Sialic acid modified CuS anti-influenza virus H3N2 nanomaterial and its preparation method and application

Abstract

The present invention belongs to the field of nano-functional materials, in particular to a sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial and its preparation method and application. The prepared sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial is based on CuS, NH2-PEG-SH is loaded on its surface, lactobionic acid molecules are connected by bonding reaction, lactobionic acid molecules are obtained, and sialic acid molecules are transferred to the surface of lactobionic CuS by sialic acid substrate and sialyltransferase; the functional nanomaterial can better achieve antiviral effect, the sialic acid molecules loaded by the material can target influenza virus hemagglutinin, competitively bind to influenza virus with host cells, achieve high targeting ability, can more effectively inactivate viruses, and prevent virus transmission in daily protective products such as masks, protective clothing, and disinfectant mouthwash. The functional nanomaterial has the advantages of good biosafety, strong targeting effect, and strong antiviral ability.

Description

Technical Field

[0001] The invention relates to the field of nano functional materials, in particular to a sialic acid modified CuS anti-influenza virus H3N2 nano material and a preparation method and application thereof. Background Art

[0002] Influenza virus is a respiratory infectious virus. A few strains can cause pandemics worldwide, which is extremely harmful. Influenza virus infection poses a threat to human health not only in the form of severe infection or even death, but also because influenza virus can mutate frequently, mutating into highly pathogenic strains or acquiring the potential threat of rapid transmission between animals and humans, and between people. At present, drugs for influenza virus include neuraminidase inhibitors and adamantane, but due to the high variability of the virus, it has developed widespread resistance to virus inhibitors. Therefore, vaccination is particularly important. The existing vaccines lack specificity for the prevention of new virus strains, resulting in poor clinical prevention or treatment effects of influenza virus. In addition, the production of specific influenza virus vaccines still cannot meet the urgent global demand. At the same time, the complications and uncertainties caused by vaccines force some people to refuse vaccination, further exacerbating the spread of the epidemic. In contrast, "prevention is better than cure", and curbing the spread of the virus is a more economical and effective prevention and control measure. The best method is physical protection and the use of protective equipment, such as masks, protective clothing, and disinfectant mouthwash to prevent the spread of the virus in the community.

[0003] Influenza virus hemagglutinin (HA) is a key protein that mediates the virus's invasion of host cells and is also the main target of anti-influenza virus neutralizing antibodies. Studies have shown that influenza viruses must first enter host cells through hemagglutinin (HA) and bind to sialic acid (Sa) receptors on the host cell membrane before they can replicate and spread. Therefore, sialic acid (Sa) is selected as a modifier to reduce viral infection and spread by competitively binding influenza viruses with host cells, opening up new ideas for the treatment of viral infections.

[0004] Therefore, in order to make protective materials have antiviral and self-disinfection functions, adding nanomaterials with anti-influenza virus properties to masks, protective clothing, disinfectant mouthwash and other materials provides a new idea for curbing the spread of influenza viruses. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial and a preparation method and application thereof.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] One of the purposes of the present invention is to provide a method for preparing a sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial, comprising the following steps:

[0008] S1. Add a 300 mmol / L copper chloride dihydrate solution and a 1 mol / L sodium sulfide nonahydrate solution in a volume ratio of 10:3 to a 100 mg / mL polyvinyl pyrrolidone solution twice the volume of the copper chloride dihydrate solution, and stir magnetically at room temperature for 2 to 10 minutes to obtain a mixed solution;

[0009] S2, the mixed solution was transferred into a 90°C water bath and magnetic stirring was continued for 10-30 min, the obtained mixed solution was transferred into an ice water bath for 5-10 min, and the precipitate was collected by centrifugation to obtain CuS nanoparticles;

[0010] S3, dissolving CuS nanoparticles in ultrapure water to prepare a 1 mg / mL CuS solution, mixing the NH2-PEG-SH solution with the CuS solution at a volume ratio of 1:5, standing at low temperature for 24-48 hours, and collecting the precipitate by centrifugation to obtain CuS-PEG;

