WO2022126857A1 - 二维纳米材料在抑制冠状病毒的应用 - Google Patents

二维纳米材料在抑制冠状病毒的应用 Download PDF

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WO2022126857A1
WO2022126857A1 PCT/CN2021/077226 CN2021077226W WO2022126857A1 WO 2022126857 A1 WO2022126857 A1 WO 2022126857A1 CN 2021077226 W CN2021077226 W CN 2021077226W WO 2022126857 A1 WO2022126857 A1 WO 2022126857A1
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nanosheets
hcov
coronavirus
sars
cov
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English (en)
French (fr)
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李洋
王黎明
张国芳
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深圳先进技术研究院
中国科学院高能物理研究所
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Priority to US18/267,783 priority Critical patent/US20240091167A1/en
Publication of WO2022126857A1 publication Critical patent/WO2022126857A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/42—Phosphorus; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7007—Drug-containing films, membranes or sheets
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/34—Shaped forms, e.g. sheets, not provided for in any other sub-group of this main group
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/26—Phosphorus; Compounds thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A41—WEARING APPAREL
    • A41D—OUTERWEAR; PROTECTIVE GARMENTS; ACCESSORIES
    • A41D31/00—Materials specially adapted for outerwear
    • A41D31/04—Materials specially adapted for outerwear characterised by special function or use
    • A41D31/30—Antimicrobial, e.g. antibacterial
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/44—Elemental carbon, e.g. charcoal, carbon black
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/24—Phosphorous; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/10—Washing or bathing preparations
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D39/00—Filtering material for liquid or gaseous fluids
    • B01D39/14—Other self-supporting filtering material ; Other filtering material
    • B01D39/20—Other self-supporting filtering material ; Other filtering material of inorganic material, e.g. asbestos paper, metallic filtering material of non-woven wires
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D46/00—Filters or filtering processes specially modified for separating dispersed particles from gases or vapours
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
    • B65D65/00—Wrappers or flexible covers; Packaging materials of special type or form
    • B65D65/38—Packaging materials of special type or form
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
    • C09D5/14—Paints containing biocides, e.g. fungicides, insecticides or pesticides
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/02—Inorganic compounds ; Elemental compounds
    • C11D3/12—Water-insoluble compounds
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/48—Medical, disinfecting agents, disinfecting, antibacterial, germicidal or antimicrobial compositions
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of anti-virus, in particular to the application of a two-dimensional nanomaterial in inhibiting coronavirus.
  • Nanomaterials refer to materials whose scales in a certain, two- or three-dimensional direction reach the nanometer scale. 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 (planar motion) on the nanoscale in two dimensions, such as boron nitride (BN) nanosheets, molybdenum disulfide (MoS2) nanosheets, tungsten disulfide (WS 2 ) nanosheets, Mxene materials.
  • Two-dimensional nanomaterials have many peculiar properties due to their carrier migration and heat diffusion being confined in two-dimensional planes, and are widely used in the field of electronics. However, there are few applications in medicine.
  • Coronaviruses are a large family of viruses known to cause colds as well as more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • the 2019 new coronavirus (2019-nCoV, which causes the new coronavirus pneumonia COVID-19) is the seventh known type, the first 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.
  • one aspect of the present invention proposes the application of two-dimensional nanomaterials in the preparation of medicines for treating or preventing diseases caused by coronaviruses, wherein the two-dimensional nanomaterials are indium-doped copper phosphide A combination of any one or more of nanosheets (CIPS), graphene oxide nanosheets, molybdenum disulfide nanosheets or black scale nanosheets.
  • CIPS nanosheets
  • graphene oxide nanosheets graphene oxide nanosheets
  • molybdenum disulfide nanosheets or black scale nanosheets.
  • the medicament further comprises a pharmaceutically acceptable carrier or excipient.
  • the medicine further includes the administration of a therapeutically effective amount of at least one other therapeutic agent or its composition
  • the other therapeutic agent or its composition is selected from corticosteroids, anti-inflammatory drugs Inflammatory signaling modulators, beta2-adrenoceptor agonist bronchodilators, anticholinergics, mucolytics, hypertonic saline, and other drugs used in the treatment of coronavirus infections; or mixtures thereof.
  • a second aspect of the present invention provides a pharmaceutical composition for treating or preventing diseases caused by coronavirus, wherein the pharmaceutical composition uses a two-dimensional nanomaterial as an active ingredient, and the two-dimensional nanomaterial is an indium-doped sulfur
  • the pharmaceutical composition uses a two-dimensional nanomaterial as an active ingredient, and the two-dimensional nanomaterial is an indium-doped sulfur
  • the two-dimensional nanomaterial is an indium-doped sulfur
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition further includes a second active ingredient selected from the group consisting of corticosteroids, anti-inflammatory signal transduction regulators, ⁇ 2 -Adrenoreceptor agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline, and other drugs used to treat coronavirus infections; or mixtures thereof.
  • a second active ingredient selected from the group consisting of corticosteroids, anti-inflammatory signal transduction regulators, ⁇ 2 -Adrenoreceptor agonists bronchodilators, anticholinergics, mucolytics, hypertonic saline, and other drugs used to treat coronavirus infections; or mixtures thereof.
  • the formulation form of the pharmaceutical composition is selected from oral preparations, injection preparations, mucosal administration preparations, inhalation preparations, and external preparations.
  • a third aspect of the present invention provides a method for treating or preventing coronavirus infection, comprising administering to a subject a therapeutically effective amount of a two-dimensional nanomaterial, wherein the two-dimensional nanomaterial is indium-doped sulfur phosphide
  • a combination of any one or more of copper nanosheets (CIPS), graphene oxide nanosheets, molybdenum disulfide nanosheets or black scale nanosheets comprising administering to a subject a therapeutically effective amount of a two-dimensional nanomaterial, wherein the two-dimensional nanomaterial is indium-doped sulfur phosphide
  • CIPS copper nanosheets
  • graphene oxide nanosheets graphene oxide nanosheets
  • molybdenum disulfide nanosheets or black scale nanosheets.
  • 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.
  • 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-adrenoceptor agonist bronchodilators, anticholinergics, mucolytics, hypertonic saline, and other drugs used to treat coronavirus infections; or mixtures thereof.
  • a second active ingredient selected from the group consisting of corticosteroids, anti-inflammatory signal transduction Modulators, beta2-adrenoceptor agonist bronchodilators, anticholinergics, mucolytics, hypertonic saline, and other drugs used to treat coronavirus infections; or mixtures thereof.
  • the fourth aspect of the present invention provides the use of two-dimensional nanomaterials in the preparation of a preparation for inhibiting coronavirus and reducing the binding of SARS-CoV-2 virus Spike protein to its host cell receptor ACE2; the two-dimensional nanomaterials are indium-doped A combination of any one or more of hetero copper phosphide nanosheets (CIPS), graphene oxide nanosheets, molybdenum disulfide nanosheets or black scale nanosheets; preferably, two-dimensional nanomaterials Inhibits its binding to its host cell receptor ACE2 by competitively binding to the RBD binding site of the coronavirus Spike protein.
  • CIPS hetero copper phosphide nanosheets
  • the present invention also provides the use of the two-dimensional nanomaterial in the preparation of a formulation that competitively binds to the RBD binding site of the coronavirus Spike protein.
  • the fifth aspect of the present invention provides the use of two-dimensional nanomaterials in the preparation of medicines for promoting the decomposition of SARS-CoV-2 virus proteins; the two-dimensional nanomaterials are indium-doped copper thiophosphide nanosheets (CIPS). ), graphene oxide nanosheets, molybdenum disulfide nanosheets or black scale nanosheets any one or a combination of more.
  • CIPS copper thiophosphide nanosheets
  • the sixth aspect of the present invention provides the use of two-dimensional nanomaterials in the preparation of medicines for promoting the decomposition of SARS-CoV-2 viral RNA;
  • the two-dimensional nanomaterials are indium-doped copper phosphide nanosheets ( A combination of any one or more of CIPS), graphene oxide nanosheets, molybdenum disulfide nanosheets or black scale nanosheets.
  • the seventh aspect of the present invention provides the use of two-dimensional nanomaterials in the preparation of materials for inhibiting coronavirus infection;
  • the two-dimensional nanomaterials are indium-doped copper sulfur phosphide nanosheets (CIPS), graphene oxide Any one or a combination of nanosheets, molybdenum disulfide nanosheets or black scale nanosheets.
  • CIPS copper sulfur phosphide nanosheets
  • graphene oxide Any one or a combination of nanosheets, molybdenum disulfide nanosheets or black scale nanosheets.
  • the eighth aspect of the present invention provides a material for inhibiting coronavirus infection, the material comprises a two-dimensional nanomaterial and a matrix, and the two-dimensional nanomaterial is an indium-doped copper phosphide nanosheet (CIPS) , a combination of any one or more of graphene oxide nanosheets, molybdenum disulfide nanosheets or black scale nanosheets.
  • CIPS copper phosphide nanosheet
  • the material is a material for preparing protective equipment, preferably the protective equipment is a mask, protective clothing, protective face shield, and protective cap.
  • the material is a kind of paint.
  • the material is a disinfectant or a personal care product.
  • the material is a packaging material.
  • the material is a filter material.
  • a ninth aspect of the present invention provides an article, the article includes two-dimensional nanomaterials, and the two-dimensional nanomaterials are indium-doped copper phosphide nanosheets (CIPS), graphene oxide nanosheets, A combination of any one or more of the molybdenum disulfide nanosheet layer or the black scale nanosheet layer;
  • CIPS copper phosphide nanosheets
  • graphene oxide nanosheets A combination of any one or more of the molybdenum disulfide nanosheet layer or the black scale nanosheet layer;
  • the article is selected from protective articles, coatings, disinfectants, personal care articles, packaging materials or filtration devices;
  • the protective equipment is a mask, protective clothing, protective face shield, protective cap;
  • the coating is a suspension containing two-dimensional nanomaterials, whether the coating can impart a coating with two-dimensional nanomaterials on the surface of the attached object;
  • the disinfectant is an environmental disinfectant, hand sanitizer, hand sanitizer, and detergent; more preferably, the disinfectant is used for surface disinfection of packaging materials, medical instruments, oral appliances, and beauty appliances;
  • the packaging material is a coating containing two-dimensional nanomaterials, or the packaging material is a composite material containing two-dimensional nanomaterials; more preferably, the packaging material is a packaging material for medicine or food , especially medicines or foodstuffs for cold chain transportation;
  • the filter device is a device for air filtration, a device for water body filtration, and a device for mask filtration; more preferably, the filter device is an air filter, and the surface of the air filter has Two-dimensional nanomaterials; more preferably, the filter device is an air filter cotton, and two-dimensional nanomaterials are attached to the fibers of the air filter cotton.
  • the coronavirus is selected from one of HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, SARS-CoV2 or MERS-CoV.
  • the coronavirus is preferably selected from SARS-CoV, SARS-CoV2 or HCoV-NL63.
  • the coronavirus is preferably selected from SARS-CoV or SARS-CoV2.
  • the disease caused by coronavirus is a disease caused by coronavirus infection.
  • the two-dimensional nanomaterial is a material whose length in one dimension in space is nanometer size; preferably, it is a material whose length in one dimension in space is 1-100 nm, more preferably , is a material with a length of 1-10 nm in one dimension in space, more preferably, a material with a length of 1-10 nm in one dimension in space, a material with a length of 100-500 nm in the other dimension, or, for example, other
  • the lengths of the dimensions are 100 nm, 200 nm, 300 nm, 400 nm, 500 nm.
  • the present invention achieves the following beneficial effects:
  • Two-dimensional nanomaterials such as CIPS nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets or black scale nanosheets can inhibit the cell infection of coronavirus on the one hand, and on the other hand, can specifically adsorb and combine
  • the target of coronavirus infection can reduce the infection efficiency of the virus, thereby reducing the replication of the virus in the host, reducing the number of coronaviruses, and can be used as a therapeutic drug for patients with coronavirus.
  • CIPS nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets or black scale nanosheets can specifically bind to coronaviruses, they can inhibit the infection of host cells by SARS-CoV-2 virus. Specific adsorption and immobilization for viruses. It can be used for the preparation of medicines, and can be used for meltblown cloth, cold storage coating and outer packaging coating or spray that can be applied to masks. It can absorb the new coronavirus and reduce the number of infectable viruses, which can prevent new coronary pneumonia. effect.
  • Two-dimensional nanomaterials such as CIPS nanosheets have no effect on the survival rate of HEK-293T and Vero-E6 cells, and do not cause hemolysis of red blood cells in mice, indicating that the nanomaterials have high biological safety and low toxicity.
  • the two-dimensional nanomaterial of the present invention can be stored at room temperature to -20°C for a long time, which is convenient for storage.
  • the two-dimensional nanomaterial of the present invention has high stability, and can be mixed or compounded with any auxiliary materials to form medicines and composite materials, and is not affected by factors such as temperature and pH.
  • the present invention adopts inorganic nanomaterials, the source of materials is not limited, it is suitable for large-scale mass production, the cost is low, and the dosage is small.
  • the biofilm interference experiment of the present invention was carried out in solid and liquid environments, and it was confirmed that CIPS can strongly bind to the RBD region of the coronavirus S protein (see Figure 8).
  • the results of the computer simulation also show that binding is possible at 5.7ns (Fig. 11).
  • the present invention adopts the two-dimensional nanomaterial and the coronavirus to pre-incubate for 2 hours before carrying out the evaluation of the infection experiment with the host cell. It can be seen that the material of the present invention can achieve a long time with the coronavirus.
  • the binding of SARS-CoV-2 can be used as a material to adsorb the coronavirus and prevent it from binding to host cells. That is, the material of the present invention can instantly realize the combination with coronavirus, long-term binding, and inhibit the combination of virus and host cell after binding.
  • Figure 1 is an image of a CIPS crystal sheet
  • Figure 2 is an atomic force microscope image of a CIPS crystal sheet
  • FIG. 3 shows the biosafety detection of CIPS
  • Figure 4 shows the infection results of 293T (ACE2/293T) cells transfected with ACE2-GFP by the pseudovirus (SC2-P) of the Spike protein of SARS-CoV-2 in the presence of CIPS nanosheets; ANOVA was used for statistical analysis ,**:P ⁇ 0.01,***:P ⁇ 0.001;
  • Figure A is the confocal microscope observation of SC2-P infection of ACE2/293T cells
  • the first row (ACE2) is the ACE2/293T cell channel, which is green fluorescence
  • the second row (SC2-P) is SC2- Channel P, red fluorescence
  • row 3 (Merge) is the overlay of row 1 and row 2, indicating the number and distribution of SC2-P entering ACE2/293T cells.
  • the different columns in Figure A represent different amounts of CIPS nanosheets added, calculated with the concentration of CIPS nanosheets added.
