agent (negative control). Copper (Cu) was chosen as the positive control because of its well-known ability to inactivate a variety of microorganisms, including viruses. Aluminum nitride was included in the test because of its combination with Si ₃ N ₄ As such, it is a nitrogen-based compound whose surface hydrolysis in aqueous solution results in elution of nitrogen and a consequent increase in pH. Due to the equivalent antiviral and antibacterial phenomenaIs believed to be effective for all nitride-based compounds, and thus the use of AlN provides additional insight into the anti-pathogenic mechanism of nitrogen-containing inorganic materials.

The continued presence of human coronaviruses on common materials (e.g., metal, plastic, paper, and fabric) and contact surfaces (e.g., knobs, handles, rails, tables, and desktops) can lead to hospital and social transmission of the disease. Warnes et al report that pathogenic human coronavirus 229E (HuCoV-229E) remains infectious in the lung cell model after sustained survival for at least 5 days on a variety of materials such as Teflon, polyvinyl chloride, tile, glass, stainless steel and silicone rubber at room temperature with humidity of 30% -40%. These researchers also showed that HuCoV-229E deactivated rapidly (within minutes) for simulated fingertip contamination on the Cu surface. Viral inactivation involves Cu ion release and the generation of Reactive Oxygen Species (ROS); and an increase in contact time with copper and brass surfaces results in a more non-specific cleavage of viral RNA, indicating irreversible viral inactivation. Recently, duplex Lei Malun (Doremalen) et al showed surface stability on plastic, cardboard, stainless steel and even Cu surfaces for 4-72 hours after application of both SARS-CoV-1 and SARS-CoV-2 viruses. Although the breathable N95 mask can trap the particles before they are inhaled, the activity of the SARS-CoV-2 virus particles in the mask filter can be maintained for up to 7 days. Thus, contact killing viruses (as observed on Cu surfaces) is of renewed interest as a disease-mitigation strategy.

Surprisingly, compounds capable of releasing endogenous nitrogen, such as Si ₃ N ₄ And AlN, can inactivate SARS-CoV-2 virus at least as effectively as Cu. Without being limited to any one theory, a variety of antiviral mechanisms may be effective, such as RNA fragmentation, and in the case of Cu and AlN, direct metal ion toxicity; however, although Cu and AlN supernatants showed cell lysis, si ₃ N ₄ Metabolic changes may not be caused. Exposure to Si ₃ N ₄ The raman spectrum of VeroE6 cells of the virus supernatant was similar to that of the uninfected sham group. These findings indicate that despite Si ₃ N ₄ Cu and AlN can be inactivatedSARS-CoV-2 Virus, but Si ₃ N ₄ Is the safest.

Antiviral effects may be associated with electrical attraction (in the case of influenza viruses, including "competitive binding" to the envelope glycoprotein hemagglutinin) and viral RNA cleavage by Reactive Nitrogen Species (RNS). These phenomena are due to the formation of ammonia (NH) from nitrogen ₃ ) And ammonium (NH) ₄ ⁺ ) Part of Si ₃ N ₄ The surface slowly and controllably elutes while free electrons and negatively charged silanol are released in the aqueous solution.

In the case of SARS-CoV-2 virus inactivation, si ₃ N ₄ Plays a fundamental role in two important aspects of surface chemistry: (i) Si (Si) ₃ N ₄ Protonated amino Si-NH at the surface of (C) ₃ ⁺ N-terminal C-NH to lysine on virus ₃ ⁺ Similarity between; (ii) due to Si ₃ N ₄ Hydrolysis to elute gaseous ammonia. SARS-CoV-2 and Si ₃ N ₄ A schematic of the interaction between the surfaces is given in fig. 6 (middle diagram). The similarity is depicted in the left hand drawing of this figure. It triggered an extremely effective "competitive binding" method for SARS-CoV-2 inactivation, which resulted from several other successful examples, such as Hepatitis B (hepatis B) and Influenza A (Influenza A). Eluted (gaseous) NH ₃ Due to its penetration into virions and its reaction with the RNA backbone. RNA undergoes basic transesterification by hydrolysis of its phosphodiester bonds. RNA phosphodiester bond cleavage is schematically depicted in the right panel of fig. 6. The results of the RT-PCR and fluorescence microscopy of this study showed that both mechanisms contributed to the inactivation of SARS-CoV-2, consistent with earlier studies. TCID (TCID) ₅₀ The results are shown in FIGS. 1A to 1D and FIGS. 2A to 2D from the supernatant or Si ₃ N ₄ RT-PCR data of viral RNA harvested from particles provides important information about these mechanisms. Although exposed to Si ₃ N ₄ After lasting 1 minute realize>99% inactivation (FIG. 1B), but only partial viral RNA cleavage was observed for the supernatant (FIG. 2A), from Si ₃ N ₄ RNA from particle harvest (FIG. 2B) was essentially completeBreaking. Note that for Cu, the opposite effect was found. This indicates Si ₃ N ₄ The inactivation mechanism of (as depicted in the left panel of fig. 6) has successive "competitive binding" and ammonia poisoning events-a "capture and kill" scenario. Exposure to Si ₃ N ₄ Complete RNA fragmentation for 10 minutes suggests that nitrogen elution is a critical process triggering a cascade of reactions leading to viral inactivation (see right panel in fig. 6).

In some embodiments, a subject, article, or composition comprising silicon nitride or aluminum nitride may be operable to sequentially bind a virus (e.g., SARS-Cov-2) and then inactivate the virus.

Si ₃ N ₄ May be equivalent to Cu. Although Cu is a trace element essential for human health, cu is also changed ⁺ And Cu ²⁺ Electron donors/acceptors for several key enzymes in the redox state in between, but these properties may also lead to cell damage. Its use as an antiviral agent is limited by allergic dermatitis, hypersensitivity reactions and multiple organ dysfunction. In contrast, experimental and clinical data fully demonstrate Si ₃ N ₄ Safety as a permanent implant material during spinal fusion procedures. Thus, a subject, article, or composition comprising silicon nitride may be as effective as Cu in inactivating viruses without the negative effects of Cu.