[0011] S4, dissolving CuS-PEG in ultrapure water to prepare a CuS-PEG solution with a concentration of 1 mg / mL, mixing the CuS-PEG solution with a volume ratio of 50:2:7, the lactobionic acid solution with a concentration of 100 mmol / L, and the EDC solution with a concentration of 1 mg / mL, and placing the mixture in a constant temperature shaker for reaction for 1 to 5 hours, and collecting the precipitate by centrifugation to obtain CuS-Gla;

[0012] S5. Dissolve CuS-Gla in ultrapure water to prepare a 1 μM CuS-Gla solution. Mix the CuS-Gla solution with a volume ratio of 5:2:5, a sialic acid substrate with a concentration of 3 mmol / L, and a sialyltransferase with a concentration of 1 U / mL. Place the mixture in a constant temperature shaker to react for 2 to 8 hours. Collect the precipitate by centrifugation to obtain the nanomaterial CuS-Sa.

[0013] In the present invention, the molecular formula of lactobionic acid is: 12 H 22 O 12 , the structural formula is as follows:

[0014] ;

[0015] In the present invention, the molecular formula of the sialic acid substrate (CMP-Neu5Ac) is: 20 H 31 N4NaO 16 P, the structural formula is as follows:

[0016] ;

[0017] In the present invention, the molecular formula of the sialyltransferase (α-2,3-Sialytransferase) is: 28 H 45 NO6.

[0018] Preferably, the temperature of the constant temperature shaker is 37°C.

[0019] Preferably, the centrifugal speed is 12000 rpm, the centrifugal temperature is 4°C, and the centrifugal time is 20 min.

[0020] The second purpose of the present invention is to provide a sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial prepared by the above method.

[0021] The third object of the present invention is to provide a sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial for use in the preparation of medical protective masks and / or protective clothing and / or disinfectant mouthwash.

[0022] Compared with the prior art, the sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial prepared by the present invention is based on CuS as the core, NH2-PEG-SH is loaded on its surface, lactobionic acid molecules are connected through a bonding reaction to obtain lactobionic CuS, and sialic acid molecules are transferred to the surface of lactobionic CuS through sialic acid substrates and sialyltransferases; the functional nanomaterial can better achieve the antiviral effect, the sialic acid molecules loaded on the material can target influenza virus hemagglutinin, competitively bind to influenza virus with host cells, achieve high targeting ability, and can more effectively inactivate the virus, and prevent virus transmission in daily protective products such as masks, protective clothing, and disinfectant mouthwash. The functional nanomaterial has the advantages of good biosafety, strong targeting effect, and strong antiviral ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Scanning electron microscope (SEM) images of CuS, CuS-Gla, and CuS-Sa: A, B, and C are CuS, CuS-Gla, and CuS-Sa, respectively.

[0024] Figure 2 This is the X-ray diffraction (XRD) pattern of CuS.

[0025] Figure 3 The hydrated particle size diagram of CuS, CuS-PEG, CuS-Gla, and CuS-Sa was measured by dynamic light scattering (DLS) technology.

[0026] Figure 4 Zeta potential diagram of CuS, CuS-PEG, CuS-Gla, and CuS-Sa.

[0027] Figure 5 Energy dispersive X-ray spectroscopy (EDS) diagram of CuS nanoparticles: a is the Cu element distribution diagram; b is the S element distribution diagram; c is the surface total spectrum of Cu and S elements.

[0028] Figure 6 Energy dispersive X-ray spectroscopy (EDS) diagram of CuS-PEG nanoparticles: a is the Cu element distribution diagram; b is the S element distribution diagram; c is the C element distribution diagram; d is the N element distribution diagram; e is the O element distribution diagram; f is the surface total spectrum of Cu, S, C, N, and O elements.

[0029] Figure 7 Energy dispersive X-ray spectroscopy (EDS) diagram of CuS-Gla nanoparticles: a is the Cu element distribution diagram; b is the S element distribution diagram; c is the C element distribution diagram; d is the N element distribution diagram; e is the O element distribution diagram; f is the surface total spectrum of Cu, S, C, N, and O elements.

[0030] Figure 8 Energy dispersive X-ray spectroscopy (EDS) diagram of CuS-Sa nanoparticles: a is the Cu element distribution diagram; b is the S element distribution diagram; c is the C element distribution diagram; d is the N element distribution diagram; e is the O element distribution diagram; f is the surface total spectrum of Cu, S, C, N, and O elements.

