Nano-silver tetrahedron lysozyme compound and preparation method thereof

Abstract

The invention relates to a nano-silver tetrahedron lysozyme compound as well as a preparation method and application thereof. The compound is a nano particle formed by chemical connection of silver tetrahedron (Ag-t) nano particles and sulfydryl (-SH) modified lysozyme in an aqueous medium through silver-sulfydryl interaction. According to the compound, the antibacterial performance of the lysozyme can be remarkably improved through the silver tetrahedron, the antibacterial spectrum of the lysozyme is expanded, and the compound also has a good antibacterial effect on drug-resistant bacteria and can be applied to skin wounds infected by bacteria to promote wound healing.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly to a silver nanometer tetrahedron lysozyme complex and a preparation method thereof. Background Art

[0002] Bacterial infection refers to the invasion and colonization of pathogenic bacteria or opportunistic pathogens in the blood circulation system, followed by reproduction and release of toxins and other metabolites, thereby triggering acute systemic infection, which may threaten life in severe cases. At present, antibiotics are still the main treatment means for dealing with bacterial infections. However, the excessive and improper use of antibiotics has led to the emergence of some drug-resistant bacteria. With the spread of drug-resistant bacteria, people have gradually realized that if antibacterial drugs that are not easily resistant to bacteria cannot be developed, there may be a situation where there is no medicine to treat in the future. In this case, lysozyme, as a natural antibacterial component, has attracted the attention of scientific researchers.

[0003] Lysozyme (abbreviated as Lys), also known as muramidase or N-acetylmuramide glycanohydrlase, is a naturally occurring antibacterial protein that is widely distributed in human body fluids such as saliva, tears, nasal mucus, and breast milk, and also exists in many animals and plants. It kills bacteria by breaking the β-1,4 glycosidic bond of peptidoglycan in the bacterial cell wall, resulting in the leakage of cell contents. Lysozyme has a particularly strong antibacterial effect on Gram-positive bacteria. However, due to the different compositions of cell walls, although lysozyme has a good antibacterial effect on Gram-positive bacteria when used alone, its killing effect on Gram-negative bacteria is very poor, and its killing effect on some drug-resistant bacteria is also relatively limited.

[0004] Therefore, improving the antibacterial ability of lysozyme and expanding its antibacterial spectrum are crucial for expanding the application range and effect of lysozyme, especially for enhancing the antibacterial effect on drug-resistant bacteria. Summary of the Invention

[0005] In view of the above deficiencies of the prior art, the present invention provides a silver nanometer tetrahedron lysozyme complex and a preparation method thereof. The complex can significantly improve the antibacterial performance of lysozyme by using silver tetrahedrons, expand its antibacterial spectrum, and has a good antibacterial effect on drug-resistant bacteria. At the same time, it can be applied to skin wounds infected with bacteria to promote wound healing.

[0006] To achieve the above object, a first aspect of the present invention provides a nano silver tetrahedron lysozyme complex, which is a nanoparticle formed by connecting silver tetrahedron (Ag-t) nanoparticles and mercapto (-SH)-modified lysozyme through silver-mercapto interaction. Among them, the mercapto group is introduced through a modifying molecule NHS-PEG 1000 -SH, and NHS- is an N-hydroxysuccinimide functional group.

[0007] The lysozyme includes plant lysozyme, animal lysozyme, microbial lysozyme, and egg white lysozyme, preferably egg white lysozyme.

[0008] Furthermore, the mercapto-modified lysozyme is SH-PEG 1000 -Lys, where Lys represents lysozyme.

[0009] A second aspect of the present invention provides a method for preparing the above nano silver tetrahedron lysozyme complex, and the method includes the following steps:

[0010] S1. Prepare a uniformly dispersed silver sol wrapped with sodium tartrate;

[0011] S2. Add sodium citrate to the prepared silver sol and perform light irradiation to obtain silver tetrahedron nanoparticles (Ag-t);

[0012] S3. Modification reaction: React the NHS-functional group of the modifying molecule NHS-PEG 1000 -SH with the amino functional group outside the lysozyme to obtain the mercapto-modified lysozyme SH-PEG 1000 -Lys; Linking reaction: Link by mixing and reacting the silver tetrahedron nanoparticles and the mercapto-modified lysozyme to obtain the nano silver tetrahedron lysozyme complex.

