Gold nanoclusters and preparation method and application thereof

Abstract

The application provides a kind of gold nanocluster and its preparation method and application.The gold nanocluster of the application, the gold nanocluster is tiopronin modified gold nanocluster, the gold nanocluster has gold atom and sulfur atom.The gold nanocluster of the application, the gold nanocluster is tiopronin modified gold nanocluster, the gold nanocluster has gold atom and sulfur atom, can be widely used with antibiotic, has synergistic antibacterial effect, for example, against carbapenem-resistant gram-negative pathogens.The application also provides the preparation method and application of gold nanocluster.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibacterial materials, and particularly relates to a gold nanocluster and a preparation method and application thereof. BACKGROUND

[0002] The emergence and spread of carbapenem-resistant gram-negative pathogens is a serious public health problem, and the development of multidrug resistance has seriously hindered the progress of antibiosis. The production of metallo-beta-lactamases (MBLs) is considered to be the mechanism by which pathogenic bacteria acquire resistance, especially the New Delhi metallo-beta-lactamase (NDM-1) which has caused serious bacterial resistance in recent years. The resistance gene encoding NDM-1 (blaNDM-1) can be expressed in different bacteria through plasmids and integrins, conferring NDM-1 and other MBLs bacteria with resistance to almost all available beta-lactam antibiotics, resulting in poor clinical effect of conventional antibiotic therapy.

[0003] Currently, the development cycle of new antibiotics cannot cope with the ongoing infection crisis, and new materials need to be developed to make drug-resistant bacteria sensitive to existing antibiotics again. SUMMARY

[0004] The present application aims to at least solve one of the above technical problems existing in the prior art. To this end, the present application provides a gold nanocluster having good biocompatibility and enhanced antibacterial activity of antibiotics.

[0005] The present application also provides a preparation method of the gold nanocluster.

[0006] The present application also provides an application of the gold nanocluster.

[0007] The present application also provides an antibacterial composition.

[0008] The first aspect of the present application provides a gold nanocluster, which is a tiopronin-modified gold nanocluster, and the gold nanocluster has gold atoms and sulfur atoms.

[0009] One of the technical solutions of the present application regarding the gold nanocluster has at least the following beneficial effects:

[0010] Nanomaterials have unique physical and chemical properties, such as large specific surface area, high degree of surface functionalization, etc. Among them, gold nanoclusters, copper nanoclusters, metal oxide nanoclusters, silver nanoclusters, and carbon materials including fullerenes and graphene are found to have certain antibacterial activity. Compared with other antibacterial materials, antibacterial gold nanomaterials have many unique properties such as good biocompatibility, excellent antibacterial activity and fast synthesis method on the premise of having the above excellent properties, which provides a new opportunity for screening antibiotic nanoadjuvants. The gold nanoclusters of the present application are thiamine-modified gold nanoclusters, which have gold atoms and sulfur atoms, can be widely used with antibiotics, have synergistic antibacterial effect, and are resistant to carbapenem-resistant gram-negative pathogens.

[0011] The gold nanoclusters of the present application have good biocompatibility and enhanced antibacterial activity of antibiotics.

[0012] According to some embodiments of the present application, the gold atoms and the sulfur atoms are covalently bonded.

[0013] The gold nanoclusters described above form a stable covalent bond structure between gold atoms and sulfur atoms, which endows the gold nanoclusters with functional groups that impart antibacterial properties. The gold nanoclusters described above have good antibacterial effect and good biocompatibility.

[0014] According to some embodiments of the present application, the particle size of the gold nanoclusters is in the range of 1 nm to 20 nm.

[0015] According to some embodiments of the present application, the particle size of the gold nanoclusters is in the range of 1 nm to 15 nm.

[0016] According to some embodiments of the present application, the particle size of the gold nanoclusters is in the range of 1 nm to 10 nm.

[0017] According to some embodiments of the present application, the particle size of the gold nanoclusters is in the range of 1 nm to 5 nm.

[0018] According to some embodiments of the present application, the thiamine-modified gold nanoclusters also have C, N, O and Na elements.

