Application of two-dimensional nanomaterials in inhibiting coronavirus

Abstract

The application provides application of two-dimensional nanomaterials in inhibition of coronavirus, and specifically discloses application of two-dimensional nanomaterials in preparation of a drug for treating or preventing a disease caused by coronavirus or in adhesion or inhibition of coronavirus, wherein the two-dimensional nanomaterials are any one or a combination of graphene oxide nanosheets, molybdenum disulfide nanosheets or black scale nanosheets; the coronavirus is selected from one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, SARS-CoV2 or MERS-CoV; and the disease caused by coronavirus is a disease caused by coronavirus infection. The material of the application is simple to prepare, has high biological safety and excellent effect.

Description

[0001] This application is a divisional application of the Chinese patent application with the application number 202110183434.X, the application date of February 10, 2021, the priority date of December 17, 2020, the priority number of 202011495254.7, and the invention name of "Application of two-dimensional nanomaterial in inhibiting coronavirus". TECHNICAL FIELD

[0002] The present application relates to the field of antiviral technology, in particular to the application of two-dimensional nanomaterial in inhibiting coronavirus. BACKGROUND

[0003] Nanomaterial refers to a material with a scale of nanometers in one, two or three dimensions. Nanomaterials can be divided into zero-dimensional materials, one-dimensional materials, two-dimensional materials and three-dimensional materials. Two-dimensional materials refer to materials in which electrons can only move freely in two dimensions (planar motion), such as boron nitride (BN) nanosheet, molybdenum disulfide (MoS2) nanosheet, tungsten disulfide (WS2) nanosheet and Mxene material. Two-dimensional nanomaterials have unique properties due to the limitation of carrier migration and heat diffusion in two dimensions, and are widely used in the field of electronics. However, there are few applications in the field of medicine.

[0004] Coronavirus is a large virus family, which is known to cause colds and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). 2019-nCoV is the seventh known virus, and the previous six are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing Middle East respiratory syndrome). So far, there is no effective drug.

[0005] At present, there is still a lack of nanomedicines and related preparations that can significantly inhibit coronavirus cell infection and are convenient and low-cost in source. Moreover, few drugs can simultaneously play the role of treatment and prevention. SUMMARY

[0006] In view of the defects in the prior art, one aspect of the present application provides the application of two-dimensional nanomaterial in the preparation of drugs for treating or preventing coronavirus-induced diseases. The two-dimensional nanomaterial is any one or a combination of indium-doped copper phosphorus sulfide nanosheet (CIPS), graphene oxide nanosheet, molybdenum disulfide nanosheet or black scale nanosheet.

[0007] In the technical solution of the present application, the drug further comprises a pharmaceutically acceptable carrier or excipient in the above application.

[0008] In the technical scheme of the present application, in the above application, the drug further comprises administration of a therapeutically effective amount of at least one other therapeutic agent or a composition thereof, which is selected from the group consisting of corticosteroids, anti-inflammatory signal transduction modulators, beta2-adrenergic receptor agonists bronchodilators, anticholinergics, mucolytic agents, hypertonic saline and other drugs for treating Coronaviridae virus infection; or their mixtures.

[0009] The second aspect of the present application provides a pharmaceutical composition for treating or preventing a disease caused by a coronavirus, wherein a two-dimensional nanomaterial is used as an active ingredient, and the two-dimensional nanomaterial is any one or a combination of indium-doped copper phosphorus sulfide nanosheet (CIPS), graphene oxide nanosheet, molybdenum disulfide nanosheet or black scale nanosheet.

[0010] In the technical scheme of the present application, in the above pharmaceutical composition, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0011] In the technical scheme of the present application, in the above pharmaceutical composition, the pharmaceutical composition further comprises a second active ingredient, which is selected from the group consisting of corticosteroids, anti-inflammatory signal transduction modulators, beta2-adrenergic receptor agonists bronchodilators, anticholinergics, mucolytic agents, hypertonic saline and other drugs for treating Coronaviridae virus infection; or their mixtures.

[0012] In the technical scheme of the present application, in the above pharmaceutical composition, the preparation form of the pharmaceutical composition is selected from oral preparations, injection preparations, mucosal administration preparations, inhalants, and external preparations.

[0013] The third aspect of the present application provides a method for treating or preventing a coronavirus infection, which comprises administering a therapeutically effective amount of a two-dimensional nanomaterial to a subject, wherein the two-dimensional nanomaterial is any one or a combination of indium-doped copper phosphorus sulfide nanosheet (CIPS), graphene oxide nanosheet, molybdenum disulfide nanosheet or black scale nanosheet.

[0014] In the technical scheme of the present application, in the above method, the therapeutically effective amount of the two-dimensional nanomaterial is administered in the form of a preparation, and the preparation further comprises a pharmaceutically acceptable carrier or excipient.

[0015] In the technical solution of the present application, in the above method, the method further comprises the step of administering to the subject a therapeutically effective amount of a second active ingredient selected from the group consisting of corticosteroids, anti-inflammatory signal transduction modulators, beta2-adrenergic receptor agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline, and other drugs for treating Coronaviridae virus infection; or mixtures thereof.

[0016] The fourth aspect of the present application provides the use of two-dimensional nanomaterials in the preparation of a preparation for inhibiting the binding of the Spike protein of SARS-CoV-2 virus to its host cell receptor ACE2; the two-dimensional nanomaterials are any one or a combination of indium-doped copper sulfide phosphide nanosheet layers (CIPS), graphene oxide nanosheet layers, molybdenum disulfide nanosheet layers, or black scale nanosheet layers; preferably, the two-dimensional nanomaterials inhibit the binding of the Spike protein of SARS-CoV-2 virus to its host cell receptor ACE2 by competitively binding to the binding site of the RBD of the Spike protein of SARS-CoV-2 virus.

[0017] The present application also provides the use of two-dimensional nanomaterials in the preparation of a preparation for competitively binding to the binding site of the RBD of the Spike protein of SARS-CoV-2 virus.

[0018] The fifth aspect of the present application provides the use of two-dimensional nanomaterials in the preparation of a drug for promoting the decomposition of the protein of SARS-CoV-2 virus; the two-dimensional nanomaterials are any one or a combination of indium-doped copper sulfide phosphide nanosheet layers (CIPS), graphene oxide nanosheet layers, molybdenum disulfide nanosheet layers, or black scale nanosheet layers.

[0019] In the technical solution of the present application, in the above method, the two-dimensional nanomaterials are used in the preparation of a drug for promoting the decomposition of the Spike protein of SARS-CoV-2 virus protein of the coronavirus.

[0020] The sixth aspect of the present application provides the use of two-dimensional nanomaterials in the preparation of a drug for promoting the decomposition of the RNA of SARS-CoV-2 virus; the two-dimensional nanomaterials are any one or a combination of indium-doped copper sulfide phosphide nanosheet layers (CIPS), graphene oxide nanosheet layers, molybdenum disulfide nanosheet layers, or black scale nanosheet layers.

[0021] The seventh aspect of the present application provides the use of two-dimensional nanomaterials in the preparation of a material for inhibiting the infection of the coronavirus; the two-dimensional nanomaterials are any one or a combination of indium-doped copper sulfide phosphide nanosheet layers (CIPS), graphene oxide nanosheet layers, molybdenum disulfide nanosheet layers, or black scale nanosheet layers.

[0022] The eighth aspect of the present application provides a material for inhibiting coronavirus infection, the material comprising a two-dimensional nanomaterial and a matrix, the two-dimensional nanomaterial being any one or a combination of indium-doped copper phosphorus sulfide nanosheet (CIPS), graphene oxide nanosheet, molybdenum disulfide nanosheet or black-scaled nanosheet.

[0023] In the technical solution of the present application, in the above-mentioned material, the material is a material for preparing protective articles, preferably the protective articles are masks, protective clothing, protective masks, protective hats.

[0024] In the technical solution of the present application, in the above-mentioned material, the material is a coating.

[0025] In the technical solution of the present application, in the above-mentioned material, the material is a disinfectant or personal care article.

[0026] In the technical solution of the present application, in the above-mentioned material, the material is a packaging material.

[0027] In the technical solution of the present application, in the above-mentioned material, the material is a filtering material.

[0028] The ninth aspect of the present application provides an article, the article comprising a two-dimensional nanomaterial, the two-dimensional nanomaterial being any one or a combination of indium-doped copper phosphorus sulfide nanosheet (CIPS), graphene oxide nanosheet, molybdenum disulfide nanosheet or black-scaled nanosheet.

