CN115996635A - System and method for rapid inactivation of SARS-COV-2 by silicon nitride and aluminum nitride - Google Patents

Abstract

Various embodiments are disclosed herein relating to systems, methods, and articles of manufacture for rapid inactivation of SARS-CoV-2 by silicon nitride and aluminum nitride.

Description

System and method for rapid inactivation of SARS-COV-2 by silicon nitride and aluminum nitride

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/045,355, filed on 29 th 6/2020; the contents of the U.S. provisional application are incorporated by reference herein in their entirety.

Technical Field

The present disclosure relates generally to rapid inactivation of viruses, and in particular to systems and methods for rapid capture and inactivation of SARS-CoV-2 by silicon nitride and/or aluminum nitride.

Background

The novel coronavirus SARS-CoV-2 has caused epidemic situation worldwide and has led to interest in the surface-mediated transmission of viral diseases. Respiratory aerosols and spray and contaminated surfaces promote the transmission of viruses from person to person, thus suggesting social distance maintenance, mask wear, hand washing and periodic surface disinfection. The data indicate that SARS-CoV-2 virus survives for 4-72 hours after contact with copper, plastic, steel and cardboard surfaces and up to 7 days on surgical masks. The continued presence of viruses on these and other materials presents a risk to the social and hospital transmission of SARS-CoV-2-caused disease, COVID-19. Current methods of viral inactivation involve surface application of chemicals such as ethanol in combination with hydrogen peroxide or sodium hypochlorite. Irradiation of surfaces with ultraviolet light is another virus disinfection strategy. These and other proposed antiviral methods are ultimately limited by their cytotoxicity to humans. As a practical solution, surfaces that are safe for human contact and that are capable of spontaneously inactivating viruses are desirable for controlling the spread of viral diseases.

It is with respect to these observations that various aspects of the present disclosure have also been contemplated and developed.

Disclosure of Invention

Provided herein are embodiments of a method for capturing and inactivating SARS-CoV-2 virus by contacting the virus with a subject comprising silicon nitride and/or aluminum nitride.

In some aspects, the subject may comprise silicon nitride or aluminum nitride, wherein the silicon nitride or aluminum nitride sequentially binds (i.e., traps) the virus, and then inactivates the virus. For example, the silicon nitride or aluminum nitride may be present as a coating on the surface of the object or may be incorporated into the object. In some examples, the silicon nitride is present at a concentration of about 1wt.% to about 30 wt.%.

In some aspects, the subject may be contacted with the virus for at least one minute or for at least 30 minutes. For example, the virus may be at least 75% inactivated after contact with the subject for at least 1 minute. In some aspects, the object may comprise paper, cardboard, polymer, fabric, plastic, ceramic, stainless steel, and/or metal. In some aspects, the subject is a protective garment, body suit, headgear, shoe cover, face mask, face shield, goggles, glove, surgical gown, surgical drape, or a compartment curtain. In some further aspects, the subject is a mask filter, a respirator filter, an air filter, or an air ventilation filter. In further aspects, the subject is a knob, handle, railing, bed frame, bed tray, table, chair, equipment rack, or cart. In some further aspects, the subject may be a composition, such as a slurry, suspension, gel, paint, or toothpaste.

Also provided herein are embodiments of articles of Personal Protection Equipment (PPE) having antiviral and antimicrobial properties. In some aspects, the article comprises silicon nitride or aluminum nitride. The silicon nitride or aluminum nitride may be incorporated into the article or may be coated onto the surface of the article. In some aspects, the silicon nitride or aluminum nitride may be present at a concentration of about 1wt.% to about 30 wt.%. In some aspects, the article may be a body cover, a headgear, a shoe cover, a face mask, a mask, and goggles or gloves. In some examples, the preparation is operable to bind/capture and then inactivate the SARS-CoV-2 virus upon contact with the virus.

Drawings

FIGS. 1A-1D are diagrams showing the use of 15wt.% Cu, alN and Si in an aqueous medium at room temperature ₃ N ₄ Powder treatment was performed for 1 min and 10 min on a graph of the inactivation of SARS-CoV-2 by nitride powder, wherein the control virus was treated identically without any powder addition. 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 /50μL (FIGS. 1A and 1B) and decrease% (FIGS. 1C and 1D). Statistics are given in the inset according to the unpaired double tailed Student's t-test (n=3).

Figures 2A-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 The powder was maintained for 1 minute 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-3E are diagrams illustrating 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 10 min UTE virions of AlN (FIG. 3B), alN (FIG. 3C) and Cu (FIG. 3D) 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 Phillidine (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. By unpaired two-tailed schwann t-test (n=3), ^* and ^** p is respectively<0.05 and 0.01; n.s. =non-salient.