[0031] Fig. 9 The figure shows the cytotoxicity assay of CuS, CuS-PEG, CuS-Gla and CuS-Sa on MDCK cells using MTT.

[0032] Fig.10 These are the results of the virus hemagglutination test: A is the virus hemagglutination test, B is the virus hemagglutination inhibition test of CuS-Sa at different concentrations, and C is the comparison of the virus hemagglutination inhibition of CuS, CuS-Gla and CuS-Sa.

[0033] Fig.11The results of CuS-Sa inhibiting the cytopathic effect (CPE) of MDCK cells caused by H3N2: A is the normal cell control group; B is the CuS-Sa-treated virus group; C is the virus control group; D is the culture medium-treated virus group.

[0034] Fig.12 This is the MTT result diagram of direct inactivation of viruses by CuS, CuS-Gla, and CuS-Sa in vitro.

[0035] Fig.13 This is the MTT result diagram of the influenza virus adsorption to cells after the action of CuS, CuS-Gla, and CuS-Sa.

[0036] Fig.14 This is the MTT result diagram of the influenza virus invading cell stage after the action of CuS, CuS-Gla, and CuS-Sa.

[0037] Fig.15 This is the MTT result diagram of the influenza virus progeny replication stage in cells after the action of CuS, CuS-Gla, and CuS-Sa.

[0038] Fig.16 Transmission Electron Microscope (TEM) images of CuS, CuS-Gla, CuS-Sa and virus: A is the TEM negative staining image of CuS; B is the TEM negative staining image of CuS-Gla; C is the TEM negative staining image of CuS-Sa; D is the TEM negative staining image of H3N2.

[0039] Fig.17 These are transmission negative staining electron micrographs of the effects of CuS, CuS-Gla, CuS-Sa and virus: A is the TEM negative staining image of the virus after 30 minutes of CuS-Sa action; B is the TEM negative staining image of the virus after 60 minutes of CuS-Sa action; C is the TEM negative staining image of the virus after 120 minutes of CuS-Sa action; D is the TEM negative staining image of the virus after 120 minutes of CuS action; E is the TEM negative staining image of the virus after 120 minutes of CuS-Gla action. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0041] Example 1

[0042] This example provides the synthesis of CuS.

[0043] Dissolve 1g PVP in 10mL ultrapure water for later use; dissolve 0.25g copper chloride dihydrate in 5mL ultrapure water; add 5mL copper chloride dihydrate solution to the PVP solution; dissolve 0.36g sodium sulfide nonahydrate in 1.5mL ultrapure water; add 1.5mL sodium sulfide nonahydrate solution to the above PVP solution, stir magnetically at room temperature for 5min, transfer to a 90℃ water bath and continue magnetic stirring for 15min to obtain a dark green solution, then transfer the solution to an ice water bath for 5min, centrifuge at 4℃, 12000 rpm for 15min to collect the precipitate, wash three times with ultrapure water, and dry in a vacuum oven at 40℃ for 48h to obtain a black powder, which is CuS.

[0044] The CuS nanoparticles of the present invention were observed microscopically using a scanning electron microscope. Figure 1 As shown in A, the CuS nanoparticles are spherical, aggregated on the surface, and relatively uniform in size.

[0045] Example 2

[0046] This example provides the synthesis of CuS-PEG.

[0047] 2 mg of NH2-PEG-SH was dissolved in 10 mL of 1 mg / mL CuS solution and allowed to stand at 4°C for 36 h. The solution was centrifuged at 12,000 rpm for 15 min at 4°C, washed three times with ultrapure water, and stored at 4°C for later use.

[0048] Example 3

[0049] This example provides the synthesis of CuS-Gla.

[0050] Dissolve 20 mL of CuS-PEG in ultrapure water for later use; dissolve 10 mg of lactobionic acid in 10 mL of ultrapure water; dissolve 10 mg of EDC in 10 mL of ultrapure water; add 200 μL of lactobionic acid solution and 700 μL of EDC solution to 5 mL of CuS-PEG solution, mix well, react at 37°C for 2 hours in a constant temperature shaker, centrifuge at 12000 rpm, 4°C for 20 min, wash three times with ultrapure water, dissolve in 10 mL of ultrapure water and store at 4°C for later use.