[0013] Furthermore, in step S1, preparing a uniformly dispersed silver sol wrapped with sodium tartrate includes: mixing sodium tartrate with an aqueous silver nitrate solution, stirring, adding an aqueous NaBH 4 solution, continuing to stir, then performing light irradiation, adding polyvinylpyrrolidone (PVP), and then standing still; where

[0014] the concentration of silver nitrate in the aqueous silver nitrate solution is 0.1 mM, and the concentration of NaBH 4 in the aqueous NaBH 4 solution is 1 mM, and the average molecular weight of PVP is 30,000; the molar ratio of silver nitrate, sodium tartrate, NaBH 4 and PVP used is 1:(18 - 22):0.1:(2.8 - 3.2), where the amount of substance of PVP is calculated in units of its monomer.

[0015] In a preferred embodiment, in step S1, the molar ratio of silver nitrate, sodium tartrate, NaBH 4 and PVP is 1:20:0.1:3. The stirring is carried out at 400 r / min for 10 min, and the illumination is carried out under a 70 W sodium lamp for 8 - 10 h, preferably for 9 h.

[0016] Furthermore, in step S2, the molar ratio of sodium citrate added to silver nitrate is (0.8 - 1.5):1, and the illumination is carried out under a 70 W sodium lamp for 18 - 22 h.

[0017] In a preferred embodiment, in step S2, the molar ratio of sodium citrate added to silver nitrate is (1 - 1.2):1, and the illumination time is 20 h.

[0018] Furthermore, in step S3, after reacting through the NHS-functional group with the amino functional group on the outer side of lysozyme, filtration is carried out through an ultrafiltration centrifugal tube to remove unreacted NHS-PEG 1000 -SH molecules; after mixing and reacting silver tetrahedral nanoparticles with thiol-modified lysozyme, filtration is carried out through an ultrafiltration centrifugal tube to remove thiol-modified lysozyme molecules not connected to the silver tetrahedral nanoparticles.

[0019] Furthermore, in step S3, the molar ratio of lysozyme to NHS-PEG 1000 -SH molecules is 1:2, and the molar ratio of silver tetrahedral nanoparticles to thiol-modified lysozyme is 1:(1 - 3), preferably 1:1; the modification reaction is carried out at 27 °C for 30 min, and the molecular weight of the ultrafiltration centrifugal tube used is 3 KD; the coupling reaction is carried out at 4 °C for 3 d, and the molecular weight of the ultrafiltration centrifugal tube used is 30 KD.

[0020] The third aspect of the present invention provides the use of the above-mentioned silver tetrahedral lysozyme complex or the silver tetrahedral lysozyme complex prepared by the above-mentioned preparation method in the preparation of antibacterial drugs and drugs for promoting wound healing, and the antibacterial includes anti-Gram-negative bacteria and anti-Gram-positive bacteria.

[0021] The beneficial effects of the present invention include:

[0022] 1. The silver tetrahedral-lysozyme antibacterial complex prepared by the present invention is nanoparticles of about 80 nm, which can efficiently remove bacteria on the wound surface, promote rapid wound healing, and exhibit excellent antibacterial effects and wound repair capabilities; it is non-toxic to the human body and has good biocompatibility.

[0023] 2. The present invention uses a photoinduced method to prepare silver tetrahedral nanoparticles. The obtained particles have regular shapes, uniform dispersibility, and excellent stability, and are more likely to bind to mercapto-modified lysozyme. The preparation method used is simple and easy to implement, with the advantages of high stability and easy mass production, and at the same time, the drug bioavailability is relatively high.