  • Figure B shows the quantitative results of SC2-P infection on ACE2/293T cells, where the abscissa of the left figure is the amount of CIPS nanosheets added, in terms of the added concentration; the ordinate is the amount of SC2-P entering the cells.
  • the right picture shows that the abscissa is the amount of CIPS nanosheets added, in terms of the concentration after the addition; the ordinate is the relative fluorescence intensity.
  • Figure 5 shows the infection efficiency of pseudovirus SC2-P on Vero-E6 and ACE2/293T cells detected by luciferase activity; ANOVA was used for statistical analysis, *: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001;
  • Figure 6 shows the quantitative detection of the infection efficiency of the true new coronavirus SARS-CoV-2 on Vero-E6 cells; ANOVA was used for statistical analysis, *: P ⁇ 0.05, **: P ⁇ 0.01, ***: P ⁇ 0.001;
  • a in Figure 6 shows the infection rate of different concentrations of CIPS, B shows the amount of virus quantitatively detected by the expression of ORF1ab/RdRp gene, and C in Figure 6 shows the nucleocapsid protein The amount of gene expression is the amount of virus quantitatively detected by the index;
  • Figure 7 shows the adsorption and reduction of pseudovirus SC2-P by CIPS
  • FIG 8 shows the quantitative measurement results of Biofilm Interferometry (BLI).
  • A is the affinity and interaction between the CIPS material at different concentrations and the RBD domain protein of the new coronavirus Spike protein
  • B is the affinity and interaction between the CIPS material and the protein in serum
  • C is the molybdenum disulfide nanosheet (MoS 2 ) and the new crown virus.
  • Virus Spike protein RBD domain protein affinity and interaction D is the affinity and interaction between graphene oxide nanosheets (GO) and 2019-nCoV Spike protein RBD domain protein; in the ACD results, the curves from top to bottom are respectively Concentrations of 200nM, 100nM, 50nM, 25nM and 12.5nM; B results from top to bottom are RBD, Fg, FBS, IgG, HDL, Tf, HAS, respectively.
  • Figure 9 shows the affinity change between the RBD domain of the new coronavirus Spike protein and the ACE2 protein before and after the quantitative determination of biofilm interference (BLI) and CIPS;
  • Figure 10 is a mathematical model to simulate the combination of RBD and CIPS or ACE2;
  • Figure 11 is a mathematical model to simulate the combination of RBD and CIPS at different time points.
  • the computer simulation results show that the binding time is 5.7ns (the binding time is 5.7ns)
  • Figure 12 shows the adsorption and inhibition of SC2-P by CIPS in the protein mixture of SC2-P and FBS.
  • A means that CIPS can bind to SC2-P in the protein mixture added with 1, 3 or 10 times the volume of FBS of SC2-P;
  • B means that CIPS can still inhibit the invasion of SC2-P in the protein mixture. dyeing efficiency.
  • Figure 13 is the result of Example 11, showing the quantitative PCR result of CIPS adsorption of the true virus SARS-CoV-2.
  • FIG 14 shows the results of Example 12, CIPS reduces the amount of the true virus SARS-CoV-2.
  • Figure 15 shows the effect of pseudovirus SC2-P on ACE2/293T in the presence of different two-dimensional nanomaterials CIPS nanosheets, GO (graphene oxide nanosheets), MoS 2 (molybdenum disulfide nanosheets) or black scale nanosheets Infection results of cells.
  • Figure 16 shows the infection efficiency of the pseudovirus SARS-P of SARS inhibited by CIPS.
  • 2D nanomaterials include graphene, transition metal dichalcogenides, transition metal carbides, nitrides and carbonitrides, black phosphorus nanosheets, layered double hydroxides, 2D MOFs, and other types of 2D nanosheets.
  • graphene oxide is a two-dimensional sp2 carbon atomic layer containing oxygen functional groups, which has been widely used in nanomedicine and other fields.
  • indium-doped copper phosphide nanosheets (CIPS), graphene oxide nanosheets, molybdenum disulfide nanosheets and black scale nanosheets in two-dimensional nanomaterials are effective in inhibiting coronavirus infection.
  • infection especially the cell infection of SARS virus and new coronavirus pneumonia virus SARS-CoV-2 is effective.
  • indium-doped copper phosphide nanosheets (CIPS), graphene oxide nanosheets, molybdenum disulfide nanosheets and black phosphorus nanosheets in the present invention have high biological safety and low toxicity, they can be used in in many fields such as medicine.
  • indium-doped copper phosphide has the same meaning as CIPS or CuInP 2 S 6 .
  • the terms “nanosheet” and “two-dimensional nanomaterial” have similar meanings, and both refer to a material with a nanoscale dimension in space, such as a nanomaterial with a thickness of 1-100 nm.
  • the two-dimensional nanomaterial is selected from any one of indium-doped copper phosphide nanosheets (CIPS), graphene oxide nanosheets, molybdenum disulfide nanosheets and black scale nanosheets or a combination of more than one.
  • the size of each two-dimensional nanomaterial is selected as follows: the thickness (one dimension in space) is 1-10 nm. The dimensions of the nanosheets perpendicular to the thickness direction (other dimensions in space) are 100-300 nm.
  • nanosheet layer can be obtained by exfoliating the nanosheet material by a conventional method in the art, for example, it can be obtained by mechanical grinding (eg ball milling), ultrasonic and other methods.
  • “Pharmaceutically acceptable carrier or excipient” includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surface active agent agents, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifying agents that have been approved by the Food and Drug Administration as acceptable for use in humans or livestock.
  • “Pharmaceutical composition” refers to a formulation of a compound of the present invention and a vehicle generally accepted in the art for delivering a biologically active compound to a mammal (eg, a human). Such vehicles include all pharmaceutically acceptable excipients for this purpose.
  • an “effective amount” or “therapeutically effective amount” refers to an amount of a compound according to the present invention which, when administered to a patient in need thereof, is sufficient to effect the treatment of a disease state, condition or disorder for which the two-dimensional nanomaterial is of utility. Such an amount will be sufficient to elicit the biological or medical response of the tissue system or patient sought by the researcher or clinician.
  • the amount of a compound according to the invention that constitutes a therapeutically effective amount will vary depending on factors such as the compound and its biological activity, 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 treatment duration the type and severity of the disease state or disorder, the drugs used in combination or in consonance with the compounds of the present invention, and the patient's age, weight, general health, sex, and diet.
  • Such therapeutically effective amounts can be routinely determined by those of ordinary skill in the art based on their own knowledge, prior art, and this disclosure.
  • treating refers to reversing, alleviating, inhibiting the progression of, or preventing the disorder or condition to which the term applies, or one or more of such disorders or conditions symptom.
  • treatment refers to the act of treating, as “treatment” is as immediately described above.
  • treating is intended to mean administering a two-dimensional nanomaterial or a composition thereof according to the present invention to alleviate or eliminate symptoms of coronavirus infection and/or reduce viral load in a patient.
  • Prevention refers to any treatment of a disease or disorder that results in the absence of clinical symptoms of the disease or disorder.
  • the term “prophylaxis” also includes the administration of a therapeutically effective amount of a compound or composition according to the invention (eg, pre-exposure prophylaxis) prior to exposure of the individual to the virus to prevent the development of symptoms of disease and/or to prevent the virus from becoming detectable in the blood Level.
  • subject refers to an animal, such as a mammal (including a human), that has been or will be the subject of treatment, observation, or experimentation.
  • the methods described herein can be used in human therapy and/or veterinary applications.
  • the subject is a mammal (or patient).
  • subjects (or patients) are humans, livestock (eg, dogs and cats), farm animals (eg, cattle, horses, sheep, goats, and pigs) and/or laboratory animals (eg, mice) , rats, hamsters, guinea pigs, pigs, rabbits, cats, dogs and monkeys).
  • the subject (or patient) is a human.
  • a "person (or patient) in need thereof” refers to a person who may have or is suspected of having a disease or disorder that would benefit from certain treatments; or a pharmaceutical composition containing the above-mentioned ingredients.
  • the two-dimensional nanomaterials of the present invention are formulated with conventional carriers and excipients, which will be selected according to conventional practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form and are expected to be generally isotonic for delivery other than by oral administration.
  • the active ingredient two-dimensional nanomaterials can be administered alone, it may be preferable to present them as a pharmaceutical formulation.
  • the formulations for veterinary and human use of the present invention comprise at least one active ingredient as defined above (the active ingredient is a two-dimensional nanomaterial: indium-doped copper phosphide nanosheets (CIPS), graphite oxide alkene nanosheets, molybdenum disulfide nanosheets, black scale nanosheets, the same below) and one or more acceptable carriers and optional other therapeutic ingredients, such as corticosteroids, anti-inflammatory signal transduction modulators , beta2-adrenoceptor agonist bronchodilators, anticholinergics, mucolytics, hypertonic saline, and other drugs used to treat coronavirus infections; or mixtures thereof.
  • the active ingredient is a two-dimensional nanomaterial: indium-doped copper phosphide nanosheets (CIPS), graphite oxide alkene nanosheets, molybdenum disulfide
  • Formulations of the invention suitable for oral administration may be presented in discrete unit form such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as powders or granules; as solutions in aqueous or non-aqueous liquids or suspension; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. Or prepared into oral administration formulations by other known techniques.
  • a tablet is made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. Tablets may optionally be coated or scored and optionally formulated to provide slow or controlled release of the active ingredient therefrom.
  • the pharmaceutical composition of the present invention may be an external preparation such as an ointment for topical administration.
  • the formulation is preferably as a topical ointment or cream containing the active ingredient.
  • the active ingredient can be used with a paraffin or water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base.
  • topical formulations may include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected area. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.
  • the oily phase of the emulsions of the present invention may be constituted in a known manner from known ingredients.
  • the oily phase may contain only emulsifiers, but it may also contain at least one emulsifier in admixture with fat or oil or both.
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer.
  • Oils and fats are also preferably included.
  • compositions of the present invention may be in the form of sterile injectable preparations, such as sterile injectable aqueous or oily suspensions.
  • compositions of the present invention may be formulations suitable for topical administration to the eye and also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.
  • compositions of the present invention may be in formulations suitable for oral topical administration including lozenges containing the active ingredients in a flavored base, usually sucrose and acacia or tragacanth; in an inert base such as gelatin and glycerol or sucrose and A lozenge containing the active ingredient in acacia; and a mouthwash containing the active ingredient in a suitable liquid carrier.
  • compositions of the present invention may be formulated for rectal administration, which may be presented as a suppository with a suitable base containing, for example, cocoa butter or salicylates.
  • compositions of the present invention may be formulations suitable for intrapulmonary or intranasal administration, such formulations typically have particle sizes in the range of 0.1-500 microns, such as 0.5, 1, 30, 35 microns, etc., which pass through the nasal passages Administer by rapid inhalation or by oral inhalation to reach the alveoli.
  • the active ingredients of the present invention have dimensions that can be used for intrapulmonary or intranasal administration, such as inhalation.
  • compositions of the present invention may be in formulations suitable for parenteral administration including aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents and are isotonic with the blood of the intended recipient. solutes; and aqueous and non-aqueous sterile suspensions which may contain suspending and thickening agents.
  • Formulations of the pharmaceutical compositions of the present invention are presented in unit-dose or multi-dose containers (eg, sealed ampoules and vials) and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier immediately before use (eg water for injection). 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 recited above, or an appropriate fraction thereof, of the active ingredient.
  • the pharmaceutical composition of the present invention may be a veterinary composition comprising at least one active ingredient as defined above together with a veterinary carrier.
  • the compounds of the present invention are useful in providing controlled release pharmaceutical formulations containing one or more active ingredients of the present invention as active ingredients, wherein the release of the active ingredients is controlled and regulated to allow less frequent dosing or to improve the pharmacokinetics of a given active ingredient Kinetic or toxicity profile.
  • the medicament, pharmaceutical composition or active ingredient of the present invention can also be used in combination with other active ingredients.
  • other active ingredients are active against coronavirus infections, especially SARS or SARS-CoV-2 infections.
  • Non-limiting examples of these other active therapeutic agents are corticosteroids, anti-inflammatory signaling modulators, beta2-adrenoceptor agonist bronchodilators, anticholinergics, mucolytics, hypertonic saline and others for Drugs for the treatment of coronavirus infections; or mixtures thereof.
  • the medicaments, pharmaceutical compositions, or active ingredients of the present invention may also be administered to a patient simultaneously or sequentially in combination with one or more other active ingredients in unit dosage form.
  • Combination therapy can be administered as a simultaneous or sequential regimen.
  • the combination may be administered in two or more administrations.
  • Co-administration of a medicament, pharmaceutical composition, or active ingredient of the present invention with one or more other active ingredients generally refers to the simultaneous or sequential administration of a compound of the present invention and one or more other active therapeutic agents such that a therapeutically effective amount of the present invention is administered
  • the drug, pharmaceutical composition, or active ingredient and one or more other active ingredients are present in the patient.
  • Combination therapy can provide "synergistic effect” and “synergistic effect”, that is, the effect obtained when used together is greater than the sum of the effects produced by the compounds used alone. Synergistic effects can be achieved when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined formulation; (2) delivered alternately or in parallel as separate formulations; or (3) by other regimens.
  • An application scheme of the present invention provides the use of two-dimensional nanomaterials in the preparation of materials for inhibiting coronavirus infection;
  • the two-dimensional nanomaterials are indium-doped copper sulfur phosphide nanosheets (CIPS), graphite oxide A combination of any one or more of the alkene nanosheet layer, the molybdenum disulfide nanosheet layer, and the black scale nanosheet layer.
  • CIPS copper sulfur phosphide nanosheets
  • graphite oxide A combination of any one or more of the alkene nanosheet layer, the molybdenum disulfide nanosheet layer, and the black scale nanosheet layer are indium-doped copper sulfur phosphide nanosheets (CIPS), graphite oxide A combination of any one or more of the alkene nanosheet layer, the molybdenum disulfide nanosheet layer, and the black scale nanosheet layer.
  • An application solution of the present invention is to provide a material for inhibiting coronavirus infection, the material comprises a two-dimensional nanomaterial and a matrix, and the two-dimensional nanomaterial is an indium-doped copper phosphide nanosheet (CIPS). ), graphene oxide nanosheet layer, molybdenum disulfide nanosheet layer, black scale nanosheet layer any one or a combination of more.
  • CIPS copper phosphide nanosheet
  • the material may also include a matrix, and the matrix can be changed according to the application. Due to the high stability of the two-dimensional nanomaterial of the present invention, it can be adapted to various matrices.
  • the material of the corresponding matrix can be obtained by the method, including but not limited to compounding the two-dimensional nanomaterial with the matrix through the following methods, adsorbing the two-dimensional nanomaterial to the surface of the matrix by adsorption; or blending the two-dimensional nanomaterial with the matrix , and then solidify the matrix to obtain and so on.
  • the material is a material for preparing protective equipment, preferably the protective equipment is a mask, protective clothing, protective face shield, and protective cap.