[0029] The article is selected from protective articles, coatings, disinfectants, personal care articles, packaging materials or filtering devices.

[0030] Preferably, the protective articles are masks, protective clothing, protective masks, protective hats.

[0031] Preferably, the coating is a suspension comprising a two-dimensional nanomaterial, and the coating can impart a coating layer of the two-dimensional nanomaterial to the surface of the adherend.

[0032] Preferably, the disinfectant is an environmental disinfectant, a no-rinse hand sanitizer, a hand sanitizer, a detergent; more preferably, the disinfectant is used for surface disinfection of packaging materials, medical devices, oral appliances, cosmetic appliances.

[0033] Preferably, the packaging material has a coating layer comprising a two-dimensional nanomaterial, or the packaging material is a composite material comprising a two-dimensional nanomaterial; more preferably, the packaging material is a packaging material for drugs or food, especially for cold-chain transportation of drugs or food.

[0034] Preferably, the filter device is a device for air filtration, a device for water body filtration, a device for mask filtration; more preferably, the filter device is an air filter screen, the surface of which has two-dimensional nanomaterials; more preferably, the filter device is an air filter cotton, the fibers of which are attached with two-dimensional nanomaterials.

[0035] In the technical scheme of the present application, the coronavirus is selected from one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, SARS-CoV2 or MERS-CoV.

[0036] In the technical scheme of the present application, the coronavirus is preferably selected from SARS-CoV, SARS-CoV2 or HCoV-NL63.

[0037] In the technical scheme of the present application, the coronavirus is preferably selected from SARS-CoV or SARS-CoV2.

[0038] In the technical scheme of the present application, the coronavirus-induced disease is a disease caused by coronavirus infection.

[0039] In the technical scheme of the present application, the two-dimensional nanomaterial is a material with a length of nanometer size in one dimension in space; preferably, it is a material with a length of 1-100 nm in one dimension in space, more preferably, it is a material with a length of 1-10 nm in one dimension in space, more preferably, it is a material with a length of 1-10 nm in one dimension in space, and a length of 100-500 nm in other dimensions, or for example, a length of 100 nm, 200 nm, 300 nm, 400 nm, 500 nm in other dimensions.

[0040] In summary, compared with the prior art, the present application has the following beneficial effects:

[0041] 1. Two-dimensional nanomaterials such as CIPS nanosheet layers, graphene oxide nanosheet layers, molybdenum disulfide nanosheet layers or black scale nanosheet layers can inhibit the cell infection of coronaviruses on one hand, and can specifically adsorb and bind to the target points of coronavirus infection, reduce the infection efficiency of viruses, and further reduce the replication of viruses in the host body, reduce the number of coronaviruses, and can be used for patient treatment drugs.

[0042] 2. The inhibitory effect of CIPS on SARS-CoV-2 virus infection is also applicable to SARS virus, indicating that CIPS has broad-spectrum inhibition on coronaviruses.

[0043] 3. The CIPS nanosheet, graphene oxide nanosheet, molybdenum disulfide nanosheet or black scale nanosheet can specifically bind to the coronavirus, inhibit the invasion of the SARS-CoV-2 virus to the host cell, and achieve specific adsorption and fixation of the virus. It can be used for the preparation of drugs, and can be applied to melt-blown cloth, cold storage coating and outer packaging coating or spray applied to masks, can adsorb the new coronavirus and reduce the number of infectious viruses, and play a preventive role.

[0044] 4. The two-dimensional nanomaterial such as the CIPS nanosheet has no effect on the survival rate of HEK-293T and Vero-E6 cells, and does not cause hemolysis of red blood cells in mice, indicating that the nanomaterial has high biological safety and low toxicity.

[0045] 5. The two-dimensional nanomaterial of the present application can be stored at room temperature to -20℃ for a long time, which is convenient for storage. The two-dimensional nanomaterial of the present application has high stability, can be mixed with any excipient, compounded, etc. to prepare drugs and composite materials, and is not affected by temperature and pH and other factors.

[0046] 6. The present application adopts inorganic nanomaterials, which are not limited in source, suitable for large-scale production, low cost and small amount.

[0047] 7. The biological membrane interference experiment of the present application is carried out in solid and liquid environment, which proves that CIPS can strongly bind to the RBD region of coronavirus S protein (see Figure 8 ). The computer simulation results also show that it can be combined at 5.7 ns ( Figure 11 ). Moreover, from the verification results of the embodiments of the present application, the two-dimensional nanomaterial of the present application is pre-incubated with coronavirus for 2 hours before the infection experiment with host cells is carried out, which shows that the material of the present application can combine with coronavirus for a long time, and can be used as a material for adsorbing coronavirus and preventing it from combining with host cells. That is, the material of the present application can realize instantaneous combination, long-time combination and combination inhibition of virus and host cells. BRIEF DESCRIPTION OF DRAWINGS

[0048] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0049] Figure 1 is the image of CIPS crystal sheet;

[0050] Figure 2For CIPS crystal sheet atomic force microscope imaging;

[0051] Figure 3 For the biosafety detection of CIPS;

[0052] Figure 4 For the invasion results of SARS-CoV-2 Spike protein pseudovirus (SC2-P) on ACE2 / 293T cells transfected with ACE2-GFP (ACE2 / 293T) in the presence of CIPS nanosheets; statistical analysis used ANOVA, **: P<0.01, ***: P<0.001;

[0053] Wherein, Figure A is the observation of the invasion of SC2-P on ACE2 / 293T cells by confocal microscopy, the first row (ACE2) is the channel of ACE2 / 293T cells, which is green fluorescence, the second row (SC2-P) is the channel of SC2-P, which is red fluorescence; the third row (Merge) is the superposition of the first row and the second row, which shows the number and distribution of SC2-P entering ACE2 / 293T cells. Different columns in Figure A represent different amounts of CIPS nanosheets added, calculated based on the concentration after adding CIPS nanosheets.

[0054] Figure B is the quantitative results of the invasion of SC2-P on ACE2 / 293T cells, wherein the left graph has the horizontal coordinate of the added amount of CIPS nanosheets, calculated based on the concentration after adding; the vertical coordinate is the number of SC2-P entering the cells. The right graph has the horizontal coordinate of the added amount of CIPS nanosheets, calculated based on the concentration after adding; the vertical coordinate is the relative fluorescence intensity.

[0055] Figure 5 For the detection of the invasion efficiency of pseudovirus SC2-P on Vero-E6 and ACE2 / 293T cells by luciferase activity; statistical analysis used ANOVA, *: P<0.05, **: P<0.01, ***: P<0.001;

[0056] Figure 6 For the quantitative detection of the invasion efficiency of real new coronavirus SARS-CoV-2 on Vero-E6 cells; statistical analysis used ANOVA, *: P<0.05, **: P<0.01, ***: P<0.001; Figure 6 A in Figure shows the invasion rate of different concentrations of CIPS, B shows the virus amount quantitatively detected by taking the expression amount of ORF1ab / RdRp gene as an index, and Figure 6 C in Figure shows the virus amount quantitatively detected by taking the expression amount of nucleocapsid protein as an index;

[0057] Figure 7 For the adsorption and reduction of pseudovirus SC2-P by CIPS;

[0058] Figure 8 Quantitative determination of bio-layer interferometry (BLI). Wherein A is the affinity and interaction of CIPS material with SARS-CoV-2 Spike protein RBD domain protein at different concentrations; B is the affinity and interaction of CIPS material with proteins in serum; C is the affinity and interaction of molybdenum disulfide nanosheet (MoS2) with SARS-CoV-2 Spike protein RBD domain protein; D is the affinity and interaction of graphene oxide nanosheet (GO) with SARS-CoV-2 Spike protein RBD domain protein; wherein in the results of ACD, from top to bottom, the curves are respectively at concentrations of 200 nM, 100 nM, 50 nM, 25 nM and 12.5 nM; in the B result, from top to bottom, they are RBD, Fg, FBS, IgG, HDL, Tf, HAS.