[0024] 3. The nano silver tetrahedron-lysozyme antibacterial complex provided by the present invention can well improve the disadvantage that lysozyme has poor killing effect on Gram-negative bacteria through the combination of the two. It can not only achieve the killing effect on Gram-negative bacteria but also enhance the antibacterial effect on Gram-positive bacteria, and can effectively realize the dual functions of antibacterial and promoting wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the preparation process of the silver tetrahedral nanoparticles (Ag-t) and the nano silver tetrahedron-lysozyme complex (Ag-t-Lys) prepared in Example 1 of the present invention;

[0026] Figure 2 From left to right are the SEM characterization diagrams of the silver tetrahedral nanoparticles and the nano silver tetrahedron-lysozyme complex prepared in Example 1 of the present invention;

[0027] Figure 3 It is the DLS dynamic light scattering diagram of Ag-t, Lys, and Ag-t-Lys prepared in Example 2 of the present invention. Figure 3 Among them, the three curves from left to right represent Lys, Ag-t, and Ag-t-Lys in sequence;

[0028] Figure 4 It is the zeta potential diagram of Ag-t, Lys, and Ag-t-Lys prepared in Example 3 of the present invention;

[0029] Figure 5 It is the ultraviolet absorption spectrum diagram of Ag-t, Lys, and Ag-t-Lys prepared in Example 3 of the present invention; at a wavelength of 600 nm, the three curves in the figure represent Ag-t, Ag-t-Lys, and Lys from top to bottom in sequence.

[0030] Figure 6 It is the plate colony counting diagram of treating methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli O157:H7) with Ag-t, Lys, and Ag-t-Lys groups in Example 4 of the present invention, where Control represents the control group;

[0031] Figure 7 It is the comparison diagram of the antibacterial effects of Ag-t, Lys, and Ag-t-Lys against MRSA in Example 4 of the present invention (***p < 0.001, data are expressed as mean ± SD).

[0032] Figure 8 This is a comparison graph of the effects of Ag-t, Lys, and Ag-t-Lys against E. coli O157:H7 in Example 4 of the present invention (***p < 0.001, data are represented as mean ± SD).

[0033] Figure 9 This is a confocal graph of live / dead bacteria staining after Ag-t, Lys, and Ag-t-Lys in Example 5 of the present invention were used to treat MRSA and E. coli O157:H7; untreated represents the untreated bacterial sample used as a control group, and Merge represents the merged staining results of SYTO9 and PI;

[0034] Figure 10 This is a graph of the hemolysis rate of deionized water, Ag-t, Lys, and Ag-t-Lys on rabbit red blood cells in Example 6 of the present invention;

[0035] Figure 11 This is a morphological characterization graph of PBS, Ag-t, Lys, and Ag-t-Lys after incubation with rabbit red blood cells under an inverted microscope in Example 6 of the present invention;

[0036] Figure 12 This is a graph of the survival rate of L929 cells after co-culture with Ag-t, Lys, and Ag-t-Lys for 24 h in Example 6 of the present invention;

[0037] Figure 13 This is a live / dead cell staining graph of L929 in Example 7 of the present invention;

[0038] Figure 14 This is a morphological characterization graph of the control group (i.e., untreated bacteria) and Ag-t, Lys, and Ag-t-Lys after incubation with MRSA and E. coli O157:H7 respectively in Example 7 of the present invention;

[0039] Figure 15 This is a graph of the wound healing conditions of the control group (i.e., untreated wound) and Ag-t, Lys, and Ag-t-Lys acting on mouse wounds on the first day and the seventh day respectively in Example 8 of the present invention. Detailed implementation manners

[0040] The present invention provides a nano silver tetrahedron lysozyme complex and a preparation method thereof. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can make changes or appropriate alterations and combinations to the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0041] To further illustrate the present invention, the following is a detailed description of a nano silver tetrahedron lysozyme complex and a preparation method thereof provided by the present invention in conjunction with embodiments.