  • the material is a kind of paint.
  • the material is a disinfectant.
  • the material is a packaging material.
  • the material is a filter material.
  • 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 contains the two-dimensional nanomaterial of the present invention.
  • the material for inhibiting coronavirus infection is a material for preparing masks, and the material is a non-woven fabric, preferably a spunbond non-woven fabric or a melt-blown non-woven fabric Cloth; the surface or interior of the non-woven fabric contains the two-dimensional nanomaterial of the present invention.
  • the two-dimensional nanomaterials and the matrix materials can be composited by conventional methods in the art.
  • the melt-blown material can be compounded with the two-dimensional nanomaterial before melt-blown spinning, and then melt-blown, or the obtained melt-blown cloth can be compounded with the two-dimensional nanomaterial.
  • the material for inhibiting coronavirus infection is a coating
  • the coating is a suspension containing two-dimensional nanomaterials.
  • the coating further includes at least one of a surfactant and a thickener.
  • the coating can impart two-dimensional nanomaterials to surfaces of objects, such as packaging surfaces, medical device surfaces, cosmetic device surfaces, etc., so that a coating comprising two-dimensional nanomaterials can be formed on the surfaces of objects.
  • the coating imparts a coating layer with two-dimensional nanomaterials on the surface of the outer or inner packaging of food or medicine.
  • the food or medicine needs to be transported in a cold chain.
  • the material for inhibiting coronavirus infection is a disinfectant
  • the disinfectant is a suspension containing two-dimensional nanomaterials.
  • the disinfectant is an environmental disinfectant, a hand sanitizer, a hand sanitizer, and a detergent.
  • the disinfectant is capable of being used in packaging materials, medical instruments (eg, catheters, injection needles, surgical instruments, surgical masks, and other medical devices), oral appliances (eg, dentures, protective tapes, fillers, palatal dilators), surface disinfection of cosmetic appliances (eg, cosmetic instruments, orthopedic devices).
  • the concentration of the two-dimensional nanomaterial in the disinfectant is 1 ⁇ g/mL-1000 ⁇ g/mL, preferably 2.5-160 ⁇ g/mL, more preferably 20 ⁇ g/mL.
  • the material for inhibiting coronavirus infection is a packaging material
  • the packaging material is used for cold chain transportation or storage of medicines or foods.
  • the surface of the packaging material has a coating layer comprising two-dimensional nanomaterials, or the packaging material is a composite material comprising two-dimensional nanomaterials.
  • the material for inhibiting coronavirus infection is a filter material
  • the filter material is a material for air filtration, a material for water filtration, and a material for mask filtration.
  • the filter material is an air filter
  • the surface of the air filter has two-dimensional nanomaterials.
  • the filter material is air filter cotton, and the fibers of the air filter cotton are attached with two-dimensional nanomaterials.
  • the filter material can be used for air conditioner filtration, air purifier filtration, fresh air system filtration and any other device or equipment that needs to filter air. The scenarios used are not limited to homes, offices, laboratories, factories, etc.
  • the material is a solid, liquid or semi-solid material.
  • the liquid material is a coating comprising two-dimensional nanomaterials, a disinfectant comprising two-dimensional nanomaterials, and a personal care product comprising two-dimensional nanomaterials.
  • the solid material is a device or a packaging material containing two-dimensional nanomaterials on the surface.
  • the semi-solid material is a gel containing two-dimensional nanomaterials.
  • the present invention verifies the effect of nanomaterial indium-doped copper phosphide nanosheets (CIPS or CuInP 2 S 6 ) on cell viability through specific examples, CIPS nanosheets, graphene oxide nanosheets, Inhibition of SARS-CoV-2 virus cell infection by molybdenum disulfide nanosheets and black scale nanosheets, adsorption of CIPS to SARS-CoV-2 pseudovirus SC2-P, specific adsorption of CIPS to RBD domain of SARS-CoV-2 Spike protein, And mathematical simulation to analyze the effect of CIPS adsorption on the RBD region of Spike protein on ACE2 recognition.
  • CIPS nanomaterial indium-doped copper phosphide nanosheets
  • graphene oxide nanosheets Inhibition of SARS-CoV-2 virus cell infection by molybdenum disulfide nanosheets and black scale nanosheets
  • adsorption of CIPS to SARS-CoV-2 pseudovirus SC2-P specific adsorption of C
  • the experimental materials used in the following examples include: HEK-293T cells, ACE2/293T cells, Vero-E6 cells, SARS-CoV-2 pseudovirus SC2-P, and SARS pseudovirus SARS-P.
  • the materials, reagents, vectors, strains, etc. used in the following examples can be purchased through commercial channels unless otherwise specified.
  • HEK-293T cell a derivative of human embryonic kidney cell 293, is a cell line derived from human embryonic kidney cells. It has the characteristics of high transfection efficiency and easy culture. Cell lines have become a powerful tool for researchers to study gene function.
  • ACE2/293T refers to the expression of ACE2 in HEK-293T cells.
  • ACE2 also known as ACEH
  • angiotensin-converting enzyme 2 is called angiotensin-converting enzyme 2.
  • the protein encoded by this gene belongs to the angiotensin-converting enzyme family of dipeptidyl carboxydipeptidase, and has considerable homology with human angiotensin-converting enzyme 1.
  • the protein encoded by this gene is a functional receptor for the Spike glycoprotein of SARS, SARS-CoV-2, and HCoV-NL63 human coronaviruses.
  • Vero-E6 cell is an African green monkey kidney cell line, 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 drug on the replication rate of the new coronavirus, to test whether the new coronavirus exists or to cultivate the new coronavirus for research purposes.
  • SC2-P is a pseudovirus expressing the Spike protein of the new coronavirus SARS-CoV-2.
  • Pseudoviruses are a class of chimeric virus particles that express recombinant glycoproteins of another virus on the surface of one replication-defective virus (viral vector). Pseudoviruses have been widely used in vaccine research and development, antibody neutralization research, simulated virus-infected cell function experiments, and positive reference of detection kits because of their advantages of biological safety and stability.
  • pseudovirus systems related to SARS-CoV-2 there are mainly two kinds of pseudovirus systems related to SARS-CoV-2: one is the use of lentiviral protein coat constructed by lentiviral system to wrap the ORF1a/b, N, E and other genes of SARS-CoV-2, which can be used for nucleic acid Positive control for quasi-detection kits.
  • the other is to use the Spike protein of SARS-CoV-2 to wrap marker genes such as Luciferase/GFP to simulate virus infection of cells, detect the expression of marker genes, and then study the infection efficiency.
  • the SC2-P used in the following examples can be ordered from Goldwisdom Corporation.
  • SARS-P is a pseudovirus of SARS, and it is also a commercial product, so I won't repeat it here.
  • CIPS single crystals were obtained by purchasing or known methods, and the CIPS nanosheets were obtained by exfoliating experiments by mechanical methods such as mechanical grinding.
  • the X axis is the length and the Y axis is the thickness, indicating that the material thickness is about 1- 10nm.
  • the size of the nanosheets is non-uniform in the direction perpendicular to the thickness, distributed in the range of 100-300 nm.
  • Vero-E6 cells and ACE2/293T cells were seeded into 96-well plates at a density of 1 ⁇ 10 4 cells/well, and cultured overnight at 37°C, 5% CO 2 .
  • the medium was changed to medium containing different concentrations of CIPS nanosheets (0, 2.5, 5, 10, 20, 40, 80, 160 ⁇ g/mL), and incubated at 37°C for 24 h.
  • FIG. 3 is the cell viability detected by CCK-8 after incubation of Vero-E6 cells with different concentrations (0-160 ⁇ g/mL) of CIPS nanosheets for 24 h.
  • B in Figure 3 shows the cell viability detected by CCK-8 after ACE2/293T cells were incubated with different concentrations (0-160 ⁇ g/mL) of CIPS for 24 h.
  • the results showed that the CIPS nanosheets of 0-160 ⁇ g/mL had no cytotoxicity to Vero-E6 and ACE2/293T cells, and the cell viability was not significantly reduced.
  • hemolysis index (Hem)% (A sample -A PBS )/(A pure water -A PBS ) ⁇ 100%, the result of C in Figure 3 shows that the hemolysis rate of CIPS nanosheets is 0, and its biological Better compatibility and higher security.
  • the 14mm cell slides were placed in a 24-well plate, inoculated with HEK-293T cells, cultured overnight at 37°C, 5% CO 2 , and transfected with ACE2-GFP for 24h, then mixed with 0/10/20/40 ⁇ g Cells were infected with SC2-P/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:
  • the cells on the 24-well plate were fixed with 4% paraformaldehyde at room temperature for 15 min, and washed 3 times with PBS.
  • the cells were blocked with 3% BSA diluted in PBS for 30 min, and washed three times with PBS.
  • Anti-Flag (1:500 for rabbit) and anti-GFP (1:500 for mouse) were incubated for 2 h, and washed 3 times with PBS.
  • FIG. 4A is the result of confocal microscope observation of SC2-P infection of ACE2-GFP/HEK-293T cells. After SC2-P was pre-incubated with CIPS nanosheets of different concentrations (0, 10, 20 and 40 ⁇ g/mL) for 2 h, ACE2-GFP/HEK-293T cells were infected, and the SC2-P infection was detected by immunofluorescence.
  • Figure 4B is ImageJ statistics of the number and fluorescence intensity of SC2-P entering cells. RFI stands for relative fluorescence intensity.
  • the ACE2 channel is green fluorescence
  • the SC2-P channel is red fluorescence
  • Merge is the superposition of the two, shown in grayscale in the figure.
  • Example 4 The effect of luciferase reporter gene detection of CIPS nanosheets on SC2-P infection of Vero-E6 and ACE2/293T
  • Vero-E6 cells and ACE2/293T cells were seeded into 96-well plates at a density of 1 ⁇ 10 4 cells/well, respectively, and cultured overnight at 37°C, 5% CO 2 .
  • Cells were infected with SC2-P mixed with 0/2.5/5/10/20/40/80 ⁇ g/mL CIPS nanosheets for 2h, washed once in DMEM, and incubated with DMED medium for 40-48h, and then luciferase was detected. activity.
  • the present invention uses the true virus SARS-CoV-2 to detect the infection and obtains similar results.
  • Vero-E6 cells were infected with SARS-CoV-2 virus mixed with 0/2.5/5/10/20/40/80/160 ⁇ g/mL CIPS nanosheets for 1 h, and the virus in the medium was quantitatively detected 48 h later. quantity.
  • the concentration of CIPS nanosheets increased, the infection efficiency of the true virus SARS-CoV-2 gradually decreased. It shows that CIPS nanosheets can not only effectively inhibit the infection of host cells by SC2-P, but also inhibit the infection of the new coronavirus SARS-CoV-2.
  • a in Figure 6 shows the infection rate of different concentrations of CIPS
  • B shows the amount of virus quantitatively detected by the expression of ORF1ab/RdRp gene
  • C in Figure 6 shows the nucleocoat
  • the gene expression level of capsid protein is the amount of virus quantitatively detected by the index.
  • Example 6 The characterization results of the physicochemical properties of CIPS nanosheets show that CIPS can adsorb SC2-P
  • the binding ability of CIPS nanosheets to SC2-P was detected.
  • the CIPS-SC2-P suspension was obtained after co-incubating the CIPS nanosheets with SC2-P for 2 h.
  • the CIPS-SC2-P suspension, the CIPS nanosheet suspension and the SC2-P suspension were tested Liquid UV-VIS and Z-potential characterization. The results are shown in Fig. 7A-B, Fig.
  • FIG. 7A shows the UV-VIS results of the CIPS-SC2-P suspension, the suspension of CIPS nanosheets alone, and the SC2-P suspension, it can be seen that CIPS and SC2- The absorption peaks of P are different, and the absorption peaks of the CIPS-SC2-P suspension after 2h incubation are shifted relative to the absorption peaks of the CIPS nanosheet suspension and SC2-P suspension, and the absorption intensity also changes. .
  • Figure 7B is the Z-potential of the CIPS-SC2-P suspension, the suspension of CIPS nanosheets alone, and the SC2-P suspension, wherein the CIPS results are the Z-potentials of the suspension of CIPS nanosheets alone potential, SC2-P is the Z-potential of SC2-P suspension, CIPS+SC2-P is the Z-potential of CIPS-SC2-P suspension.
  • the results showed that CIPS nanosheets could adsorb SC2-P.
  • the CIPS nanosheets and SC2-P were directly detected by Western blot.
  • the results are shown in the left panel of C in Figure 7.
  • the proteins in SC2-P can be clearly displayed on the Western blot results, while the CIPS nanosheets Since there is no protein, no corresponding bands are displayed in the SC2-P suspension.
  • 0 ⁇ g, 10 ⁇ g and 20 ⁇ g of CIPS nanosheets were added to the SC2-P suspension, incubated for 120 minutes, and then centrifuged at the same speed for separation, using Western blot
  • the results are shown in the right image of 7C.
  • the first column of results is the suspension containing only SC2-P. Since the virus is too small to be separated by centrifugation, the Western blot does not have any bands.
  • the results in the three columns are the results of adding 10 ⁇ g and 20 ⁇ g of CIPS nanosheets respectively. Since the CIPS nanosheets have adsorption effect on SC2-P, they can be obtained by centrifugation, and there are corresponding bands in the Western blot. The amount of adsorption increased with the increase in the amount of lamellae.
  • Example 8 CIPS nanosheets compete with ACE2 for binding to the receptor binding domain (RBD, receptor binding domain) domain of the Spike protein of the new coronavirus SARS-CoV-2
  • Biofilm interferometry was used to quantitatively determine the affinity and interaction of CIPS nanosheets with the RBD domain protein of 2019-nCoV Spike protein, the protein in serum, and the model protein BSA.
  • KD values for different systems were calculated. It shows that the CIPS nanosheet has a strong affinity with the RBD domain of the S protein, but has a weak affinity with other proteins in serum. suggestive of the selective adsorption of RBD by CIPS nanosheets.
  • This experiment confirmed that the CIPS nanosheets can act on the virus alone and specifically bind to the RBD domain protein of the virus S protein.
  • the experimental results show that CIPS nanosheets can be used for ACE2 protein binding and front, and can be used for the prevention of coronavirus.
  • MoS 2 and GO also have strong affinity to the RBD domain. It shows that MoS 2 and GO nanosheets can also adsorb RBD, and the experiments confirm that MoS2 and GO nanosheets can act on viruses alone and specifically bind to the RBD domain protein of virus S protein. The experimental results show that both MoS 2 and GO nanosheets can be used for ACE2 protein binding and front, and can be used for the prevention of coronavirus.
  • biofilm interferometry quantitatively determined the change in the affinity of RBD protein to ACE2 protein before and after binding to CIPS nanosheets.
  • RBD and ACE2 in 200nM RBD protein solution, the sensor was chemically coupled and immobilized RBD protein for 300s, and desorbed for 600s; the sensor continued in 100nM ACE2 solution, adsorbed for 600s, and desorbed for 300s.