[0059] Figure 9 Quantitative determination of bio-layer interferometry (BLI) of the affinity change of SARS-CoV-2 Spike protein RBD domain and ACE2 protein before and after binding with CIPS;

[0060] Figure 10 Mathematical model simulation of the binding of RBD with CIPS or ACE2;

[0061] Figure 11 Mathematical model simulation of the binding of RBD with CIPS at different time points. The results of computer simulation show that it can be combined at 5.7 ns (binding time is 5.7 ns)

[0062] Figure 12 Adsorption and inhibition of infection of CIPS on SC2-P in the protein mixture of SC2-P and FBS. Wherein A is that CIPS can bind with SC2-P in the protein mixture of 1, 3 or 10 times the volume of FBS added with SC2-P; B is that CIPS can still inhibit the infection efficiency of SC2-P in the protein mixture.

[0063] Figure 13 Results of Example 11, showing the quantitative PCR results of CIPS adsorbing SARS-CoV-2 real virus.

[0064] Figure 14 Results of Example 12, CIPS reducing the amount of SARS-CoV-2 real virus.

[0065] Figure 15 Results of the infection of pseudovirus SC2-P on ACE2 / 293T cells in the presence of different two-dimensional nanomaterials CIPS nanosheet, GO (graphene oxide nanosheet), MoS2 (molybdenum disulfide nanosheet) or black scale nanosheet.

[0066] Figure 16 To inhibit the infection efficiency of SARS pseudovirus SARS-P by CIPS. DETAILED DESCRIPTION

[0067] In order to enable persons skilled in the art to better understand the technical scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by persons skilled in the art without creative labor should belong to the protection scope of the present application.

[0068] Two-dimensional nanomaterials include graphene, transition metal disulfide, transition metal carbide, nitride and carbonitride, black phosphorus nanosheet, layered double hydroxide, two-dimensional MOF and other types of two-dimensional nanosheet. Among them, graphene oxide is a two-dimensional sp2 carbon atom layer containing oxygen functional groups, which has been widely used in the fields of nanomedicine and the like. Currently, there is no report that indium-doped copper phosphorus sulfide nanosheet layer (CIPS), graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer and black phosphorus nanosheet layer in two-dimensional nanomaterials are effective in inhibiting coronavirus infection, especially SARS virus and new coronavirus SARS-CoV-2 cell infection. Since the indium-doped copper phosphorus sulfide nanosheet layer (CIPS), graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer and black phosphorus nanosheet layer in the present application have high biological safety and low toxicity, they can be used in multiple fields such as drugs.

[0069] Terminology

[0070] In the present application, indium-doped copper phosphorus sulfide has the same meaning as CIPS or CuInP2S6.

[0071] In the present application, the terms "nanosheet layer" and "two-dimensional nanomaterial" have similar meanings, both referring to materials having one dimension in the nanometer scale in space, for example, nanomaterials with a thickness size of 1-100 nm. In the present application, the two-dimensional nanomaterial is selected from any one or a combination of indium-doped copper phosphorus sulfide nanosheet layer (CIPS), graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer and black phosphorus nanosheet layer. In a preferred embodiment, the size of each two-dimensional nanomaterial is selected as follows: thickness (one dimension in space) is 1-10 nm. The size of the nanosheet layer in the direction perpendicular to the thickness (other dimensions in space) is 100-300 nm.

[0072] The "nanosheet layer" can be obtained by conventional methods in the art to obtain nanosheet layer materials, for example, mechanical grinding (such as ball milling), ultrasonic and the like can be used.

[0073] A "pharmaceutically acceptable carrier or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening, diluting, preserving, dye / colorant, flavoring, surface-active, wetting, dispersing, suspending, stabilizing, isotonic, solvent, or emulsor agent approved by the Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0074] A "pharmaceutical composition" refers to a preparation of a compound of the present application and a vehicle generally accepted in the art for the delivery of biologically active compounds to mammals (e.g., humans). Such vehicles include all pharmaceutically acceptable excipients therefor.

[0075] An "effective amount" or "therapeutically effective amount" refers to the amount of a compound according to the present application which, when administered to a patient in need thereof, is sufficient to effect treatment for a disease state, condition, or disorder for which two-dimensional nanomaterials are useful. Such amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is being sought by a researcher or clinician. The amount of a compound according to the present application which constitutes a therapeutically effective amount will vary depending on factors such as the compound and its bioactivity, the composition used for administration, the time of administration, the route of administration, the rate of excretion of the compound, the duration of treatment, the type of disease state or condition being treated and its severity, drugs used in combination or coincidental with the compound of the present application, and the age, body weight, general health, gender, and diet of the patient. Such therapeutically effective amounts can be determined routinely by those of ordinary skill in the art, using only routine experimentation and in light of the present disclosure.

[0076] The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing a disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treatment" as used herein means the act of treating as "treating" is defined immediately above. In some embodiments, the term "treating" is intended to mean the administration of a two-dimensional nanomaterial according to the present application or a composition thereof to alleviate or eliminate symptoms of a coronavirus infection and / or to reduce viral load in a patient.

[0077] "Prevention" or "preventing" refers to any treatment of a disease or condition that results in the non-development of clinical symptoms of the disease or condition. The term "prevention" also includes the administration of a therapeutically effective amount of a compound or composition according to the present application prior to exposure of an individual to a virus (e.g., pre-exposure prophylaxis) to prevent the development of symptoms of the disease and / or prevent the virus from reaching detectable levels in the blood.

[0078] The terms "subject" or "patient" refer to an animal, such as a mammal (including a human), that has been or will be a subject of treatment, observation, or experimentation. The methods described herein can be used for human treatment and / or veterinary applications. In some embodiments, the subject is a mammal (or patient). In some embodiments, the subject (or patient) is a human, livestock (e.g., dogs and cats), farm animals (e.g., cattle, horses, sheep, goats, and pigs), and / or laboratory animals (e.g., mice, rats, hamsters, guinea pigs, pigs, rabbits, cats, dogs, and monkeys). In some embodiments, the subject (or patient) is a human. "A person in need (or patient)" means a person who may have or is suspected of having a disease or condition that would benefit from certain treatments; for example, treatment with the two-dimensional nanomaterials disclosed herein or compositions thereof, or pharmaceutical compositions comprising the aforementioned components, according to this application.

[0079] Pharmaceutical formulations

[0080] The two-dimensional nanomaterials of this invention are formulated using conventional carriers and excipients, which will be selected according to conventional practice. Tablets will contain excipients, flow aids, fillers, binders, etc. Aqueous formulations are prepared aseptically and are intended for delivery, typically isotonic, other than orally.

[0081] Although the active ingredient, two-dimensional nanomaterials, can be applied alone, it may be preferred to present them as pharmaceutical formulations. The formulations of the present invention for veterinary and human use comprise at least one active ingredient as defined above (the active ingredient being a two-dimensional nanomaterial: indium-doped copper thiophosphide nanosheets (CIPS), graphene oxide nanosheets, molybdenum disulfide nanosheets, black scale nanosheets, hereinafter the same) and one or more acceptable carriers and optional other therapeutic ingredients, such as corticosteroids, anti-inflammatory signal transduction modulators, β2-adrenergic receptor agonists, bronchodilators, anticholinergics, mucolytics, hypertonic saline, and other drugs for treating coronavirus infections; or mixtures thereof.

[0082] The formulations of the present invention suitable for oral administration can be presented in discrete unit form, such as capsules, flat capsules, or tablets, each containing a predetermined amount of the active ingredient; as powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. Alternatively, they can be prepared into oral administration formulations using other known techniques.

[0083] Tablets are made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets can be made by molding, in a suitable machine, a mixture of the powdered active ingredient moistened with a liquid diluent. Tablets can optionally be coated or scored and optionally are formulated to provide slow or controlled release of the active ingredient therefrom.

[0084] The pharmaceutical compositions of this application can be in a form suitable for topical administration in the form of ointments or the like. For use in the eye or other external tissues, for example mouth and skin, the formulations are preferably applied as a topical ointment or cream containing the active ingredient. When formulated in an ointment, the active ingredient can be employed in combination with either a solid or a liquid emollient. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water emulsion base or a water-in-oil base. If desired, the topical formulations can include a compound to enhance absorption or penetration of the active ingredient through the skin or other affected area. Examples of such dermal penetration enhancers include dimethyl sulfoxide and related analogs. The oil phase of emulsions of the present application can be constituted of known ingredients in a known form. The oil phase can include at least one emulsifying agent well known in the art. Preferably, the emulsions also include at least one sweetening or flavoring agent and at least one preservative. The topical formulations of the present application can be for use in the form of a suppository or pessary, or they can be in the form of a spray. The pharmaceutical compositions of this application can be in unit dosage form or multiple dosage form. Each unit dose or multiple dose will normally contain from about 1 mg to about 100 mg of the active ingredient of the present application.