[0042] Example 1

[0043] Preparation of silver tetrahedron nanoparticles

[0044] (1) Preparation of silver tetrahedron nanoparticles Ag-t, including steps such as preparation of silver sol wrapped with sodium tartrate and preparation of silver tetrahedron nanoparticles. The specific steps are as follows:

[0045] (1) Preparation of silver sol wrapped with sodium tartrate: Add 0.0388 g of sodium tartrate to 99 mL of 0.1 mM silver nitrate solution under vigorous stirring, and stir for 10 min. At this time, the molar ratio of AgNO 3 :Na 2 C 4 H 4 O 6 is 1:20. Drop freshly prepared NaBH 4 solution (1 mL, 1 mM) into the above mixed solution so that the molar ratio of the three is 1:20:0.1. The mixed solution immediately turns into a yellow sol after introducing NaBH 4 . Continue to stir for 2 min to obtain the required silver sol. Irradiate the silver sol with a 70 W high-pressure sodium lamp for 9 h, and the solution color turns into turbid yellow. Then add 0.0033 g of PVP (average molecular weight = 30000) to the sol and let it stand for 12 hours to make the silver sol wrapped with sodium tartrate uniformly dispersed.

[0046] (2) Synthesis of silver tetrahedron nanoparticles: Add 0.0029 g of sodium citrate to the silver sol prepared above. At this time, the molar ratio of each component is:

[0047] AgNO 3 :Na 2 C 4 H 4 O 6 :NaBH 4 :PVP:Na 3C 6 H 5 O 7 was 1:20:0.1:3:1. Continuing the light irradiation for 20 h, a series of changes occurred in the solution color, and the turbid yellow gradually changed to turbid green, thus obtaining silver tetrahedron nanoparticles (see Figure 1 ).

[0048] (II) Verification of the prepared silver tetrahedron nanoparticles

[0049] The morphology of the prepared silver tetrahedron nanoparticles was characterized using a scanning electron microscope.

[0050] The method is as follows:[[]]END]]

[0051] The freshly prepared silver tetrahedron nanoparticles (Ag-t) were dropped on a clean silicon wafer at an appropriate concentration (40 μL, 40 nM), and reacted overnight at room temperature until the surface of the silicon wafer was dry. The silicon wafer with the sample was pasted on the SEM sample stage using conductive glue, and its morphology was characterized using a super-high-resolution field emission scanning electron microscope (Hitachi Regulus 8100). Since the silver tetrahedron is a silver nanoparticle with good conductivity, an experiment was carried out at a voltage of 20 KV (the results are as Figure 2 shown).

[0052] Example 2

[0053] After the silver tetrahedron was prepared in Example 1, subsequent experiments on complexing with lysozyme were carried out. The specific steps are as follows:[[]]END]]

[0054] (1) Using a double-end modified NHS-PEG 1000 -SH molecule with a molecular weight of 1000 to link lysozyme to the silver tetrahedron. First, the NHS-PEG 1000 -SH modified molecule was reacted with egg white lysozyme at room temperature for 0.5 h at a molar ratio twice that of lysozyme. The NHS functional group of the modified molecule can react with the outer amino functional group of lysozyme, and then filtered using an ultrafiltration centrifugal tube with a molecular weight of 3 KD to remove the unreacted NHS-PEG 1000 -SH molecules. The thiol-modified lysozyme (SH-PEG 1000 -Lys) was reacted with the silver tetrahedron at a molar ratio of 1:1 at 4 °C for 3 d, and then filtered using an ultrafiltration centrifugal tube with a molecular weight of 30 KD to remove the unlinked thiol-modified lysozyme molecules, thus obtaining the Ag-t-Lys complex (see Figure 1 ).

[0055] (2) Characterize the prepared silver tetrahedron-lysozyme complex. Use DLS (Dynamic Light Scattering) analysis to characterize whether the silver tetrahedron-lysozyme complex is successfully assembled into nanoparticles. Pipette 100 μL of the prepared silver tetrahedron-lysozyme complex and place it in a nanoparticle size analyzer for detection. The results are as Figure 3 shown. The diameter of the nanoparticles is about 80 nm, which is larger than the sizes of free Ag-t nanoparticles (68 nm) and Lys (6 nm), and is close to the sum of the two, proving its successful assembly (see Figure 3 ).

