A medicine for anti-coronavirus and anti-bacteria and a preparation method thereof

Abstract

This invention provides a drug for combating SARS-CoV-2 and antibacterial agents, and a method for preparing the same. The method involves reacting L-cysteine ​​and copper sulfate under alkaline conditions to prepare negatively charged nano-Cu2S. This nano-Cu2S reacts with the main protease M of SARS-CoV-2. pro and papain PL pro The nano-Cu2S binds to the main protease M of the SARS-CoV-2 virus to inhibit viral replication. pro and papain PL pro The dissociation constants between the components are all less than 0.001 nM. Simultaneously, nano-Cu2S exhibits antibacterial activity, inhibiting the proliferation of various bacteria. The synthesis process of the drugs for combating COVID-19 and antibacterial purposes according to this invention is simple and suitable for large-scale production. Nano-Cu2S has low toxicity and good biosafety, and can be widely used in the coatings and disinfection fields to inhibit the spread of viruses and bacteria.

Description

Technical Field

[0001] This invention relates to the field of antiviral and antibacterial infection technology, and in particular to a drug for fighting COVID-19 and antibacterial agents, and a method for preparing the same. Background Technology

[0002] Current research has found that the novel coronavirus (SARS-CoV-2) has four main structural proteins: spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein), and envelope protein (E protein). The S protein has two subunits: S1 and S2. The receptor binding site (RBD) is located on the S1 subunit. Its main function is to recognize receptors on the surface of host cells. It can specifically recognize human angiotensin-converting enzyme (ACE2) to mediate fusion with host cells.

[0003] Co-infection with COVID-19 and bacteria is a significant cause of worsening symptoms and even death in patients. However, currently available anti-COVID-19 drugs, such as Pfizer's Paxlovid, can only be used to treat viral infections, and antibiotics such as cephalosporins and methicillin can only be used to treat bacterial infections. There is a lack of drugs that can simultaneously combat two different types of pathogens: viruses and bacteria.

[0004] With the development of nanotechnology and nanomedicine, researchers have developed nanomedicines with different preventive and therapeutic functions against SARS-CoV-2 and bacteria. For example, mRNA vaccines based on lipid nanoparticles have been widely used to prevent SARS-CoV-2 infection; ACE2 nanodecoys derived from human lung cells can bind to and neutralize SARS-CoV-2, triggering a phagocytic response in macrophages, thereby accelerating viral elimination and protecting host lung cells from infection; furthermore, graphene nanoparticles modified with sulfonated polyglycidol and hydrophobic fatty acid segments can interact with SARS-CoV-2 particles through electrostatic interactions, completely destroying the structure of SARS-CoV-2 and rendering it infective. These nanomedicines, either directly or by stimulating the body to produce neutralizing antibodies, can efficiently recognize and bind to the virus extracellularly, thereby inhibiting SARS-CoV-2 infection. In addition to the above extracellular therapies against SARS-CoV-2, researchers are also dedicated to developing intracellular therapies, such as designing gold nanoclusters that inhibit the activity of the SARS-CoV-2 main protease, which can directly inhibit viral replication intracellularly and significantly reduce lung inflammation caused by the virus. However, SARS-CoV-2 is constantly mutating, and existing strategies often rely on a single antiviral mechanism, making it difficult to effectively combat the proliferation of variant strains, and still cannot achieve simultaneous anti-infection against two different types of pathogens: viruses and bacteria.

[0005] Therefore, this invention is proposed. Summary of the Invention

[0006] To address the limitation of existing technologies in simultaneously achieving anti-infection against two different types of pathogens—COVID-19 and bacteria—this invention provides a drug for both anti-COVID-19 and antibacterial purposes, along with its preparation method. The nano-Cu2S prepared through a specific method exhibits dual activity against both COVID-19 and bacterial infections, simultaneously inhibiting the replication and growth of multiple COVID-19 viruses and various bacteria.

[0007] Specifically, in a first aspect, the present invention provides a method for preparing a drug for combating the novel coronavirus and for fighting bacteria, comprising: reacting L-cysteine ​​and copper sulfate under alkaline conditions to prepare negatively charged nano-Cu2S.