  • the results show that the KD value of RBD and ACE2 is about 4 nM; Figure 9 shows that in the presence of CIPS nanosheets, the KD value is 41 nM. This indicates that the CIPS nanosheets lead to the weakening of the affinity of RBD to ACE2. This suggests that CIPS nanosheets can reduce the binding of the SARS-CoV-2 virus Spike protein to its host cell receptor ACE2, thereby inhibiting virus infection.
  • FIG. 10A the site where CIPS nanosheets bound to RBD
  • FIG. 10B the site where ACE2 bound to RBD
  • FIG. 10C the site where ACE2 bound to RBD
  • CIPS nanosheets have stronger affinity with RBD, so CIPS nanosheets can compete with ACE2 to bind RBD.
  • the CIPS nanosheets occupy the binding site of ACE2, thereby preventing the binding of RBD to ACE2, thereby inhibiting the infection of host cells by the virus SARS-CoV-2.
  • the abc in the lower part of Figure 10 respectively represent the binding site of ACE2, the binding site of CIPS and the overlapping result of the two. It can be seen from the overlapping simulation diagram that the binding site of ACE2 and CIPS overlap.
  • Snapshots of typical trajectories of RBD and CIPS adsorption in the system based on molecular dynamics (MD) simulations The bottom row shows the dynamic process of RBD adsorption on the CIPS surface.
  • the upper row is a basemap of the RBD amino acid sites that bind to CIPS, and the VDW spheres are the amino acid residues that contact CIPS. Red balls are polar residues, green are hydrophobic residues, blue are positively charged residues, purple are negatively charged residues, and yellow are cystines with disulfide bonds.
  • Example 10 CIPS can adsorb SC2-P in the protein mixture of SC2-P and FBS, and inhibit its infection efficiency
  • Example 12 Quantitative PCR detection of CIPS nanosheets to reduce the amount of true virus SARS-CoV-2
  • ACE2/293T cells were seeded into 96-well plates at a density of 1 ⁇ 10 4 cells/well and cultured overnight at 37°C, 5% CO 2 .
  • Cells were infected with SC2-P mixed with 20 ⁇ g/mL CIPS nanosheets, GO (graphene oxide) nanosheets, MoS 2 (molybdenum disulfide) nanosheets or black scale nanosheets for 2 h, washed with DMEM. The activity of luciferase was detected after adding DMED medium for 40-48 hours.
  • the results of the luciferase activity detection in Figure 15 show that different two-dimensional nanomaterials have the ability to inhibit the infection of host cells by the new coronavirus pseudovirus SC2-P.
  • the Ctrl group was a control group that only applied SC2-P without adding nanosheets. When CIPS nanosheets or black scale nanosheets were applied, the amount of SC2-P infecting ACE2/293T cells was significantly higher than that of the control group.
  • Example 14 The infection-inhibiting effect of CIPS nanosheets on SARS pseudovirus particle SARS-P shows that its effect of inhibiting coronavirus has a broad spectrum.
  • FIG. 16A shows the infection of ACE2-GFP/HEK-293T cells by SARS-P under a confocal microscope. After pre-incubating SARS-P with 20 ⁇ g/mL CIPS nanosheets for 2 h, ACE2-GFP/HEK-293T cells were infected for 2 h, and the infection of SARS-P was detected by immunofluorescence.
  • Figures 16B-16C are ImageJ statistics of the number and fluorescence intensity of SARS-P entering cells. RFI stands for relative fluorescence intensity.
  • Figures 16D-16E are the luciferase activity to detect the infection efficiency of SARS-P on ACE2/293T and Vero-E6 cells. After pre-incubating SARS-P with 20 ⁇ g/mL CIPS nanosheets for 2 h, Vero-E6 and ACE2/293T cells were infected for 2 h, and the infection efficiency of SARS-P was detected by luciferase activity after 40 h. The CIPS nanosheets were also able to inhibit SARS-P infection. It shows that the inhibition of virus infectivity by CIPS nanosheets is not only for SARS-CoV-2 virus, but has a broad spectrum for coronavirus.
  • the present invention provides a nanomedicine for inhibiting SARS-CoV-2 virus cell infection.
  • the nanomedicine comprises indium-doped copper phosphide nanosheets, graphene oxide nanosheets and molybdenum disulfide nanosheets any one or more of them.
  • Embodiment 1 Oral administration with any one or more of indium-doped copper phosphide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black scale nanosheets as medicinal components liquid;
  • oral liquid is a new dosage form developed on the basis of decoction and injection. It has the advantages of small dose, fast absorption, stable quality, convenient carrying and taking, and easy storage. It contains a variety of active ingredients. Great impact on quality and taste. On the premise of not changing the structure and function of the main active ingredients, how to retain the active ingredients to the maximum extent 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.
  • excipients for oral liquid are: solvent, fragrance, flavoring agent, clarifying agent, preservative, etc. These excipients can be added at the same time, or one of them can be added. Among them, the solvent must be added, and water can be used. Different adjuvants are combined with sweeteners, aromatics, clarifying agents or preservatives, or a combination of sweeteners and preservatives, preferably a combination of sweeteners and preservatives. Some excipients have the functions of sweetening and flavoring, and only one kind of excipient can be added at this time.
  • the sweetener is selected from one or more of protein sugar, xylitol, aspartame and sucralose.
  • the preservative is selected from one or more of parabens, butylated hydroxyanisole, butylated hydroxytoluene and sorbic acid.
  • Preservatives can be selected from parabens, butylated hydroxytoluene or sorbic acid, preferably butylated hydroxytoluene. Combinations of hydroxybenzoates and butylated hydroxytoluene are also possible, or butylated hydroxytoluene and sorbic acid, or parabens and sorbic acid, or parabens , a combination of butylated hydroxytoluene and sorbic acid.
  • the fragrance is fruit essence.
  • the clarifying agent is one or both of chitosan and gelatin.
  • Embodiment 2 A sheet containing any one or more of indium-doped copper phosphide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black scale nanosheets as medicinal components agent;
  • the tablet has the advantages of accurate dosage, stable quality, convenient taking, carrying and transportation.
  • the formulation auxiliary materials include one or more of diluents, binders, lubricants and disintegrants, preferably a combination of diluents, binders, lubricants and disintegrants.
  • the diluent is one or more of cellulose and inorganic salts.
  • cellulose and inorganic salts Such as microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate and medicinal calcium carbonate, mannitol, etc., to increase the volume of raw materials to help them shape.
  • the binder is one of water, ethanol, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, gelatin, polyvinylpyrrolidone, etc. or more.
  • the lubricant is one or more of magnesium stearate, micropowder silica gel, talc, hydrogenated vegetable oil, polyethylene glycol and magnesium lauryl sulfate.
  • the disintegrant is one or more of low-substituted hydroxypropyl, cross-linked polyvinyl pyrrolidone, and cross-linked sodium carboxymethyl cellulose.
  • Embodiment 3 Capsules with any one or more of indium-doped copper phosphide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black scale nanosheets as medicinal components ;
  • the capsule mainly improves the stability and bioavailability of the drug.
  • the preparation auxiliary material is a capsule shell, and the capsule shell is a hard capsule shell or a soft capsule shell.
  • Embodiment 4 Any one or more of indium-doped copper phosphide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black scale nanosheets are particles of medicinal effect ingredients agent;
  • Granules can be swallowed directly, or can be washed into water with warm water to drink, it is more convenient to apply and carry, and the dissolution and absorption speed are faster.
  • the formulation auxiliary materials used in granules are similar to those of tablets, involving one or more of fillers, binders, wetting agents, disintegrants, lubricants and film coating materials.
  • the filler is one or more of cellulose and inorganic salts.
  • cellulose and inorganic salts Such as microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate and medicinal calcium carbonate, mannitol, etc., to increase the volume of raw materials to help them shape.
  • the binder is one of water, ethanol, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, gelatin, polyvinylpyrrolidone, etc. or more.
  • the wetting agent is water or ethanol or a mixture of both.
  • the wetting agent is water or ethanol or a mixture of both.
  • the disintegrant is one or more of low-substituted hydroxypropyl, cross-linked polyvinyl pyrrolidone, and cross-linked sodium carboxymethyl cellulose.
  • the film coating material is one or more of hydroxypropyl methylcellulose, polyethylene glycol, cellulose acetate phthalate and polyvinyl acetal diethylamine acetate .
  • Embodiment 5 Powder formulations using any one or more of indium-doped copper phosphide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black scale nanosheets as medicinal components .
  • the present invention can also be made into powder, and the powder is convenient to be divided into doses and taken.
  • composition of the present invention can be made into beverages with different flavors, which will be very popular as daily beverages.
  • the preparation auxiliary materials used in the beverage are at least one of clarifying agent, preservative and flavoring agent.
  • composition of the present invention can also be made into other powders, such as functional milk powder, and the added auxiliary materials are mainly milk powder, such as skimmed milk powder, non-fat sugar-free milk powder.
  • the above multiple embodiments can adjust the amount of medicine in a unit product to suit different uses, such as medicine, health care product, food and so on.
  • a preferred embodiment of the present invention provides a mask melt-blown cloth infected with SARS-CoV-2 virus cells, in which indium-doped copper thiophosphide nanosheets and graphene oxide nanosheets are added to the mask meltblown cloth Any one or more of the molybdenum disulfide nanosheet layer, the black scale nanosheet layer.
  • a preferred embodiment of the present invention provides a cold storage coating or an outer packaging coating for inhibiting the infection of SARS-CoV-2 virus cells, wherein indium-doped copper phosphide is added to the cold storage coating or the outer packaging coating Any one or more of nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black scale nanosheets.
  • a preferred embodiment of the present invention provides a disinfectant spray for inhibiting the infection of SARS-CoV-2 virus cells, and the spray is added with indium-doped copper sulfur phosphide nanosheets, graphene oxide nanosheets, Any one or more of the molybdenum disulfide nanosheet layer and the black scale nanosheet layer.
  • the spray also contains a solvent.
  • the solvent is water, without toxicity, and the effective concentration of indium-doped copper thiophosphide nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black scale nanosheets is 2.5-160 ⁇ g/mL ( As shown in Figure 3), the inhibitory effect is not obvious if the concentration is too low, and the cost is increased if the concentration is too high, which is not conducive to commercial production.
  • the preferred concentration is 20 ⁇ g/mL.
  • the present invention verifies that the two-dimensional nanomaterials CIPS nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets and black scale nanosheets can all inhibit the infection efficiency of the SARS-CoV-2 virus.
  • CIPS can also inhibit SARS-P infection. It shows that the inhibition of virus infectivity by CIPS is not only for SARS-CoV-2 virus, but also has broad-spectrum adaptability to coronaviruses.
  • the present invention also verifies the effect of CIPS on cell viability.
  • the experimental results showed that CIPS (1-160 ⁇ g/mL) had no effect on the activity of Vero-E6 and ACE2/293T cells, and did not have cell hemolysis, indicating that it has high biological safety and low toxicity.
  • the present invention also verifies the inhibition of CIPS on the infection efficiency of SARS-CoV-2 virus.
  • CIPS inhibited the infection efficiency of SARS-CoV-2 pseudovirus (SC2-P) against ACE2-GFP/HEK-293T (ACE2-GFP was expressed in HEK-293T) and Vero-E6.
  • SC2-P SARS-CoV-2 pseudovirus
  • ACE2-GFP was expressed in HEK-293T
  • Vero-E6 was confirmed by experiments that CIPS could inhibit the infection efficiency of the true virus SARS-CoV-2 on Vero-E6.
  • the present invention also verifies the adsorption effect of CIPS on SC2-P. It was confirmed by experiments that CIPS could adsorb and reduce SC2-P. Compared with other proteins, CIPS has a stronger binding effect on the receptor binding domain (RBD) domain of the Spike protein of SARS-CoV-2 virus, and the presence of CIPS can inhibit the binding of the virus to the receptor. .
  • RBD receptor binding domain
  • the nanomaterials of the present invention can be prepared into nanomedicines for the treatment of novel coronavirus pneumonia, or added to the meltblown cloth of masks, cold storage coatings, outer packaging coatings and sprays to inhibit the spread of novel coronavirus pneumonia and achieve prevention. effect.
  • the inhibition of virus infectivity by the two-dimensional nanomaterial of the present invention is not only aimed at SARS-CoV-2 virus, but has a broad spectrum of coronaviruses.
  • the two-dimensional nanomaterials CIPS nanosheets, graphene oxide nanosheets, molybdenum disulfide nanosheets, and black scale nanosheets can be applied individually or stacked.