[0085] The pharmaceutical compositions of this application can be in the form of a sterile injectable aqueous or oleaginous suspension.

[0086] The pharmaceutical compositions of this application can be in the form of a sterile injectable aqueous or oleaginous suspension.

[0087] The pharmaceutical compositions of this application can be in the form of a sterile injectable aqueous or oleaginous suspension.

[0088] The pharmaceutical compositions of this application can be in the form of a sterile injectable aqueous or oleaginous suspension.

[0089] The pharmaceutical compositions of the present application can be in a formulation to be administered internally or intranasally, such formulations generally have a particle size for rapid inhalation through the nasal passages or by mouth inhalation to the alveoli, which is in the range of 0.1 to 500 microns, such as 0.5, 1, 30, 35 microns, etc. The active ingredients of the present application are of a size that can be used for internal or intranasal administration, for example, inhalants.

[0090] The pharmaceutical compositions of the present application can be in a formulation to be administered parenterally including aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents.

[0091] The formulations of the pharmaceutical compositions of the present application are presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and can be preserved under freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above described, in an amount suitable for the subject to be treated.

[0092] The pharmaceutical compositions of the present application can be veterinary compositions comprising at least one active ingredient as defined above and a veterinary carrier.

[0093] The compounds of the present application are used to provide controlled release pharmaceutical formulations containing as active ingredient one or more active ingredients of the present application wherein the release of the active ingredients is controlled and modulated to allow less frequent dosing or to improve the pharmacokinetic or toxicity profile of a given active ingredient.

[0094] Combination therapy

[0095] The medicaments, pharmaceutical compositions or active ingredients of the present application are also used in combination with other active ingredients. For the treatment of a coronavirus infection, the other active ingredients are active against a coronavirus infection, particularly a SARS or SARS-CoV-2 infection. Non-limiting examples of these other active therapeutic agents are corticosteroids, anti-inflammatory signal transduction modulators, beta2-adrenoreceptor agonist bronchodilators, anticholinergics, mucolytics, hypertonic saline and other drugs for the treatment of a coronavirus family virus infection; or mixtures thereof.

[0096] The medicaments, pharmaceutical compositions or active ingredients of the present application can also be administered to a patient simultaneously or sequentially with one or more other active ingredients in a unit dosage form. The combination therapy can be administered as a simultaneous or sequential regimen. When administered sequentially, the combination can be given in two or more administrations.

[0097] Co-administration of the drug, pharmaceutical composition or active ingredient of the present application with one or more other active ingredients generally refers to simultaneous or sequential administration of a compound of the present application and one or more other active therapeutic agents such that therapeutically effective amounts of the drug, pharmaceutical composition or active ingredient of the present application and the one or more other active ingredients are both present in the body of a patient.

[0098] Combination therapy can provide "synergy" and "potentiation" such that the effect of the compounds, when used together, is greater than the sum of the effects of the compounds used separately. Synergistic effects can occur when active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered by alternation (sequential delivery, e.g. one after the other, or in parallel, e.g. in separate formulations); or (3) by some other regimen.

[0099] Composite material

[0100] One application of the present application provides the use of two-dimensional nanomaterials in the preparation of a material for inhibiting the infection of coronavirus; the two-dimensional nanomaterials are any one or a combination of indium-doped copper phosphorus sulfide nanosheet layer (CIPS), graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer, and black scale nanosheet layer.

[0101] One application of the present application provides a material for inhibiting the infection of coronavirus, the material comprising two-dimensional nanomaterials and a matrix, the two-dimensional nanomaterials being any one or a combination of indium-doped copper phosphorus sulfide nanosheet layer (CIPS), graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer, and black scale nanosheet layer.

[0102] In the above-mentioned material for inhibiting coronavirus, the material can further comprise a matrix, which can be changed according to the use, since the two-dimensional nanomaterials of the present application have high stability and can adapt to various matrices, the corresponding matrix material can be obtained according to the conventional method in the art, including but not limited to the following ways: the two-dimensional nanomaterials are adsorbed on the surface of the matrix by adsorption; or the two-dimensional nanomaterials are blended with the matrix, and then the matrix is solidified to obtain, etc.

[0103] In the technical scheme of the present application, in the above-mentioned material, the material is a material for preparing protective articles, preferably the protective articles are masks, protective clothing, protective masks, and protective hats.

[0104] In the technical scheme of the present application, in the above-mentioned material, the material is a coating.

[0105] In the technical scheme of the present application, in the above-mentioned material, the material is a disinfectant.

[0106] In the technical scheme of the present application, in the above-mentioned material, the material is a packaging material.

[0107] In the technical scheme of the present application, in the above-mentioned material, the material is a filter material.

[0108] In a specific preferred embodiment, the material for inhibiting coronavirus infection is a material for preparing a mask, and the material is a fabric, and the surface or interior of the fabric comprises the two-dimensional nanomaterials of the present application. In a variable preferred embodiment, the material for inhibiting coronavirus infection is a material for preparing a mask, and the material is a non-woven fabric, preferably a spun-bond non-woven fabric or a melt-blown non-woven fabric; the surface or interior of the non-woven fabric comprises the two-dimensional nanomaterials of the present application. The preparation method of the above-mentioned material can be that the two-dimensional nanomaterials are compounded with the matrix material by using conventional methods in the art. For example, the melt-blown material can be compounded with the two-dimensional nanomaterials before melt-blown spinning, and then melt-blown spinning is performed, or the obtained melt-blown fabric can be compounded with the two-dimensional nanomaterials.

[0109] In a specific preferred embodiment, the material for inhibiting coronavirus infection is a coating, and the coating is a suspension comprising the two-dimensional nanomaterials. Preferably, the coating further comprises at least one of a surfactant and a thickening agent. The coating can impart the two-dimensional nanomaterials to the surface of an object, such as the surface of a packaging, the surface of a medical device, the surface of a cosmetic device, etc., so that a coating layer comprising the two-dimensional nanomaterials is formed on the surface of the object. In a specific embodiment, the coating imparts a coating layer with the two-dimensional nanomaterials to the surface of the outer packaging or the inner packaging of a food or a drug, and preferably, the food or the drug needs to be transported in a cold chain.

[0110] In a specific preferred embodiment, the material for inhibiting coronavirus infection is a disinfectant, and the disinfectant is a suspension comprising the two-dimensional nanomaterials. In a specific preferred embodiment, the disinfectant is an environmental disinfectant, a no-rinse hand sanitizer, a hand sanitizer, or a detergent. In a specific preferred embodiment, the disinfectant can be used for surface disinfection of packaging materials, medical devices (such as catheters, injection needles, surgical instruments, surgical masks, and other medical devices), oral devices (such as dentures, protective bands, fillers, and palatal expanders), and cosmetic devices (such as cosmetic instruments and plastic devices). In a specific preferred embodiment, the concentration of the two-dimensional nanomaterials in the disinfectant is 1 μg / mL to 1000 μg / mL, preferably 2.5 μg / mL to 160 μg / mL, and more preferably 20 μg / mL.

[0111] In a specific preferred embodiment, the material for inhibiting coronavirus infection is a packaging material, and the packaging material is used for cold-chain transported or stored medicines or food. The surface of the packaging material has a coating containing two-dimensional nanomaterials, or the packaging material is a composite material containing two-dimensional nanomaterials.

[0112] In a specific preferred embodiment, the material for inhibiting coronavirus infection is a filter material, which is a material for air filtration, a material for water body filtration, a material for mask filtration. In a specific preferred embodiment, the filter material is an air filter screen, and the surface of the air filter screen has two-dimensional nanomaterials. In a specific preferred embodiment, the filter material is air filter cotton, and the fibers of the air filter cotton are attached with two-dimensional nanomaterials. For example, the filter material can be used in air conditioner filtration, air purifier filtration, fresh air system filtration, etc. in any device or equipment that needs to filter air. The use scenarios are not limited to home, office, laboratory, factory, etc.

[0113] In the technical solution of the present application, in the above uses, the material is a solid, liquid or semi-solid material.

[0114] In the technical solution of the present application, in the above uses, the liquid material is a coating containing two-dimensional nanomaterials, a disinfectant containing two-dimensional nanomaterials, a personal care product containing two-dimensional nanomaterials.

[0115] In the technical solution of the present application, in the above uses, the solid material is a surface containing two-dimensional nanomaterials.