[0008] Verification has shown that nano-Cu2S can efficiently bind to the receptor-binding domains of multiple variant SARS-CoV-2 strains (including the OmicronBQ.1 strain), inhibiting the binding of SARS-CoV-2 to cellular receptors and thus blocking SARS-CoV-2 infection of cells. Simultaneously, nano-Cu2S can also inhibit the activity of two enzymes related to virus replication: the SARS-CoV-2 main protease and papain, thereby inhibiting SARS-CoV-2 replication within cells. These multiple antiviral mechanisms enable nano-Cu2S to significantly combat multiple variant SARS-CoV-2 strains. Furthermore, nano-Cu2S can also inhibit the growth of various bacteria, including Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus, demonstrating dual activity against both SARS-CoV-2 and bacterial infections.

[0009] According to the preparation method of the drug for anti-COVID-19 and antibacterial purposes provided by the present invention, the average particle size of the nano-Cu2S is 15-25 nm. Specifically, the size of Cu2S can be 15 nm, 18 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm or other particle sizes, or any combination of these particle sizes, such as 18-24 nm, 20-25 nm, 18-23 nm, etc., which are not listed here. The size of nano-Cu2S is smaller than that of the COVID-19 virus, which is more conducive to its binding to the spike protein on the surface of the COVID-19 virus.

[0010] According to the preparation method of the drug for anti-COVID-19 and antibacterial purposes provided by the present invention, the copper sulfate is copper sulfate pentahydrate; the molar ratio of L-cysteine ​​to copper sulfate pentahydrate is (3-5):1; Preferably, the reaction temperature is 50-70°C; the reaction time is 4-6 hours.

[0011] According to the preparation method of the drug for anti-COVID-19 and antibacterial purposes provided by the present invention, the L-cysteine ​​and copper sulfate are reacted in an aqueous solution with a pH of 9-11 and then dialyzed to obtain the nano-Cu2S. Preferably, the dialysis medium is water, and the dialysis time is 40-60 hours.

[0012] In the method for preparing nano-Cu2S, the molar ratio of L-cysteine ​​to copper sulfate pentahydrate can be 3:1, 4:1, 5:1, or other molar ratios, or any combination of these molar ratios, such as (3-4):1, (4-5):1; the pH can be 9, 10, 11, or other pH values, or any combination of these pH values, such as 9-10, 10-11, etc.; the temperature can be 50℃, 55℃, 60℃, 65℃, 70℃, or other temperatures, or any combination of these temperatures, such as 55-65℃, 60-70℃, 50-60℃, etc.; the reaction time can be 4 hours, 5 hours, or 6 hours; the dialysis time can be 40 hours, 42 hours, 44 hours, 45 hours, 46 hours, 48 ​​hours, 50 hours, etc., or any combination of these dialysis times, forming a time range; for the sake of simplicity, the specific point values ​​and their arbitrary combinations are not listed here.

[0013] In the preparation of nano-Cu2S, an excess of L-cysteine ​​is used, and some of the cysteine ​​reduces copper sulfate to Cu. + It provides S to synthesize Cu2S, and another part of L-cysteine ​​coordinates with copper atoms through thiol groups to form copper-sulfur bonds, thereby adsorbing onto the surface of nano-Cu2S to improve the biocompatibility of nano-Cu2S.

[0014] According to the preparation method of the drug for anti-COVID-19 and antibacterial purposes provided by the present invention, the zeta potential of the nano-Cu2S is -20 to -35 mV. Preferably, the zeta potential is -20 to -35 mV. The zeta potential can be -20 mV, -25 mV, -30 mV, -35 mV, etc., or any combination of these values, such as -20 to -30 mV, -25 to -35 mV, etc. The surface of the nano-Cu2S carries a negative charge, enabling it to efficiently bind to the positively charged RBD domain on the surface of the SARS-CoV-2 spike protein. By competitively binding the RBD to the ACE2 receptor binding region, the spike protein is unable to recognize the ACE2 receptor of the host cell, thus inhibiting the invasion of the SARS-CoV-2 virus and its variants into host cells.

[0015] According to the preparation method of the drug for anti-COVID-19 and antibacterial purposes provided by the present invention, the nano-Cu2S inhibits viral infection of the host by binding to the RBD protein of COVID-19.

[0016] According to the preparation method of the drug for anti-COVID-19 and antibacterial purposes provided by the present invention, the nano-Cu2S reacts with the main protease M of the COVID-19 virus. proand papain PL pro The nano-Cu2S binds to the main protease M of the SARS-CoV-2 virus to inhibit viral replication. pro and papain PL pro The dissociation constants between them are all less than 0.001 nM.