Fig. 5A-5G are graphical representations of raman spectra of: (a) Uninfected cells (FIG. 5A) (i.e., not exposed to virions) and (b) Si ₃ N ₄ (FIG. 5B), (C) AlN (FIG. 5C) and (D) Cu (FIG. 5D) for 10 minutes in cells infected with SARS-CoV-2 virion; 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 the 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 basic transesterification by hydrolysis). Note that the similarity between the N-terminal ends of protonated amine and lysine may trigger an extremely efficient "competitive binding" mechanism for SARS-CoV-2 virion inactivation, while eluted ammonia fatally degrades virion RNA in a combined "capture and kill" effect.

Corresponding reference numerals indicate corresponding elements in 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.

Several definitions will now be presented that apply throughout this disclosure. Reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the 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. Furthermore, various features are described which may be exhibited by some embodiments and not by others.

As used herein, the terms "include," "have," "include," and "contain" 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 "mixtures thereof" also relates to "mixtures thereof".

As used herein, "about" refers to a value, including an integer, fraction, percentage, etc., whether or not explicitly indicated. The term "about" generally refers to a range of values that would be considered equivalent to the recited value (e.g., having the same function or result), for example, ±0.5-1%, ±1-5% or ±5-10% of the recited value.

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

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

As used herein, the terms "object," "apparatus," or "component" include materials, compositions, devices, surface coatings, and/or composites. In some examples, the instrument may comprise various medical devices or equipment, inspection stations, 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.

Within the context of the present disclosure and in the specific context of use of each term, the term as used in this specification generally has its 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 specification.

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

Provided herein 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) Inactivation of human and animal viruses, bacteria and fungi; and 4) the polymer or metal matrix composite, natural or man-made fibers, polymer or metal containing silicon nitride powder retains key silicon nitride bone restoration, bacteriostatic, antiviral and antifungal properties.

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 Implant finenessThe bacterial infection rate 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 basic transesterification, which results in loss of genome integrity and viral inactivation. This may also reduce the activity of hemagglutinin. Ammonia elution and the consequent increase in pH inactivate viruses, bacteria and fungi. As shown in the examples, it was surprisingly found that each of 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 Can 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. The test contained aluminum nitride because of the 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. Since comparable antiviral and antibacterial phenomena are believed to be effective for all nitride-based compounds, the use of AlN provides additional insight into the anti-pathogenic mechanisms 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, ceramic 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, 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 inactivate SARS-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, involving "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 Two important aspects of surface chemistry play a fundamental role: (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 and 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 the study show thatBoth mechanisms contribute to the inactivation of SARS-CoV-2, consistent with earlier work. TCID (TCID) ₅₀ The results are shown in FIGS. 1A-1D and in FIGS. 2A-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 The RNA harvested from the particles (FIG. 2B) was essentially completely fragmented. 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 at 10 min indicated that nitrogen elution was the key process to trigger the cascade 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). In addition to its proven record as a bioimplant, 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-bearing body 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 fabrics 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 comprise a silicon nitride and/or aluminum nitride composition on at least a portion of the surface of the subject. The silicon nitride or aluminum nitride coating may be applied as a powder 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 comprise 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 comprise about 10wt.% to about 20wt.% silicon nitride or aluminum nitride. In at least one example, the coating comprises 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., permeation limit) on the amount of silicon nitride and/or aluminum nitride that can 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 comprise 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 comprise about 10wt.% to about 20wt.% silicon nitride or aluminum nitride. In at least one example, the coating comprises 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.

Examples

Example 1:

preparation of test materials

Si ₃ N ₄ Cu and AlN powders are obtained from commercial sources. Preparation of Si by mixing with Water and spray drying inorganic Components ₃ N ₄ Powder (nominal composition 90wt.% Si) ₃ N ₄ 、6wt.％Y ₂ O ₃ And 4wt.% Al ₂ O ₃ ) The spray dried granules were then sintered (about 1700 ℃ for about 3 hours), hot isostatic pressed (under N ₂ About 1600 c, 2 hours, 140 MPa), water-based comminution and freeze-drying. The average particle diameter of the obtained powder 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 Equivalent particle size. The average particle diameter of the AlN powder was 1.2.+ -. 0.6. Mu.m, which was obtained by mixing with Si ₃ N ₄ Equivalent.