[0116] In the technical solution of the present application, in the above uses, the semi-solid material is a gel containing two-dimensional nanomaterials.

[0117] Hereinafter, the present application verifies the influence of nanomaterial indium-doped copper phosphorus sulfide nanosheet layer (CIPS or CuInP2S6) on cell survival rate through specific examples, the inhibition of CIPS nanosheet layer, graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer, and black scale nanosheet layer on SARS-CoV-2 virus cell infection, the adsorption of CIPS to the new crown pseudo-virus SC2-P, the specific adsorption of CIPS to the RBD domain of the Spike protein of the new crown virus, and the mathematical simulation and analysis of the influence of CIPS on the recognition of ACE2 after adsorbing the RBD region of the Spike protein.

[0118] The experimental materials used in the following examples include: HEK-293T cells, ACE2 / 293T cells, Vero-E6 cells, SARS-CoV-2 pseudovirus SC2-P, SARS pseudovirus SARS-P. The materials, reagents, vectors and strains used in the following examples, unless otherwise specified, can be purchased through commercial channels.

[0119] HEK-293T cells are a derivative strain of human embryonic kidney cells 293, a cell line derived from human embryonic kidney cells, with high transfection efficiency, easy to culture and other characteristics, and are a very commonly used cell strain for expressing foreign genes, becoming a powerful tool for researchers to study gene function.

[0120] ACE2 / 293T refers to the expression of ACE2 in HEK-293T cells.

[0121] ACE2 is also known as ACEH, referred to as angiotensin-converting enzyme 2. The protein encoded by this gene belongs to the dipeptidyl carboxy dipeptidase angiotensin-converting enzyme family, and has considerable homology with human angiotensin-converting enzyme 1. The protein encoded by this gene is a functional receptor for SARS and SARS-CoV-2, HCoV-NL63 human coronavirus Spike glycoprotein.

[0122] Vero-E6 cells are African green monkey kidney cell lines, which are one of the verified cells that can be infected by the new coronavirus, and can be used as a cell host for culturing the new coronavirus. For example: to determine the effect of a certain drug on the replication rate of the new coronavirus, to test for the presence of the new coronavirus, or to culture the new coronavirus for research purposes.

[0123] SC2-P is a pseudovirus expressing the Spike protein of SARS-CoV-2. Pseudovirus is a type of chimeric virus particle, which is a chimeric virus particle expressing a recombinant glycoprotein of another virus on the surface of a replication-defective virus (viral vector). Pseudovirus has been widely used in vaccine development, antibody neutralization research, simulation of virus infection cell function experiments, detection kit positive reference, etc. due to its biological safety and stability.

[0124] Current SARS-CoV-2-related pseudovirus systems mainly have two types: one is to use lentivirus system to construct lentivirus protein shell to wrap SARS-CoV-2 ORF1a / b, N, E, etc. genes, which can be used as positive control for nucleic acid detection reagent. The other is to use SARS-CoV-2 Spike protein to wrap Luciferase / GFP marker genes, simulate virus infection of cells, detect expression of marker genes, and further study infection efficiency, etc.

[0125] The SC2-P used in the following examples can be ordered from Genewiz.

[0126] SARS-P is a pseudovirus of SARS and a commercially available product, which will not be discussed further here.

[0127] Example 1: Preparation of CIPS Nanosheets

[0128] CIPS single crystals were obtained through purchase or known methods, and CIPS nanosheets were obtained through exfoliation experiments using mechanical methods such as mechanical polishing. Figure 2 As shown, the X-axis represents length and the Y-axis represents thickness, indicating that the material thickness is approximately 1-10 nm. The nanosheets are not uniform in size along the thickness direction, and are distributed between 100-300 nm.

[0129] Example 2: Testing the biosafety of CIPS nanosheets

[0130] The specific steps are as follows:

[0131] Vero-E6 cells and ACE2 / 293T cells were seeded into 96-well plates at a density of 1×10⁶ cells. 4 Cells / well, incubated overnight at 37°C with 5% CO2.

[0132] The culture medium was replaced with a medium containing different concentrations of CIPS nanosheets (0, 2.5, 5, 10, 20, 40, 80, 160 μg / mL), and incubated at 37°C for 24 h.

[0133] Add 10 μL of CCK-8 solution to each well at a ratio of 1:10, incubate for 30-60 min, and measure the absorbance at 450 nm.

[0134] Calculate cell viability using the following formula: Cell viability = (A 450 Processing Group-A 450 Blank group) / (A 450 Control group - A 450 (blank group) × 100%, where A 450 This indicates the absorbance value at 450nm.

[0135] The results are as follows Figure 3 As shown, Figure 3 In the figure, A represents Vero-E6 cells incubated with different concentrations (0-160 μg / mL) of CIPS nanosheets for 24 h, and the cell viability was detected by CCK-8 assay. Figure 3B in the figure is the cell activity detected by CCK-8 after ACE2 / 293T cells were incubated with different concentrations (0-160 μg / mL) of CIPS for 24 h. The results show that 0-160 μg / mL of CIPS nanosheets has no cytotoxicity to Vero-E6 and ACE2 / 293T cells, and the cell survival rate has no obvious decrease. The above results show that CIPS nanosheets have good biocompatibility and high safety.

[0136] The 1 mL fresh blood of healthy C57BL / 6 mice was collected in an anticoagulant tube by orbital bleeding. The upper serum and lower red blood cells were separated by centrifugation at 3000 rpm for 10 min at 4°C, and the leukocytes and platelets on the surface of the red blood cells were removed. The red blood cell suspension was prepared by washing with pre-cooled PBS for 3 times. 100 μL of 4% red blood cells was added with an equal volume of CIPS nanosheets (40, 80, 160 μg / mL), and incubated at 37°C for 4 h. The OD value at 540 nm wavelength was measured. Pure water was used as a positive control (100% hemolysis), and PBS was used as a negative control (0% hemolysis) to calculate the hemolysis rate. The formula is: Hemolysis index (Hem) % = (A 样品 -A PBS ) / (A 纯水 -A PBS ) × 100%, Figure 3 The results show that the hemolysis rate of CIPS nanosheets is 0, which has good biocompatibility and high safety.

[0137] Example 3 Immunofluorescence detection of the effect of CIPS nanosheets on SC2-P infection of ACE2-GFP / HEK-293T

[0138] After the 14 mm cell climbing sheet was placed in a 24-well plate, HEK-293T cells were inoculated, cultured at 37°C, 5% CO2 overnight, and transfected with ACE2-GFP for 24 h. The cells were infected with SC2-P mixed with 0 / 10 / 20 / 40 μg / mL of CIPS nanosheets for 2 h, and the amount of SC2-P infected cells was detected by immunofluorescence. The specific method is as follows:

[0139] The cells on the cell climbing sheet in the 24-well plate were fixed with 4% paraformaldehyde at room temperature for 15 min, and washed with PBS for 3 times.

[0140] Membrane rupture was performed with 0.1% Triton X-100 for 15 min, and washed with PBS for 3 times.

[0141] 3% BSA diluted with PBS was blocked for 30 min, and washed with PBS for 3 times.

[0142] Anti-Flag (rabbit source 1:500) and anti-GFP (mouse source 1:500) were incubated for 2 h, and washed with PBS for 3 times.

[0143] 488fluorescently labeled secondary mouse antibody and 555fluorescently labeled secondary rabbit antibody were incubated for 1 h, washed 4 times with PBS, mounted with antifluorescence quenching agent, and observed by fluorescence confocal microscopy.