[0019] Thiamine mainly plays the role of a surface ligand to maintain the stability of the structure.

[0020] The actual form of the gold nanoclusters can be an aqueous solution.

[0021] The second aspect of the present application provides a method for preparing the gold nanoclusters, which comprises mixing a gold source, thiamine and a reducing agent and then reacting to obtain the gold nanoclusters.

[0022] The application relates to a technical scheme of a gold nanocluster preparation method.

[0023] The gold nanocluster preparation method is simple, does not require expensive equipment, complex procedures and harsh reaction conditions, and can be easily produced on a large scale.

[0024] According to some embodiments of the application, the gold source comprises chloroauric acid.

[0025] According to some embodiments of the application, the chloroauric acid is chloroauric acid trihydrate.

[0026] According to some embodiments of the application, the reducing agent comprises sodium borohydride.

[0027] According to some embodiments of the application, the reducing agent can be a sodium borohydride solution.

[0028] According to some embodiments of the application, the concentration of the sodium borohydride solution is 2 mg / mL-3 mg / mL.

[0029] According to some embodiments of the application, the molar ratio of the gold source to tiopronin is 1:0.5-10.

[0030] According to some embodiments of the application, the molar ratio of the gold source to tiopronin is 1:1-10.

[0031] According to some embodiments of the application, the molar ratio of the gold source to tiopronin is 1:2-10.

[0032] According to some embodiments of the application, the molar ratio of the gold source to tiopronin is 1:4-10.

[0033] According to some embodiments of the application, the method comprises the following steps:

[0034] S1: mixing and dissolving a gold source and tiopronin in a solvent to obtain a mixture;

[0035] S2: adding a reducing agent to the mixture under stirring to obtain a crude product after reaction;

[0036] S3: purifying the crude product to obtain the gold nanocluster.

[0037] According to some embodiments of the application, in step S1:

[0038] The solvent comprises methanol and / or water.

[0039] Methanol can be replaced by water, but the antibacterial performance will decrease, because there is a difference between the surface structure of methanol and water, and the reduction reaction and esterification reaction will occur during the synthesis of gold nanoclusters using methanol, which can change the valence of gold and thus improve the antibacterial performance.

[0040] According to some embodiments of the present application, in step S2:

[0041] The reducing agent is added to the mixture, and a redox reaction occurs, and the BH4 - ion in sodium borohydride reacts with the AuCl4 - ion, and the gold is reduced from the positive trivalent to a low valence. The final reaction product is boric acid and hydrogen.

[0042] The stirring condition is that the stirring speed is 1000 rpm-2000 rpm.

[0043] The stirring condition is that the stirring speed is 1500 rpm-2000 rpm.

[0044] During the reaction, the color of the solution changes to yellow, and the reaction is continued for a period of time under this reaction condition.

[0045] According to some embodiments of the present application, in step S3:

[0046] The boric acid in the crude product can be removed by dialysis bag for a period of time. For example, dialysis for 48 h.

[0047] The molecular weight cut-off of dialysis can be 14 kDa MW.

[0048] The third aspect of the present application provides the use of the gold nanoclusters or the gold nanoclusters prepared by the method in the preparation of antibacterial drugs.

[0049] The technical solution of the present application in the use of gold nanoclusters in the preparation of antibacterial drugs has at least the following beneficial effects:

[0050] The gold nanoclusters of the present application are thioctic acid modified gold nanoclusters, and the gold nanoclusters have gold atoms and sulfur atoms, can be widely used with antibiotics, have synergistic antibacterial effect, and for example, can resist carbapenem-resistant gram-negative pathogens. The gold nanoclusters of the present application have good biocompatibility and enhanced antibacterial activity of antibiotics. Therefore, they have good application prospect in the preparation of antibacterial drugs.

[0051] The fourth aspect of the present application provides an antibacterial composition comprising the gold nanoclusters or the gold nanoclusters prepared by the method.

[0052] The technical solution of the present application in the technical solution of the antibacterial composition has at least the following beneficial effects:

[0053] The antibacterial composition of the present application, including the gold nanoclusters or the gold nanoclusters prepared by the method, thereby has all the beneficial effects of the gold nanoclusters. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 is a schematic diagram of the appearance of the gold nanoclusters prepared in the examples.