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Abstract

提供了一种二维纳米材料在抑制冠状病毒中的应用，具体公开了二维纳米材料在制备治疗或预防冠状病毒所致疾病的药物或用于粘附或抑制冠状病毒的材料中的应用，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种；冠状病毒所致疾病为由冠状病毒感染导致的疾病。所述材料制备简单，生物安全性高，且效果优异。

Description

二维纳米材料在抑制冠状病毒的应用 技术领域
本发明涉及抗病毒技术领域，特别涉及一种二维纳米材料在抑制冠状病毒中的应用。
背景技术
纳米材料是指材料在某一维、二维或三维方向上的尺度达到纳米尺度的材料。纳米材料可以分为零维材料、一维材料、二维材料、三维材料。二维材料，是指电子仅可在两个维度的纳米尺度上自由运动(平面运动)的材料，例如氮化硼(BN)纳米片层、二硫化钼(MoS2)纳米片层、二硫化钨(WS 2)纳米片层、Mxene材料。二维纳米材料具有因其载流子迁移和热量扩散都被限制在二维平面内，使得这种材料展现出许多奇特的性质，被广泛的用于电子领域。但是关于医药方面的应用较少。
冠状病毒是一个大型病毒家族，已知可引起感冒以及中东呼吸综合征(MERS)和严重急性呼吸综合征(SARS)等较严重疾病。2019新型冠状病毒(2019-nCoV，引发新型冠状病毒肺炎COVID-19)是目前已知的第7种，前6种分别是HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV(引发重症急性呼吸综合征)和MERS-CoV(引发中东呼吸综合征)，截止到目前还没有有效的药物出现。
现阶段，尚缺少能够显著抑制冠状病毒细胞侵染且来源方便、成本低廉的纳米药物和相关制剂。并且，极少有药物能同时起到治疗加预防的功效。
发明内容
针对现有技术中的缺陷，本发明一个方面提出了二维纳米材料在制备治疗或预防冠状病毒所致疾病的药物中的应用，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合。
在本发明的技术方案中，上述应用中，所述的药物中进一步包含药学上可接受的载体或赋形剂。
在本发明的技术方案中，上述应用中，所述的药物中进一步包括施用治疗有效量的至少一种其他治疗剂或其组合物，所述其他治疗剂或其组合物选自皮质类固醇、抗炎信 号转导调节剂、β2-肾上腺受体激动剂支气管扩张剂、抗胆碱能药、粘液溶解剂、高渗盐水和其他用于治疗冠状病毒科病毒感染的药物；或它们的混合物。
本发明第二方面提供了一种治疗或预防冠状病毒所致疾病的药物组合物，所述药物组合物中以二维纳米材料作为活性成分，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合。
在本发明的技术方案中，上述药物组合物中，所述的药物组合物中还包含药学上可接受的载体或赋形剂。
在本发明的技术方案中，上述药物组合物中，所述的药物组合物中还包括第二种活性成分，所述第二种活性成分选自皮质类固醇、抗炎信号转导调节剂、β2-肾上腺受体激动剂支气管扩张剂、抗胆碱能药、粘液溶解剂、高渗盐水和其他用于治疗冠状病毒科病毒感染的药物；或它们的混合物。
在本发明的技术方案中，上述药物组合物中，所述的药物组合物的制剂形式选自口服制剂、注射制剂、粘膜给药制剂、吸入剂、外用制剂。
本发明第三方面提供了一种治疗或预防的冠状病毒感染的方法，其包括向受试者施用治疗有效量的二维纳米材料，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合。
在本发明的技术方案中，上述方法中，所述的治疗有效量的二维纳米材料以制剂形式给与，制剂中还包含药学上可接受的载体或赋形剂。
在本发明的技术方案中，上述方法中，所述的还包括向受试者施用治疗有效量第二种活性成分的步骤，所述第二种活性成分选自皮质类固醇、抗炎信号转导调节剂、β2-肾上腺受体激动剂支气管扩张剂、抗胆碱能药、粘液溶解剂、高渗盐水和其他用于治疗冠状病毒科病毒感染的药物；或它们的混合物。
本发明第四方面提供了二维纳米材料在制备抑制冠状病毒减少SARS-CoV-2病毒Spike蛋白与其宿主细胞的受体ACE2的结合的制剂中的用途；所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；优选地，二维纳米材料通过竞争性结合冠状病毒Spike蛋白的RBD结合的位点抑制其与其宿主细胞的受体ACE2的结合。
本发明还提供了二维纳米材料在制备竞争性结合冠状病毒Spike蛋白的RBD结合 的位点的制剂中的用途。
本发明第五个方面提供了二维纳米材料在制备促进SARS-CoV-2病毒蛋白分解的药物中的用途；所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合。
在本发明的技术方案中，上述方法中，二维纳米材料在制备促进冠状病毒分解SARS-CoV-2病毒蛋白中Spike蛋白的药物中的用途。
本发明第六个方面提供了二维纳米材料在制备促进SARS-CoV-2病毒RNA的分解的药物中的用途；所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合。
本发明第七方面提供了二维纳米材料在制备抑制冠状病毒侵染的材料中的用途；所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合。
本发明第八方面提供了一种抑制冠状病毒侵染的材料，所述材料包含二维纳米材料和基质，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合。
在本发明的技术方案中，在上述材料中，所述材料为制备防护用品的材料，优选所述的防护用品为口罩、防护服、防护面罩、防护帽。
在本发明的技术方案中，在上述材料中，所述材料为一种涂料。
在本发明的技术方案中，在上述材料中，所述材料为一种消毒剂或个人护理用品。
在本发明的技术方案中，在上述材料中，所述材料为一种包装材料。
在本发明的技术方案中，在上述材料中，所述材料为一种过滤材料。
本发明第九方面提供了一种用品，所述用品中包含二维纳米材料，所述二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
所述用品选自防护用品、涂料、消毒剂、个人护理用品、包装材料或过滤装置；
优选地，所述的防护用品为口罩、防护服、防护面罩、防护帽；
优选地，所述的涂料为包含二维纳米材料的混悬剂，所述的涂料能否赋予被附着物表面具有二维纳米材料的涂层；
优选地，所述的消毒剂为环境消毒剂、免洗洗手液、洗手液、洗涤剂；更优选地，所述消毒剂用于包装材料、医用器械、口腔器具、美容器具的表面消毒；
优选地，所述的包装材料为具有包含二维纳米材料的涂层，或者所述包装材料为包含二维纳米材料的复合材料；更优选地，包装材料为用于的药物或食品的包装材料，尤其是用于冷链运输的药物或食品；
优选地，所述过滤装置为用于空气过滤的装置、用于水体过滤的装置、用于口罩过滤的装置；更优选地，所述的过滤装置为空气过滤网，所述空气过滤网表面具有二维纳米材料；更优选地，所述的过滤装置为空气过滤棉，所述空气过滤棉纤维上附着二维纳米材料。
在本发明的技术方案中，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
在本发明的技术方案中，所述的冠状病毒优选自SARS-CoV、SARS-CoV2或HCoV-NL63。
在本发明的技术方案中，所述的冠状病毒优选自SARS-CoV或SARS-CoV2。
在本发明的技术方案中，冠状病毒所致疾病为由冠状病毒感染导致的疾病。
在本发明的技术方案中，所述的二维纳米材料为在空间中一个维度的长度为纳米尺寸的材料；优选地，为在空间中一个维度的长度为1-100nm的材料，更优选地，为在空间中一个维度的长度为1-10nm的材料，更优选地，为在空间中一个维度的长度为1-10nm的材料，其他维度的长度为100-500nm的材料，或者例如，其他维度的长度为100nm、200nm、300nm、400nm、500nm。
综上，与现有技术相比，本发明达到了以下有益效果：
1.二维纳米材料如CIPS纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层一方面能够抑制冠状病毒的细胞侵染，另一方面可特异吸附并结合冠状病毒侵染靶点，减少病毒的侵染效率，进而减少病毒在宿主体内的复制，减少冠状病毒的数量，可用于冠状病人的治疗药物。
2.CIPS的抑制SARS-CoV-2病毒侵染效果同样适用于SARS病毒，说明CIPS对抑制冠状病毒具有广谱性。
3.CIPS纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层由于可以与冠状病毒特异性结合，抑制SARS-CoV-2病毒对宿主细胞的侵染，实现对于病毒的特异性吸附固定。可以用于制备药物外，可以用于可以应用于口罩的熔喷布、冷库涂层和外包装涂层或喷剂，可以吸附新冠病毒并减少可侵染病毒的数量，起到预防新冠肺炎的作用。
4.二维纳米材料如CIPS纳米片层对HEK-293T和Vero-E6细胞存活率无影响，并且不会造成小鼠的血红细胞溶血，说明该纳米材料的生物安全性高，毒性比较低。
5.本发明的二维纳米材料可以在室温到-20℃长期保存，便于储存。且本发明的二维纳米材料稳定性高，可以与任何辅料混合、复配等制成药物和复合材料，不受温度和pH等因素的影响。
6.本发明采用的是无机纳米材料，取材来源不受限制，适合大规模量产，成本低，且用量少。
7.本发明生物膜干涉实验为在固体和液体环境下进行的，证实了CIPS可以极强的结合冠状病毒S蛋白的RBD区(见图8)。计算机模拟的结果也显示，5.7ns的时候可以结合(图11)。而且从本发明实施例验证结果看，本发明采用二维纳米材料与冠状病毒预孵育2小时后才与宿主细胞进行侵染实验评价，可以看出，本发明的材料能够与冠状病毒实现长时间的结合，能够作为吸附冠状病毒，并阻止其与宿主细胞结合的材料。即本发明材料可以瞬间实现与冠状病毒的结合、长时间的结合以及结合后抑制病毒与宿主细胞结合。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为CIPS晶体片图像；
图2为CIPS晶体片原子力显微镜成像；
图3为CIPS的生物安全性检测；
图4为CIPS纳米片层的存在下SARS-CoV-2的Spike蛋白的假病毒(SC2-P)对转染ACE2-GFP的293T(ACE2/293T)细胞的侵染结果；统计学分析使用ANOVA，**：P<0.01,***:P<0.001；
其中，图A为共聚焦显微镜观察SC2-P对ACE2/293T细胞的侵染结果，第1行(ACE2)为ACE2/293T细胞通道，为绿色荧光，第2行(SC2-P)为SC2-P通道，为红色荧光；第3行(Merge)为第1行和第2行叠加，表明SC2-P进入ACE2/293T细胞的数量和分布。图A中不同列代表不同加入量的CIPS纳米片层，以CIPS纳米片层加 入后浓度计算。
图B为SC2-P对ACE2/293T细胞的侵染定量结果，其中左图横坐标为CIPS纳米片层加入量，以加入后的浓度计；纵坐标为SC2-P进入细胞的数量。右图为横坐标为CIPS纳米片层加入量，以加入后的浓度计；纵坐标为相对荧光强度。
图5为荧光素酶活性检测假病毒SC2-P对Vero-E6和ACE2/293T细胞的侵染效率；统计学分析使用ANOVA，*：P<0.05，**：P<0.01,***:P<0.001；
图6为定量检测真新冠病毒SARS-CoV-2对Vero-E6细胞的侵染效率；统计学分析使用ANOVA，*：P<0.05，**：P<0.01,***:P<0.001；图6中的A显示的是不同浓度CIPS的侵染率，B显示的是以ORF1ab/RdRp基因的表达量为指标定量检测的病毒量，而图6中的C显示的是以核衣壳蛋白的基因表达量为指标定量检测的病毒量；
图7为CIPS吸附并减少假病毒SC2-P；
图8为生物膜干涉(BLI)定量测定结果。其中A为不同浓度的CIPS材料与新冠病毒Spike蛋白RBD结构域蛋白亲和力及相互作用；B为CIPS材料与血清中的蛋白亲和力及相互作用；C为二硫化钼纳米片层(MoS 2)与新冠病毒Spike蛋白RBD结构域蛋白亲和力及相互作用；D为氧化石墨烯纳米片层(GO)与新冠病毒Spike蛋白RBD结构域蛋白的亲和力及相互作用；其中ACD结果中，从上至下曲线分别为浓度200nM、100nM、50nM、25nM和12.5nM；B结果中自上而下分别为RBD、Fg、FBS、IgG、HDL、Tf、HAS.
图9为生物膜干涉(BLI)定量测定与CIPS结合前后，新冠病毒Spike蛋白RBD结构域与ACE2蛋白的亲和力变化；
图10为数学模型模拟RBD与CIPS或ACE2的结合；
图11为数学模型模拟不同时间点RBD与CIPS结合情况。计算机模拟的结果显示，5.7ns的时候可以结合(结合时间为5.7ns)
图12为CIPS在SC2-P和FBS的蛋白混合液中对SC2-P的吸附及抑制侵染作用。其中A为在加了SC2-P的1、3或10倍体积的FBS的蛋白混合液中，CIPS能与SC2-P结合；B为在蛋白混合液中，CIPS依然能抑制SC2-P的侵染效率。
图13为实施例11结果，显示CIPS吸附真病毒SARS-CoV-2的定量PCR结果。
图14为实施例12结果，CIPS减少真病毒SARS-CoV-2的量。
图15为不同二维纳米材料CIPS纳米片层、GO(氧化石墨烯纳米片层)、MoS 2(二硫化钼纳米片层)或黑鳞纳米片层存在下假病毒SC2-P对ACE2/293T细胞的侵染结果。
图16为CIPS抑制SARS的假病毒SARS-P的侵染效率。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
二维纳米材料包括石墨烯、过渡金属二硫化物、过渡金属碳化物、氮化物和碳氮化物、黑磷纳米片、层状双氢氧化物、二维MOF和其他类型的二维纳米片。其中氧化石墨烯是一种含氧官能团的二维sp2碳原子层，在纳米医药等领域得到了广泛的应用。目前并没有报道发现二维纳米材料中的铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层和黑鳞纳米片层对于抑制冠状病毒侵染，尤其是SARS病毒和新冠肺炎病毒SARS-CoV-2的细胞侵染有效果。由于本发明中铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层和黑磷纳米片层的生物安全性高，毒性比较低，可以用于药物等多个领域。
术语
在本发明中，铟掺杂的硫磷化铜与CIPS或CuInP 2S 6具有相同的含义。
在本发明中，术语“纳米片层”、“二维纳米材料”具有类似含义，均是指在空间中有一个维度是纳米尺度的材料，例如厚度尺寸在1-100nm的纳米材料。在本发明中，二维纳米材料选自铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层和黑鳞纳米片层中的任意一种或多种的组合。在一个优选的实施例中，选择各二维纳米材料的尺寸为：厚度(在空间中的一个维度)为1-10nm。纳米片层在垂直于厚度方向上的尺寸(在空间中其他维度)为100-300nm。