[0051] The CuS-Gla nanoparticles of the present invention were observed microscopically using a scanning electron microscope. Figure 1 As shown in B, CuS-Gla nanoparticles are also spherical and have a relatively uniform size.

[0052] Example 4

[0053] This example provides the synthesis of CuS-Sa.

[0054] Add 20 μL (3 mmol / L) sialic acid substrate CMP-Neu5Ac and 50 μL sialyltransferase (α-2,3-Sialytransferase) (1U / mL) to 50 μL CuS-Gla (1 μM) solution, mix well, react at 37°C in a constant temperature shaker for 4 hours, centrifuge at 12000 rpm, 4°C for 20 min, and wash three times with ultrapure water to obtain CuS-Sa nanoparticles.

[0055] The CuS-Sa nanoparticles of the present invention were observed microscopically using a scanning electron microscope. Figure 1 As shown in Figure C, CuS-Sa nanoparticles are also spherical and have a relatively uniform size.

[0056] Example 5

[0057] This example provides the characterization of CuS-Sa.

[0058] The same mass of CuS, CuS-Gla and CuS-Sa were weighed and dissolved in the same volume of ultrapure water. The morphology and size distribution of the nanoparticles were observed by scanning electron microscopy. Figure 1 It can be seen that the three nanoparticles of CuS, CuS-Gla and CuS-Sa are spherical and uniform in size. After the PEG groups are introduced on the surface of CuS nanoparticles, the aggregation is improved.

[0059] Weigh a certain amount of CuS and dissolve it in ultrapure water, and obtain its diffraction peak by X-ray diffractometer. Figure 2 The diffraction peaks shown are similar to those of the standard Joint Committee Card 79-2321. The relatively broad diffraction peaks reflect the small size of the CuS crystals. No obvious impurity peaks were detected, indicating that high-quality CuS was obtained.

[0060] The same mass of CuS, CuS-PEG, CuS-Gla, and CuS-Sa were weighed and dissolved in the same volume of ultrapure water. The hydrated particle size and distribution of the four nanoparticles were measured by dynamic light scattering. Figure 3 It can be seen that the hydrated particle size of CuS is 78 nm, the hydrated particle size of CuS-PEG is 58 nm, the hydrated particle size of CuS-Gla is 68 nm, and the hydrated particle size of CuS-Sa is 91 nm. Compared with CuS nanoparticles, the surface of CuS-PEG nanoparticles is modified with PEG groups, which makes the hydrated particle size smaller. This may be because the CuS nanoparticles themselves are aggregated. After being modified with PEG groups, the aggregation of nanoparticles is improved and the dispersibility is improved. As the number of modified groups increases, the hydrated particle size of nanoparticles continues to increase, which indirectly proves that the groups are modified on the surface of CuS nanoparticles.

[0061] Weigh the same mass of CuS, CuS-PEG, CuS-Gla, and CuS-Sa, dissolve them in the same volume of ultrapure water, and measure the potential of the four nanoparticles using a Zeta potential analyzer. Figure 4 It can be seen that the potential of CuS is -4 mV, the potential of CuS-PEG is -6 mV, the potential of CuS-Gla is -24 mV, and the potential of CuS-Sa is -26 mV. After the surface of CuS nanoparticles is modified with PEG, the absolute value of the potential becomes larger. With the successive modification of lactobionic acid and sialic acid molecules (acidic groups carry negative charges), the negative potential continues to increase, which indirectly proves that the groups are modified on the surface of CuS nanoparticles.

[0062] Weigh the same mass of CuS, CuS-PEG, CuS-Gla, and CuS-Sa and dissolve them in the same volume of ultrapure water. Use scanning electron microscopy to analyze the elemental composition of the four nanoparticles. Figure 5 It can be seen that the Cu element accounts for about 75% and the S element accounts for about 25% in the CuS nanoparticles; Figure 6 It can be seen that the CuS-PEG nanoparticles contain 9.64% Cu, 4.64% S, 39.66% C, 7.23% N, and 38.83% O. Figure 7 It can be seen that the CuS-Gla nanoparticles contain 13.25% Cu, 5.08% S, 56.86% C, 10.32% N and 14.5% O elements. Figure 8 It can be seen that the Cu element detected in the CuS-Sa nanoparticles accounts for 2.35%, the S element accounts for 1.92%, the C element accounts for 55.54%, the N element accounts for 19.62%, and the O element accounts for 20.58%. CuS nanoparticles only contain Cu and S elements. As PEG modifies the surface of CuS nanoparticles with C, N, and O elements, it shows that PEG is modified on the surface of CuS. Lactobionic acid and sialic acid are introduced according to the bond reaction. The element content on the surface of the nanoparticles changes continuously. The hydrated particle size and potential results prove that the nanoparticles are successfully synthesized and the sialic acid groups are successfully modified on the surface of the CuS nanoparticles.