[0056] Example 3

[0057] Evaluate the stability of the silver tetrahedron-lysozyme complex prepared in Example 2, and evaluate its performance by Zeta potential and ultraviolet-visible absorption spectrum. The specific method is as follows:

[0058] Ultrasonically treat the sample to be tested to make the Ag-t nanoparticles evenly dispersed. Use a ZETA potential analyzer to measure its potential. The potential of Ag-t is ~ -22.5 mV; since the Zeta potential of lysozyme is about +4.5 mV, the Zeta potential of Ag-t-Lys (~ -18.5 mV) is higher than that of Ag-t (~ -22.5 mV) (as Figure 4 shown), so the potential shift of the Ag-t-Lys complex confirms the successful complexation of Lys and Ag-t.

[0059] Further characterize the Ag-t-Lys complex by ultraviolet spectrum. Detect the prepared silver tetrahedron-lysozyme complex with an ultraviolet-visible spectrophotometer. The characteristic absorption peak of Lys near 280 nm was observed in the ultraviolet spectrum of the Ag-t-Lys complex, and a slight red shift occurred in the characteristic absorption peak of Ag-t at 642 nm (as Figure 5 shown), indicating the successful coupling of Lys and Ag-t. In summary, these results indicate the successful construction of Ag-t and Ag-t-Lys complexes.

[0060] Example 4

[0061] Evaluate the in vitro antibacterial performance of the silver tetrahedron-lysozyme antibacterial complex nanoparticles prepared in Example 2. The specific method is as follows:

[0062] To observe the effects of Ag-t, Lys, and Ag-t-Lys on bacterial growth, co-culture different concentrations of the drug with bacteria for 24 h and measure the absorbance of the bacteria at a wavelength of 600 nm. The control group uses PBS with the same volume as the drug. The initial concentration of bacteria is adjusted to 5×10 5CFU / mL, and the minimum inhibitory concentration (MIC) of different experimental groups was tested on a 96-well plate. Subsequently, on the 96-well plate with a drug concentration of 1×MIC Ag-t-Lys , a sterilized pipette tip was used to aspirate the diluted bacterial solution and inoculated onto an MH agar plate, and cultured in an incubator at 37 °C for 16 h for plate counting.

[0063] The antibacterial abilities of Ag-t, Lys, and Ag-t-Lys complexes with different concentrations against MRSA and E. coli O157:H7 were studied, and their minimum inhibitory concentrations were compared. The results showed that when Ag-t was used alone, its minimum inhibitory concentrations against MRSA and E. coli O157:H7 were 24.2 μg / mL and 12.6 μg / mL, respectively; the minimum inhibitory concentration of Lys against the Gram-positive bacterium MRSA was 1300 μg / mL, and it had almost no obvious antibacterial effect on the Gram-negative bacterium E. coli O157:H7; while the minimum inhibitory concentration of the Ag-t-Lys complex against MRSA was 5.8 μg / mL, and the minimum inhibitory concentration against the Gram-negative bacterium E. coli O157:H7 was 5.1 μg / mL. To further evaluate the killing ability of Ag-t-Lys against suspended bacteria, the plate colony counting method was used to count the colonies after incubation of Ag-t, Lys, and Ag-t-Lys (all at the minimum inhibitory concentration of Ag-t-Lys) with bacteria. The results were as Figure 6 shown. Compared with the control group (i.e., without adding Ag-t, Lys, and Ag-t-Lys complexes), the number of colonies in the Ag-t-Lys group was significantly reduced, and compared with the experimental groups of Ag-t and Lys, the number of colonies in the Ag-t-Lys group was also significantly reduced. Further quantitative analysis of the number of colonies (as Figure 7 and Figure 8 shown) showed that the bactericidal ability of Ag-t-Lys was significantly better than that of Ag-t and Lys, proving that the prepared silver tetrahedron-lysozyme nanoparticles had good antibacterial performance in vitro.