Si ₃ N ₄ It is well known for its ability to be an industrial material. Bearing Si ₃ N ₄ Artificial hip bearings and spinal fusion implants were originally due to Si ₃ N ₄ Excellent strength and toughness. Later studies showed that Si ₃ N ₄ Other characteristics of the implant are welcome in orthopedic implant designs such as enhanced bone conduction, bacteriostasis, improved radiolucence, no implant subsidence, and wear resistance. Thus, si is ₃ N ₄ The surface chemistry, topography, and hydrophilicity of (a) contribute to the dual role (i.e., up-regulating osteogenic activity to promote spinal fusion while preventing bacterial adhesion and biofilm formation). Except that it has been demonstrated as a bioimplantIn addition to the recording of Si ₃ N ₄ One advantage of (2) is its versatility of manufacture. Si (Si) ₃ N ₄ Has been incorporated into other materials such as polymers, other ceramics, bioglass and metals to create a solid Si ₃ N ₄ Is a composite structure of osteogenic and antibacterial properties. Si (Si) ₃ N ₄ Three-dimensional additive deposition of (c) may enable the fabrication of protective surfaces in healthcare, thereby reducing the transmission of contaminant-mediated microbial disease. Si is mixed with ₃ N ₄ The incorporation of particles into the fabric of personal protective equipment such as masks, protective apparel, and surgical drapes may aid in the safety of medical personnel and patients.

Si ₃ N ₄ SARS-CoV-2 virus can be inactivated within minutes after exposure. Without being limited by 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.

In some embodiments, the subject for binding and inactivating 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 the surface of the subject. Silicon nitride or aluminum nitride coatings may be applied as powders to the surface of the object. In some examples, a silicon nitride or aluminum nitride powder may be filled, embedded, or impregnated in at least a portion of the object. In some embodiments, the powder may have particles in the micrometer, submicron, or nanometer size range. The average particle size may be in the range of about 100nm to about 5 μm, about 300nm to about 1.5 μm, or about 0.6 μm to about 1.0 μm. In other embodiments, silicon nitride or aluminum nitride may be incorporated into the device. For example, the object may incorporate silicon nitride and/or aluminum nitride powder into the body of the object. In one embodiment, the device may be made of silicon nitride. In another embodiment, the object may be made of aluminum nitride. In yet another embodiment, the object may comprise 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 subjects may include gowns, drapes, shoe covers, compartment curtains, tubing, clothing, gloves, goggles, masks including surgical masks and face masks, PPEs, tables such as hospital tables and tables, chairs, bed frames, bed trays, desks, fixtures, cabinets, equipment racks, carts, handles, knobs, balustrades, toys, water filters, and air filters, such as mask filters, respirator filters, air filters, and air ventilation filters or air conditioning filters. In some examples, the filter may be within a filtering device of an anesthesia machine, ventilator, or CPAP machine, such that an antimicrobial surface layer in the filter may trap lung pathogens as air enters and exits the infected lung. In various embodiments, the 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, and other similar devices.

In other embodiments, the object may be a composition in which silicon nitride or aluminum nitride powder is incorporated, including but not limited to a slurry, suspension, gel, spray, paint, or toothpaste. For example, adding silicon nitride or aluminum nitride to a slurry, such as a paint, and then applying it to a surface can provide an antibacterial, antifungal, and antiviral surface. In other embodiments, silicon nitride or aluminum nitride may be mixed with water along with any suitable dispersants and slurry stabilizers and then applied by spraying the slurry onto various surfaces. An example of a dispersant is Dolapix a88.

The silicon nitride or aluminum nitride coating may be present on the surface of the object at a concentration of about 1wt.% to about 100 wt.%. Silicon nitride and/or aluminum nitride may be coated onto or layered into the object. In various embodiments, the coating may include about 1wt.%, 2wt.%, 5wt.%, 7.5wt.%, 8.3wt.%, 10wt.%, 15wt.%, 16.7wt.%, 20wt.%, 25wt.%, or about 30wt.% silicon nitride powder or aluminum nitride powder. In some examples, the coating may include about 10wt.% to about 20wt.% silicon nitride or aluminum nitride. In at least one example, the coating includes about 15wt.% silicon nitride or aluminum nitride. In some embodiments, silicon nitride or aluminum nitride may be embedded (as a filler) in or on the surface of the object at a concentration of about 1wt.% to about 100 wt.%. In various embodiments, the subject may comprise about 1wt.%, 2wt.%, 5wt.%, 7.5wt.%, 8.3wt.%, 10wt.%, 15wt.%, 16.7wt.%, 20wt.%, 25wt.%, 30wt.%, 33.3wt.%, 35wt.%, 40wt.%, 50wt.%, 60wt.%, 70wt.%, 80wt.%, 90wt.% to 100wt.% silicon nitride or aluminum nitride. In some examples, the silicon nitride or aluminum nitride may be located on the surface of the object at a concentration of about 10wt.% to about 20 wt.%. In at least one example, the silicon nitride or aluminum nitride may be located 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, polymer, stainless steel, and/or metal. In some embodiments, the base material of the object may be a polymer, and the polymer has a practical limit (i.e., penetration limit) on the amount of silicon nitride and/or aluminum nitride that may be incorporated into the object.

In some embodiments, the object may be a monolithic component composed of silicon nitride or aluminum nitride. Such objects may be fully dense, having no internal porosity, or the objects may be porous, having a porosity in the range of about 1% to about 80%. The unitary object may be used as a medical device or may be used in an instrument where it may be desirable to inactivate viruses.

In some embodiments, the subject may be exposed to SARS-CoV-2 virus for a limited period of time. The subject may be contacted with the SARS-CoV-2 virus for a period of about 1 minute to about 2 hours to inactivate the virus. In various examples, the subject may be contacted 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 example, the subject may be permanently implanted within the patient. In at least one example, the object may be externally worn by a user. In another example, the subject may be permanently implanted within the patient. In yet another example, the object may be a high contact surface. In further examples, the subject may be in continuous or continuous contact with the body fluid of the patient. The body fluid may be blood or gas (e.g., 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, at least 95% inactivated, or at least 99% inactivated after contacting with the subject for at least 1 minute, at least 5 minutes, or at least 30 minutes. In at least one example, the virus is at least 85% inactivated after contact with the subject for at least 1 minute. In another example, the virus is at least 99% inactivated after contact with the subject for at least 30 minutes. In yet another example, the virus is at least 99% inactivated after contact with the subject for at least 1 minute.