[0055] Figure 2 is a UV-visible spectrum diagram of the gold nanoclusters prepared in the examples.

[0056] Figure 3 is a graph of the average diameter of the gold nanoclusters prepared in the examples.

[0057] Figure 4 is a zeta potential test diagram of the gold nanoclusters prepared in the examples.

[0058] Figure 5 is a chessboard dilution method determination result diagram of the gold nanoclusters prepared in the examples.

[0059] Figure 6 is a graph of the effect of the combined use of the gold nanoclusters and imipenem on gram-negative bacteria prepared in the examples.

[0060] Figure 7 is a bactericidal kinetics test result of the gold nanoclusters prepared in the examples on gram-negative bacteria.

[0061] Figure 8 is a scanning electron microscope diagram of the gold nanoclusters prepared in the examples.

[0062] Figure 9 is a cytotoxicity evaluation result of the gold nanoclusters prepared in the examples.

[0063] Figure 10 is a hemolysis performance evaluation result of the gold nanoclusters prepared in the examples.

[0064] Figure 11 is a graph of the effect of the gold nanoclusters prepared in the examples on the animal model of carbapenem-resistant Klebsiella pneumoniae infection treated with antibiotics.

[0065] Figure 12 is a graph of the effect of the gold nanoclusters prepared in the examples on the animal model of other carbapenem-resistant Enterobacteriaceae bacterial infection treated with antibiotics.

[0066] Figure 13 is a graph of the effect of the gold nanoclusters prepared in the examples on the animal model of other carbapenem-resistant Acinetobacter baumannii infection treated with antibiotics. DETAILED DESCRIPTION

[0067] The following are specific embodiments of the present application, and the technical solutions of the present application are further described in combination with the embodiments, but the present application is not limited to these embodiments.

[0068] In some embodiments of the present application, the gold nanocluster is a tiopronin-modified gold nanocluster, and the gold nanocluster has gold atoms and sulfur atoms.

[0069] It can be understood that nanomaterials have unique physical and chemical properties, such as large specific surface area, high degree of surface functionalization, and the like. Among them, gold nanoclusters, copper nanoclusters, metal oxide nanoclusters, silver nanoclusters, and carbon materials including fullerenes and graphene are found to have certain antibacterial activity. Compared with other antibacterial materials, antibacterial gold nanomaterials have many unique properties such as good biocompatibility, excellent antibacterial activity and fast synthesis method on the premise of having the above excellent properties, which provides new opportunities for screening antibiotic nanoadjuvants. The gold nanocluster of the present application is a tiopronin-modified gold nanocluster, and the gold nanocluster has gold atoms and sulfur atoms, which can be widely used with antibiotics, has a synergistic antibacterial effect, and is resistant to carbapenem-resistant gram-negative pathogens.

[0070] It can also be understood that the gold nanocluster of the present application has good biocompatibility and enhances the antibacterial activity of antibiotics.

[0071] In some embodiments of the present application, the gold atoms and the sulfur atoms are covalently bonded.

[0072] The above gold nanocluster forms a stable covalent bond structure between the gold atoms and the sulfur atoms, which gives the gold nanocluster a functional group that gives the gold nanocluster antibacterial properties. And the above gold nanocluster has good antibacterial effect and good biocompatibility.

[0073] In some embodiments of the present application, the particle size range of the gold nanocluster is 1 nm to 20 nm.

[0074] In some embodiments of the present application, the particle size range of the gold nanocluster is 1 nm to 15 nm.

[0075] In some embodiments of the present application, the particle size range of the gold nanocluster is 1 nm to 10 nm.

[0076] In some embodiments of the present application, the particle size range of the nanocluster is 1 nm to 5 nm.

[0077] In some embodiments of the present application, the tiopronin-modified gold nanocluster also has C, N, O and Na elements.

[0078] Tiopronin mainly plays the role of a surface ligand to maintain the stability of the structure.

[0079] The actual form of the gold nanocluster can be an aqueous solution.