[0064] Example 5

[0065] The bacterial killing ability of the silver tetrahedron-lysozyme antibacterial composite nanoparticles prepared in Example 2 was evaluated, and the specific method was as follows:

[0066] The mixture after co-incubation of Ag-t, Lys, and Ag-t-Lys with bacteria was stained using the SYTO9 / PI live / dead bacterial staining kit. The concentrations used for staining were SYTO9 (5 μM) and PI (5 μM). Incubation was carried out in the dark for 15 min, and then the bacterial suspension was resuspended three times with sterile PBS to remove free background fluorescent dyes in the solution. Finally, the samples were resuspended in PBS buffer. 10 μL of the sample was dropped onto a cleaned glass slide and covered with a clean ultra-thin coverslip, and immediately observed using a laser confocal microscope. The results are as Figure 9 shown. Live bacteria were labeled with green fluorescence by the SYTO9 dye; PI can penetrate the damaged cell membrane and label dead bacteria, showing red fluorescence. Compared with the blank control group, the drug Ag-t group, and the Lys group, the Ag-t-Lys group had a more significant killing effect on MRSA and E. coli O157:H7. It was proved that the prepared silver tetrahedron-lysozyme complex had a strong ability to kill bacteria.

[0067] Example 6

[0068] The biosafety of the silver tetrahedron-lysozyme antibacterial complex nanoparticles prepared in Example 2 was evaluated, and the specific method was as follows:

[0069] (1) Hemolysis experiment procedure: 1 mL of 2 v / v% rabbit red blood cells was incubated with different drugs Ag-t, Lys, and Ag-t-Lys (drug concentration was 2×MIC Ag-t-Lys ) in a 37 °C constant temperature incubator for 3 h. The 2 v / v% rabbit red blood cell normal saline suspension and the 2 v / v% rabbit red blood cell ultrapure water (DI) suspension were set as the negative control group and the positive control group. After incubation, different groups were centrifuged (3000 rpm, 5 min), and 100 μL of the supernatant was taken and placed in a 96-well plate. The absorbance at a wavelength of 545 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and all samples were measured more than 3 times. As Figure 10 shown, at a drug concentration of 2×MIC Ag-t-Lys , the hemolysis rates of Ag-t, Lys, and Ag-t-Lys were all significantly lower than 5%, indicating their good biocompatibility; the morphology after incubation of different drugs with rabbit red blood cells was characterized by an inverted microscope. As Figure 11 shown, its morphology was consistent with that of the PBS group, showing a typical biconcave disc structure, further indicating its good biocompatibility.

[0070] (2) Cytotoxicity experiment: The CCK-8 method was used to evaluate the biocompatibility of Ag-t-Lys on mouse fibroblasts (L929). First, L929 cells (10 4 cells / well) were inoculated in a 96-well plate. The 96-well plate inoculated with cells was placed in CO 2Cultivate at 37°C in a cell incubator. After 24 hours of cultivation, remove the liquid in the culture plate and add different drugs Ag-t, Lys, and Ag-t-Lys (drug concentration is 2×MIC Ag-t-Lys ) cell culture medium, and continue co-culturing at 37°C for another 24 hours. Incubate cells with pure cell culture medium and ultrapure water as the negative control group and the positive control group. After 24 hours of incubation, add the same amount of CCK-8, and then continue culturing in a 37°C CO 2 cell incubator for 1.5 hours. Measure the absorbance at a wavelength of 450 nm using a microplate reader, and measure all samples more than 3 times. As Figure 12 shown, after co-incubation with different drugs Ag-t, Lys, and Ag-t-Lys (drug concentration is 2×MIC Ag-t-Lys ), compared with the control group, the cell viability was not significantly affected.

[0071] (3) L929 live / dead cell staining: Use the Calcein-AM / PI kit to further examine the cytotoxicity of Ag-t-Lys. Inoculate L929 cells (10 4 cells / cm 2 ) into an 8-well Glass Slid cell culture chamber and culture for 24 hours. Remove the liquid in the chamber, add different drugs Ag-t, Lys, and Ag-t-Lys (drug concentration is 2×MIC Ag-t-Lys ) cell culture medium, and continue co-culturing at 37°C for another 24 hours. Remove the cell culture medium, wash 3 times with PBS, 10 minutes each time; add Calcein-AM and PI with a final concentration of 2 μM, incubate in the dark for 15 minutes, wash 3 times with PBS, and remove the free staining agent; fix the cells with 4% paraformaldehyde for 15 minutes, wash 3 times with PBS, 10 minutes each time, and image using a Nikon laser confocal microscope. As attached Figure 13 shown, the results show that after co-incubation with different drugs Ag-t, Lys, and Ag-t-Lys (drug concentration is 2×MIC Ag-t-Lys ), the cells are mainly green, indicating very low toxicity.