[0144] Results are shown in Figure 4 A shows the results of confocal microscopy observation of SC2-P infection of ACE2-GFP / HEK-293T cells. SC2-P was pre-incubated with CIPS nanosheets at different concentrations (0, 10, 20, and 40 μg / mL) for 2 h, and then infected ACE2-GFP / HEK-293T cells. Immunofluorescence was used to detect the infection of SC2-P. Figure 4 B shows the number of SC2-P entering the cells and the fluorescence intensity, which was statistically analyzed by ImageJ. RFI represents relative fluorescence intensity. ACE2 channel is green fluorescence, SC2-P channel is red fluorescence, and Merge is the superposition of the two, which is represented by grayscale in the figure. By observing the infection of SC2-P by immunofluorescence, it can be observed that SC2-P can enter the host cells ACE2-GFP / HEK-293T, and Figure 4 B shows the results of statistical analysis. It can also be seen that with the treatment of CIPS nanosheets at different concentrations, the number of SC2-P entering the host cells and the fluorescence intensity are both reduced, and the degree of reduction is dose-dependent. When the amount of CIPS nanosheets is only 10 μg / mL, it can already produce a statistically significant difference compared with the negative control (i.e. the amount of CIPS nanosheets added is 0), and when the concentration reaches 40 μg / mL, the infection amount can be only 30% of the negative control. The above results show that CIPS nanosheets can effectively inhibit the entry of SC2-P into the host cells. Figure 4 B shows the results of statistical analysis. It can also be seen that with the treatment of CIPS nanosheets at different concentrations, the number of SC2-P entering the host cells and the fluorescence intensity are both reduced, and the degree of reduction is dose-dependent. When the amount of CIPS nanosheets is only 10 μg / mL, it can already produce a statistically significant difference compared with the negative control (i.e. the amount of CIPS nanosheets added is 0), and when the concentration reaches 40 μg / mL, the infection amount can be only 30% of the negative control. The above results show that CIPS nanosheets can effectively inhibit the entry of SC2-P into the host cells.

[0145] Example 4: Luciferase reporter gene detection of the effect of CIPS nanosheets on SC2-P infection of Vero-E6 and ACE2 / 293T

[0146] Vero-E6 cells and ACE2 / 293T cells were respectively inoculated into 96-well plates at a density of 1 × 10 4 The cells were cultured at 37°C with 5% CO2 overnight.

[0147] SC2-P mixed with 0 / 2.5 / 5 / 10 / 20 / 40 / 80 μg / mL of CIPS nanosheets was used to infect the cells for 2 h, and then the cells were washed once with DMEM and cultured in DMED medium for 40-48 h before detecting the luciferase activity.

[0148] Figure 5The results of luciferase activity detection show that SC2-P can integrate the genome into the host cell. When CIPS nanosheet layer treatment is applied, the amount of SC2-P invading Vero-E6 cells and ACE2 / 293T cells is significantly reduced compared with the control, and the degree of reduction is also dose-dependent. The above results show that the CIPS nanosheet layer can effectively inhibit the invasion of SC2-P to the host cell.

[0149] Example 5 Detection of CIPS nanosheet layer inhibiting SARS-CoV-2 true virus invasion of cells

[0150] At the same time, the present application also obtains similar results by using SARS-CoV-2 true virus to detect the invasion experiment. Vero-E6 cells are infected with SARS-CoV-2 virus mixed with 0 / 2.5 / 5 / 10 / 20 / 40 / 80 / 160 μg / mL CIPS nanosheet layer for 1 h, and the amount of virus in the culture medium is quantitatively detected after 48 h. As shown in Figure 6 , with the increase of the concentration of CIPS nanosheet layer, the invasion efficiency of SARS-CoV-2 true virus gradually decreases. It shows that CIPS nanosheet layer can not only effectively inhibit the invasion of SC2-P to the host cell, but also can inhibit the invasion of SARS-CoV-2 true virus. Among them, Figure 6 A shows the invasion rate of CIPS at different concentrations, B shows the virus amount quantitatively detected by taking the expression amount of ORF1ab / RdRp gene as an index, and Figure 6 C shows the virus amount quantitatively detected by taking the expression amount of nucleocapsid protein as an index.

[0151] Example 6 CIPS nanosheet layer physicochemical property characterization results show that CIPS can adsorb SC2-P

[0152] The binding capacity of CIPS nanosheet layer to SC2-P is detected. CIPS-SC2-P suspension is obtained after CIPS nanosheet layer is incubated with SC2-P for 2 h, and the UV-VIS and Z-potential characterization of CIPS-SC2-P suspension, CIPS nanosheet layer suspension and SC2-P suspension are detected respectively. The results are shown in Figure 7 A-B, Figure 7 A is the UV-VIS result of CIPS-SC2-P suspension, CIPS nanosheet layer suspension and SC2-P suspension. It can be seen that the absorption peaks of CIPS and SC2-P are different, and the absorption peaks of CIPS-SC2-P suspension after incubation for 2 h are shifted relative to the absorption peaks of CIPS nanosheet layer suspension and SC2-P suspension, and the absorption intensity also changes. Figure 7B represents the Z-potentials of the CIPS-SC2-P suspension, the CIPS nanosheet suspension alone, and the SC2-P suspension. The CIPS result represents the Z-potential of the CIPS nanosheet suspension alone, the SC2-P result represents the Z-potential of the SC2-P suspension, and the CIPS+SC2-P result represents the Z-potential of the CIPS-SC2-P suspension. The results indicate that CIPS nanosheets can adsorb SC2-P.

[0153] Example 7: Western blot detection of SC2-P adsorption and reduction of SC2-P in CIPS nanosheets

[0154] First, Western blot was used to directly detect CIPS nanosheets and SC2-P, and the results are shown in [Figure number missing]. Figure 7 In the left image of C, the protein in SC2-P is clearly visible in the Western blot results, while the CIPS nanosheets, lacking protein, show no corresponding bands. Subsequently, 0 μg, 10 μg, and 20 μg of CIPS nanosheets were added to the SC2-P suspension, respectively, and incubated for 120 minutes. Separation was then performed by centrifugation at the same speed, and the results were detected by Western blot. (See image). Figure 7 The right side of Figure C shows the results in the first column, which is a suspension containing only SC2-P. Because the virus is too small to be separated by centrifugation, there are no bands in the Western blot. The results in the second and third columns are the results after adding 10 μg and 20 μg of CIPS nanosheets, respectively. Since CIPS nanosheets have an adsorption effect on SC2-P, they can be obtained by centrifugation and corresponding bands are shown in the Western blot. The adsorption amount also increases with the increase of CIPS nanosheets.

[0155] Meanwhile, after incubating SC2-P with different amounts (0 / 5 / 10 / 20 / 40 μg) of CIPS nanosheets for two hours, the total amount of SC2-P was detected by Western blot. Figure 7 D shows that as the amount of CIPS nanosheets gradually increases, the amount of Spike protein (the full-length 180Kd protein) gradually decreases. Figure 7 D) The experimental results show that CIPS nanosheets can promote the degradation of the full-length SC2-P protein Spike, and the amount of pseudovirus SC2-P decreases with the degradation of Spike protein. This indicates that CIPS nanosheets can not only adsorb SC2-P, but also reduce the amount of SC2-P.

[0156] Example 8: CIPS nanosheets competitively bind to the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2.

[0157] Biomembrane interference (BLI) was used to quantitatively determine the affinity and interaction between CIPS nanosheets and the RBD domain of the SARS-CoV-2 Spike protein, serum proteins, and the model protein BSA. Figure 8 As shown, the KD values ​​for different systems were calculated. This indicates that CIPS nanosheets have a strong affinity for the RBD domain of the S protein, but a weak affinity for other proteins in serum. This suggests selective adsorption of RBD by CIPS nanosheets. This experiment confirms that CIPS nanosheets can act alone on the virus and specifically bind to the RBD domain of the viral S protein. These results indicate that CIPS nanosheets can be used for binding to and before the ACE2 protein, and may be used for coronavirus prevention. Furthermore, MoS2 and GO also have a strong affinity for the RBD domain. This indicates that MoS2 and GO nanosheets can also adsorb RBD, and the experiment confirms that MoS2 and GO nanosheets can act alone on the virus and specifically bind to the RBD domain of the viral S protein. These results indicate that both MoS2 and GO nanosheets can be used for binding to and before the ACE2 protein, and may be used for coronavirus prevention.

[0158] Furthermore, biomembrane interferometry (BLI) was used to quantitatively determine the changes in affinity between RBD and ACE2 proteins before and after binding with CIPS nanosheets. Regarding the interaction between RBD and ACE2: in a 200 nM RBD protein solution, the sensor chemically coupled and immobilized the RBD protein for 300 s, followed by desorption for 600 s; the sensor was then placed in a 100 nM ACE2 solution, where it adsorbed for 600 s and desorbed for 300 s. The results indicate that the KD value between RBD and ACE2 is approximately 4 nM. Figure 9 The results showed that the KD value was 41 nM in the presence of CIPS nanosheets. This indicates that the CIPS nanosheets weaken the affinity of RBD for ACE2. This suggests that the CIPS nanosheets can reduce the binding of the SARS-CoV-2 virus spike protein to its host cell receptor ACE2, thereby inhibiting viral infection.

