Synthesis method of bismuth silver sulfide nanomaterials and their antiviral applications

Abstract

This invention discloses a synthesis method for a bismuth silver sulfide nanomaterial and its antiviral application. While conventional synthesis reactions typically require high temperatures, this invention overcomes this problem by enabling room temperature synthesis, shortening the reaction time, and simplifying the process. The antiviral activity of bismuth silver sulfide has not been previously demonstrated in the art, but this invention demonstrates the antiviral activity of bismuth silver sulfide nanomaterials for the first time.

Description

Technical Field

[0001] The present invention belongs to the fields of nanomaterials and virology, and more specifically, relates to a synthesis method of bismuth silver sulfide nanomaterials and their antiviral application. Background Art

[0002] New biomaterials based on nanomaterials have demonstrated unique advantages in the biomedical field and have attracted widespread attention from researchers. Bismuth (Bi)-based nanomaterials primarily refer to inorganic nanomaterials containing the element bismuth. Due to their excellent biocompatibility and exceptional optical, electrical, and other physicochemical properties, these materials have been studied and reported in biomedical fields such as tissue engineering, biosensing, drug delivery, and tumor therapy. In particular, they show broad potential in bioimaging and cancer diagnosis and treatment.

[0003] A wide variety of bismuth-based nanomaterials have been reported. Their morphologies primarily include nanoparticles, nanorods, nanoflowers, nanotubes, nanosheets, and mesoporous structures. Their composition primarily includes elemental bismuth (including bismuth nanoparticles and bismuthene nanosheets), bismuth oxides (including Bi2O3, BiO2-x, (BiO)2CO3), bismuth chalcogenides (Bi2S3, Bi2Se3), bismuth halides (including BiOCl, BiOBr, and BiOI), bismuth-based polymetallic compounds (Bi2WO6, BiP5W30, BiFeO3, BiGdO3, Cu3BiS3, Cu3BiSe3), and bismuth-based complexes. Different compositions, structures, morphologies, and sizes result in diverse optical, electrical, and magnetic properties. Therefore, the design and preparation of bismuth-based nanomaterials with diverse functionalities can address applications in bioimaging, drug delivery, antimicrobial treatment, tissue engineering, biosensing, and tumor therapy.

[0004] Methods for synthesizing bismuth-based nanomaterials include hydrothermal / solvothermal methods, high-temperature thermal decomposition, ion exchange, sol-gel methods, microemulsion methods, chemical reduction, and microwave / ultrasound / light-mediated synthesis. Different preparation methods can produce bismuth-based nanomaterials with completely different structures and properties.

[0005] Bismuth silver sulfide nanomaterials are a bismuth-based nanomaterial. Existing preparation methods for this material are complex, requiring high-temperature treatment (e.g., 100°C). Synthesis efficiency and product quality need to be improved, and few methods have yet achieved particle sizes within the nanometer range. Furthermore, current research and demonstration of the applications of bismuth silver sulfide nanomaterials are limited.

[0006] Therefore, there is a need in the art to optimize the preparation method of bismuth silver sulfide nanomaterials and develop new application fields thereof. Summary of the Invention

[0007] The present invention aims to provide a synthesis method of a bismuth silver sulfide nanomaterial and its antiviral application.

[0008] In a first aspect of the present invention, a method for preparing bismuth silver sulfide nanomaterial is provided, the method comprising:

[0009] (1) mixing bismuth nitrate and silver nitrate and reacting;

[0010] (2) adding polyvinyl pyrrolidone or polyethylene glycol to the reaction system of (1) and continuing the reaction;

[0011] (3) adding thiourea to the reaction system of (2) to obtain bismuth silver sulfide nanomaterials after reaction;

[0012] The reactions (1) to (3) are carried out at 15 to 35°C.

[0013] In one or more preferred embodiments, in (1), bismuth nitrate and silver nitrate are mixed in an organic solvent.

[0014] In one or more preferred embodiments, the organic solvent is ethylene glycol.

[0015] In one or more preferred embodiments, in (1), when added, the final concentration of bismuth nitrate in the reaction system is 0.001-0.1 mol / L, preferably 0.003-0.01 mol / L, such as 0.0061 mol / L.

[0016] In one or more preferred embodiments, when added, the final concentration of silver nitrate in the reaction system is 0.001-0.1 mol / L, preferably 0.003-0.01 mol / L, such as 0.0061 mol / L.