[0072] The above three experiments all show that the prepared silver tetrahedron-lysozyme complex has good biocompatibility.

[0073] Example 7

[0074] Characterize the morphology of the silver tetrahedron-lysozyme antibacterial complex nanoparticles prepared in Example 2 after their interaction with bacteria to explore their bactericidal ability. The specific method is as follows:

[0075] The morphological changes of the silver nanowire tetrahedron lysozyme complex after incubation with methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli O157:H7) were observed using a scanning electron microscope (SEM). Bacteria in the control group were treated with PBS. First, 1 mL of bacterial suspension (10 8 CFU / mL) was interacted with the silver nanowire tetrahedron lysozyme complex at the same concentration at 37 °C for 12 h. Subsequently, centrifugation was performed (10000 rpm, 5 min), and the pellet was washed three times with PBS, then resuspended with adherent bacteria in 4% paraformaldehyde, thoroughly mixed, and fixed at room temperature for 2 h. Then, centrifugation was performed again (10000 rpm, 5 min), and the pellet was washed three times with PBS. Subsequently, the bacteria were resuspended with ethanol at gradient concentrations, mixed well, dehydrated in ethanol for 15 min, and then centrifuged. Finally, 50 μL of absolute ethanol was added, dropped on a clean silicon wafer, and air-dried naturally. After the samples were sputter-coated with gold, SEM scanning imaging was performed. (As shown in Figure 14 the appendix), the results showed that the morphology of Lys was almost the same as that of the control group after interaction with bacteria and was not damaged; Ag-t caused partial damage to E. coli, but had no obvious killing effect on MRSA, and Ag-t adhered to the surface of bacteria; while Ag-t-Lys caused severe damage to the morphology of MRSA bacteria, with pores appearing on the surface; Ag-t-Lys caused E. coli to shrink and shrivel, and the bacteria ruptured and the contents leaked out, indicating that the prepared silver nanowire tetrahedron-lysozyme complex had a strong killing effect on bacteria (including Gram-positive and Gram-negative bacteria).

[0076] Example 8

[0077] The ability of the silver tetrahedron-lysozyme antibacterial composite nanoparticles prepared in Example 2 to promote wound healing was explored by investigating the wound healing effect on the wound surface of mice. The specific method was as follows:

[0078] An MRSA infection model was established on the back of female Kunming mice (6 - 8 weeks old, 20 - 40 g) to evaluate the antibacterial effect of the drug. After the standard anesthesia procedure with isoflurane, the back hair of each mouse was shaved off, and the shaved area was further treated with depilatory cream. Subsequently, the skin was cleaned and disinfected with alcohol cotton balls, and a circular full-thickness skin wound with a diameter of 8 mm was made. Then, 20 μL of MRS (1×10 8 CFU / mL) was dropped on the wound. There were five mice in each group. The first drug treatment was performed 24 h after the wound was established, and each group was treated with the corresponding drug once and photographed. As shown in Figure 15 the appendix, on the 7th day after trauma, all wound surfaces were significantly reduced, but the unhealed wound surface in the control group was still larger than that in other groups; the wound surface in the drug Ag-t-Lys group was covered by epidermis and almost completely healed. It can be seen that the silver nanowire tetrahedron-lysozyme complex can effectively promote wound healing.

[0079] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Without departing from the principle of the present invention, those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention, and these changes, modifications, substitutions, and variations should also be regarded as falling within the protection scope of the present invention.

Claims

1. A nanosilver tetrahedral lysozyme complex, characterized in that: The complex is a nanoparticle formed by connecting silver tetrahedron (Ag-t) nanoparticles and lysozyme modified with thiol (-SH) through silver-thiol interaction, wherein the thiol is modified by NHS-PEG 1000 -SH is introduced, and NHS- is N-hydroxysuccinimide functional group.