[0017] According to the method for preparing drugs for combating SARS-CoV-2 and antibacterial agents provided by the present invention, the SARS-CoV-2 virus is selected from at least one of wild-type SARS-CoV-2, Delta variant SARS-CoV-2, Omicron BA.5 variant SARS-CoV-2, Omicron BF.7 variant SARS-CoV-2, and Omicron BQ.1 variant SARS-CoV-2.

[0018] According to the method for preparing drugs for combating COVID-19 and antibacterial purposes provided by the present invention, the bacteria are selected from at least one of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0019] Secondly, the present invention also provides a drug for fighting the novel coronavirus and for fighting bacteria, prepared by the method described above.

[0020] Furthermore, in this invention, the provided nano-Cu2S can be applied to coatings, wherein the amount of nano-Cu2S added to the coating is 0.1% to 2%. Specifically, the coating is used to coat at least one of packaging materials, building materials, and medical protective materials.

[0021] Specifically, the packaging material is selected from at least one of plastic, paper, metal, and glass; the shape of the packaging material is selected from at least one of bag and box; the packaging material is used for at least one of food, express delivery, masks, and protective clothing, especially for the outer packaging bags / boxes of frozen food. The spread of COVID-19 and bacteria in daily life can be prevented by coating the surface of the packaging material with the coating.

[0022] Specifically, the nano-Cu2S can be used in building materials, such as the walls of biosafety laboratories, to avoid contamination of the experimental environment.

[0023] In this invention, the provided nano-Cu2S can be used in disinfectants, wherein the amount of nano-Cu2S added to the disinfectant is 0.1% to 1%. The dosage form of the disinfectant is selected from at least one of gel and spray. The disinfectant can be applied by means of smearing or other methods to disinfect, cut off the transmission path of the novel coronavirus and bacteria, thereby inhibiting or even preventing the spread of the novel coronavirus and bacteria.

[0024] The present invention has the following effects: (1) Nano Cu2S can adsorb and bind to the novel coronavirus, thereby inhibiting the binding of the virus to the receptor on the surface of the host cell, thus effectively inhibiting the infection of the novel coronavirus and its various variants; moreover, compared with other common plasma proteins, nano Cu2S has a stronger interaction with the receptor binding domain of the novel coronavirus and variants.

[0025] (2) Nano Cu2S can simultaneously inhibit the activity of two enzymes related to virus replication, namely the main protease of SARS-CoV-2 and papain, thus nano Cu2S can effectively inhibit the replication of SARS-CoV-2 and its various variants.

[0026] (3) Nano Cu2S has antibacterial activity and can inhibit the proliferation of a variety of bacteria; Nano Cu2S also has anti-drug-resistant bacteria activity and can inhibit the proliferation of methicillin-resistant Staphylococcus aureus, and can be used as an alternative to antibiotics.

[0027] (4) Nano Cu2S has low toxicity and good biological safety.

[0028] (5) The synthesis process of nano Cu2S is simple, and it can be stored at 4℃ for a short period of time and at -20℃ for a long period of time, making it suitable for large-scale preparation and production.

[0029] (6) Nano Cu2S can be widely used in coatings, disinfectants and other fields to inhibit the spread of viruses and bacteria. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 This is one of the transmission electron microscope images of nano-Cu2S provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0032] Figure 2 This is one of the hydration particle size distribution test results of nano-Cu2S provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0033] Figure 3 This is one of the Zeta potential test results of nano-Cu2S provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0034] Figure 4This is one of the bar charts showing the inhibitory effect of nano-Cu2S on cells infected by SARS-CoV-2 and its variants, provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection according to the present invention.

[0035] Figure 5 This is one of the transmission electron microscopy images of nano-Cu2S bound to the surface of the novel coronavirus, provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0036] Figure 6 This is one of the binding affinity test results between nano-Cu2S and wild-type SARS-CoV-2 RBD provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0037] Figure 7 This is one of the binding affinity test results between nano-Cu2S and Delta variant virus RBD provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0038] Figure 8 This is one of the binding affinity test results between nano-Cu2S and Omicron BA.1 mutant virus RBD provided in Example 1 of the application of nano-Cu2S in antiviral and antibacterial infection of the present invention.