[0017] In one or more preferred embodiments, the reactions of (1) to (3) are carried out at 18 to 32°C; preferably at 20 to 30°C; more preferably at 22 to 28°C, such as at 23, 24, 25, 26, or 27°C.

[0018] In one or more preferred embodiments, the reactions (1) to (3) are carried out at room temperature.

[0019] In one or more preferred embodiments, in (2), when added, the amount of polyvinyl pyrrolidone is 10-20 mg / mL solution, preferably 12-18 mg / mL solution, such as 15 mg / mL solution.

[0020] In one or more preferred embodiments, in (2), when added, the amount of polyethylene glycol is 5 to 16 mg / mL, preferably 7 to 14 mg / mL, such as 10 mg / mL.

[0021] In one or more preferred embodiments, in (3), when added, the final concentration of thiourea in the reaction system is 0.005-0.05 mol / L, preferably 0.007-0.03 mol / L, such as 0.01 mol / L.

[0022] In one or more preferred embodiments, thiourea is dissolved in an organic solvent and added dropwise to the reaction system.

[0023] In one or more preferred embodiments, the organic solvent is a mixed solvent of ethanol and ethylene glycol.

[0024] In one or more preferred embodiments, the volume ratio of ethanol to ethylene glycol in the mixed solvent is 2:(2.5-5), such as 2:2.8, 2:3, 2:3.5, 2:4, 2:4.5, etc.

[0025] In one or more preferred embodiments, in (1), the reaction time is 3 to 30 min, such as 5, 10, 15, 20, or 25 min.

[0026] In one or more preferred embodiments, in (3), the reaction time is 10 to 30 min, such as 12, 15, 20, or 25 min, preferably 15±3 min.

[0027] In another aspect of the present invention, a bismuth silver sulfide nanomaterial is provided, which is prepared by any of the methods described above.

[0028] In one or more preferred embodiments, the average particle size of the bismuth silver sulfide nanomaterial is 18.6 nm, and the particle shape is irregularly round.

[0029] In another aspect of the present invention, there is provided a use of any of the aforementioned methods to prepare a bismuth silver sulfide nanomaterial for preparing a composition for inhibiting viral infection.

[0030] In one or more preferred embodiments, the viruses include: RNA viruses and DNA viruses; preferably, the viruses include (but are not limited to): influenza virus, coronavirus, parainfluenza virus, enterovirus, adenovirus, herpes virus, norovirus, dengue virus, Zika virus, human immunodeficiency virus; preferably, the influenza virus includes (but is not limited to): influenza virus subtype A, subtype B, subtype C; preferably, the coronavirus includes (but is not limited to): coronavirus α subtype (229E) and β subtype (OC43), coronavirus HKU1 and NL63, severe acute respiratory syndrome virus, Middle East respiratory syndrome virus and new coronavirus.

[0031] In another aspect of the present invention, a composition is provided, comprising: a bismuth silver sulfide nanomaterial prepared by any of the aforementioned methods; and a biologically or pharmaceutically compatible carrier or excipient.

[0032] In another aspect of the present invention, a method for inhibiting viruses is provided, comprising: (a) providing the bismuth silver sulfide nanomaterial or the composition; (b) treating a virus or an object carrying the virus with the bismuth silver sulfide nanomaterial or composition of (a), thereby inhibiting the virus.

[0033] In one or more embodiments, the method of inhibiting viruses is a method that is not directly intended to treat a disease, or is a non-therapeutic method.

[0034] In one or more embodiments, the object targeted by the virus inhibition may be a place, object, or vessel to which the virus is attached.

[0035] In one or more embodiments, the object of virus inhibition may be an in vitro cell culture.

[0036] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 , a flow chart showing the method for synthesizing bismuth silver sulfide nanoparticles.

[0038] Figure 2 , a schematic diagram showing the antiviral application of bismuth silver sulfide nanoparticles synthesized by the method of the present invention.

[0039] Figure 3 AC, the inhibitory effect of nanomaterials on influenza virus subtype A during the virus and cell pre-culture stage.

[0040] Figure 4 (a) TEM image of AgBiS2 nanomaterials.

[0041] Figure 4 (b) XRD pattern of AgBiS2 nanomaterials.

[0042] Figure 4 (c) Infrared analysis of AgBiS2, Bi2S3, and Ag2S. DETAILED DESCRIPTION

[0043] Green synthesis of nanomaterials is becoming a mainstream trend in the field of synthetic materials. Current synthesis methods often require high temperatures and long reaction times. After in-depth research, the present inventors have discovered a novel synthesis method for bismuth silver sulfide nanomaterials and their application in antiviral applications.