Nursing animalsPreparation of physical and viral cells

VeroE6/TMPRSS2 mammalian cells were used in the virus assay. Cells were grown in Dulbecco's modified Eagle minimal 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 in a 5% CO2/95% wet environment at 37 ℃. SARS-CoV-2 virus stock was propagated using VeroE6/TMPRSS2 cells at 37℃for 2 days. By median tissue culture infection dose (TCID 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 in 20. Mu.L ⁵ TCID ₅₀ ). 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 precipitated by centrifugation (2400 rpm for 2 minutes) and subsequently filtered through 0.1 μm medium. Collecting supernatant and performing TCID ₅₀ Assay, real-time RT-PCR testing, and fluorescence imaging. Experiments were performed in triplicate, which contained sham supernatants 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. Plates were incubated in a 5% CO2/95% humid environment at 37℃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 powders. RNA purification was performed by using QIAamp viral RNA Mini kit. Using a ReverTra

An aliquot of 16. Mu.L of 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 at Room Temperature (RT) for 10 min, followed by a TBS permeabilization membrane containing 0.1% Triton X at RT for 5 min. Cells were blocked with TBS containing 2% skim milk for 60 min at RT 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. Virus adsorption was continued for 1 hour at 37℃Thereafter, the infected cells are combined 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:

median tissue culture infection dose

15wt.％Si ₃ N ₄ TCID of Cu and AlN powders ₅₀ The measurement results are shown in FIGS. 1A to 1D. The inactivation times of 1 minute and 10 minutes are shown in figures 1A and 1B and figures 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. Riot (E)After 10 minutes of 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 precipitated 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-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, inoculation comes from Si ₃ N ₄ The cells of the supernatant from AlN and, to a lesser extent, the cells inoculated with the supernatant from AlN exhibit almost normal functions and are 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 From Si ₃ N ₄ And cells of AlN supernatant were only 2% and 8%, respectively, infected. The cells inoculated with Cu supernatant could not be quantitatively assessed 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-5G show (a) uninfected VeroE6/TMPRSS2 cells and seed 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 of 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, the intensity of the tryptophan bands (T1 and T2) is an indicator 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-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-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).

From the foregoing, it will be appreciated that, although specific embodiments have been shown and described, various modifications may be made thereto without departing from the spirit and scope of the invention, as will be apparent to those skilled in the art. Such changes and modifications are within the scope and teachings of the present invention as defined in the appended claims.

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 (17)

1. A method for inactivating a SARS-Cov-2 virus, the method comprising contacting the SARS-Cov-2 virus with a subject comprising silicon nitride and/or aluminum nitride, wherein the silicon nitride or aluminum nitride sequentially binds and inactivates the virus.

2. The method of claim 1, wherein the silicon nitride and/or aluminum nitride is incorporated into the object or is present as a coating on a surface of the object.

3. The method of claim 1 or 2, wherein the silicon nitride and/or aluminum nitride is present at a concentration of about 1wt.% to about 30 wt.%.

4. A method according to any one of claims 1 to 3, wherein the object further comprises paper, cardboard, fabric, plastic, ceramic, stainless steel, metal or a combination thereof.

5. The method of any one of claims 1 to 4, wherein the subject is a protective suit, a body suit, a head suit, a shoe cover, a face mask, a face shield, goggles, a glove, a surgical suit, a surgical drape, or a compartmental drape.

6. The method of any one of claims 1 to 4, wherein the subject is a mask filter, a respirator filter, an air filter, or an air ventilation filter.

7. The method of any one of claims 1 to 4, wherein the subject is a knob, handle, railing, bed frame, bed tray, table, chair, equipment rack, cabinet, or cart.

8. The method of any one of claims 1-7, wherein the subject is contacted with the SARS-CoV-2 virus for at least one minute.

9. The method of any one of claims 1-8, wherein at least 75% of the SARS-CoV-2 virus is inactivated after contact with the subject.

10. The method of any one of claims 1 to 9, wherein at least 99% of the SARS-CoV-2 virus is inactivated after contact with the subject for at least 1 minute.

11. A method for inactivating a SARS-Cov-2 virus, the method comprising contacting the SARS-Cov-2 virus with a composition comprising silicon nitride or aluminum nitride, wherein the silicon nitride and/or aluminum nitride sequentially binds and inactivates the virus.

12. The method of claim 11, wherein the composition comprises a slurry, suspension, gel, paint, or toothpaste.

13. The method of claim 11, wherein the composition is a slurry sprayed onto a high contact surface.

14. An article of Personal Protection Equipment (PPE) having antiviral and antimicrobial properties, the article comprising silicon nitride or aluminum nitride embedded in or coated onto a surface of the article, wherein the silicon nitride or aluminum nitride is operable to sequentially bind and inactivate SARS-CoV-2 virus.

15. The article of claim 14, wherein the silicon nitride and/or aluminum nitride is present at a concentration of about 1wt.% to about 30 wt.%.

16. The article of claim 14 which is a body cover, a headgear, a shoe cover, a face mask, a mask, and goggles or gloves.

17. The article of manufacture of claim 14, wherein the article of manufacture is operable to inactivate the SARS-CoV-2 virus when the article of manufacture contacts the virus for at least 1 minute.

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