“纳米片层”可以通过本领域常规的方法剥离获得纳米片层材料，例如可以采用机械研磨(例如球磨)、超声等方法获得。
“药学上可接受载体或赋形剂”包括但不限于任何佐剂、载体、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增味剂、表面活性剂、润湿剂、分散剂、悬浮剂、稳定剂、等渗剂、溶剂或乳化剂，其已被食品和药物管理局批准为可接受用于人或家畜。
“药物组合物”是指本发明化合物以及本领域通常接受的用于递送生物活性化合物至哺乳动物(例如人类)的媒介物的制剂。此种媒介物包括为此的所有药学上可接受的赋形剂。
“有效量”或“治疗有效量”是指根据本发明的化合物的量，其当施用至有此需要的患者时，足以实现对于二维纳米材料具有效用的疾病状态、病症或障碍的治疗。这样的量将足以引起研究人员或临床医生所寻求的组织系统或患者的生物或医学反应。构成治疗有效量的根据本发明的化合物的量将根据诸如以下的因素而变化：化合物及其生物活性、用于施用的组合物、施用时间、施用途径、化合物排泄速率、治疗持续时间、所治疗的疾病状态或病症的类型及其严重程度、与本发明化合物组合或一致使用的药物、以及患者的年龄、体重、一般健康、性别和饮食。本领域普通技术人员可以根据他们自身的知识、现有技术以及本公开而常规地确定此种治疗有效量。
除非另有说明，否则如本文所用的术语“治疗(treating)”是指逆转、减轻、抑制其进展、或预防此术语所适用的障碍或病症，或此种障碍或病症的一种或多种症状。如本文所用的术语“治疗(treatment)”是指治疗的行为，因为“治疗”如上当即所述。在一些实施方式中，术语“治疗”旨在意指施用根据本发明的二维纳米材料或其组合物以减轻或消除冠状病毒感染的症状和/或减少患者中的病毒载量。
“预防”(prevention或preventing)是指导致疾病或病症的临床症状不发展的疾病或病症的任何治疗。术语“预防”还包括在个体暴露于病毒前施用治疗有效量的根据本发明的化合物或组合物(例如，暴露前预防)以预防出现疾病的症状和/或预防病毒在血液中达到可检测的水平。
术语“受试者”或“患者”是指已经为或将要为治疗、观察或实验对象的动物，例如哺乳动物(包括人)。本文描述的方法可用于人类治疗和/或兽医应用。在一些实施方式中，受试者是哺乳动物(或患者)。在一些实施方式中，受试者(或患者)是人、家畜(例如狗和猫)、农场动物(例如，牛、马、绵羊、山羊和猪)和/或实验室动物(例如，小鼠、大鼠、仓鼠、豚鼠、猪、兔、猫、狗和猴)。在一些实施方式中，受试者(或患者)是人。“有此需要的人(或患者)”是指可能患有或怀疑患有将受益于某些治疗的疾病或病症的人；例如，根据本申请用本文公开的二维纳米材料或其组合物或包含上述成分的药物组合物治疗。
药物制剂
用常规载体和赋形剂配制本发明的二维纳米材料，载体和赋形剂将根据常规实践 进行选择。片剂将含有赋形剂、助流剂、填充剂、粘合剂等。含水制剂以无菌形式制备，并且预期通过口服以外的递送通常是等渗的。
尽管活性成分二维纳米材料可以单独施用，但可能优选将它们作为药物制剂呈现。本发明的用于兽用和人用的制剂包含至少一种如上所定义的活性成分(所述活性成分为二维纳米材料：铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层，下同)以及一种或多种可接受的载体和任选的其他治疗成分，例如皮质类固醇、抗炎信号转导调节剂、β2-肾上腺受体激动剂支气管扩张剂、抗胆碱能药、粘液溶解剂、高渗盐水和其他用于治疗冠状病毒科病毒感染的药物；或它们的混合物。
适用于口服给药的本发明制剂可以以离散单位形式呈现，例如胶囊、扁囊剂或片剂，每个含有预定量的活性成分；作为粉末或颗粒；作为水性或非水性液体中的溶液或悬浮液；或者作为水包油液体乳剂或油包水液体乳剂。或者通过其他已知技术制备成口服给药制剂。
片剂通过压制或模制制备，任选地具有一种或多种辅助成分。压制的片剂可以通过在合适的机器中压制任选与粘合剂、润滑剂、惰性稀释剂、防腐剂、表面活性剂或分散剂混合的自由流动形式的活性成分如粉末或颗粒来制备。模制片剂可以通过在合适的机器中模制用惰性液体稀释剂润湿的粉状活性成分的混合物来制备。片剂可以任选地被包衣或刻痕并任选地被配制以提供活性成分从其中缓慢或受控地释放。
本发明的药物组合物可以是局部给药的软膏等外用制剂。对于眼睛或其他外部组织例如口腔和皮肤的感染，所述制剂优选作为含有活性成分的局部软膏剂或霜剂。当配制成软膏时，活性成分可与石蜡或水混溶性软膏基质一起使用。或者，活性成分可以用水包油乳膏基质配制成霜剂。如果需要，局部制剂可以包括增强活性成分通过皮肤或其他受影响区域的吸收或渗透的化合物。这种皮肤渗透促进剂的例子包括二甲基亚砜和相关的类似物。本发明乳液的油相可以由已知成分以已知方式构成。油相可以仅包含乳化剂，但其也可以包含至少一种乳化剂与脂肪或油或与脂肪和油两者的混合物。优选地，亲水乳化剂与充当稳定剂的亲脂性乳化剂一起包含在内。还优选包括油和脂肪。
本发明的药物组合物可以是无菌注射制剂的形式，例如无菌注射用水性或油性悬浮液。
本发明的药物组合物可以是适用于眼部局部给药的制剂还包括滴眼剂，其中活性 成分溶解或悬浮于合适的载体中，特别是用于活性成分的含水溶剂。
本发明的药物组合物可以是适用于口腔局部给药的制剂包括锭剂，其含有调味基质中的活性成分，通常为蔗糖和阿拉伯胶或黄蓍胶；在惰性基质如明胶和甘油或蔗糖和阿拉伯胶中包含活性成分的锭剂；和在合适的液体载体中含有活性成分的漱口剂。
本发明的药物组合物可以是用于直肠给药的制剂，该制剂可以以具有含有例如可可脂或水杨酸盐的合适的基质的栓剂呈现。
本发明的药物组合物可以是适用于肺内或鼻内给药的制剂，这类制剂通常具有0.1-500微米范围内的粒径，诸如0.5、1、30、35微米等，其通过鼻腔通道快速吸入或通过口腔吸入施用以达到肺泡。本发明的活性成分具有可以用于肺内或鼻内给药的尺度，可以用于肺内或鼻内给药，例如吸入剂。
本发明的药物组合物可以是适用于肠胃外给药的制剂包括含水和非水无菌注射溶液，其可含有抗氧化剂，缓冲剂，抑菌剂和使制剂与预期接受者的血液等渗的溶质；以及可包含悬浮剂和增稠剂的水性和非水性无菌悬浮液。
本发明的药物组合物的制剂在单位剂量或多剂量容器(例如密封的安瓿和小瓶)中呈现，并且可以在冷冻干燥(冻干)条件下保存，仅需要在使用前立即添加无菌液体载体(例如注射用水)。即时注射溶液和悬浮液由前述种类的无菌粉末、颗粒和片剂制备。优选的单位剂量制剂是含有如上所述的每日剂量或单位每日亚剂量或其适当部分的活性成分的制剂。
本发明的药物组合物可以是兽用组合物，其包含至少一种如上定义的活性成分以及兽用载体。
本发明化合物用于提供含有作为活性成分的一种或多种本发明活性成分的控释药物制剂，其中控制和调节活性成分的释放以允许较少频率给药或改善给定活性成分的药代动力学或毒性特征。
联合治疗
本发明的药物、药物组合物或者活性成分还与其他活性成分组合使用。为了治疗冠状病毒感染，其他活性成分对冠状病毒感染特别是SARS或SARS-CoV-2感染是有活性的。这些其他活性治疗剂的非限制性实例是皮质类固醇、抗炎信号转导调节剂、β2-肾上腺受体激动剂支气管扩张剂、抗胆碱能药、粘液溶解剂、高渗盐水和其他用于治疗冠状病毒科病毒感染的药物；或它们的混合物。
还可以将本发明的药物、药物组合物或者活性成分与一种或多种其他活性成分以 单位剂型组合同时或顺序施用于患者。联合治疗可以作为同时或顺序方案施用。当按顺序施用时，组合可以是以两次或更多次施用给予。
本发明药物、药物组合物或者活性成分与一种或多种其他活性成分的共同施用通常是指同时或顺序施用本发明化合物和一种或多种其他活性治疗剂，使得治疗有效量的本发明药物、药物组合物或者活性成分和一种或多种其他活性成分都存在于患者体内。
联合治疗可以提供“协同作用”和“协同增效作用”，即当一起使用时获得的效果大于单独使用化合物产生的作用总和。当活性成分：(1)共同配制并在组合制剂中同时施用或递送时；(2)作为单独的制剂交替或平行递送；或(3)通过其他方案时，可获得协同增效效应。
复合材料
本发明一个应用方案为提供了二维纳米材料在制备抑制冠状病毒侵染的材料中的用途；所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种的组合。
本发明一个应用方案为提供了一种抑制冠状病毒侵染的材料，所述材料包含二维纳米材料和基质，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种的组合。
在上述抑制冠状病毒的材料的方案中，材料中还可以包含基质，所述基质能够根据用途进行改变，由于本发明的二维纳米材料稳定性高，能够适应各种基质，根据本领域常规的方法即可获得相应基质的材料，包括但不限于通过以下途径将二维纳米材料与基质进行复合，将二维纳米材料通过吸附方式吸附与基质表面；或者将二维纳米材料与基质进行共混，然后将基质固化获得等。
在本发明的技术方案中，在上述材料中，所述材料为制备防护用品的材料，优选所述的防护用品为口罩、防护服、防护面罩、防护帽。
在本发明的技术方案中，在上述材料中，所述材料为一种涂料。
在本发明的技术方案中，在上述材料中，所述材料为一种消毒剂。
在本发明的技术方案中，在上述材料中，所述材料为一种包装材料。
在本发明的技术方案中，在上述材料中，所述材料为一种过滤材料。
在一个具体的优选实施例中，所述的抑制冠状病毒侵染的材料为一种用于制备口罩的材料，所述材料为织物，织物表面或内部包含本发明所述的二维纳米材料。在一个可 变的优选实施例中，所述的抑制冠状病毒侵染的材料为一种用于制备口罩的材料，所述材料为无纺布，优选为纺粘无纺布或熔喷无纺布；无纺布表面或内部包含本发明所述的二维纳米材料。上述材料的制备方法，可以采用本领域常规的方法将二维纳米材料与基质材料进行复合。例如可以在熔喷纺丝之前将熔喷材料与二维纳米材料复合，然后进行熔喷，也可以将已获得的熔喷布与二维纳米材料复合。
在一个具体的优选实施例中，所述的抑制冠状病毒侵染的材料为一种涂料，该涂料为包含二维纳米材料的混悬剂。优选地，所述涂料中还包括表面活性剂、增稠剂中的至少一种。所述涂料能够将二维纳米材料赋予物体表面，例如包装表面、医用装置表面、美容装置表面等，使其在物体表面形成包含二维纳米材料的涂层。在一个具体的实施例中，所述的涂料赋予食品或药品外包装或内包装表面一层具有二维纳米材料的涂层，优选地，所述的食品或药品需要进行冷链运输。
在一个具体的优选实施例中，所述的抑制冠状病毒侵染的材料为消毒剂，该消毒剂为包含二维纳米材料的混悬剂。在一个具体的优选实施例中，消毒剂为环境消毒剂、免洗洗手液、洗手液、洗涤剂。在一个具体的优选实施例中，消毒剂为能够用于包装材料、医用器械(例如，导管、注射针、手术器具、外科口罩、和其他医学装置)、口腔器具(例如，假牙、保护带、填料、腭扩张器)、美容器具(例如、美容仪、整形装置)的表面消毒。在一个具体的优选实施例中，消毒剂中二维纳米材料的浓度为1μg/mL-1000μg/mL，优选为2.5-160μg/mL，更优选为20μg/mL。
在一个具体的优选实施例中，所述的抑制冠状病毒侵染的材料为包装材料，且所述包装材料用于冷链运输或储存的药物或食品。所述包装材料表面具有包含二维纳米材料的涂层，或者所述包装材料为包含二维纳米材料的复合材料。
在一个具体的优选实施例中，所述的抑制冠状病毒侵染的材料为过滤材料，所述过滤材料为用于空气过滤的材料、用于水体过滤的材料、用于口罩过滤的材料。在一个具体的优选实施例中，所述的过滤材料为空气过滤网，所述空气过滤网表面具有二维纳米材料。在一个具体的优选实施例中，所述的过滤材料为空气过滤棉，所述空气过滤棉纤维上附着二维纳米材料。例如，所述的过滤材料可以用于空调过滤、空气净化器过滤、新风系统过滤等任意需要过滤空气的装置或设备。使用的场景不限于家庭、办公、实验室、工厂等。
在本发明的技术方案中，在上述用途中，所述材料为固体、液体或半固体材料。
在本发明的技术方案中，在上述用途中，液体材料为包含二维纳米材料的涂料、包 含二维纳米材料的消毒剂、包含二维纳米材料的个人护理用品。
在本发明的技术方案中，在上述用途中，固体材料为表面包含二维纳米材料器具、包装材料。
在本发明的技术方案中，在上述用途中，半固体材料为包含二维纳米材料的凝胶。
以下，本发明通过具体实施例来验证纳米材料铟掺杂的硫磷化铜纳米片层(CIPS或CuInP 2S 6)对细胞存活率的影响，CIPS纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层对SARS-CoV-2病毒细胞侵染的抑制，CIPS对新冠假病毒SC2-P的吸附作用，CIPS对新冠病毒Spike蛋白RBD结构域的特异吸附，及数学模拟解析CIPS吸附Spike蛋白RBD区域后对ACE2识别的影响。
以下实施例中所用的实验材料包括：HEK-293T细胞、ACE2/293T细胞、Vero-E6细胞、SARS-CoV-2假病毒SC2-P、SARS假病毒SARS-P。下述实施例中所用的材料、试剂、载体和菌株等，如无特殊说明，均可通过商业途径购买。
HEK-293T细胞，是人胚胎肾细胞293的一个衍生株，是一个衍生自人胚胎肾细胞的细胞系，具有转染效率高，易于培养等特点，是一个很常用的表达研究外源基因的细胞株，成为广大研究者研究基因功能的一个强大工具。
ACE2/293T，指的是在HEK-293T细胞中表达ACE2。
ACE2也称为ACEH，称为血管紧张素转化酶2。该基因编码的蛋白属于二肽基羧基二肽酶的血管紧张素转换酶家族，与人血管紧张素转换酶1具有相当大的同源性。该基因编码蛋白是SARS和SARS-CoV-2、HCoV-NL63人类冠状病毒Spike糖蛋白的功能受体。
Vero-E6细胞是非洲绿猴肾细胞系，是被验证的可被新冠病毒侵染的细胞之一，可作为培养新冠病毒的细胞宿主。比如：测定某种药物对新冠病毒复制速度的影响，检验是否存在新冠病毒或者为了研究目的培养新冠病毒。
SC2-P是表达新冠肺炎病毒SARS-CoV-2的Spike蛋白的假病毒。假病毒是一类嵌合型病毒颗粒，是在一种复制缺陷型病毒(病毒载体)的表面上表达另一种病毒的重组糖蛋白的嵌合病毒颗粒。假病毒因为其生物安全性和稳定性等优点，已被广泛应用到疫苗研发、抗体中和研究、模拟病毒侵染细胞功能实验、检测试剂盒阳性参照等。
目前的SARS-CoV-2相关的假病毒系统主要有两种：一种是利用慢病毒系统构建的慢病毒蛋白外壳包裹SARS-CoV-2的ORF1a/b,N,E等基因，可用于核酸类检测试剂 盒的阳性对照。另外一种是利用SARS-CoV-2的Spike蛋白包裹Luciferase/GFP等标记基因，模拟病毒侵染细胞，检测标记基因的表达，进而研究侵染效率等。
以下实施例中所用的SC2-P可由金唯智公司订购。
SARS-P是SARS的假病毒，也是商业化的产品，此处不再赘述。
实施例1 CIPS纳米片层的制备
通过购买或已知方法获得CIPS单晶，并通过机械研磨等力学方法剥离实验，得到了CIPS纳米片层，如图2所示，X轴是长度，Y轴是厚度，说明材料厚度约1-10nm。纳米片层在垂直于厚度方向上的尺寸不均匀，分布在100-300nm。