[0044] In the prior art, synthesis reactions generally require high temperatures, and no synthesis scheme for bismuth silver sulfide under mild conditions has been found. The method of the present invention is carried out at room temperature, has a short reaction time, and is simple to handle.

[0045] In the existing technology in this field, there has been no research on the antiviral effects of bismuth silver sulfide on influenza virus and coronavirus, which is a blank. The present invention is the first to conduct research on the anti-influenza and anti-coronavirus effects of bismuth silver sulfide nanomaterials.

[0046] The method for preparing bismuth silver sulfide nanomaterials of the present invention comprises: (1) mixing bismuth nitrate and silver nitrate and reacting them; (2) adding polyvinyl pyrrolidone or polyethylene glycol to the reaction system of (1) and continuing the reaction; (3) adding thiourea to the reaction system of (2) and obtaining the bismuth silver sulfide nanomaterials after the reaction.

[0047] In all the above steps, the reaction can be carried out at 15-35°C without the need for additional high-temperature treatment. In other words, all steps can be completed at room temperature.

[0048] Bismuth nitrate and silver nitrate are mixed in an organic solvent; an organic solvent suitable for dissolving bismuth nitrate and silver nitrate and not affecting the two and their reaction can be used in the present invention. As a preferred embodiment of the present invention, the organic solvent is ethylene glycol, which serves only as a solvent.

[0049] As a preferred embodiment of the present invention, when reacting, bismuth nitrate and silver nitrate are mixed in a ratio (molar ratio) of 0.9 to 1.2:1 and reacted, which is conducive to the complete reaction, improving reaction efficiency and controlling costs. As a preferred embodiment of the present invention, when added, the final concentration of bismuth nitrate in the reaction system is 0.001 to 0.1 mol / L; and / or the final concentration of silver nitrate in the reaction system is 0.001 to 0.1 mol / L.

[0050] As a preferred embodiment of the present invention, the amount of polyvinyl pyrrolidone added during the reaction is 10-20 mg / mL. Such a concentration is compatible with the reaction product in the previous step and helps ensure the efficiency of the reaction.

[0051] As a preferred embodiment of the present invention, the final concentration of thiourea in the reaction system is 0.005-0.05 mol / L; preferably, thiourea is dissolved in an organic solvent and added dropwise to the reaction system; preferably, the organic solvent is a mixed solvent of ethanol and ethylene glycol; more preferably, in the mixed solvent, the ratio of ethanol to ethylene glycol is 2:(2.5-5) (e.g., 2:2.8, 2:3, 2:3.5, 2:4, 2:4.5, etc.) by volume.

[0052] The method provided by the present invention synthesizes nano-sized materials, so bismuth silver sulfide crystals are formed at ambient temperature and wrapped with polyvinyl pyrrolidone, which is not a strictly regular metal crystal structure.

[0053] It is understood that the bismuth nitrate or silver nitrate is a bismuth salt or a silver salt. In the present invention, other bismuth salts or silver salts, such as bismuth sulfate or silver sulfate, may also be used, and such bismuth salts or silver salts are also included in the present invention. It is understood that the bismuth salt or silver salt may also be an analog or derivative thereof.

[0054] It is understood that polyvinyl pyrrolidone or polyethylene glycol or their analogs or derivatives can be used in the present invention because they can act as amphiphilic macromolecules to serve as templates and have good biocompatibility.

[0055] The present invention also includes bismuth silver sulfide nanomaterials prepared by the methods described herein. Those skilled in the art will appreciate that different reaction methods can produce significantly different nanomaterials. Therefore, given the uniqueness of the methods of the present invention, the resulting nanomaterials are also unique.

[0056] The present invention also includes a composition containing a bismuth-silver sulfide nanomaterial prepared by the method described herein. For example, a composition of the present invention can be obtained by mixing the bismuth-silver sulfide nanomaterial prepared by the method described herein with a suitable biocompatible carrier or excipient. In the composition, the bismuth-silver sulfide nanomaterial prepared by the method described herein is the sole active ingredient (active component) or the primary active ingredient (active component).

[0057] In the present invention, the terms “contain”, “have” or “include” include “comprise”, “mainly consist of”, “substantially consist of”, and “consist of”; “mainly consist of”, “substantially consist of” and “consist of” are subordinate concepts of “contain”, “have” or “include”.