Also provided herein is an article of personal protective equipment having antiviral and antimicrobial properties. 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 at a concentration of about 1wt.% to about 100 wt.%. In various embodiments, the coating may include about 1wt.%, 2wt.%, 5wt.%, 7.5wt.%, 8.3wt.%, 10wt.%, 15wt.%, 16.7wt.%, 20wt.%, 25wt.%, or about 30wt.% silicon nitride powder or aluminum nitride powder. In some examples, the coating may include about 10wt.% to about 20wt.% silicon nitride or aluminum nitride. In at least one example, the coating includes about 15wt.% silicon nitride or aluminum nitride. In some embodiments, silicon nitride or aluminum nitride may be embedded (as a filler) in or on the surface of the article at a concentration of about 1wt.% to about 100 wt.%. In various embodiments, the subject may comprise about 1wt.%, 2wt.%, 5wt.%, 7.5wt.%, 8.3wt.%, 10wt.%, 15wt.%, 16.7wt.%, 20wt.%, 25wt.%, 30wt.%, 33.3wt.%, 35wt.%, 40wt.%, 50wt.%, 60wt.%, 70wt.%, 80wt.%, 90wt.% to 100wt.% silicon nitride or aluminum nitride. In some examples, the silicon nitride or aluminum nitride may be located on the surface of the article at a concentration of about 10wt.% to about 20 wt.%. In at least one example, the silicon nitride or aluminum nitride may be located 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.

In some embodiments, the article is PPE. In some aspects, the article is a body cover, headgear, shoe cover, face mask, and goggles or gloves. In some aspects, the preparation is operable to inactivate SARS-CoV-2 virus when the preparation is contacted with the virus.

Nitride-based antipathogenic compositions and devices and methods of use thereof

Provided herein are anti-pathogenic compositions and devices comprising nitrides for inactivating viruses, bacteria, and fungi. As used herein, a nitride is a compound of nitrogen, where the nominal oxidation state of nitrogen is 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. The nitride may have a high inherent nitrogen content. For example, silicon nitride (Si ₃ N ₄ ) Contains about 40wt.% nitrogen, boron Nitride (BN) has about 56wt.% nitrogen, aluminum nitride (AlN) has 34wt.% nitrogen, and titanium nitride (TiN) has about 22wt.% nitrogen.

Nitrides may generally be antipathogenic due to the release of nitrogen-containing species when in contact with aqueous media or biological fluids and tissues. For example, the silicon nitride non-surface hydrolysis chemistry may be as follows:

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 may be as follows:

BN+3H ₂ O→B ₂ O ₃ +2NH ₃

furthermore, in the presence of water, ammonia and ammonium are present in a pH dependent equilibrium as follows:

ammonia elution, its protonation of the ammonium and hydroxide ions, and the consequent increase in pH inactivate viruses, bacteria and fungi. Like silicon nitride, other nitride materials may exhibit anti-pathogenic properties due to surface hydrolysis of nitrogen and the consequent formation of ammonia and ammonium. It was previously thought that only beta-silicon nitride exhibited anti-pathogenic properties. However, it was unexpectedly found that other nitride materials also exhibit similar surface chemistry. These results are unexpected in view of the fact that all nitrides are substantially insoluble in water and their hydrolysis was previously thought to be insufficient to produce antipathogenic properties.

In one embodiment, the nitride-based antipathogenic composition may exhibit the following elution kinetics: (i) Slowly but continuously eluting ammonia from a solid state rather than from a generally gaseous state; (ii) no harm or negative effect on eukaryotic cells; and (iii) smart elution increases with decreasing pH.

The antipathogenic compositions and devices disclosed herein may comprise one or more of the following: 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. In some embodiments, the compositions and devices may further comprise silicon nitride, such as alpha silicon nitride. In other embodiments, the nitride may be alpha silicon nitride. In some embodiments, the nitride may be aluminum nitride. In still other embodiments, the anti-pathogenic compositions and devices can include a mixture of nitrides, such as a mixture of AlN and BN. Surprisingly, it has been found that these compositions are capable of inactivating viruses.

The nitride-based composition may be a powder, particle, slurry, suspension, coating, film, and/or composite. In some embodiments, the composition may comprise micro-or nanoparticles of nitride. The average particle size of the nitride may be in the range of about 100nm to about 5 μm, about 300nm to about 1.5 μm, or about 0.6 μm to about 1.0 μm. In another embodiment, the composition may comprise a slurry or suspension of nitride particles.

In some embodiments, the anti-pathogenic composition may be a monolithic component composed of a nitride. Such an assembly may be fully dense, having no internal porosity, or such an assembly may be porous, having a porosity in the range of about 1% to about 80%. The unitary assembly may be used as a medical device or may be used in an apparatus where it may be desirable to inactivate viruses, bacteria and/or fungi.

In another embodiment, the nitride-based anti-pathogenic composition may be incorporated into a device or into a coating on the surface of the device 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, colonoscopy, and other similar devices. The device or apparatus may be metallic, polymeric, and/or ceramic.

In some embodiments, the nitride may be incorporated into or applied as a coating to materials or devices having anti-pathogenic 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 examination and surgical tables, service consoles, fixtures, handles, knobs, toys, and filters such as air conditioning filters, or toothbrushes.

Nitride-based coatings may be applied as powders to the surface of the device. In some examples, the nitride powder may be embedded or impregnated in at least a portion of the device. In some embodiments, the size of the powder may be on the order of microns or nanometers. In other embodiments, silicon nitride may be incorporated into the device. For example, the device may incorporate nitride powder into the body of the device.

In some embodiments, the nitride-based antipathogenic composition may be a slurry of nitride powder in an aqueous solution. The aqueous medium may be water, brine, buffered brine or phosphate buffered brine. In other embodiments, the nitride-based composition may be a suspension or emulsion of the nitride powder and a suitable vehicle (e.g., a cream, gel, or lotion) for topical application. The nitride powder may be present in the composition at a concentration of about 0.1vol.% to about 20 vol.%. In various embodiments, the slurry may include about 0.1vol.%, 0.5vol.%, 1vol.%, 1.5vol.%, 2vol.%, 5vol.%, 10vol.%, 15vol.%, or 20vol.% silicon nitride. In some embodiments, the concentration of the nitride may be effective to inactivate pathogens.