[0063] Example 6

[0064] This example provides the cytotoxic effect of CuS-Sa on MDCK cells.

[0065] The MTT method was used to detect the toxicity of nanoparticles to MDCK cells. Four groups were set up: CuS, CuS-PEG, CuS-Gla, and CuS-Sa. The results are shown in Fig. 9When the concentration of CuS, CuS-PEG, CuS-Gla, and CuS-Sa nanoparticles was 100 μg / mL, the cell viability was above 50%, and the toxic side effects of the four nanoparticles on MDCK cells increased with increasing concentration.

[0066] Example 7

[0067] This example provides a hemagglutination test of influenza virus H3N2 on red blood cells.

[0068] The hemagglutination titer of influenza virus was determined by influenza virus hemagglutination test. First, add 50 μL of saline to each column of the hemagglutination plate, then add 50 μL of H3N2 virus solution to the first column and mix by pipetting, and then dilute each column from the first column. Take 50 μL of liquid from the first column and add it to the next column and mix by pipetting, and then dilute to the eleventh column in sequence. Take 50 μL of liquid from the eleventh column and discard it. The twelfth column is used as a blank control, and only 50 μL of saline is added. Finally, add 50 μL of 1% chicken red blood cells to each column, shake immediately for 1 minute, and observe the results after standing for 30 minutes. Results are shown in Fig.10 In A, the influenza virus hemagglutination titer is 1:16.

[0069] Example 8

[0070] This example provides an influenza virus hemagglutinin inhibition assay.

[0071] The inhibition of H3N2 hemagglutination by nanoparticles was determined by the influenza virus hemagglutination inhibition experiment. The influenza virus solution in the hemagglutination plate was diluted in multiples as in Example 7. Different dilutions of CuS-Sa nanoparticles were added to each column and gently mixed. After 120 min, an equal amount of 1% chicken red blood cells was added to each column, and a blank control was set up: only red blood cells and PBS were added. The results are shown in Fig.10 In Figure B, 100 μg / mL and 75 μg / mL of CuS-Sa nanoparticles completely inhibited the agglutination of H3N2 to erythrocytes, 25 μg / mL and below did not inhibit the agglutination of H3N2 to erythrocytes, and 50 μg / mL of CuS-Sa nanoparticles did not inhibit the agglutination of influenza virus H3N2 to erythrocytes, which may be due to the different amounts of sialic acid molecules loaded on different CuS-Sa nanoparticles; Fig.10 C in the figure is the result of CuS, CuS-Gla and CuS-Sa nanoparticles at a concentration of 100 μg / mL inhibiting the agglutination of H3N2 to erythrocytes. It can be seen that CuS and CuS-Gla nanoparticles at the maximum concentration cannot inhibit the agglutination of H3N2 to erythrocytes.

[0072] Example 9

[0073] This example provides a graph showing the results of CuS-Sa inhibiting H3N2-induced MDCK cell pathological changes. After 100 μg / mL of CuS-Sa and H3N2 were used for 2 hours, the supernatant was taken to infect cells, and the pathological changes of MDCK were observed under a fluorescence microscope. A normal cell group, a virus control group, and a cell group in which the supernatant was taken after the virus was treated with a single culture medium were set up. The results are shown in Fig.11 No obvious pathological changes were observed in MDCK in the normal cell group and the CuS-Sa treated group. However, obvious pathological changes were observed in MDCK in the virus control group and the virus treated group with culture medium alone. The cells became round, the gaps widened, and they dissolved and died, showing obvious grape-like appearance.