[0039] Figure 9 This is one of the binding affinity test results between nano-Cu2S and Omicron BA.2 variant virus RBD provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0040] Figure 10 This is one of the binding affinity test results between nano-Cu2S and Omicron BA.5 variant virus RBD provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0041] Figure 11 This is one of the binding affinity test results between nano-Cu2S and other plasma proteins provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0042] Figure 12 This is one of the binding affinity test results between ACE2 protein and RBD involved in the application of nano-Cu2S provided by this invention in anti-COVID-19 infection and anti-bacterial infection.

[0043] Figure 13This is one of the results of the inhibition of the binding between different concentrations of RBD and ACE2 by nano-Cu2S provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection.

[0044] Figure 14 This is one of the results of the nano-Cu2S provided in Example 1 of the application of the present invention in anti-COVID-19 infection and anti-bacterial infection, which inhibits the replication of COVID-19 and its variants in cells.

[0045] Figure 15 This is one of the binding affinity results between nano-Cu2S and the main protease of the novel coronavirus, provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0046] Figure 16 This is one of the binding affinity results between nano-Cu2S and papain provided in Example 1 of the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection provided by the present invention.

[0047] Figure 17 This is one of the results of the effect of different concentrations of nano-Cu2S provided in Example 1 on the main protease activity of the novel coronavirus in the application of nano-Cu2S in anti-COVID-19 infection and anti-bacterial infection.

[0048] Figure 18 This is one of the results of the effect of different concentrations of nano-Cu2S provided in Example 1 on the papain activity of SARS-CoV-2, which is the application of nano-Cu2S provided by the present invention in anti-SARS-CoV-2 infection and anti-bacterial infection.

[0049] Figure 19 This is one of the minimum inhibitory concentration (MIC) test results of nano-Cu2S against Staphylococcus aureus provided in Example 1, which describes the application of nano-Cu2S provided by this invention in the fight against COVID-19 infection and bacterial infection.

[0050] Figure 20 This is one of the minimum inhibitory concentration (MIC) test results of nano-Cu2S against methicillin-resistant Staphylococcus aureus (MRSA) provided in Example 1, which describes the application of nano-Cu2S provided by this invention in the fight against COVID-19 infection and bacterial infection.

[0051] Figure 21 This is one of the minimum inhibitory concentration (MIC) test results of nano-Cu2S against Bacillus subtilis provided in Example 1, which describes the application of nano-Cu2S provided by this invention in the fight against COVID-19 infection and bacterial infection.

[0052] Figure 22 This is one of the minimum inhibitory concentration (MIC) test results of nano-Cu2S against Escherichia coli (E. coli) provided in Example 1, which describes the application of nano-Cu2S in the fight against COVID-19 infection and bacterial infection.

[0053] Figure 23 This is one of the minimum inhibitory concentration (MIC) test results of nano-Cu2S against Klebsiella pneumoniae provided in Example 1, which describes the application of nano-Cu2S provided by this invention in anti-COVID-19 infection and anti-bacterial infection.

[0054] Figure 24 This is one of the minimum inhibitory concentration (MIC) test results of nano-Cu2S against Acinetobacter baumannii provided in Example 1, which describes the application of nano-Cu2S provided by this invention in anti-COVID-19 infection and anti-bacterial infection.

[0055] Figure 25 This is one of the minimum inhibitory concentration (MIC) test results of nano-Cu2S against Pseudomonas aeruginosa provided in Example 1, which describes the application of nano-Cu2S provided by this invention in anti-COVID-19 infection and anti-bacterial infection at different concentrations.

[0056] Figure 26 This is one of the results of the effect of different concentrations of nano-Cu2S on the viability of Vero-E6 cells provided in Example 1 of the application of nano-Cu2S provided by the present invention in anti-COVID-19 infection and anti-bacterial infection. Detailed Implementation

[0057] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0058] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0059] In the following embodiments of the present invention, the information of each raw material / reagent is shown in the table below.

[0060] The instrument information used is as follows: Transmission electron microscope: Tecnai G2 20 S-TWIN, FEI Corporation, USA; Dynamic light scattering instrument: DLS, Malvern, UK; Biomolecular interaction analyzer: Octet RED96e, FortéBio, USA; Real-time PCR instrument: Eppendorf GmbH, Germany; Multi-mode microplate detection system: PerkinElmer, USA; Inductively coupled plasma mass spectrometry: ICP-MS, Thermo Fisher Scientific, USA; High-speed centrifuge: Thermo Fisher Scientific, USA; Carbon dioxide constant temperature incubator: Thermo Fisher Scientific, USA.