In other embodiments, the nitride-based coating may be present on the surface of the device or within the device at a concentration of about 1wt.% to about 100 wt.%. In various embodiments, the coating may include about 1wt.%, 2wt.%, 5wt.%, 7.5wt.%, 8.3wt.%, 10wt.%, 15wt.%, 16.7wt.%, 20wt.%, 25wt.%, 30wt.%, 33.3wt.%, 35wt.%, or 40wt.% nitride powder. In at least one example, the coating includes about 15wt.% nitride. In some embodiments, the nitride may be present in or on the surface of the device or apparatus at a concentration of about 1wt.% to about 100 wt.%. In various embodiments, the device or apparatus may include about 1wt.%, 2wt.%, 5wt.%, 7.5wt.%, 8.3wt.%, 10wt.%, 15wt.%, 16.7wt.%, 20wt.%, 25wt.%, 30wt.%, 33.3wt.%, 35wt.%, 40wt.%, 50wt.%, 60wt.%, 70 wt.%, 80wt.%, 90wt.% to 100wt.% nitride. In some embodiments, the concentration of the nitride may be effective to inactivate pathogens.

In some embodiments, the nitride-based anti-pathogenic composition or device may inactivate or reduce the transmission 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 nitride-based anti-pathogenic composition include coronaviruses (e.g., SARS-CoV-2), rhinoviruses, influenza viruses (A, B, C, D), and feline caliciviruses. The nitride-based anti-pathogenic composition or device may kill both gram-positive and gram-negative bacteria. Examples of fungi that can be dissolved include, but are not limited to, those that cause downy mildew (downy mildew), powdery mildew (powdery mildew), gray mold (Botrytis rot), fusarium rot, rust, damping off (Rhizoctonia rot), sclerotinia rot (Sclerotinia rot), sclerotium rot, or other agriculturally related diseases.

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

Also provided herein are methods of inactivating pathogens by contacting a virus, bacterium, and/or fungus with an antipathogenic nitride-based composition disclosed herein. In an embodiment, the method may include coating a device or apparatus with a nitride-based composition and contacting the coated apparatus with a virus, bacteria, or fungus. In another embodiment, the method may comprise contacting a virus, bacteria, and/or fungus with a composition comprising a nitride-based composition. The composition may be a slurry comprising nitride powders or particles. The composition may be a suspension or emulsion comprising a nitride powder.

Also provided herein is a method of treating or preventing a pathogen at a location in a human patient. For example, the pathogen may be a virus, a bacterium, or a fungus. The method may include contacting the patient with a device, apparatus, or composition comprising a nitride-based composition. The apparatus, device, or composition may comprise about 1wt.% to about 100wt.% nitride. In some examples, the device or apparatus may include about 1wt.% to about 100wt.% nitride on a surface of the device or apparatus. In one embodiment, the device or apparatus may be a monolithic nitride-based ceramic. In another embodiment, the device or apparatus may include a nitride coating, such as a nitride powder coating. In another embodiment, the device or apparatus may incorporate nitride into the body of the device. For example, the nitride powder may be incorporated or impregnated into the body of the device or apparatus using methods known in the art.

In some embodiments, the apparatus or device 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 example, the apparatus or device may be permanently implanted in the patient. In at least one example, the apparatus or device may be externally worn by a user.

Exemplary embodiment

Example 1: a method for inactivating a pathogen comprising contacting the pathogen with a composition comprising an effective concentration of a nitride selected from the group consisting of: 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, wherein the effective concentration of the nitride inactivates the pathogen.

Example 2: the method of embodiment 1, wherein the composition further comprises silicon nitride.

Example 3: the method of embodiment 1, wherein the composition further comprises aluminum nitride.

Example 4: the method of embodiment 1 wherein the nitride is boron nitride.

Example 5: the method of embodiment 1, wherein the composition comprises a slurry of nitride particles in an aqueous medium.

Example 6: the method of embodiment 5, wherein the effective concentration of the nitride is about 0.1vol.% to about 20vol.%.

Example 7: the method of embodiment 1 wherein the composition comprises a powder of the nitride.

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

Example 9: the method of embodiment 8, wherein the effective concentration of the nitride is about 1wt.% to about 100wt.%.

Example 10: the method of embodiment 8 or 9, wherein the device is an orthopedic implant, spinal implant, pedicle screw, dental implant, indwelling catheter, endotracheal tube, colonoscope, surgical gown, mask, filter, or catheter.

Example 11: the method of any one of embodiments 1-10, wherein the pathogen is a virus, a bacterium, or a fungus.

Example 12: the method of embodiment 11, wherein the virus is a coronavirus.

Example 13: the method of any one of embodiments 1 to 12, wherein the pathogen is on a surface or in a human, animal or plant.

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

Example 15: a nitride-based composition for inactivating pathogens, the composition comprising: a slurry of nitride particles in an aqueous medium, wherein the nitride is present at a concentration of about 0.1vol.% to about 20vol.%, and wherein the concentration is effective to inactivate the pathogen.

Example 16: the nitride-based composition of embodiment 15, wherein the nitride is selected from the group consisting of 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.

Example 17: the nitride-based composition of embodiment 16, wherein the composition further comprises silicon nitride.

Example 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 bacterium, or a fungus.

Example 21: the nitride-based composition of embodiment 20, wherein the virus is a coronavirus.

Example 22: the nitride-based composition according to any one of embodiments 15-21, wherein the pathogen is on a surface or in a human, animal, or plant.

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

Example 24: a nitride-based device for inactivating pathogens, the device comprising: a powder of a nitride coated on at least a portion of a surface of and/or incorporated into the device, wherein the nitride is present at a concentration of about 10wt.% to about 30wt.%, and wherein the concentration is effective to inactivate the pathogen.

Example 25: the nitride-based device of embodiment 24, wherein the nitride is selected from the group consisting of 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.

Example 26: the nitride based device of embodiment 25 wherein the device further comprises silicon nitride.

Example 27: the nitride based device of embodiment 25 wherein the device further comprises aluminum nitride.

Example 28: the nitride-based device of embodiment 25, wherein the nitride is boron nitride.