2. The nanosilver tetrahedral lysozyme complex according to claim 1, characterized in that: The thiol-modified lysozyme is SH-PEG 1000 -Lys, Lys means lysozyme.

3. The method for preparing the nanosilver tetrahedral lysozyme complex according to claim 1 or 2, characterized in that: The method comprises the following steps: S1, preparing uniformly dispersed silver sol wrapped with sodium tartrate; S2, adding sodium citrate to the prepared silver sol, and irradiating the sol with light to obtain silver tetrahedral nanoparticles (Ag-t); S3. Modification reaction: by modifying the molecule NHS-PEG 1000 The NHS-functional group of -SH reacts with the amino functional group on the outside of lysozyme to obtain thiol-modified lysozyme SH-PEG 1000 -Lys; Linking reaction: silver tetrahedral nanoparticles are linked by mixed reaction with thiol-modified lysozyme to obtain a nano silver tetrahedral lysozyme complex.

4. The method for preparing the nanosilver tetrahedral lysozyme complex according to claim 3, characterized in that: In step S1, preparing uniformly dispersed sodium tartrate-coated silver sol includes: mixing sodium tartrate with an aqueous silver nitrate solution, stirring, adding an aqueous NaBH4 solution, continuing to stir, then irradiating with light, then adding polyvinyl pyrrolidone (PVP), and then standing; wherein The concentration of silver nitrate in the silver nitrate aqueous solution is 0.1 mM, the concentration of NaBH4 in the NaBH4 aqueous solution is 1 mM, and the average molecular weight of PVP is 30,000; the molar ratio of the silver nitrate, sodium tartrate, NaBH4 and PVP used is 1:(18-22):0.1:(2.8-3.2), wherein the amount of PVP is calculated based on its monomer unit.

5. The method for preparing the nanosilver tetrahedral lysozyme complex according to claim 4, characterized in that: In step S1, the molar ratio of silver nitrate, sodium tartrate, NaBH4 and PVP used is 1:20:0.1:3, the stirring is 10 min at 400 r / min, and the illumination is 8-10 h under a 70W sodium lamp.

6. The method for preparing the nanosilver tetrahedral lysozyme complex according to claim 4, characterized in that: In step S2, the molar ratio of the added sodium citrate to silver nitrate is (0.8-1.5):1, and the illumination is performed under a 70W sodium lamp for 18-22 hours.

7. The method for preparing the nanosilver tetrahedral lysozyme complex according to claim 6, characterized in that: In step S2, the molar ratio of the added sodium citrate to silver nitrate is (1-1.2):1, and the illumination time is 20 hours.

8. The method for preparing the nanosilver tetrahedral lysozyme complex according to claim 6, characterized in that: In step S3, after the NHS-functional group reacts with the amino functional group on the outside of the lysozyme, the unreacted NHS-PEG is removed by filtration through an ultrafiltration centrifuge tube. 1000 -SH molecules; after the silver tetrahedral nanoparticles and the thiol-modified lysozyme are mixed and reacted, they are filtered through an ultrafiltration centrifuge tube to remove the thiol-modified lysozyme molecules that are not connected to the silver tetrahedral nanoparticles.

9. The method for preparing the nanosilver tetrahedral lysozyme complex according to claim 8, characterized in that: In step S3, lysozyme is reacted with NHS-PEG 1000 The mass ratio of -SH molecules is 1:2, and the mass ratio of silver tetrahedral nanoparticles and thiol-modified lysozyme is 1:(1-3); the modification reaction is carried out at 27°C for 30 minutes, and the molecular weight of the ultrafiltration centrifuge tube used is 3KD; the connection reaction is carried out at 4°C for 3 days, and the molecular weight of the ultrafiltration centrifuge tube used is 30KD.

10. Use of the nanosilver tetrahedral lysozyme complex according to claim 1 or 2 or the nanosilver tetrahedral lysozyme complex prepared by the preparation method according to any one of claims 3-9 in the preparation of antibacterial drugs and drugs for promoting wound healing, wherein the antibacterial drugs include anti-Gram-negative bacteria and anti-Gram-positive bacteria.

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