[0061] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0062] Example 1: Synthesis and Characterization of Cu2S This embodiment provides a method for preparing nano-Cu2S, specifically, nano-Cu2S is synthesized using copper sulfate pentahydrate and L-cysteine ​​as raw materials. Specifically, L-cysteine ​​(4.5 × 10⁻⁶) is used as a raw material... -4 mol), copper sulfate pentahydrate (1.5 × 10⁻⁶) -4 45.5 mL of ultrapure water and sodium hydroxide solution were added to a three-necked flask. The mixture was stirred at room temperature for 15 min to ensure complete dissolution. Sodium hydroxide solution was then added dropwise to adjust the pH to 10. Stirring continued for 10 minutes to ensure thorough mixing. The temperature was then raised to 60 °C, and the reaction was continued for 4 hours. The successful synthesis of nano-Cu₂S was indicated by a brownish-red color of the solution. The synthesized nano-Cu₂S was dialyzed against triple-distilled water for 48 hours, and the product was finally stored at 4 °C.

[0063] Transmission electron microscopy revealed that the nano-Cu₂S particles were spherical, with a size of approximately 20 nm. Figure 1 The particle size distribution and zeta potential of Cu₂S nanoparticles were detected using DLS, and the results showed that the average size of Cu₂S nanoparticles was approximately 21.66 ± 2.24 nm. Figure 2 Its surface is negatively charged, with an average Zeta potential of -26.7 mV. Figure 3 ).

[0064] Experiment 1: Verification of the Inhibition of SARS-CoV-2 Infection by Nano-Cu2S Experimental subjects: nano-Cu2S prepared in Example 1, wild-type SARS-CoV-2, Delta variant virus, OmicronBA.5 variant virus, Omicron BF.7 variant virus and Omicron BQ.1 variant virus, and Vero-E6 cells.

[0065] Experimental Methods: The ability of the nano-Cu2S provided in the examples to inhibit the infection of host cells by real SARS-CoV-2 and its variants was tested in a biosafety level 3 laboratory. The viral strains used for testing included wild-type SARS-CoV-2, Delta variant, Omicron BA.5 variant, Omicron BF.7 variant, and Omicron BQ.1 variant. Vero-E6 cells were simultaneously infected with the virus after mixing serum-free DMEM cell culture medium solutions with different concentrations of nano-Cu2S (0 μg / mL, 0.078 μg / mL, 0.156 μg / mL, 0.3125 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL). The viral load was 50 times the tissue half-maximal infectious dose (TCID). 50 After incubating in a cell culture incubator in a biosafety level 3 laboratory for 1 hour, viruses and nano-Cu2S were simultaneously removed. After incubating in a cell culture incubator in a biosafety level 3 laboratory for 24 hours, the content of viral RNA in the cells was detected using a real-time PCR instrument to evaluate the ability of nano-Cu2S to prevent viral infection of cells.

[0066] Experimental results: The results are as follows Figure 4 As shown, the nano-Cu2S provided in Example 1 can significantly inhibit the infection of cells by wild-type SARS-CoV-2 and four variant strains: Delta, Omicron BA.5, Omicron BF.7, and Omicron BQ.1.

[0067] Experimental Example 2: Mechanism of SARS-CoV-2 Inhibition by Nanoparticle Cu2S To investigate the mechanism by which nano-Cu2S inhibits the invasion of host cells by the novel coronavirus, the following is a detailed study.

[0068] Experimental subject: The system in Experiment 1, after mixing 10 μg / mL of nano-Cu2S serum-free DMEM cell culture medium solution with the virus and treating it for 1 hour in a cell culture incubator in a biosafety level 3 laboratory.

[0069] Test method: (1) First, the state of nano-Cu2S and the novel coronavirus was observed by TEM at 200 kV and magnified 80,000 times.