[0058] When the composition further comprises the bismuth silver sulfide nanomaterial of the present invention as an active component, the composition can exert a virus-inhibiting function.

[0059] When the composition described herein is used as a medicine, it can also be referred to as a pharmaceutical composition, which further comprises a pharmaceutically acceptable excipient. A "pharmaceutically acceptable excipient" is a pharmaceutically or food-acceptable carrier, solvent, suspending agent or excipient for delivering the active ingredient (e.g., bismuth silver sulfide nanomaterial) in the composition of the present invention to an animal or human. Exemplary excipients can be liquid or solid, including but not limited to: pH regulators, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in combination with the pharmacological compound. More specifically, suitable excipients can be excipients commonly used in the art for small molecule compound administration. Examples of excipients include various lactoses, mannitol, oils such as corn oil, buffers such as PBS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as albumin, and detergents such as Tween 80, monosaccharides and oligopolysaccharides such as glucose, lactose, cyclodextrin and starch.

[0060] Typically, the composition contains a therapeutically effective amount of the active ingredients described herein. A therapeutically effective amount refers to a dose that can achieve treatment, prevention, alleviation and / or relief of a disease or condition in a subject. The effective amount of the bismuth silver sulfide nanomaterial described in the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the bismuth silver sulfide nanomaterial, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. The therapeutically effective amount can be determined based on factors such as the patient's age, gender, the disease and its severity, and the patient's other physical conditions. The therapeutically effective amount can be administered as a single dose, or it can be administered in multiple doses according to an effective treatment regimen. Herein, a subject or patient generally refers to a mammal, particularly a human. Illustratively, the composition contains an active component (eg, bismuth silver sulfide nanomaterial) in a weight ratio of, for example, 0.001-50%, preferably 0.01-30%, more preferably 0.05-10%.

[0061] The pharmaceutical composition or mixture of the present invention can be made into any conventional dosage form by conventional methods. The dosage form can be varied, as long as it is a dosage form that can effectively allow the active ingredient to reach the mammalian body. For example, it can be selected from: injection, infusion, tablet, capsule, pill. The active component (such as bismuth silver sulfide nanomaterial) can be present in a suitable solid or liquid carrier or diluent. The mixture or pharmaceutical composition of the active component of the present invention can also be stored in a sterilizer suitable for injection or infusion. The effective dose of the active component (such as bismuth silver sulfide nanomaterial) in the composition can vary with the mode of administration and the severity of the disease to be treated, which can be based on the experience and advice of the clinician.

[0062] The present invention also includes a kit or a medicine box containing the bismuth silver sulfide nanomaterial prepared by the method described herein; or a kit or a medicine box containing the above-mentioned composition of the present invention. Typically, the kit or medicine box comprises an appropriate container or packaging for containing the bismuth silver sulfide nanomaterial or the composition.

[0063] In addition, the kit or medicine box may also include an instruction manual or operating manual describing the method of using the bismuth silver sulfide nanomaterial or the composition containing the same, so as to facilitate the use by those skilled in the art.

[0064] The present invention also includes the use of the bismuth silver sulfide nanomaterial or a composition containing the same for inhibiting viruses. The virus can be an RNA virus or a DNA virus. As a preferred embodiment of the present invention, the virus includes influenza virus, coronavirus, parainfluenza virus, enterovirus, adenovirus, herpes virus, norovirus, dengue virus, Zika virus, human immunodeficiency virus, etc.

[0065] The present invention also provides a method for inhibiting viruses, comprising treating a virus or an object carrying a virus with the bismuth silver sulfide nanomaterial or composition of the present invention, thereby inhibiting the virus. The method for inhibiting viruses can be a method that is not directly intended to treat a disease that is not easily treated, or a non-therapeutic method. The object targeted by the virus inhibition can be a place, object, vessel, etc. where the virus is attached. The object targeted by the virus inhibition can be an in vitro cell culture. It is understood that in the present invention, there is no particular limitation on the object to be inhibited by the virus, and any object that is potentially infected or attached to the virus can be used as the object of the virus inhibition of the present invention.

[0066] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0067] Example 1, Synthesis Method 1 of Bismuth Silver Sulfide Nanomaterial (Applying PVP)

[0068] In this embodiment, the synthesis of bismuth silver sulfide nanomaterials is carried out in three steps, as shown in the following diagram: Figure 1 ):

[0069] First, add bismuth nitrate and silver nitrate to 20 mL of ethylene glycol at room temperature (25°C) to form a homogeneous solution. The final concentration of bismuth nitrate is 0.0061 mol / L, and the final concentration of silver nitrate is 0.0061 mol / L. Allow to react for 5 minutes.