[0074] Example 10

[0075] This example provides a study on the mechanism of CuS-Sa against influenza virus H3N2

[0076] Nanoparticles directly inactivate viruses in vitro. Mix different dilutions of CuS, CuS-Gla, and CuS-Sa with an equal volume of 100TCID50 H3N2 virus solution for 120 minutes, then take 100 μL of the mixture per well and add it to a 96-well plate filled with MDCK cells. Gently shake the culture plate every 15 minutes, discard the supernatant after 120 minutes of full action, wash it three times with PBS, add 100 μL of cell maintenance solution to each well of the 96-well plate for culture, set up a solvent control group (cells infected after the culture medium and virus reacted) and a virus control group, and observe that the cell pathological changes in the virus control group reached more than 75%, and the cell viability was determined by the MTT method. Results are shown in Fig.12 After the action of CuS, CuS-Gla, and CuS-Sa nanoparticles, all three nanoparticles showed concentration-dependent antiviral effects.

[0077] Nanoparticles inhibit H3N2 adsorption to cells. 100 μL of CuS, CuS-Gla, and CuS-Sa of different dilutions were added to each well of a 96-well plate filled with MDCK cells. After 120 minutes, the supernatant was discarded, and the cells were washed 3 times with PBS. 100 μL of 100TCID50 H3N2 was added to each well of the 96-well plate for 120 minutes, the supernatant was discarded, and the cells were washed 3 times with PBS. 100 μL of cell maintenance solution was added to each well of the 96-well plate for culture. The cell lesions in the virus control group were observed to be more than 75%, and the cell viability was determined by the MTT method. The results are shown in Fig.13After the cells were pretreated with CuS-Sa nanoparticles before the virus adsorbed to the cells, the cell survival rates of 100 μg / mL-10 μg / mL were all above 80%, and the cell survival rate at a concentration of 100 μg / mL reached 92%; the cell survival rates of CuS and CuS-Gla nanoparticles at each concentration were all above 50%, and the cell survival rates at a concentration of 100 μg / mL reached 70% and 72%, respectively, indicating that the three nanoparticles have excellent preventive effects on the H3N2 adsorption cell stage.

[0078] Nanoparticles inhibit the invasion of H3N2 into cells. 100 μL of 100TCID50 H3N2 was added to each well of a 96-well plate filled with MDCK cells for 60 minutes, the supernatant was discarded, and the cells were washed three times with PBS. 100 μL of CuS, CuS-Gla, and CuS-Sa of different dilutions were added to each well of the 96-well plate for 120 minutes, the supernatant was discarded, and the cells were washed three times with PBS. 100 μL of cell maintenance solution was added to each well of the 96-well plate for culture. The cell lesions in the virus control group were observed to be more than 75%, and the cell viability was determined by the MTT method. Results are shown in Fig.14 CuS-Sa nanoparticles were used to treat cells after the virus adsorbed the cells. The cell survival rates of 100 μg / mL-10 μg / mL were all above 55%, and the cell survival rate of 100 μg / mL concentration reached 74%; the cell survival rates of CuS and CuS-Gla nanoparticles at various concentrations were all above 45%, and the cell survival rates of 100 μg / mL concentration reached 53% and 55%, respectively, indicating that the three nanoparticles had general therapeutic effects on the H3N2 invasion stage of cells.

[0079] Nanoparticles inhibit the replication stage of H3N2 in cells. 100 μL of 100TCID50 H3N2 was added to each well of a 96-well plate filled with MDCK cells for 120 min, the supernatant was discarded, and the cells were washed 3 times with PBS. 100 μL of CuS, CuS-Gla, and CuS-Sa of different dilutions were added to each well of the 96-well plate for 120 min, the supernatant was discarded, and the cells were washed 3 times with PBS. 100 μL of cell maintenance solution was added to each well of the 96-well plate for culture. The cell lesions in the virus control group were observed to be more than 75%, and the cell viability was determined by the MTT method. The results are shown in Fig.15After treatment with CuS-Sa nanoparticles, the cell survival rates of 100 μg / mL-10 μg / mL were all above 40%, and the cell survival rate at 100 μg / mL reached 60%; the cell survival rates of CuS and CuS-Gla nanoparticles at each concentration were all above 35%, the cell survival rate at 10 μg / mL was below 40%, and the cell survival rates at 100 μg / mL reached 47% and 48%, respectively, indicating that the therapeutic effects of the three nanoparticles on the replication stage of H3N2 in cells are poorer than those on adsorption and invasion.