[0070] (2) The binding affinity of Cu2S with five viral RBDs and several common proteins in plasma (such as low-density lipoprotein, high-density lipoprotein, fibrinogen, immunoglobulins, human serum albumin, and transferrin) was detected using BLI. The specific method is as follows: PBSP (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, and 0.05% P20 surfactant) was used as the buffer for detecting the interaction between CIPS and proteins. First, Cu2S (50 μg / mL) was immobilized on the surface of the AR2G sensor chip for 180 s, with a fixation height of approximately 3 nm. Then, the sensor chip was immersed in different protein solutions (including the RBD of wild-type SARS-CoV-2, the RBDs of four variant SARS-CoV-2, HSA, FG, IgG, Tf, and HDL proteins). Subsequently, a binding time of 300 s and a dissociation time of 600 s were set to evaluate the binding affinity between them. In addition, the binding between ACE2 and RBD, as well as mixtures of different concentrations of nano-Cu2S and RBD, was detected using BLI. The specific method is as follows: 10 μg / mL of ACE2 was immobilized on the AR2G sensor via an amino coupling procedure. Then, the sensor was immersed in solutions of different concentrations of RBD (6.25, 12.5, 25, 50, 100 nM) or Cu2S-RBD complexes (the Cu2S concentration in the complexes was 1 μg / mL, and the RBD concentrations were 6.25, 12.5, 25, 50, 100, and 200 nM, respectively). The binding time was set to 150 s and the dissociation time to 300 s to evaluate the binding between them.

[0071] Experimental results: First, the state of nano-Cu2S mixed with the novel coronavirus was observed using TEM, such as... Figure 5 As shown, a large amount of nano-Cu2S is attached to the surface of the novel coronavirus, suggesting a potentially strong binding between the virus and nano-Cu2S. Furthermore, the binding affinity between nano-Cu2S and the RBDs of five viruses was detected using BLI, such as... Figure 6-11 As shown, nano-Cu2S resists wild-type SARS-CoV-2 RBD (… Figure 6 Delta variant virus RBD ( Figure 7 Omicron BA.1 variant virus RBD ( Figure 8 ), Omicron BA.2 variant virus RBD ( Figure 9 ) and Omicron BA.5 variant virus RBD ( Figure 10 All of them exhibited very high affinity for the target organism. K DThe values ​​are all less than 0.001 nM. However, the binding affinity between several common plasma proteins, such as low-density lipoprotein, high-density lipoprotein, fibrinogen, immunoglobulins, human serum albumin, and transferrin, and Cu2S is much weaker than that between nano-Cu2S and RBD. Figure 11 The protein concentration was 100 nM. This result confirms that nano-Cu2S can selectively and efficiently bind to the SARS-CoV-2 RBD.

[0072] The binding of ACE2 with mixtures of nano-Cu2S and RBD of different concentrations is as follows: Figure 12 and Figure 13 As shown. From Figure 12 It can be seen from this that there is a strong interaction between ACE2 and RBD. Figure 13 As can be seen, when nano-Cu2S at a concentration of 1 μg / mL was added to the RBD, the binding height between RBD and ACE2 at all concentrations decreased significantly, indicating that nano-Cu2S weakens the affinity between RBD and ACE2. This also shows that the nano-Cu2S provided in Example 1 can reduce the binding of the SARS-CoV-2 RBD to the receptor ACE2 of its host cells, thereby inhibiting viral infection.

[0073] Experimental Example 3: Verification of the Inhibition of SARS-CoV-2 Replication by Nanoparticle Cu2S Experimental subjects: Nano-Cu2S provided in Example 1, wild-type SARS-CoV-2, Delta variant virus, OmicronBA.5 variant virus, Omicron BF.7 variant virus and Omicron BQ.1 variant virus, and Vero-E6 cells.

[0074] Experimental method: One hour after infecting Vero-E6 cells with the virus, the virus was removed from the cell supernatant, and then Cu2S was added and incubated with the cells for 24 hours. After that, the viral RNA content in the cells was detected to evaluate the inhibitory effect of Cu2S on viral replication.

[0075] Test results: such as Figure 14 As shown, nano-Cu2S can significantly inhibit the replication of five strains of SARS-CoV-2, including wild-type SARS-CoV-2, Delta variant, Omicron BA.5 variant, Omicron BF.7 variant, and Omicron BQ.1 variant, in cells, but its effective antiviral concentration is very low.

[0076] Example 4: Mechanism of Cu2S in inhibiting SARS-CoV-2 replication Test subject: The novel coronavirus treated with nano-Cu2S in Test Example 3.