实施例2检测CIPS纳米片层的生物安全性
具体步骤如下：
将Vero-E6细胞和ACE2/293T细胞分别接种至96孔板中，密度为1×10 4细胞/孔，37℃，5％CO 2培养过夜。
将培养基换为含不同浓度CIPS纳米片层(0、2.5、5、10、20、40、80、160μg/mL)的培养基，37℃培养孵育24h。
按照1:10每孔加入10μL CCK-8溶液，孵育30-60min，测定450nm处吸光值。
根据下列公式计算细胞活性：细胞活性＝(A 450处理组-A 450空白组)/(A 450对照组-A 450空白组)×100％，其中A 450表示在450nm处的吸光值。
结果如图3所示，图3中的A为Vero-E6细胞与不同浓度(0-160μg/mL)CIPS纳米片层孵育24h后，CCK-8检测细胞活性。图3中的B为ACE2/293T细胞与不同浓度(0-160μg/mL)CIPS孵育24h后，CCK-8检测细胞活性。结果显示，0-160μg/mL的CIPS纳米片层对Vero-E6和ACE2/293T细胞没有细胞毒性，细胞存活率无明显降低，以上结果表明CIPS纳米片层的生物相容性较好，安全性较高。
通过眼眶取血，收集健康C57BL/6小鼠1mL新鲜血液于抗凝管中。4℃3000rpm离心10min，分离上层血清与下层红细胞，去除红细胞表层的白细胞及血小板。用预冷的PBS洗3次制备红细胞悬液。取4％的红细胞100μL加等体积的CIPS纳米片层(40、80、160μg/mL)，37℃孵育4h，测定540nm波长的OD值。用纯水作为阳性对照(100％溶血)，PBS为阴性对照(0％溶血)计算溶血率。公式为：溶血指数(Hem)％＝(A 样 品-A PBS)/(A 纯水-A PBS)×100％，图3中的C结果表明CIPS纳米片层的溶血率为0，其生物相容性较好，安全性较高。
实施例3免疫荧光检测CIPS纳米片层对SC2-P侵染ACE2-GFP/HEK-293T的影响
将14mm的细胞爬片，放到24孔板中后，接种HEK-293T细胞，37℃，5％CO 2培养过夜，转染ACE2-GFP 24h后，分别用混有0/10/20/40μg/mL的CIPS纳米片层的SC2-P侵染细胞2h，免疫荧光检测SC2-P侵染细胞的量，具体方法如下：
将24孔板中细胞爬片上的细胞，用4％的多聚甲醛室温固定15min，PBS洗3次。
加0.1％的Triton X-100破膜15min，PBS洗3次。
PBS稀释的3％BSA封闭30min，PBS洗3次。
Anti-Flag(兔源1:500)和anti-GFP(鼠源1:500)孵育2h，PBS洗3次。
488荧光标记的鼠源二抗和555荧光标记的兔源二抗孵育1h，PBS洗4次，抗荧光淬灭剂封片，荧光共聚焦观察。
结果如图4中A所示，图4A为共聚焦显微镜观察SC2-P对ACE2-GFP/HEK-293T细胞的侵染结果。SC2-P与不同浓度(0、10、20和40μg/mL)的CIPS纳米片层预孵育2h后，侵染ACE2-GFP/HEK-293T细胞，免疫荧光检测SC2-P的侵染。图4B为ImageJ统计SC2-P进入细胞的数目和荧光强度。RFI代表相对荧光强度。ACE2通道为绿色荧光，SC2-P通道为红色荧光，Merge为两者的叠加，图中以灰度表示。通过免疫荧光观察SC2-P的侵染，可以观察到SC2-P能够进入到宿主细胞ACE2-GFP/HEK-293T中，并且图4B统计的结果也显示，不同浓度的CIPS纳米片层处理下，SC2-P能够进入到宿主细胞的数目和荧光强度都有所减少，并且减少的程度呈剂量依赖，其中当CIPS纳米片层的用量只有10μg/mL时已经可以与阴性对照(即CIPS纳米片层添加量为0)时产生统计学差异，且当浓度达到40μg/mL可以使侵染量仅为阴性对照的30％。以上结果表明，CIPS纳米片层能够有效抑制SC2-P进入到宿主细胞内。
实施例4荧光素酶报告基因检测CIPS纳米片层对SC2-P侵染Vero-E6和ACE2/293T的影响
分别将Vero-E6细胞和ACE2/293T细胞接种至96孔板中，密度为1×10 4细胞/孔，37℃，5％CO 2培养过夜。
分别用混有0/2.5/5/10/20/40/80μg/mL的CIPS纳米片层的SC2-P侵染细胞2h，DMEM洗一次后加DMED培养基培养40-48h后检测荧光素酶的活性。
图5的荧光素酶活性检测的结果显示，SC2-P能够将基因组整合到宿主细胞中。当施加CIPS纳米片层处理时，SC2-P侵染Vero-E6细胞和ACE2/293T细胞的量与对照相比，均显著降低，且减少的程度也呈剂量依赖。以上结果表明，CIPS纳米片层能够有效抑制SC2-P对宿主细胞的侵染。
实施例5检测CIPS纳米片层抑制SARS-CoV-2真病毒对细胞的侵染
同时，本发明利用真病毒SARS-CoV-2检测侵染实验，也得到类似结果。分别用混有0/2.5/5/10/20/40/80/160μg/mL的CIPS纳米片层的SARS-CoV-2病毒侵染Vero-E6细胞1h，48h后定量检测培养基中病毒的量。如图6所示，随着CIPS纳米片层浓度增加，真病毒SARS-CoV-2的侵染效率逐渐降低。表明CIPS纳米片层不仅能够有效抑制SC2-P对宿主细胞的侵染，也能抑制新冠真病毒SARS-CoV-2的侵染。其中，图6中的A显示的是不同浓度CIPS的侵染率，B显示的是以ORF1ab/RdRp基因的表达量为指标定量检测的病毒量，而图6中的C显示的是以核衣壳蛋白的基因表达量为指标定量检测的病毒量。
实施例6 CIPS纳米片层理化性质表征结果显示CIPS能够吸附SC2-P
检测CIPS纳米片层对SC2-P的结合能力。将CIPS纳米片层与SC2-P共同孵育2h后得到CIPS-SC2-P混悬液，分别检测了CIPS-SC2-P混悬液、单独CIPS纳米片层的混悬液以及SC2-P混悬液的UV-VIS和Z-电位等表征。结果如图7A-B所示，图7A为CIPS-SC2-P混悬液、单独CIPS纳米片层的混悬液以及SC2-P混悬液的UV-VIS结果，可以看出CIPS和SC2-P的吸收峰不同，而孵育2h以后的CIPS-SC2-P混悬液的吸收峰相对于CIPS纳米片层的混悬液以及SC2-P混悬液的吸收峰有所位移，吸收强度也有变化。图7B为CIPS-SC2-P混悬液、单独CIPS纳米片层的混悬液以及SC2-P混悬液的Z-电位，其中，CIPS结果为单独CIPS纳米片层的混悬液的Z-电位，SC2-P为SC2-P混悬液的Z-电位，CIPS+SC2-P为CIPS-SC2-P混悬液Z-电位。结果表明CIPS纳米片层能够吸附SC2-P。
实施例7 Western blot检测CIPS纳米片层吸附SC2-P和减少SC2-P
首先，利用Western blot分别直接检测CIPS纳米片层和SC2-P，结果见图7中的C的左图，SC2-P中的蛋白能够在Western blot结果上有清晰的显示，而CIPS纳米片层中由于没有蛋白，所以没有对应条带显示，随后分别在SC2-P混悬液中分别加入0μg、10μg和20μg的CIPS纳米片层，孵育120分钟，然后采用相同速度离心进行分离，利用Western blot检测，结果见图7C的右图，其中第一列结果为仅包含SC2-P的混悬液，由于病毒过小，无法通过离心方式分离，所以Western blot没有任何条带，而第2和第3列结果分别为加入10μg和20μg的CIPS纳米片层的结果，由于CIPS纳米片层对SC2-P有吸附作用，所以可以通过离心获得，并在Western blot有对应条带显示，随着CIPS纳米片层用量的增加吸附量也有所增加。
同时，将SC2-P与不同量的(0/5/10/20/40μg)CIPS纳米片层孵育两小时后，以Westerm blot检测SC2-P的总量。图7D显示，随着CIPS纳米片层量的逐渐增加，Spike蛋白(180Kd的全长蛋白)量逐渐减少(图7D)，由该实验结果可以看出CIPS纳米片层能够促进SC2-P全长蛋白Spike的降解，并且随着Spike蛋白的分解，假病毒SC2-P的量减少。这就表明，CIPS纳米片层不仅能够吸附SC2-P，还能够减少SC2-P的量。
实施例8 CIPS纳米片层与ACE2竞争性结合新冠病毒SARS-CoV-2的刺突(Spike)蛋白的受体结合区(RBD，receptor binding domain)结构域
利用生物膜干涉(BLI)定量测定CIPS纳米片层与新冠病毒Spike蛋白的RBD结构域蛋白、血清中的蛋白、模型蛋白BSA的亲和力及相互作用。如图8所示，计算出不同体系的KD值。说明CIPS纳米片层与S蛋白的RBD结构域亲和力很强，但是与血清中其他蛋白亲和力弱。暗示着CIPS纳米片层对RBD的选择性吸附。该实验证实CIPS纳米片层可以单独作用于病毒并特异性地和病毒S蛋白RBD结构域蛋白结合。该实验结果表明，CIPS纳米片层可以用于ACE2蛋白结和前，能够用于冠状病毒的预防。此外，MoS 2和GO与RBD结构域也有很强的亲和力。说明，MoS 2和GO纳米片层对RBD也能产生吸附，实验证实MoS 2和GO纳米片层可以分别单独作用于病毒并特异性地和病毒S蛋白RBD结构域蛋白结合。该实验结果表明，MoS 2和GO纳米片层均可以用于ACE2蛋白结和前，能够用于冠状病毒的预防。
此外，生物膜干涉(BLI)定量测定与CIPS纳米片层结合前后，RBD蛋白与ACE2蛋白的亲和力变化。针对RBD和ACE2相互作用：200nM RBD蛋白溶液中，传感器通过化学偶联并固定RBD蛋白300s，脱吸附600s；传感器继续在100nM ACE2溶液中，吸附600s，解吸附300s。结果说明，RBD与ACE2的KD值约为4nM；图9显示，在CIPS纳米片层存在情况下，KD值为41nM。说明，CIPS纳米片层导致RBD与ACE2的亲和力减弱。这就表明，CIPS纳米片层能够减少SARS-CoV-2病毒Spike蛋白与其宿主细胞的受体ACE2的结合，从而抑制病毒的侵染。
根据实验所得的数据，并利用数学模型模拟RBD与CIPS纳米片层或ACE2的结合。图10所示，CIPS纳米片层与RBD结合的位点(图10A)与ACE2与RBD结合的位点(图10B)相重合(图10C)。并且，与ACE2相比，CIPS纳米片层与RBD的亲和力更强，所以CIPS纳米片层能够与ACE2竞争性的结合RBD。CIPS纳米片层占据ACE2的结合位点，进而阻止RBD与ACE2的结合，从而抑制病毒SARS-CoV-2对宿主细胞的侵染。图10下部的abc分别代表ACE2的结合位点，CIPS的结合位点以及二者的重 合结果，从重合模拟图中可以看出，ACE2与CIPS结合的位点相重合。
实施例9基于分子动力学(MD)模拟RBD与CIPS和RBD的结合
基于分子动力学(MD)模拟的系统中RBD和CIPS吸附的典型轨迹快照。底部一行显示了RBD吸附在CIPS表面的动态过程。上一行是与CIPS结合的RBD氨基酸位点的底图，VDW球是与CIPS接触的氨基酸残基。红球为极性残基，绿色为疏水残基，蓝色为正电荷残基，紫色为负电荷残基，黄色为含二硫键的胱氨酸。
实施例10 CIPS能吸附SC2-P与FBS的蛋白混合液中的SC2-P，并抑制其侵染效率
分别将1倍、3倍和10倍SC2-P体积量的FBS与SC2-P混合。将20μg/mL的CIPS纳米片层与蛋白混合液预孵育2h后，加PBS补充到1ml，4000rpm离心5min。WB检测沉淀中SC2-P的量。结果表明，在蛋白混合液中，CIPS依然可以吸附SC2-P。
分别将1倍、3倍和10倍SC2-P体积量的FBS与SC2-P混合。将20μg/mL的CIPS纳米片层与蛋白混合液预孵育2h后，侵染ACE2/293T细胞2h，荧光素酶活性检测SARS-P对ACE2/293T细胞的侵染效率。结果表明，CIPS在复杂的蛋白液环境中，能够吸附并抑制SC2-P的侵染效率。
实施例11定量PCR检测CIPS纳米片层吸附真病毒SARS-CoV-2
在P3实验室，符合P3实验室操作规范的前提下，将6000pfu/500ml的真病毒SARS-CoV-2与不同浓度(6和12pM)的CIPS孵育2h后，4000rpm离心5min，得到上清(supernatant)和沉淀(precipitate)。分别将上清和沉淀中病毒的RNA提取后，利用定量PCR对病毒的量进行定量。如图13所示，结果表明，CIPS能够结合真病毒SARS-CoV-2，随着CIPS的量增加，离心得到的CIPS结合的病毒量也逐渐增加，并且，上清中剩余的病毒量逐渐减少。
实施例12定量PCR检测CIPS纳米片层减少真病毒SARS-CoV-2的量
在P3实验室，符合P3实验室操作规范的前提下，将6000pfu/500ml的真病毒SARS-CoV-2与不同浓度(1.5-48pM)的CIPS孵育2h后，收集所有的病毒并提取其RNA，利用定量PCR对病毒的量进行定量。图14显示，随着CIPS纳米片层量的逐渐增加，病毒RNA的量逐渐减少，由该实验结果可以看出CIPS纳米片层能够减少病毒RNA的量。这就表明，CIPS纳米片层不仅能够吸附真病毒SARS-CoV-2，还能够减少SARS-CoV-2的量。
实施例13不同二维纳米材料CIPS纳米片层、氧化石墨烯(GO)纳米片层、二硫化钼(MoS 2)纳米片层、黑鳞纳米片层(BP)存在下SC2-P对ACE2/293T细胞的侵染 结果
将ACE2/293T细胞接种至96孔板中，密度为1×10 4细胞/孔，37℃，5％CO 2培养过夜。
分别用混有20μg/mL的CIPS纳米片层、GO(氧化石墨烯)纳米片层、MoS 2(二硫化钼)纳米片层或黑鳞纳米片层的SC2-P侵染细胞2h，DMEM洗一次后加DMED培养基培养40-48h后检测荧光素酶的活性。
图15的荧光素酶活性检测的结果显示，不同的二维纳米材料均具有抑制新冠假病毒SC2-P侵染宿主细胞的能力。Ctrl组为仅施加SC2-P而未添加纳米片层的对照组，当施加CIPS纳米片层或黑鳞纳米片层处理时，SC2-P侵染ACE2/293T细胞的量与对照相比，显著降低，施加GO(氧化石墨烯)纳米片层、MoS 2(二硫化钼)纳米片层时，SC2-P侵染ACE2/293T细胞的量与对照相比，也有显著降低，但效果不如施加CIPS纳米片层或黑鳞纳米片层明显。CIPS纳米片层、GO纳米片层、MoS 2纳米片层组或黑鳞纳米片层与对照组(Ctrl组)具有显著性差异。以上结果表明，CIPS纳米片层、GO纳米片层、MoS 2纳米片层均能够有效抑制SC2-P对宿主细胞的侵染，且CIPS纳米片层效果最优。
实施例14 CIPS纳米片层对SARS假病毒颗粒SARS-P的侵染抑制效果，说明其抑制冠状病毒效果具有广谱性。
为了检测这种抑制是特异的针对SARS-CoV-2病毒的侵染还是适用于其他冠状病毒。本发明还检测了CIPS纳米片层对SARS假病毒颗粒SARS-P的侵染抑制效果。结果如图16所示，统计学分析使用ANOVA，**表示P<0.01，***表示P<0.001。图16A为共聚焦显微镜下观察SARS-P对ACE2-GFP/HEK-293T细胞的侵染。SARS-P与20μg/mL的CIPS纳米片层预孵育2h后，侵染ACE2-GFP/HEK-293T细胞2h，免疫荧光检测SARS-P的侵染。图16B-16C为ImageJ统计SARS-P进入细胞的数目和荧光强度。RFI代表相对荧光强度。图16D-16E为荧光素酶活性检测SARS-P对ACE2/293T和Vero-E6细胞的侵染效率。SARS-P与20μg/mL的CIPS纳米片层预孵育2h后，侵染Vero-E6和ACE2/293T细胞2h，40h后荧光素酶活性检测SARS-P的侵染效率。CIPS纳米片层也能够抑制SARS-P的侵染。说明CIPS纳米片层对病毒侵染性的抑制并不是只针对SARS-CoV-2病毒，而是对冠状病毒具有广谱性。