[0159] Based on the experimental data, mathematical models were used to simulate the binding of RBD with CIPS nanosheets or ACE2. Figure 10 As shown, the binding sites of CIPS nanosheets and RBD ( Figure 10 A) The binding site of ACE2 and RBD ( Figure 10 B) overlap ( Figure 10C). And, compared with ACE2, CIPS nanosheets have stronger affinity to RBD, so CIPS nanosheets can competitively bind to RBD with ACE2. CIPS nanosheets occupy the binding site of ACE2, and thus prevent the binding of RBD to ACE2, thereby inhibiting the infection of viral SARS-CoV-2 to host cells. Figure 10 The lower abc respectively represent the binding site of ACE2, the binding site of CIPS and the coincidence of the two. From the coincidence simulation diagram, it can be seen that the binding sites of ACE2 and CIPS coincide.

[0160] Example 9 Molecular dynamics (MD) simulation of RBD binding to CIPS and RBD

[0161] Typical trajectory snapshots of RBD and CIPS adsorption in the system based on molecular dynamics (MD) simulation. The bottom row shows the dynamic process of RBD adsorbed on the surface of CIPS. The bottom map of the upper row is the RBD amino acid site bound to CIPS, and the VDW ball is the amino acid residue in contact with CIPS. Red ball is polar residue, green is hydrophobic residue, blue is positive charge residue, purple is negative charge residue, and yellow is cysteine containing disulfide bond.

[0162] Example 10 CIPS can adsorb SC2-P in the protein mixture of SC2-P and FBS and inhibit its infection efficiency

[0163] FBS was mixed with SC2-P at 1-fold, 3-fold and 10-fold volume of SC2-P, respectively. 20 μg / mL CIPS nanosheets were pre-incubated with the protein mixture for 2 h, then supplemented with PBS to 1 ml, and centrifuged at 4000 rpm for 5 min. WB was used to detect the amount of SC2-P in the precipitate. The results showed that in the protein mixture, CIPS can still adsorb SC2-P.

[0164] FBS was mixed with SC2-P at 1-fold, 3-fold and 10-fold volume of SC2-P, respectively. 20 μg / mL CIPS nanosheets were pre-incubated with the protein mixture for 2 h, and then infected ACE2 / 293T cells for 2 h. Luciferase activity was used to detect the infection efficiency of SARS-P on ACE2 / 293T cells. The results showed that CIPS can adsorb and inhibit the infection efficiency of SC2-P in the complex protein solution environment.

[0165] Example 11 Quantitative PCR detection of CIPS nanosheets adsorbing real virus SARS-CoV-2

[0166] In the P3 laboratory, under the premise of meeting the P3 laboratory operation specification, 6000 pfu / 500 ml of true virus SARS-CoV-2 was incubated with different concentrations (6 and 12 pM) of CIPS for 2 h, and then centrifuged at 4000 rpm for 5 min to obtain the supernatant and precipitate. After extracting the RNA of the virus in the supernatant and precipitate, respectively, the amount of virus was quantified by quantitative PCR. As shown in Figure 13 , the results show that CIPS can bind to true virus SARS-CoV-2, and as the amount of CIPS increases, the amount of CIPS-bound virus obtained by centrifugation also gradually increases, and the amount of virus remaining in the supernatant gradually decreases.

[0167] Example 12 Quantitative PCR detection of CIPS nanosheet to reduce the amount of true virus SARS-CoV-2

[0168] In the P3 laboratory, under the premise of meeting the P3 laboratory operation specification, 6000 pfu / 500 ml of true virus SARS-CoV-2 was incubated with different concentrations (1.5-48 pM) of CIPS for 2 h, and then all the viruses were collected and their RNA was extracted, and the amount of virus was quantified by quantitative PCR. Figure 14 , as the amount of CIPS nanosheet gradually increases, the amount of viral RNA gradually decreases, and from the experimental results, it can be seen that CIPS nanosheet can reduce the amount of viral RNA. This shows that CIPS nanosheet not only can adsorb true virus SARS-CoV-2, but also can reduce the amount of SARS-CoV-2.

[0169] Example 13 Infection results of SC2-P on ACE2 / 293T cells in the presence of different two-dimensional nanomaterials CIPS nanosheet, graphene oxide (GO) nanosheet, molybdenum disulfide (MoS2) nanosheet, and black phosphorus (BP) nanosheet

[0170] ACE2 / 293T cells were seeded into a 96-well plate at a density of 1×10 4 cells / well, and cultured at 37°C, 5% CO2 overnight.

[0171] SC2-P mixed with 20 pg / mL of CIPS nanosheet, GO (graphene oxide) nanosheet, MoS2 (molybdenum disulfide) nanosheet, or black phosphorus (BP) nanosheet was used to infect cells for 2 h, and then washed once with DMEM before adding DMED culture medium for 40-48 h to detect luciferase activity.

[0172] Figure 15The results of luciferase activity detection show that different two-dimensional nanomaterials all have the ability to inhibit the infection of the new crown pseudo-virus SC2-P to host cells. The amount of SC2-P infection of ACE2 / 293T cells is significantly reduced compared with the control when CIPS nanosheets or black scale nanosheets are applied. When GO (graphene oxide) nanosheets or MoS2 (molybdenum disulfide) nanosheets are applied, the amount of SC2-P infection of ACE2 / 293T cells is also significantly reduced compared with the control, but the effect is not as obvious as that of applying CIPS nanosheets or black scale nanosheets. The CIPS nanosheet, GO nanosheet, MoS2 nanosheet group or black scale nanosheet has a significant difference with the control group (Ctrl group). The above results show that CIPS nanosheets, GO nanosheets and MoS2 nanosheets can effectively inhibit the infection of SC2-P to host cells, and CIPS nanosheets have the best effect.

[0173] Example 14 Inhibition effect of CIPS nanosheets on SARS pseudo-virus particles SARS-P, which shows that the inhibition effect of CIPS nanosheets on coronavirus has broad spectrum.

[0174] In order to detect whether this inhibition is specific to the infection of SARS-CoV-2 virus or applicable to other coronaviruses, the present application also detects the inhibition effect of CIPS nanosheets on SARS pseudo-virus particles SARS-P. The results are shown in Figure 16 Figure 16 A is the observation of SARS-P infection of ACE2-GFP / HEK-293T cells under a confocal microscope. SARS-P is pre-incubated with 20 μg / mL of CIPS nanosheets for 2 h, and then infects ACE2-GFP / HEK-293T cells for 2 h. Immunofluorescence is used to detect the infection of SARS-P. Figure 16 B-16C is the number of SARS-P entering cells and the fluorescence intensity counted by ImageJ. RFI represents relative fluorescence intensity. Figure 16 D-16E is the luciferase activity detection of the infection efficiency of SARS-P to ACE2 / 293T and Vero-E6 cells. SARS-P is pre-incubated with 20 μg / mL of CIPS nanosheets for 2 h, and then infects Vero-E6 and ACE2 / 293T cells for 2 h. The infection efficiency of SARS-P is detected by luciferase activity after 40 h. CIPS nanosheets can also inhibit the infection of SARS-P. This shows that the inhibition of CIPS nanosheets to viral infectivity is not only for SARS-CoV-2 virus, but also has broad spectrum for coronavirus.

[0175] ​The application provides a kind of nanomedicine for inhibiting SARS-CoV-2 virus cell infection, any one or more of indium-doped copper sulfide phosphide nanosheet, graphene oxide nanosheet, molybdenum disulfide nanosheet are included in the nanomedicine.

[0176] Embodiment 1: oral liquid with any one or more of indium-doped copper sulfide phosphide nanosheet, graphene oxide nanosheet, molybdenum disulfide nanosheet, black scale nanosheet as the pharmacodynamic component;

[0177] Taking oral liquid as an example, oral liquid is a new dosage form developed on the basis of decoction and injection, which has the advantages of small dose, rapid absorption, stable quality, convenient carrying and taking, and easy storage. It contains multiple effective components, and has a great influence on quality and taste. Under the premise of not changing the structure and function of the main active ingredient, how to maximize the retention of effective components and improve the taste is a difficulty in the selection of excipients. Adding excipients to oral liquid can improve taste, improve clarity, enhance stability and improve product quality.

[0178] Commonly used excipients for oral liquid include solvents, flavorings, flavorings, clarifiers, preservatives, etc. These excipients can be added at the same time or one at a time. Among them, the solvent is a must-add, and water can be used. Different combinations of excipients include sweeteners, flavorings, clarifiers or preservatives, or combinations of sweeteners and preservatives. The preferred combination is sweetener and preservative.