Example 29: the nitride-based device of embodiment 24, wherein the pathogen is a virus, a bacterium, or a fungus.

Example 30: the nitride based device of embodiment 29, wherein the virus is a coronavirus.

Example 31: the nitride based device according to any one of embodiments 24-30, wherein the pathogen is on a surface or within a human, animal or plant.

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

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

Example 34: the method of embodiment 33, wherein the slurry further comprises a nitride selected from the group consisting of: 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.

Example 35: a method for inactivating pathogens comprising contacting the pathogens with a slurry comprising an effective concentration of aluminum nitride, wherein the effective concentration of the nitride inactivates the pathogens, and wherein the effective concentration of aluminum nitride in the slurry is about 15% w/v.

Example 36: the method of embodiment 35, wherein the slurry further comprises a nitride selected from the group consisting of: 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.

Examples

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

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

Preparation of test materials

Si ₃ N ₄ The Cu and AlN powders are commercially availableObtained from a source. The inorganic components were mixed and spray dried by water followed by sintering of the spray dried particles (about 1700 ℃ for about 3 hours), hot isostatic pressing (under N ₂ About 1600 ℃,2 hours, 140 MPa), water-based comminution and freeze drying to produce Si ₃ N ₄ Powder (nominal composition 90wt.% Si) ₃ N ₄ 、6wt.％Y ₂ O ₃ And 4wt.% Al ₂ O ₃ ). The average particle size of the powder obtained was 0.8.+ -. 1.0. Mu.m. Pulverizing the obtained Cu powder (USP grade 99.5% purity) particles to obtain Si-containing powder ₃ N ₄ Comparable particle size. The average particle size of the AlN powder thus obtained was 1.2.+ -. 0.6. Mu.m, which was equal to Si ₃ N ₄ Equivalent.

Preparation of mammalian and viral cells

VeroE6/TMPRSS2 mammalian cells were used in the virus assay. Cells were grown in Du's modified eagle's minimum essential medium (DMEM) supplemented with G418 disulfate (1 mg/mL), penicillin (100 units/mL), streptomycin (100. Mu.g/mL) and 5% fetal bovine serum, and maintained at 5% CO ₂ And/95% in a humid environment at 37 ℃. SARS-CoV-2 virus stock was propagated using VeroE6/TMPRSS2 cells at 37℃for 2 days. Infection Dose (TCID) by half of tissue culture ₅₀ ) Virus titer was determined.

Example 2:

virus assay

Fifteen weight percent (15 wt.%) Si ₃ N ₄ The Cu and AlN powders were dispersed in 1mL of PBS (-) respectively, followed by addition of the virus suspension (2X 10) ⁵ TCID ₅₀ In 20 μl). Since the Cu powder has a high density, the volume fraction thereof is Si ₃ N ₄ About one third of (a) is provided. Mixing was performed by slow manual rotation at 4 ℃ for 1 min and 10 min. After exposure, the powder was granulated by centrifugation (2400 rpm for 2 minutes) followed by filtration through 0.1 μm media. Collecting supernatant and performing TCID ₅₀ Assay, real-time RT-PCR testing, and fluorescence imaging. Experiments were performed in triplicate, the experimental packageIncludes pseudo supernatant without antiviral powder. A confluent monolayer of VeroE6/TMPRSS2 cells in 96-well plates was inoculated with 50 μl/well of each virus suspension serially diluted ten times with 0.5% FBS DMEM (i.e., maintenance medium). The virus was adsorbed at 37 ℃ for 1 hour, with a tilt every 10 minutes. After that, 50. Mu.l/well of maintenance medium was added. The plates were exposed to 5% CO ₂ Incubation at 37℃in a 95% humid environment lasted for 4 days. Cytopathic effects (CPE) of the infected cells were observed under a phase contrast microscope. Cells were then fixed by addition of 10 μl/well glutaraldehyde and then stained with 0.5% crystal violet. Calculation of TCID according to Reed-Muench method ₅₀ 。

Viral RNA assay

After exposure to the powder, 140 μl of supernatant was used for viral RNA extraction. RNA was also extracted from the surface of the centrifuged and filtered powder. RNA purification was performed by using QIAamp viral RNA Mini kit. Using a ReverTra A16. Mu.L aliquot of the isolated RNA was reverse transcribed from the qPCR RT master mix. Quantitative Real-time PCR was performed on two specific viral N gene sets using a One-Step Plus Real-time PCR system primer/probe (Step-One Plus Real-Time PCR system primers/probes). Each 20. Mu.L of reaction mixture contained 4. Mu.L of cDNA, 8.8pmol of each primer, 2.4pmol of probe and 10. Mu.L of GoTaq probe qPCR master mix. The amplification protocol contained 50 cycles of denaturation at 95℃for 3 seconds and annealing and extension at 60℃for 20 seconds.

Immunochemical fluorescence assay

Vero E6/TMPRSS2 cells on coverslips were inoculated with 200. Mu.L of the virus supernatant. After virus adsorption at 37 ℃ for 1 hour, the cells were incubated with maintenance medium in a CO2 incubator for 7 hours. To detect infected cells, the cells were washed with TBS (20 mM Tris-HCl pH 7.5, 150mM NaCl) and fixed with 4% PFA for 10 min at Room Temperature (RT), followed by membrane permeation with TBS containing 0.1% Triton X for 5 min at RT. With a composition of 2 at RT% skimmed milk TBS blocked cells for 60 min and stained with anti-SARS coronavirus envelope (rabbit) antibody (dilution=1:100) for 60 min at RT. After washing with buffer, cells were incubated with Alexa 594 goat anti-rabbit IgG (H+L) (1:500) and Alexa 488 Phallloidin (1:50) for 60 minutes at RT in the dark. ProLong with DAPI ^TM The anti-fading sealing agent for diamond is used as a sealing medium. Staining was observed under a fluorescence microscope BZX 710. Total cells and infected cell counts were obtained using a Keyence BZ-X analyzer.