本发明提供一种抑制SARS-CoV-2病毒细胞侵染的纳米药物，所述纳米药物中包括铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层中的任意一种或多种。
实施方式1：以铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种为药效成分的口服液；
以口服液为例，口服液是在汤剂、注射剂基础上发展起来的新剂型，具有剂量小、吸收较快、质量稳定、携带和服用方便、易于保存的优点，其含有多种有效成分，对质量和口感有很大的影响。在不改变主要活性成分结构和功能前提下，如何能最大限度的保留有效成分、改善口感是其选用辅料的一个难点。在口服液中添加辅料可以提高口感，改善澄清度，增强稳定性，提高产品质量。
口服液常用辅料有：溶剂、芳香剂、矫味剂、澄清剂、防腐剂等，这些辅料可同时加入，也可择其一加入，其中溶剂是必加的，可以采用水。不同的辅料组合有甜味剂、芳香剂、澄清剂或防腐剂，或甜味剂和防腐剂的组合，优选甜味剂和防腐剂的组合。部分辅料兼具增甜和增香的作用，此时只需加入一种辅料即可。
针对口服液，优选地，所述甜味剂选自蛋白糖、木糖醇、阿斯巴甜和三氯蔗糖中的一种或多种。
针对口服液，优选地，所述防腐剂选自对羟基苯甲酸酯、丁基羟基茴香醚、丁基羟基甲苯和山梨酸中的一种或多种。
防腐剂可选用对羟基苯甲酸酯、丁基羟基甲苯或山梨酸，优选丁基羟基甲苯。也可以组合使用，例如羟基苯甲酸酯与丁基羟基甲苯的组合，或丁基羟基甲苯与山梨酸的组合，或对羟基苯甲酸酯与山梨酸的组合，或对羟基苯甲酸酯、丁基羟基甲苯和山梨酸的组合。
针对口服液，优选地，所述芳香剂为水果香精。
针对口服液，优选地，所述澄清剂为壳聚糖和明胶中的一种或两种混合。
实施方式2：以铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种为药效成分的片剂；
片剂具有剂量准确，质量稳定，服用、携带及运输方便等优点。
针对片剂，所述制剂辅料包括稀释剂、粘合剂、润滑剂和崩解剂中一种或多种，优选稀释剂、粘合剂、润滑剂和崩解剂的组合。
针对片剂，优选地，所述稀释剂为纤维素类和无机盐类中的一种或多种。例如微晶纤维素、硫酸钙、磷酸氢钙及药用碳酸钙、甘露醇等，以增加原料体积助其成型的。
针对片剂，优选地，所述粘合剂为水、乙醇、羧甲基纤维素钠、羟丙基纤维素、甲基纤维素、乙基纤维素、明胶和聚乙烯吡咯烷酮等中的一种或多种。
针对片剂，优选地，所述润滑剂为硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇和月桂醇硫酸镁中的一种或多种。
针对片剂，优选地，所述崩解剂为低取代羟丙基、交联聚乙烯吡咯烷酮和交联羧甲基纤维素钠等中的一种或多种。
实施方式3：以铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种为药效成分的胶囊；
在本发明中，胶囊主要提高药物的稳定性和生物利用度。所述制剂辅料为胶囊壳，所述胶囊壳为硬胶囊壳或软胶囊壳。
实施方式4：以铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种为药效成分的颗粒剂；
颗粒剂可以直接吞服，也可以用温水冲入水中饮入，应用和携带比较方便，溶出和吸收速度较快。颗粒剂所用的制剂辅料与片剂相似，涉及填充剂、粘合剂、润湿剂、崩解剂、润滑剂和薄膜包衣材料中的一种或多种。
针对颗粒剂，优选地，所述填充剂为纤维素类和无机盐类中的一种或多种。例如微晶纤维素、硫酸钙、磷酸氢钙及药用碳酸钙、甘露醇等，以增加原料体积助其成型的。
针对颗粒剂，优选地，所述粘合剂为水、乙醇、羧甲基纤维素钠、羟丙基纤维素、甲基纤维素、乙基纤维素、明胶和聚乙烯吡咯烷酮等中的一种或多种。
针对颗粒剂，优选地，所述润湿剂为水或乙醇或两者的混合。例如为硬脂酸镁、微粉硅胶、滑石粉、氢化植物油、聚乙二醇和月桂醇硫酸镁中的一种或多种。
针对颗粒剂，优选地，所述崩解剂为低取代羟丙基、交联聚乙烯吡咯烷酮和交联羧甲基纤维素钠等中的一种或多种。
针对颗粒剂，优选地，所述薄膜包衣材料羟丙基甲基纤维素、聚乙二醇、醋酸纤维素酞酸酯和聚乙烯缩乙醛二乙胺醋酸酯中的一种或多种。
实施方式5：以铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种为药效成分的散剂。
本发明也可制成散剂，散剂便于分剂量和服用。
以饮料为例，将本发明的组合物制成不同风味的饮料，作为日常饮品将深受欢迎。
饮料所用的制剂辅料为澄清剂、防腐剂和香味剂中的至少一种。
本发明的组合物也可制成其它散剂，例如制成功能性奶粉，加入的辅料主要为奶粉，例如脱脂奶粉，脱脂无糖奶粉。
以上多个实施方式可调整单位产品中的药剂量大小，以适应不同的用途，如药品、保健品、食品等。
本发明一个优选的实施例，提供了SARS-CoV-2病毒细胞侵染的口罩熔喷布，所述口罩熔喷布中添加铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种。
本发明一个优选的实施例，提供了抑制SARS-CoV-2病毒细胞侵染的冷库涂层或外包装涂层，所述冷库涂层或外包装涂层中添加铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种。
本发明一个优选的实施例，提供了抑制SARS-CoV-2病毒细胞侵染的消毒喷剂，所述喷剂中添加铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种。所述喷剂中除铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层中的任意一种或多种外，还包含溶剂，溶剂为水，没有毒性，其中铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层、黑鳞纳米片层的有效浓度为2.5-160μg/mL(如图3所示)，浓度过低抑制效果不明显，浓度过高成本增大，不利于商业化生产，优选浓度为20μg/mL。
综上，本发明验证了二维纳米材料CIPS纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层和黑鳞纳米片层均能够抑制SARS-CoV-2病毒的侵染效率。CIPS也能够抑制SARS-P的侵染。说明CIPS对病毒侵染性的抑制不仅针对SARS-CoV-2病毒，而具有对冠状病毒的广谱适应性。
以CIPS为例，本发明还验证了CIPS对细胞存活率的影响。实验结果表明，CIPS(1-160μg/mL)对Vero-E6和ACE2/293T细胞活性无影响，并且没有细胞溶血，说明其生物安全性高，毒性低。
本发明还验证了CIPS对SARS-CoV-2病毒侵染效率的抑制。通过实验证实，CIPS抑制SARS-CoV-2的假病毒(SC2-P)对ACE2-GFP/HEK-293T(在HEK-293T中表达ACE2-GFP)和Vero-E6的侵染效率。并且通过实验证实，CIPS能够抑制真病毒SARS-CoV-2对Vero-E6的侵染效率。
本发明还验证了CIPS对SC2-P的吸附作用。通过实验证实，CIPS可以吸附并减少SC2-P。并且CIPS相对于其他蛋白，对SARS-CoV-2病毒的纤突蛋白(Spike protein)的受体结合域(RBD)结构域的结合作用更强，并且CIPS的存在能够抑制病毒与受体的结合。
因此，本发明的纳米材料可以制备成纳米药物治疗新型冠状病毒肺炎，或者添加到口罩的熔喷布、冷库涂层以及外包装涂层以及喷剂中，抑制新型冠状病毒肺炎的传播，达到预防作用。本发明的二维纳米材料对病毒侵染性的抑制并不仅仅针对SARS-CoV-2病毒，而是对冠状病毒具有广谱性。二维纳米材料CIPS纳米片层、氧化石墨烯纳米片层和二硫化钼纳米片层、黑鳞纳米片层可以单独应用，也可以叠加应用。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (14)

  1. 二维纳米材料在制备治疗或预防冠状病毒所致疾病的药物中的应用，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种；冠状病毒所致疾病为由冠状病毒感染导致的疾病。
  2. 二维纳米材料在制备抑制冠状病毒Spike蛋白与其宿主细胞的受体ACE2的结合的制剂中的用途；
    所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，二维纳米材料通过竞争性结合冠状病毒Spike蛋白的RBD结合的位点抑制其与其宿主细胞的受体ACE2的结合；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
  3. 二维纳米材料在制备促进冠状病毒的蛋白的分解的药物中的用途；
    所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的蛋白至少包括Spike蛋白；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
  4. 二维纳米材料在制备促进冠状病毒的RNA的分解的药物中的用途；
    所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
  5. 二维纳米材料在制备抑制冠状病毒侵染的药物中的用途；
    所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
  6. 一种治疗或预防冠状病毒所致疾病的药物组合物，所述药物组合物中以二维纳米材料作为活性成分，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种；冠状病毒所致疾病为由冠状病毒感染导致的疾病。
  7. 根据权利要求2所述的药物组合物，所述的药物组合中进一步包括施用治疗有效量的至少一种其他治疗剂或其组合物，所述其他治疗剂或其组合物选自皮质类固醇、抗炎信号转导调节剂、β2-肾上腺受体激动剂支气管扩张剂、抗胆碱能药、粘液溶解剂、高渗盐水和其他用于治疗冠状病毒科病毒感染的药物；或它们的混合物；
    优选地，所述的药物组合物的制剂形式选自口服制剂、注射制剂、粘膜给药制剂、吸入剂、外用制剂；
    优选地，所述的药物组合物中还包含药学上可接受的载体或赋形剂。
  8. 二维纳米材料在制备抑制冠状病毒的Spike蛋白与其宿主细胞的受体ACE2的结合的制剂或材料中的用途；所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
  9. 二维纳米材料在制备抑制冠状病毒侵染或降低、杀灭冠状病毒的材料中的用途；所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二 硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
  10. 二维纳米材料在制备吸附冠状病毒、或者在制备分解冠状病毒蛋白、或者在制备分解冠状病毒RNA的材料中的用途；所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种；
    优选地，优选地，所述的病毒蛋白至少包含Spike蛋白。
  11. 一种吸附或抑制冠状病毒的材料，所述材料包含二维纳米材料和基质，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
  12. 根据权利要求8-10任一项所述的用途或权利要求11所述的材料，所述材料为制备防护用品的材料、或用于制备涂料、消毒剂或个人护理用品、包装材料或过滤材料的材料；
    优选地，所述的防护用品为口罩、防护服、防护面罩、防护帽；
    所述的涂料为包含二维纳米材料的混悬剂，所述的涂料能否赋予被附着物表面具有二维纳米材料的涂层；
    所述的消毒剂为环境消毒剂、免洗洗手液、洗手液、洗涤剂；更优选地，所述消毒剂用于包装材料、医用器械、口腔器具、美容器具的表面消毒；
    所述的包装材料为具有包含二维纳米材料的涂层，或者所述包装材料为包含二维纳米材料的复合材料；更优选地，包装材料为用于的药物或食品的包装材料，尤其是用于冷链运输的药物或食品；
    所述过滤材料为用于空气过滤的材料、用于水体过滤的材料、用于口罩过滤的材料； 更优选地，所述的过滤材料为空气过滤网，所述空气过滤网表面具有二维纳米材料；更优选地，所述的过滤材料为空气过滤棉，所述空气过滤棉纤维上附着二维纳米材料；
    优选地，所述的冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
  13. 一种用品，所述用品中包含二维纳米材料，所述二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    所述用品选自防护用品、涂料、消毒剂、个人护理用品、包装材料或过滤装置；
    优选地，所述的防护用品为口罩、防护服、防护面罩、防护帽；
    优选地，所述的涂料为包含二维纳米材料的混悬剂，所述的涂料能否赋予被附着物表面具有二维纳米材料的涂层；
    优选地，所述的消毒剂为环境消毒剂、免洗洗手液、洗手液、洗涤剂；更优选地，所述消毒剂用于包装材料、医用器械、口腔器具、美容器具的表面消毒；
    优选地，所述的包装材料为具有包含二维纳米材料的涂层，或者所述包装材料为包含二维纳米材料的复合材料；更优选地，包装材料为用于的药物或食品的包装材料，尤其是用于冷链运输的药物或食品；
    优选地，所述过滤装置为用于空气过滤的装置、用于水体过滤的装置、用于口罩过滤的装置；更优选地，所述的过滤装置为空气过滤网，所述空气过滤网表面具有二维纳米材料；更优选地，所述的过滤装置为空气过滤棉，所述空气过滤棉纤维上附着二维纳米材料。
  14. 一种治疗或预防的冠状病毒感染的方法，其包括向受试者施用治疗有效量的二维纳米材料，所述的二维纳米材料为铟掺杂的硫磷化铜纳米片层(CIPS)、氧化石墨烯纳米片层、二硫化钼纳米片层或黑鳞纳米片层中的任意一种或多种的组合；
    优选地，所述的治疗有效量的二维纳米材料以制剂形式给与，制剂中还包含药学上可接受的载体或赋形剂；
    更优选地，冠状病毒选自HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、SARS-CoV2或MERS-CoV中的一种。
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