[0077] Experimental Methods: First, the binding affinity between nano-Cu2S and the main protease (Mpro) and papain (PLpro) of SARS-CoV-2 was tested using BLI. These two enzymes are involved in the replication process of SARS-CoV-2. Then, the effect of nano-Cu2S on the activity of the two replicase enzymes was tested based on fluorescence resonance energy transfer (FRET). Specifically, the inhibitory effect of nano-Cu2S on Mpro enzyme activity was evaluated using FRET. First, nano-Cu2S containing 100 nM... pro A detection system containing 20 μM fluorescent substrate MCA-AVLQSGFR-Lys(DNP)-Lys-NH2 (Shanghai Jier Biochemical Co., Ltd.) and 100 nM PLpro and 20 μM fluorescent substrate Z-RLRGG↓-AMC ("↓" indicates the restriction site) was used to evaluate the inhibitory effect of nano-Cu2S on Mpro and PLpro enzyme activities, respectively. Subsequently, different concentrations of nano-Cu2S were pre-incubated with Mpro or PLpro for 10 minutes. After pre-incubation, the fluorescent substrate was added to the mixture, and the fluorescence signal was immediately monitored continuously for 10 minutes at 320 nm (excitation) / 405 nm (emission) wavelengths at 30-second intervals using a microplate reader. The half-maximal inhibitory concentration (IC50) against Mpro was calculated. 50 ); The reaction signal was monitored for 10 minutes at 360nm (excitation) / 460nm (emission) wavelengths, and the IC for PLpro was calculated. 50 value.

[0078] Experimental results: The affinity test results between nano-Cu2S and the main protease (Mpro) and papain (PLpro) of the novel coronavirus are as follows: Figure 15 and Figure 16 As shown in the figure, the dissociation constants between nano-Cu2S and both replicase enzymes are less than 0.001 nM, indicating a very strong interaction between them. This strong interaction is likely to affect the catalytic activity of the two replicase enzymes. Further results on the effect of nano-Cu2S on the activity of the two replicase enzymes are shown below. Figure 17 and Figure 18 As shown, nano-Cu2S can significantly inhibit the activity of two replicase enzymes.

[0079] These results demonstrate that nano-Cu2S possesses an extremely strong ability to inhibit viral replication enzyme activity.

[0080] Evaluation of the antibacterial activity of nano-Cu2S in Experiment 5 Test subjects: Nano-Cu2S provided in Example 1 and bacteria, including Gram-positive bacteria such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus subtilis, as well as Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0081] Experimental Methods: Staphylococcus aureus and methicillin-resistant Staphylococcus aureus strains were inoculated onto solid LB agar plates and incubated overnight at 37°C. Single colonies from the agar plates were then transferred to 50 mL centrifuge tubes containing 20 mL of LB broth and incubated overnight at 37°C in a shaker (180 rpm). The bacteria were then diluted 1:100 with LB broth and incubated for 3 hours at 37°C until the early logarithmic growth phase. The bacteria were then used for subsequent minimum inhibitory concentration (MIC) testing. The bacteria in the early logarithmic growth phase were diluted to 1×10⁻⁶ with LB broth. 5 The concentrations of nano-Cu2S were measured at CFU / mL, and the MICs against different bacteria were determined using a microdilution method. Nano-Cu2S concentrations were set at 0, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / mL. For each concentration, five parallel wells and one control well (containing only the same concentration of material, without bacteria) were prepared. The 96-well plates were then incubated at 37°C for 24 hours. The absorbance at 600 nm was measured using a multi-mode microplate detection system, and the MICs against different bacteria were calculated based on the results.

[0082] Test results: such as Figure 19-25 As shown, nano-Cu2S exhibited excellent antibacterial activity against all seven bacteria mentioned above. The minimum inhibitory concentration (MIC) of nano-Cu2S against Staphylococcus aureus, Bacillus subtilis, Acinetobacter baumannii, and Pseudomonas aeruginosa was 2 μg / mL, against MRSA it was 4 μg / mL, and against Escherichia coli and Klebsiella pneumoniae it was 16 μg / mL. These results indicate that the nano-Cu2S provided in Example 1 possesses broad-spectrum antibacterial activity.

[0083] Toxicity evaluation of nanometer Cu2S in Experiment Example 6 Test subjects: Nano-Cu2S and Vero E6 cells provided in Example 1.