[0070] Next, 300 mg (15 mg / mL solution) of polyvinylpyrrolidone (PVP-k30) was added and stirred evenly at room temperature.

[0071] Finally, a solution (5 mL) of thiourea (CH4N2S) in ethanol / ethylene glycol (ethanol:ethylene glycol = 2:3 by volume) was added dropwise to the mixture and stirred at room temperature for 15 minutes to obtain bismuth silver sulfide nanomaterials. In the solution, the concentration of thiourea in 5 mL of ethanol / ethylene glycol was 0.05 mol / L, and the concentration in the total system (20 + 5 ml) was 0.01 mol / L.

[0072] Morphological identification of bismuth silver sulfide nanomaterials: black, uniform particle size, average particle size of 18.6±0.3nm, irregular circle, such as Figure 4 (a).

[0073] Performance identification of bismuth silver sulfide nanomaterials: XRD confirmed that the material was bismuth silver sulfide nanomaterials, and IR was used to determine the surface PVP, which was well distributed. Figure 4 (b).

[0074] Infrared analysis of AgBiS2, Bi2S3, Ag2S Figure 4 (c).

[0075] Example 2, Synthesis Method 2 of Bismuth Silver Sulfide Nanomaterial (Application of PEG)

[0076] In this embodiment, the synthesis of bismuth silver sulfide nanomaterials is carried out in three steps, as shown in the following diagram: Figure 1 ):

[0077] First, add bismuth nitrate and silver nitrate to 20 mL of ethylene glycol at 28°C to form a homogeneous solution. The final concentration of bismuth nitrate is 0.0061 mol / L, and the final concentration of silver nitrate is 0.0061 mol / L. The reaction is allowed to proceed for 16 minutes.

[0078] Next, 200 mg of polyethylene glycol (PEG; HO(CH2CH2O) n H, n is 2000), and stirred at room temperature.

[0079] Finally, a solution of CH4N2S in ethanol / ethylene glycol (ethanol:ethylene glycol = 2:3 by volume) was added dropwise to the mixture and stirred at room temperature for 15 minutes to obtain bismuth silver sulfide nanomaterials. In the solution, the concentration of thiourea in ethanol / ethylene glycol was 0.05 mol / L.

[0080] Morphological identification of bismuth silver sulfide nanomaterials: Morphological identification of bismuth silver sulfide nanomaterials: black, uniform particle size, reaching nanometer level, irregular circle.

[0081] Performance identification of bismuth silver sulfide nanomaterials: It is expected that the material is confirmed to be bismuth silver sulfide nanomaterials through XRD, and the surface PEG is determined by IR, and its distribution state is good.

[0082] Example 3. Evaluation of anti-influenza virus effect (viral inhibition effect)

[0083] In this example, the antiviral effect (virus inhibition effect) was evaluated. A schematic diagram of the antiviral verification of bismuth sulfide silver nanoparticles is shown in FIG. Figure 2 .

[0084] Bismuth silver sulfide nanomaterials were synthesized as described in Example 1.

[0085] Virus: Influenza virus subtype A and subtype B.

[0086] Cells: Madin-Darby canine kidney cells (MDCK cells)

[0087] 1. Design different virus infection and antiviral systems to determine the antiviral effect of the materials

[0088] (1) Direct killing experiment (material pre-acts on virus)

[0089] MDCK cells were cultured overnight in a 96-well plate at a density of 2×10^4 cells / well. A mixture of virus and various concentrations of material (0, 10, 20, 50, 100, and 150 μg / mL) was prepared in serum-free medium to a final concentration of 100 TCID50 / mL and incubated at room temperature for 1 hour. The supernatant was then aspirated and discarded from the 96-well plates. The plates were washed twice with PBS (pH 7.4). 100 μL of the supernatant of the virus-material mixture was added, and the 96-well plates were incubated at 35°C for 1 hour. The plates were washed twice with PBS (pH 7.4), followed by the addition of 100 μL of viral infection medium (EMEM medium supplemented with 2% FBS and 1% double-antibody) and incubated at 35°C for 48 hours. The supernatants were collected by centrifugation, and viral RNA was extracted for quantitative fluorescence PCR and TCID50 analysis.