[0179] For oral liquid, preferably, the sweetener is selected from one or more of protein sugar, xylitol, aspartame and sucralose.

[0180] For oral liquid, preferably, the preservative is selected from one or more of p-hydroxybenzoic acid ester, butylated hydroxyanisole, butylated hydroxytoluene and sorbic acid.

[0181] The preservative can be p-hydroxybenzoic acid ester, butylated hydroxytoluene or sorbic acid, preferably butylated hydroxytoluene. It can also be used in combination, such as a combination of hydroxybenzoic acid ester and butylated hydroxytoluene, or a combination of butylated hydroxytoluene and sorbic acid, or a combination of p-hydroxybenzoic acid ester and sorbic acid, or a combination of p-hydroxybenzoic acid ester, butylated hydroxytoluene and sorbic acid.

[0182] For oral liquid, preferably, the flavoring agent is fruit essence.

[0183] For oral liquid, preferably, the clarifier is a mixture of one or both of chitosan and gelatin.

[0184] Embodiment 2: tablet with any one or more of indium-doped copper sulfide nanosheet layer, graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer, and black scale nanosheet layer as the pharmaceutical ingredient;

[0185] The tablet has the advantages of accurate dosage, stable quality, convenient taking, carrying and transporting, etc.

[0186] For the tablet, the preparation adjuvant includes one or more of diluent, binder, lubricant and disintegrant, preferably a combination of diluent, binder, lubricant and disintegrant.

[0187] For the tablet, preferably, the diluent is one or more of cellulose and inorganic salt, for example, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, pharmaceutical grade calcium carbonate, mannitol, etc., to increase the volume of the raw material to help its molding.

[0188] For the tablet, preferably, the binder is one or more of water, ethanol, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, gelatin and polyvinyl pyrrolidone, etc.

[0189] For the tablet, preferably, the lubricant is one or more of magnesium stearate, micro-powder silica, talc, hydrogenated vegetable oil, polyethylene glycol and magnesium lauryl sulfate.

[0190] For the tablet, preferably, the disintegrant is one or more of low-substituted hydroxypropyl, cross-linked polyvinyl pyrrolidone and cross-linked sodium carboxymethyl cellulose, etc.

[0191] Embodiment 3: capsule with any one or more of indium-doped copper sulfide nanosheet layer, graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer and black scale nanosheet layer as the pharmaceutical ingredient.

[0192] In the present application, the capsule mainly improves the stability and bioavailability of the drug. The preparation adjuvant is the capsule shell, and the capsule shell is a hard capsule shell or a soft capsule shell.

[0193] Embodiment 4: granules with any one or more of indium-doped copper sulfide nanosheet layer, graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer and black scale nanosheet layer as the pharmaceutical ingredient.

[0194] The granules can be directly swallowed, or can be drunk into water with warm water. The application and carrying are relatively convenient, and the dissolution and absorption speed is relatively fast. The preparation adjuvant used for the granules is similar to that of the tablet, involving one or more of filler, binder, wetting agent, disintegrant, lubricant and film coating material.

[0195] For granules, preferably, the filler is one or more of cellulose and inorganic salts. For example, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, pharmaceutical grade calcium carbonate, mannitol, etc. to increase the volume of the raw material to help its molding.

[0196] For granules, preferably, the binder is one or more of water, ethanol, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, gelatin and polyvinyl pyrrolidone, etc.

[0197] For granules, preferably, the wetting agent is water or ethanol or a mixture of the two. For example, one or more of magnesium stearate, fine silica gel, talc, hydrogenated vegetable oil, polyethylene glycol and magnesium lauryl sulfate.

[0198] For granules, preferably, the disintegrant is one or more of low-substituted hydroxypropyl, cross-linked polyvinyl pyrrolidone and cross-linked sodium carboxymethyl cellulose, etc.

[0199] For granules, preferably, the film coating material is one or more of hydroxypropyl methyl cellulose, polyethylene glycol, cellulose acetate phthalate and polyvinyl acetal diethylamine acetate.

[0200] Embodiment 5: a powder with any one or more of indium-doped copper sulfide nanosheet layers, graphene oxide nanosheet layers, molybdenum disulfide nanosheet layers, and black scale nanosheet layers as the active ingredient.

[0201] The present application can also be made into a powder, which is convenient for dosing and taking.

[0202] For example, taking beverages, the composition of the present application can be made into different flavored beverages, which will be popular as daily drinks.

[0203] The formulation adjuvants used in beverages are at least one of clarifying agents, preservatives and flavoring agents.

[0204] The composition of the present application can also be made into other powders, for example, functional milk powder, the main adjuvant added is milk powder, for example, skimmed milk powder, skimmed sugar-free milk powder.

[0205] The above multiple embodiments can adjust the amount of the medicament in the unit product to adapt to different uses, such as pharmaceuticals, health products, food, etc.

[0206] A preferred embodiment of the present application provides a melt-blown cloth for SARS-CoV-2 virus cell invasion, which adds any one or more of indium-doped copper sulfide nanosheet layers, graphene oxide nanosheet layers, molybdenum disulfide nanosheet layers, and black scale nanosheet layers.

[0207] In one preferred embodiment of the present application, a cold storage coating or an outer packaging coating for inhibiting cell infection of SARS-CoV-2 virus is provided, wherein any one or more of indium-doped copper phosphosulfide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black-scaled nanosheets are added.

[0208] In one preferred embodiment of the present application, a disinfectant spray for inhibiting cell infection of SARS-CoV-2 virus is provided, wherein any one or more of indium-doped copper phosphosulfide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black-scaled nanosheets are added. In addition to any one or more of indium-doped copper phosphosulfide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black-scaled nanosheets, the spray also contains a solvent, which is water, and is non-toxic. The effective concentration of indium-doped copper phosphosulfide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black-scaled nanosheets is 2.5-160 μg / mL (as shown in Figure 3 The concentration is preferably 20 μg / mL.

[0209] In summary, the present application verifies that two-dimensional nanomaterials CIPS nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black-scaled nanosheets can all inhibit the infection efficiency of SARS-CoV-2 virus. CIPS can also inhibit the infection of SARS-P. This indicates that the inhibition of CIPS on viral infectivity is not only directed against SARS-CoV-2 virus, but also has broad-spectrum adaptability to coronaviruses.

[0210] Taking CIPS as an example, the present application also verifies the effect of CIPS on cell survival rate. Experimental results show that CIPS (1-160 μg / mL) has no effect on the activity of Vero-E6 and ACE2 / 293T cells, and there is no cell hemolysis, indicating that it has high biological safety and low toxicity.

[0211] The present application also verifies the inhibition of CIPS on the infection efficiency of SARS-CoV-2 virus. Experiments prove that CIPS inhibits the infection efficiency of SARS-CoV-2 pseudovirus (SC2-P) on ACE2-GFP / HEK-293T (expressing ACE2-GFP in HEK-293T) and Vero-E6. And experiments prove that CIPS can inhibit the infection efficiency of real virus SARS-CoV-2 on Vero-E6.

[0212] The application also verifies the adsorption of CIPS to SC2-P. Experiments prove that CIPS can adsorb and reduce SC2-P. And CIPS has stronger binding effect on the receptor binding domain (RBD) domain of the spike protein of SARS-CoV-2 virus than other proteins, and the presence of CIPS can inhibit the binding of the virus to the receptor.

[0213] Therefore, the nanomaterials of the application can be prepared into nanodrugs for treating novel coronavirus infection, or added to the melt-blown cloth of the mask, the coating of the cold storage and the coating of the outer package, and the spray, to inhibit the spread of the novel coronavirus and achieve the prevention effect. The inhibition of the virus infectivity by the two-dimensional nanomaterials of the application is not only for SARS-CoV-2 virus, but also has broad spectrum for coronaviruses. The two-dimensional nanomaterials CIPS nanosheet, graphene oxide nanosheet and molybdenum disulfide nanosheet, and black scale nanosheet can be applied alone or stacked.

[0214] The above only describes the preferred embodiments of the application and is not intended to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. Application of two-dimensional nanomaterial in preparation of a drug for treating or preventing a disease caused by a coronavirus, wherein the two-dimensional nanomaterial is black-scaled nanosheet layers. The coronavirus is selected from one of SARS-CoV and SARS-CoV2; the disease caused by the coronavirus is a disease caused by infection with the coronavirus.

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