[0080] Embodiment 11

[0081] This example provides transmission electron microscopy images of three nanoparticles and viruses.

[0082] The same mass of CuS, CuS-Gla and CuS-Sa were weighed and dissolved in the same volume of ultrapure water. The morphology and size distribution of nanoparticles and viruses were observed by transmission electron microscopy. The results are shown in Fig.16 The three nanoparticles have good dispersion. The electron microscope scale is 100 nm, and the small size of the nanoparticles can be seen. The sizes of CuS, CuS-Gla, and CuS-Sa nanoparticles are 50±9.2 nm, 55±7.4 nm, and 62±5.5 nm, respectively. The influenza virus morphology is complete, and the viral envelope and matrix can be clearly observed. The size is about 100-130 nm, which is consistent with the morphology and characteristics of H3N2.

[0083] Example 12

[0084] This example provides transmission negative staining electron microscopy images of nanoparticles acting on viruses.

[0085] The same volume of 100 μg / mL CuS-Sa nanoparticles and H3N2 virus solution were fully reacted for 30 min, 60 min, and 120 min, and then negatively stained on a carbon film copper grid and observed by transmission electron microscopy. Fig.17 After 30 minutes of action of CuS-Sa nanoparticles and H3N2, the virus morphology remained intact. After 60 minutes of action, partial defects appeared in the H3N2 outer membrane. After 120 minutes of action, the virus morphology became blurred and the structure was destroyed. CuS-Sa nanoparticles may destroy the virus function by destroying the morphological structure of H3N2; while the virus structure remained intact after 120 minutes of action of 100 μg / mL CuS and CuS-Gla.

[0086] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial, characterized in that: The following steps are involved: S1. Add a 300 mmol / L copper chloride dihydrate solution and a 1 mol / L sodium sulfide nonahydrate solution in a volume ratio of 10:3 to a 100 mg / mL polyvinyl pyrrolidone solution twice the volume of the copper chloride dihydrate solution, and stir magnetically at room temperature for 2 to 10 minutes to obtain a mixed solution; S2, the mixed solution was transferred into a 90°C water bath and magnetic stirring was continued for 10-30 min, the obtained mixed solution was transferred into an ice water bath for 5-10 min, and the precipitate was collected by centrifugation to obtain CuS nanoparticles; S3, dissolving CuS nanoparticles in ultrapure water to prepare a 1 mg / mL CuS solution, mixing the NH2-PEG-SH solution with the CuS solution at a volume ratio of 1:5, standing at low temperature for 24-48 hours, and collecting the precipitate by centrifugation to obtain CuS-PEG; S4, dissolving CuS-PEG in ultrapure water to prepare a CuS-PEG solution with a concentration of 1 mg / mL, mixing the CuS-PEG solution with a volume ratio of 50:2:7, the lactobionic acid solution with a concentration of 100 mmol / L, and the EDC solution with a concentration of 1 mg / mL, and placing the mixture in a constant temperature shaker for reaction for 1 to 5 hours, and collecting the precipitate by centrifugation to obtain CuS-Gla; S5. Dissolve CuS-Gla in ultrapure water to prepare a 1 μM CuS-Gla solution. Mix the CuS-Gla solution with a volume ratio of 5:2:5, the sialic acid substrate CMP-Neu5Ac with a concentration of 3 mmol / L, and the sialyltransferase with a concentration of 1 U / mL. Place the mixture in a constant temperature shaker to react for 2 to 8 hours. Collect the precipitate by centrifugation to obtain the nanomaterial CuS-Sa.

2. The method for preparing the sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial according to claim 1, characterized in that: The temperature of the constant temperature shaker was 37°C.

3. The method for preparing the sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial according to claim 1, characterized in that: The centrifugal speed is 12000 rpm, the centrifugal temperature is 4°C, and the centrifugal time is 20 min.

4. A sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial prepared by the method according to any one of claims 1 to 3.

5. Use of the sialic acid-modified CuS anti-influenza virus H3N2 nanomaterial as claimed in claim 4 in the preparation of medical protective masks and / or protective clothing and / or disinfectant mouthwash.

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