[0090] (2) Co-culture inhibition experiment (co-culture of virus and material)

[0091] MDCK cells were seeded at a density of 2×10^4 cells / well in a 96-well plate and cultured overnight at 37°C. Serum-free medium was used to prepare 200 TCID50 / mL of virus and various concentrations of nanomaterial dilutions. The 96-well plate was removed, the cell supernatant was aspirated, and the plate was washed twice with PBS (pH = 7.4). Virus was added at a final concentration of 100 TCID50 / mL and the material dilutions at final concentrations of 0, 10, 20, 50, 100, and 150 μg / mL. The 96-well plate was incubated at 35°C for 1 hour, then washed twice with PBS (pH = 7.4). 100 μL of viral infection medium was added and incubated at 35°C for 48 hours. The supernatant was collected by centrifugation, and viral RNA was extracted from the supernatant for quantitative fluorescence PCR and half-tissue culture infectious dose (TCID50) analysis.

[0092] (3) Material and cell pre-culture inhibition experiment

[0093] MDCK cells were seeded at a density of 2 × 10^4 cells / well in a 96-well plate and cultured overnight at 37°C. Material dilutions at concentrations of 0, 10, 20, 50, 100, and 150 μg / mL were prepared in serum-free medium. The 96-well plate was removed, the cell supernatant was aspirated, and the plate was washed twice with PBS (pH 7.4). 100 μL of the material dilution was added and the plate was incubated at 37°C for 3 hours. The supernatant was then aspirated, the plate was washed twice with PBS (pH 7.4), and 100 μL of a 100 TCID50 / mL virus solution prepared in serum-free medium was added. The plate was incubated at 35°C for 1 hour. The plate was then washed twice with PBS (pH 7.4), 100 μL of the viral infectious medium was added, and the plate was incubated at 35°C for 48 hours. The supernatant was collected by centrifugation, and viral RNA was extracted for quantitative fluorescence PCR and TCID50 analysis.

[0094] (4) Inhibition experiment after infection (pre-culture of virus and cells)

[0095] MDCK cells were seeded at a density of 2×10^4 cells / well in a 96-well plate and cultured overnight at 37°C. The cell supernatant was then aspirated and the plate was washed twice with PBS (pH = 7.4). 100 μL of a 100 TCID50 / mL virus solution prepared in serum-free medium was added and incubated at 35°C for 1 hour. The cell supernatant was then aspirated and the plate was washed twice with PBS (pH = 7.4). 100 μL of a material dilution solution prepared using viral infectious medium at concentrations of 0, 10, 20, 50, 100, and 150 μg / mL was added and incubated at 35°C for 48 hours. Finally, the supernatant in the wells was collected by centrifugation and viral RNA was extracted from the supernatant for fluorescence quantitative PCR detection and half tissue culture infectious dose (TCID50) detection.

[0096] 2. TCID50 analysis results

[0097] The TCID50 differences of the above different virus infection and antiviral systems were measured with the virus-infected cell system without the addition of nanomaterials as the control, as shown in Table 1.

[0098] Table 1. Virus inhibition effect at different stages of virus infection (TCID50 determination; unit TCID50 / ml)

[0099]

[0100] The results indicate that the bismuth silver sulfide nanomaterial of the present invention exhibits a significant and stable infection inhibition effect under different viral infection states.

[0101] Among them, when the concentration was 150 μg / mL, the inhibitory effect of bismuth silver sulfide nanomaterials in the virus and cell pre-culture group was the most obvious, indicating that its main inhibition stage occurred during the virus invasion process and after invading the cells, and the nanomaterials interfered with the virus internalization and replication process in the cells.

[0102] 3. RT-qPCR test

[0103] For the NS1 gene fragment of influenza virus subtype B, the primer sequences used were 5'-TCCTCAACTCACTCTTCGAGCG-3' (SEQ ID NO: 1) and 5'-CGGTGCTCTTGACCAA ATTGG-3' (SEQ ID NO: 2), and the probe sequence was 5'-CCAATTCGAGCAGCTGAA ACTGCGGT-3' (SEQ ID NO: 3) and was determined according to conventional methods. For the M1 gene fragment of influenza virus subtype A, the primer sequences used were 5'-GACCRATCCTGTCACCTCTGAC-3' (SEQ ID NO: 4) and 5'-AGGGCATTYTTGACAAAKCGTCTA-3' (SEQ ID NO: 5), and the probe sequence was 5'-TGCAGTCCTCGCTCACTGGGCACG-3' (SEQ ID NO: 6) and was determined according to conventional methods.