Raman spectroscopy

Vero E6/TMPRSS2 cells were infected onto glass sites with 200. Mu.L of each virus suspension. After virus adsorption at 37℃for 1 hour, the infected cells were incubated with maintenance medium in CO ₂ Incubate for 4 hours and fix with 4% paraformaldehyde for 10 minutes at RT. 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 spectroscope with a 20 x optical lens. Which operates in a microscopic measurement mode with two-dimensional confocal imaging. A holographic notch filter within the optical circuit was used to effectively achieve a spectral resolution of 1.5cm "1 with a 532nm 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. The raman spectrum was deconvolved into Gaussian-Lorentzian (Gaussian-Lorentzian) subbands using commercially available software.

Statistical analysis

Using Prism software, the schwann t-test determines statistical significance at n=3 and p value of 0.01.

Example 3:

half of tissue culture infectious dose

15wt.％Si ₃ N ₄ TCID of Cu and AlN powders ₅₀ The measurement results are shown in fig. 1A to 1D. Inactivation time of 1 min and 10 minShown in fig. 1A and 1B and fig. 1C and 1D, respectively. All three powders were effective in inactivating SARS-CoV-2 virions over the two exposure times relative to the negative control>99％)。

RNA gene disruption

To check if viral RNA exposed to both supernatant and powder was split, RT-PCR testing was performed on the N gene set of viral RNA. The results of the 1 minute and 10 minute exposures are shown in fig. 2A and 2B and fig. 2C and 2D, respectively. Also, compared to the negative control at 1 min exposure to supernatant, almost complete cleavage of Cu RNA was observed, while AlN caused significant damage, and Si ₃ N ₄ The damage degree is light. After 10 minutes exposure to the supernatant, extensive cleavage of RNA was observed for all three materials. Although Cu still shows the largest fracture, si ₃ N ₄ Shows similar effects and AlN is substantially the same as the 1 minute exposure conditions. Based on the RNA extracted from the granular powder at 1 minute of exposure, almost no viral RNA was detected for all three materials (see fig. 2A and 2B). This result indicates that the reduction of viral RNA in the supernatant is not due to RNA adhesion to the powder, but rather to direct degradation.

Immunofluorescence test

Immunofluorescence imaging was then performed using anti-SARS coronavirus envelope antibody (red), phaliodin (green) staining F-actin in living cells, and DAPI (blue) for nuclear staining for confirmation of TCID ₅₀ Determination and gene disruption results. FIGS. 3A through 3D show fluorescence micrographs representing VeroE6/TMPRSS2 cell populations seeded with (a) unexposed virions (i.e., negative control) and (b) Si ₃ N ₄ 10 minutes of exposure of the virions to (c) AlN and (d) Cu. FIG. 3E shows cells not vaccinated with virus (labeled "pseudoinfected" cells). Red spots in the negative control (fig. 3A) indicate that virions have entered and hijacked the metabolism of Vero6E cells. This is in contrast to pseudoinfected cells (fig. 3E) which show normal metabolic function.

Notably, theIs inoculated with Si from ₃ N ₄ Cells of the supernatant from AlN and, to a lesser extent, cells inoculated with the supernatant from AlN exhibited almost normal functions and were hardly infected. In contrast, cells vaccinated with Cu supernatant were essentially dead (i.e. completely devoid of F-actin, fig. 3D), although they were likely to survive before death based on the blue-red spots present in the nuclei, as the virions appeared to hijack some of the nuclei. This suggests that cell lysis is not only the result of viral infection, but also due to the toxic effects of free Cu ions within the cell. Quantification of the colorimetric results from fig. 3A-3E is provided in fig. 4. These data indicate that about 35% of the live VeroE6 cells from the negative control were infected with virions, while vaccinated with Si from ₃ N ₄ And cells of AlN supernatant were only 2% and 8%, respectively, infected. The Cu supernatant-inoculated cells could not be quantitatively evaluated due to premature cell death.

Raman spectrum

Raman spectroscopy examined VeroE6 cells exposed to various supernatants to assess biochemical cell changes due to infection and ion (i.e., cu and Al) toxicity. FIGS. 5A to 5G show (a) uninfected VeroE6/TMPRSS2 cells and inoculation exposure to (b) Si ₃ N ₄ Raman spectra of cells of (c) AlN, (d) Cu (positive control) and (e) virosome-containing supernatant without antiviral compound (negative control) for 10 minutes in the frequency range 700-900cm "1. Of most importance are the ring respiratory and H-shear vibration bands of the indole ring of tryptophan (labeled T1 and T2 at 756 and 875cm-1, respectively). Tryptophan plays a vital role in protein synthesis and the production of various immunocompetent molecules. Stereoisomers thereof are used to anchor proteins within cell membranes and catabolites thereof have immunosuppressive functions. The catabolism of tryptophan is triggered by viral infection. This occurs through the enzymatic activity of indoleamine-2, 3-dioxygenase (IDO), which protects host cells from the effects of an excessive reactive immune response. IDO reduces tryptophan to kynurenine and then to N' -formyl-kynurenine. The increase in IDO activity depletes tryptophan. Thus, tryptophan The intensities of the bands (T1 and T2) are indicators of these biochemical changes. In addition to Cu-treated samples, the data presented in fig. 5F shows an exponential decrease in the combined tryptophan bands related to the fraction of infected cells. (the chemical structure of N' -formyl-kynurenine is given in the inset for clarity). The abnormality of copper provides additional evidence of its toxicity. VeroE6 cells deplete tryptophan to reduce Cu ²⁺ And stabilize it as Cu ⁺ 。

Raman signals due to ring stretching vibrations of adenine, cytosine, guanine and thymine are found at 725, 795, 680 and 748cm "1 and are labeled A, cy, G and Th, respectively, in fig. 5A to 5E. These bands were preserved after virus exposure. However, for cells infected with Cu-exposed virions, tyrosine at 642 and 832cm-1 labeled Ty1 and Ty2, respectively, represent an abnormality in the line. The circular respiratory tract Ty2 of tyrosine was very weak compared to other samples (see fig. 5D and 5B). In contrast, the C-C bond-related Ty1 signal is still strong. This suggests that the aromatic ring of tyrosine chelates Cu ions. This explains why only the tyrosine loop breathing pattern is reduced, while the C-C signal remains unchanged. Three possible Cu (II) chelate conformations in tyrosine are given in fig. 5G.