[0084] Experimental methods: Vero E6 cells were incubated with serum-free DMEM cell culture medium solutions containing different concentrations of nano-Cu2S (0 μg / ml, 0.625 μg / ml, 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml, and 160 μg / ml) for 24 hours, and their cytotoxicity was detected using a CCK-8 assay kit.

[0085] Test results: such as Figure 26 As shown, the results indicate that 0-160 μg / ml of nano-Cu2S had no significant toxicity to Vero E6 cells. This demonstrates that the nano-Cu2S provided in Example 1 has good biocompatibility and high safety.

[0086] In summary, the nano-Cu2S provided in Example 1 exhibits dual antiviral and antibacterial activities: the nano-Cu2S provided in Example 1 can selectively and efficiently bind to the receptor site RBD of SARS-CoV-2, thereby effectively inhibiting SARS-CoV-2 infection of host cells; the nano-Cu2S provided in Example 1 can effectively inhibit the activity of two enzymes related to virus replication, namely the main protease and papain of SARS-CoV-2, thereby effectively inhibiting the replication of SARS-CoV-2 and its various variants; the nano-Cu2S provided in Example 1 can inhibit the proliferation of various bacteria, exhibiting antibacterial activity, including activity against drug-resistant bacteria, and can inhibit the proliferation of methicillin-resistant Staphylococcus aureus, making it a viable alternative to antibiotics.

[0087] In summary, the nano-Cu2S provided in Example 1 has dual activities against the novel coronavirus and bacteria. It can be used to fight both novel coronavirus infection and bacterial infection. When used as a coating for frozen food packaging, it can effectively inhibit the replication and infectivity of the novel coronavirus, as well as effectively inhibit the replication of various bacteria, thus blocking the spread of the novel coronavirus and bacteria.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a drug for combating COVID-19 and bacteria, characterized in that, include: Negatively charged nano-Cu2S was prepared by reacting L-cysteine ​​and copper sulfate under alkaline conditions.

2. The method for preparing the drug for combating COVID-19 and antibacterial agents according to claim 1, characterized in that, The average particle size of the nano-Cu2S is 15~25nm.

3. The method for preparing the drug for combating COVID-19 and antibacterial agents according to claim 1 or 2, characterized in that, The copper sulfate is copper sulfate pentahydrate; the molar ratio of L-cysteine ​​to copper sulfate pentahydrate is (3-5):1; Preferably, the reaction temperature is 50-70°C; the reaction time is 4-6 hours.

4. The method for preparing the medicament for combating COVID-19 and antibacterial agents according to any one of claims 1-3, characterized in that, The nano-Cu2S is obtained by dialysis after reacting L-cysteine ​​and copper sulfate in an aqueous solution with a pH of 9-11. Preferably, the dialysis medium is water, and the dialysis time is 40-60 hours.

5. The method for preparing the medicament for combating COVID-19 and antibacterial agents according to any one of claims 1-4, characterized in that, The zeta potential of the nano-Cu2S is -20 to -35 mV.

6. The method for preparing the medicament for combating COVID-19 and antibacterial agents according to any one of claims 1-5, characterized in that, The nano-Cu2S inhibits viral infection of the host by binding to the RBD protein of the SARS-CoV-2 virus.

7. The method for preparing the medicament for combating COVID-19 and antibacterial agents according to any one of claims 1-6, characterized in that, The nano-Cu2S and the main protease M of the SARS-CoV-2 virus pro and papain PL pro The nano-Cu2S binds to the main protease M of the SARS-CoV-2 virus to inhibit viral replication. pro and papain PL pro The dissociation constants between them are all less than 0.001 nM.

8. The method for preparing a medicament for combating COVID-19 and antibacterial agents according to any one of claims 1-7, characterized in that, The SARS-CoV-2 virus is selected from at least one of the following: wild-type SARS-CoV-2, Delta variant SARS-CoV-2, Omicron BA.5 variant SARS-CoV-2, Omicron BF.7 variant SARS-CoV-2, and Omicron BQ.1 variant SARS-CoV-2.

9. The method for preparing a medicament for combating COVID-19 and antibacterial agents according to any one of claims 1-8, characterized in that, The bacteria are selected from at least one of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

10. A drug for treating COVID-19 and for treating bacteria, prepared by any one of claims 1-9.

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