[0104] The gene expression inhibition rate results are shown in Tables 2 and 3.

[0105] Table 2. Virus inhibition effect at different stages of virus-infected cells (RT-qPCR test)

[0106]

[0107] The results in Table 2 demonstrate that the bismuth silver sulfide nanomaterial of the present invention exhibits significant and stable infection inhibition under different influenza B virus subtype infection conditions. Specifically, at a concentration of 100-150 μg / mL, the inhibitory effect was highest during the pre-incubation phase between the virus and cells, exceeding 90%. This indicates that the primary inhibition occurs during and after viral invasion, with the nanomaterial interfering with viral internalization and intracellular replication.

[0108] Table 3. Inhibitory effect of nanomaterials on influenza virus subtype A during the virus and cell pre-culture phase (concentration gradient effect RT-qPCR determination)

[0109]

[0110] The results in Table 3 show that as the concentration of nanomaterials increases, the inhibition rate of influenza virus increases in a dose-dependent manner; however, when the concentration is higher than 100 μg / ml, the improvement is no longer significant.

[0111] As described above, the qRT-PCR results for influenza virus subtype B inhibition by the nanomaterial were consistent with those from the TCID50 assay. Furthermore, during the pre-incubation phase of the virus and cells, qRT-PCR testing for influenza virus subtype A showed that the inhibitory effect of the bismuth silver sulfide nanomaterial was consistent with its inhibitory effect against influenza virus subtype B. This result further demonstrates that the bismuth silver sulfide nanomaterial's inhibitory effect occurs during and after viral invasion.

[0112] 4. Indirect Immunofluorescence Assay

[0113] The antibodies used for the HA protein of influenza virus subtype B were Influenza B virus (B / Florida / 4 / 2006) Hemagglutinin / HA Antibody (purchased from Beijing Sino Biological Science and Technology Co., Ltd., Catalog No. 11053-T62-50) and Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (purchased from Abcam, Catalog No. ab150077). Protein expression was determined according to conventional methods. The measured protein expression results are shown in Table 4.

[0114] Table 4. Inhibitory effect of nanomaterials on influenza virus subtype A during the virus and cell pre-culture phase (indirect immunofluorescence assay)

[0115]

[0116] This result shows that the bismuth silver sulfide nanomaterial of the present invention inhibits the expression of virus-related proteins by inhibiting the gene expression of influenza virus subtype B. Its inhibitory effect on the virus is basically consistent at the gene and protein levels.

[0117] Example 4. Evaluation of anti-coronavirus effect (virus inhibition effect)

[0118] In this example, the antiviral effect (virus inhibitory effect) was evaluated.

[0119] Bismuth silver sulfide nanomaterials were synthesized as described in Example 1.

[0120] Virus: Coronavirus alpha subtype (VR-740 229E) and beta subtype (VR-1558OC43)

[0121] Cells: Human hepatocellular carcinoma Huh7 cells and rhabdomyosarcoma RD cells

[0122] Cell culture: Human hepatocellular carcinoma Huh7 cells and rhabdomyosarcoma RD cells were cultured in EMEM medium containing 10% fetal bovine serum and 1% double antibody in a constant temperature incubator at 37°C containing 5% CO2.

[0123] Virus infection: Coronavirus α subtype (229E) and β subtype (OC43) were incubated with cells in EMEM medium containing 2% FBS and 1% double antibody (virus infection medium) at 37°C and 5% CO2 for 1 hour.

[0124] During the intracellular viral replication phase (i.e., pre-incubation of the virus with cells), bismuth silver sulfide nanoparticles were added to the cell culture medium at final concentrations of 0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. After 48 hours of incubation, the culture supernatant was harvested.

[0125] The antiviral effect (viral inhibition effect) of the obtained culture supernatant was evaluated by real-time fluorescence quantitative PCR (qRT-PCR).

[0126] qRT-PCR assay: For the N gene of the VR-740 229E virus, the primers used were 5'-TCCTTCCCGGTCTCAGTCG-3' (SEQ ID NO: 7) and 5'-CTGTCACTT GAAGGATTCCGAG-3' (SEQ ID NO: 8), and the probe sequence was 5'-TCGCGGTCGTGGTGAATCCAAACCTCA-3' (SEQ ID NO: 9), and the assay was performed according to conventional methods. For the N gene of the VR-1558OC43 virus, the primers used were 5'-TCGTTCTGGTAATGGCATCCT-3' (SEQ ID NO: 10) and 5'-CTGATGGTTGCTGAGAGGTAG-3' (SEQ ID NO: 11), and the probe sequence was 5'-CTAAACTGGTCGGACTGATCGGCCCA-3' (SEQ ID NO: 12), and the assay was performed according to conventional methods. The measured gene expression results are shown in Table 5.