For VeroE6 cells exposed to virions treated with AlN (fig. 5C), tryptophan T1 and T2 bands were retained, but bands at 615 and about 700cm "1 were almost disappeared due to loop bending in the DNA cytosine (labeled Cy2 and Cy3 in fig. 5A to 5E, respectively). The disappearance of the band is due to progressive inter-nucleosome DNA cleavage or complex formation, and both are associated with toxicity. The loss of cytosine signal is interpreted as a toxic effect of Al ions, although far less important than copper. Al (Al) ₃ ⁺ Interacts with carbonyl O and/or N ring donors in nucleotide bases and selectively binds to the backbone of PO2 groups and/or to guanine N-7 sites of G-C base pairs by chelation.

Unlike exposure of VeroE6 cells to Cu and AlN supernatants (resulting in moderate to severe toxicity), si ₃ N ₄ No modification of tryptophan, tyrosine and cytosine was induced. Si (Si) ₃ N ₄ The morphology of the spectra of the virus supernatant closely matched that of the uninfected sham suspension (see fig. 5A and 5B).

Example 4: biofilm assay of aluminum nitride and boron nitride materials

This study was performed to compare the biomaterial changes of silicon nitride and other ceramic materials and the growth of staphylococcus epidermidis biofilm on PEEK as a positive control. Ceramic materials under observation included burned silicon nitride (AFSN), aluminum nitride (AlN), two grades of boron nitride (BN: AX05BN and PCBN 1000), and Shapal (a mixture of AlN and BN).

Three replicates of each sample material were selected at 24 and 48h time points for basic statistical analysis. Samples were prepared by cleaning the samples in ethanol under ultrasonic agitation for 5 minutes, followed by DI water rinse using ultrasonic agitation for 5 minutes, followed by sterilization of the samples by a centered UV-C exposure on each side for 30 minutes and leaving the samples at rest for at least 60 minutes prior to inoculation.

Bacterial media were prepared by combining 7% glucose, 1 XPBS and 10% human plasma, followed by inoculation with small aliquots of Staphylococcus epidermidis. Initial absorbance values were obtained using a spectrometer. The medium was then placed in an oscillating incubator at 37 ℃ and 175rpm for six hours, wherein the bacteria were allowed to proliferate until an absorbance value of 0.05AU was reached (corresponding to 10 according to the previously generated growth curve ⁵ Individual cells/ml).

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

Samples were removed at the appropriate time points (24 or 48 hours) and rinsed in 5ml of 1 x PBS in fresh well plates in a shaking incubator at 125rpm for 2 minutes. Each sample was rinsed by immersion in fresh 1 x PBS. The samples were placed in 10mL of 1 x PBS in a 50mL centrifuge tube and vortexed vigorously for 2 minutes.

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

Each sample biofilm solution was serially diluted in 1 XPBS to give 1×, 1/10×, 1/100×, 1/1,000× and 1/10,000× concentrations of the initial sample. Each dilution was inoculated onto Petrifilm to allow the lowest countable dilution to be used for data comparison for each sample. After 24 hours of growth in a 37℃incubation environment, bacterial colonies are expressed in Colony Forming Units (CFU). After obtaining the CFU count, the CFU for each sample is calculated by multiplying the count by the appropriate dilution factor. The total CFU is then divided by the surface area to accurately compare samples of different sizes, so the final data reported is in CFU/mm ² In units of. Statistical analysis is performed and illustrated. Table 1 and fig. 7 show the average value at 24 and 48 hours for each material. Statistical analysis was performed by applying a heteroscedastic two-tailed t-test with 95% confidence interval to all samples.

Table 1.

At 24 hours, the bacterial growth ratio of PEEK was compared to burnt beta-Si ₃ N ₄ (AFSN), alN and Shapal (all p<0.05 More than an order of magnitude. Statistical differences were found between PEEK and all materials except AX05 BN. AX05BN was also found to have a statistically significant difference in biofilm growth compared to all other materials. The comparison of biofilm growth between AlN, shapal, AFSN and PCBN1000 was not significantly different overall. It can be inferred that AlN, shapal, PCBN and AFSN contain the most effective antibacterial properties for the 24 hour time point.

At 48 hours, the serial dilution and inoculation procedure described in [0096] was performed on the remaining samples. Biofilm growth on PEEK is consistent with the results found at 24 hours, as CFU counts are still at least an order of magnitude greater than any of the ceramic materials. The representative p-value of this comparison does not reflect the visual differences due to the outliers of one PEEK sample. This outlier did not change the conclusions drawn from the overall result due to the expected biological changes. Significant differences were found for both AX05BN and PCBN1000 compared to AFSN, shapal and AlN. The P values of AFSN, alN and Shapal are p=0.032, p=0.031 and p=0.033, respectively, when compared to AX05 BN. The 48 hour results met the 24 hour trend. All materials increased consistently in terms of biofilm growth over time.

At the 24 hour and 48 hour time points, as expected, staphylococcus epidermidis was found to form a significantly denser biofilm on PEEK compared to either of the ceramic variants. AX05BN does not perform as effectively as other ceramic materials evaluated in this experiment because the resulting CFU count is not significantly different from PEEK. Shapal, alN and AFSN have similar biofilm growth highlighting their ability to resist adhesion and colonization by the surface of staphylococcus epidermidis. Although their bacterial resistance was quite similar, alN outperformed PEEK, AX05BN, PCBN1000, shape and AFSN in resisting biofilm formation in 24 hour and 48 hour time point evaluations. Shapal and AFSN are in close proximity to AlN in terms of their antibacterial properties.

These results support previous conclusions: ceramic materials are significantly superior to PEEK in terms of biofilm resistance. Comparison within ceramic samples showed that AX05BN was significantly less effective at resisting bacterial adhesion than other ceramic compositions. Although insignificant differences were seen between Shapal, alN and AFSN, alN had 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 nominal composition of the beta-silicon nitride powder is 90wt.% beta-Si ₃ N ₄ 6wt.% yttria (Y) ₂ O ₃ ) And 4wt.% alumina (Al ₂ O ₃ ). The powder was prepared by aqueous mixing and spray drying the inorganic components, respectively, followed by sintering the spray dried granules at about 1700 ℃ for about 3 hours. The sintered pellets were then hot isostatically pressed under nitrogen at 140MPa for 2 hours at about 1600 c. Pressing is followed by water-based comminution and freeze-drying.