[0127] Table 5. Inhibitory effect of nanomaterials on influenza virus subtype A during the virus and cell pre-culture phase (concentration gradient effect RT-qPCR determination)

[0128]

[0129] This result further illustrates that the virus-inhibiting effect of the bismuth silver sulfide nanomaterial of the present invention occurs during the virus invasion process and after invading cells, and its inhibitory effect increases with the increase in the concentration of the nanomaterial and exhibits a dose-dependent effect.

[0130] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. A method for preparing bismuth silver sulfide nanomaterials, characterized in that: The method comprises: (1) Mixing bismuth nitrate and silver nitrate and reacting them; the final concentration of bismuth nitrate in the reaction system is 0.001 to 0.1 mol / L; the final concentration of silver nitrate in the reaction system is 0.001 to 0.1 mol / L; (2) Adding polyvinyl pyrrolidone or polyethylene glycol to the reaction system of (1) and continuing the reaction; the amount of polyvinyl pyrrolidone when added is 10 to 20 mg / mL, or the amount of polyethylene glycol when added is 5 to 16 mg / mL; (3) adding thiourea to the reaction system of (2) to obtain bismuth silver sulfide nanomaterials after reaction; the final concentration of thiourea in the reaction system is 0.005 to 0.05 mol / L; and the reaction time is 10 to 30 minutes; The reactions (1) to (3) are carried out at 15 to 35°C.

2. The method according to claim 1, wherein In (1), bismuth nitrate and silver nitrate are mixed in an organic solvent.

3. The method according to claim 2, wherein The organic solvent is ethylene glycol.

4. The method according to claim 1 or 2, wherein (1), when added, the final concentration of bismuth nitrate in the reaction system is 0.003 to 0.01 mol / L; or (1), when added, the final concentration of silver nitrate in the reaction system is 0.003-0.01 mol / L.

5. The method according to claim 1, wherein (2) When adding polyvinylpyrrolidone, the amount is 12 to 18 mg / mL; or (2), the amount of polyethylene glycol added is 7 to 14 mg / mL.

6. The method according to claim 1, wherein (3), when added, the final concentration of thiourea in the reaction system is 0.007-0.03 mol / L.

7. The method according to claim 6, wherein Thiourea is dissolved in an organic solvent and added dropwise to the reaction system; the organic solvent is a mixed solvent of ethanol and ethylene glycol; according to the volume ratio, ethanol:ethylene glycol in the mixed solvent is 2:(2.5-5).

8. The method according to claim 1, wherein In (1), the reaction time is 3 to 30 min.

9. The method according to claim 1, wherein (3), the reaction time was 15 ± 3 min.

10. A bismuth silver sulfide nanomaterial, characterized in that: It is prepared by the method described in any one of claims 1 to 9; the bismuth silver sulfide nanomaterial is black, has a uniform particle size and an average particle size of 18.6±0.3nm, and is an irregular circle.

11. Use of the bismuth silver sulfide nanomaterial prepared by the method according to any one of claims 1 to 9 for preparing a composition for inhibiting viral infection.

12. The use according to claim 11, characterized in that The viruses include: RNA viruses and DNA viruses, specifically influenza viruses, coronaviruses, parainfluenza viruses, enteroviruses, adenoviruses, herpes viruses, and noroviruses; the coronaviruses include: coronavirus α subtype and β subtype, coronavirus HKU1 and NL63.

13. The use according to claim 12, characterized in that The influenza virus is influenza virus subtype A, subtype B or subtype C.

14. A composition, characterized in that The composition contains: The method according to any one of claims 1 to 9 prepares a bismuth silver sulfide nanomaterial, which is black, has a uniform particle size and an average particle size of 18.6±0.3 nm, and is an irregular circle; and a biologically or pharmaceutically compatible carrier or excipient.

15. A method for non-therapeutic viral suppression, characterized in that: The method comprises: (a) providing the bismuth silver sulfide nanomaterial according to claim 10 or the composition according to claim 14; (b) treating a virus or an object carrying the virus with the bismuth silver sulfide nanomaterial or composition of (a) to inhibit the virus.

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