Alpha-nitridationSilicon powder 98wt.% pure Si ₃ N ₄ About 2wt.% SiO ₂ . It is prepared by heating commercially available high purity alpha-silicon nitride in air at about 300 ℃ for about 1 hour, and then cooling it to room temperature.

To perform antiviral assays, washington variants of SARS-CoV-2 virus were diluted to a concentration of 2X 10 in DMEM growth medium ⁴ Individual virions/ml. Four milliliters of the diluted virus solution was then added to the tube containing the alpha-or beta-silicon nitride powder at 15wt.%/vol (w/v). Processing Si-free in parallel ₃ N ₄ As a control. The tube was vortexed for 30 seconds to ensure adequate contact, and then the tube was placed on a tube rotator for 30 minutes. The sample was centrifuged and the supernatant was collected and filtered through a 0.2 μm filter. The RNA in the remaining infectious virus in the clarified supernatant was isolated along with the particles and quantified by RT-qPCR method.

The supernatant was also subjected to plaque assay test methods. The results are provided in FIG. 8, showing that the genome copies of the virus-only control have about 1X 10 ⁶ Concentration of/mL, while one of the beta-silicon nitride powders showed a reduction of virions to about 4.3X10 ⁴ Two different batches of/mL (95.9%) and alpha-silicon nitride showed a reduction in virus to about 1X 10 ³ /mL (99.9%). In this same test series, granular samples from both alpha-silicon nitrides were found to be completely free of live virions.

The plaque assay test results are provided in fig. 9A. These data show that the reduction in viral activity of beta-silicon nitride powder ranges from 1.25 to 3.5log ₁₀ (about 93% to 99.97%) while the alpha-silicon nitride powder showed >4.5log ₁₀ Reduce%>99.997%). It is believed that changes in the surface hydrolysis of the beta-silicon nitride powder will lead to a range of observed results. However, comparison of RT-qPCR and plaque assay methods showed that SARS-CoV-2 virion was not as granular as silicon nitride and its RNA structure was destroyed by incubation with silicon nitride.

Plaque assay assays were performed on the efficacy of alpha-and beta-silicon nitride powders against south african variants of SARS-CoV-2 virus using the same powders and methods as described aboveAnd (5) testing. The results are shown in fig. 9B. Alpha-silicon nitride has proven to be extremely effective, where the virus has>4.5log ₁₀ Reduction (99.9997%) and the beta-silicon nitride powder showed about 1-log ₁₀ Reduction (about 90%).

Similarly, plaque assay trials were performed on the effectiveness of alpha-and beta-silicon nitride powders against uk variants of SARS-CoV-2 virus using the same materials and procedures as given above. The results are provided in fig. 9C. Alpha-silicon nitride powder reduced virus count by about 4-log ₁₀ (99.99%) and beta-silicon nitride reduced virus count by about 1-log ₁₀ (about 90%).

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

Those skilled in the art will appreciate that the presently disclosed embodiments are taught by way of example and not limitation. Accordingly, what is included in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all of the generic and specific features described herein as well as all statements of the scope of the inventive methods and systems which, as a matter of language, might be said to fall therebetween.

Claims (36)

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

2. The method of claim 1, wherein the composition further comprises silicon nitride.

3. The method of claim 1, wherein the composition further comprises aluminum nitride.

4. The method of claim 1, wherein the nitride is boron nitride.

5. 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 about 0.1vol.% to about 20vol.%.

7. The method of claim 1, wherein the composition comprises a powder of the nitride.

8. The method of claim 7, wherein the composition is coated on at least a portion of a surface of a device and/or incorporated into the device.

9. The method of claim 8, wherein the effective concentration of the nitride is about 1wt.% to about 100wt.%.

10. The method of claim 8 or 9, wherein the device is an orthopedic implant, spinal implant, pedicle screw, dental implant, indwelling catheter, endotracheal tube, colonoscope, surgical gown, mask, filter or catheter.

11. The method of any one of claims 1 to 10, wherein the pathogen is a virus, a bacterium or a fungus.

12. The method of claim 11, wherein the virus is a coronavirus.

13. The method of any one of claims 1 to 12, wherein the pathogen is on a surface or in a human, animal or plant.

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

15. A nitride-based composition for inactivating pathogens, the composition comprising: a slurry of nitride particles in an aqueous medium, wherein the nitride is present at a concentration of about 0.1vol.% to about 20vol.%, and wherein the concentration is effective to inactivate the pathogen.

16. The nitride-based composition of claim 15, wherein the nitride is selected from the group consisting of 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.

17. The nitride-based composition of claim 16, wherein the composition further comprises silicon nitride.

18. The nitride-based composition of claim 16, wherein the composition further comprises aluminum nitride.

19. The nitride-based composition of claim 16, wherein the nitride is boron nitride.

20. The nitride-based composition of claim 15 wherein the pathogen is a virus, a bacterium, or a fungus.

21. The nitride-based composition of claim 20, wherein the virus is a coronavirus.

22. The nitride-based composition according to any one of claims 15 to 21, wherein the pathogen is on a surface or in a human, animal or plant.

23. The nitride-based composition according to any one of claims 15 to 21, wherein the pathogen is on a surface of an inanimate object.

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

25. The nitride-based device of claim 24, wherein the nitride is selected from the group consisting of 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.

26. The nitride-based device of claim 25, wherein the device further comprises silicon nitride.

27. The nitride-based device of claim 25, wherein the device further comprises aluminum nitride.

28. The nitride-based device of claim 25, wherein the nitride is boron nitride.

29. The nitride-based device of claim 24, wherein the pathogen is a virus, a bacterium, or a fungus.

30. The nitride-based device of claim 29, wherein the virus is a coronavirus.

31. The nitride-based device of any one of claims 24 to 30, wherein the pathogen is on a surface or within a human, animal or plant.

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

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

34. The method of claim 33, wherein the slurry further comprises a nitride selected from the group consisting of: 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.

35. A method for inactivating pathogens comprising contacting the pathogens with a slurry comprising an effective concentration of aluminum nitride, wherein the effective concentration of the nitride inactivates the pathogens, and wherein the effective concentration of aluminum nitride in the slurry is about 15% w/v.

36. The method of claim 35, wherein the slurry further comprises a nitride selected from the group consisting of: 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.