[0080] In some embodiments of the present application, the present application provides a method for preparing the gold nanocluster of the present application, the method comprising: mixing a gold source, tiopronin and a reducing agent, and then reacting to obtain the gold nanocluster of the present application.

[0081] It can be understood that the method for preparing the gold nanocluster of the present application only needs to mix the gold source, tiopronin and reducing agent, and then react to obtain the gold nanocluster of the present application. The preparation method is simple, does not require expensive equipment, complex process and harsh reaction conditions, and is easy to mass-produce.

[0082] In some embodiments of the present application, the gold source comprises chloroauric acid.

[0083] In some embodiments of the present application, the chloroauric acid is chloroauric acid trihydrate.

[0084] In some embodiments of the present application, the reducing agent comprises sodium borohydride.

[0085] In some embodiments of the present application, the reducing agent can be a sodium borohydride solution.

[0086] In some embodiments of the present application, the concentration of the sodium borohydride solution is 2 mg / mL to 3 mg / mL.

[0087] In some embodiments of the present application, the concentration of the sodium borohydride solution is 2.4 mg / mL.

[0088] In some embodiments of the present application, the molar ratio of the gold source to tiopronin is 1:0.5 to 10.

[0089] In some embodiments of the present application, the molar ratio of the gold source to tiopronin is 1:1 to 10.

[0090] In some embodiments of the present application, the molar ratio of the gold source to tiopronin is 1:2 to 10.

[0091] In some embodiments of the present application, the molar ratio of the gold source to tiopronin is 1:4 to 10.

[0092] In some embodiments of the present application, the preparation method specifically comprises the following steps:

[0093] S1: mixing and dissolving a gold source and tiopronin in a solvent to obtain a mixture;

[0094] S2: under stirring conditions, adding a reducing agent to the mixture, and after reaction, obtaining a crude product;

[0095] S3: purifying the crude product to obtain the gold nanocluster of the present application.

[0096] In some embodiments of the present application, in step S1:

[0097] The solvent includes methanol and / or water.

[0098] The methanol can be replaced by water, but the antibacterial performance will decrease, because there is a difference in the surface structure between methanol and water, and a reduction reaction and an esterification reaction occur simultaneously in the process of synthesizing gold nanoclusters using methanol, which can change the valence of gold and thus improve the antibacterial performance.

[0099] In some embodiments of the present application, in step S2:

[0100] It should be noted that the addition of the reducing agent to the mixture causes an oxidation-reduction reaction, and the BH4 - ion in sodium borohydride reacts with the AuCl4 - ion, and the gold is reduced from a positive valence of +3 to a low valence. The final reaction product is boric acid and hydrogen.

[0101] The stirring condition is that the stirring speed is 1000 rpm to 2000 rpm.

[0102] The stirring condition is that the stirring speed is 1500 rpm to 2000 rpm.

[0103] During the reaction, the color of the solution changes to yellow, and the reaction is continued for a period of time under this reaction condition.

[0104] In some embodiments of the present application, in step S3:

[0105] The boric acid in the crude product can be removed by dialysis for a period of time, for example, for 48 h.

[0106] The molecular weight cut-off of the dialysis can be 14 kDa MW.

[0107] In some other embodiments of the present application, the present application provides the use of the gold nanoclusters of the present application or the gold nanoclusters prepared by the method of the present application in the preparation of antibacterial drugs.

[0108] It can be understood that the gold nanoclusters of the present application are gold nanoclusters modified by tiopronin, and the gold nanoclusters have gold atoms and sulfur atoms, which can be widely used in combination with antibiotics and have a synergistic antibacterial effect, for example, against carbapenem-resistant gram-negative pathogens. The gold nanoclusters of the present application have good biocompatibility and enhanced antibacterial activity of antibiotics. Therefore, they have good application prospects in the preparation of antibacterial drugs.

[0109] In some embodiments of the present application, the present application provides an antibacterial composition comprising the gold nanoclusters of the present application or the gold nanoclusters prepared by the method of the present application.

[0110] It can be understood that the antibacterial composition of the present application, including the gold nanoclusters of the present application or the gold nanoclusters prepared by the method of the present application, thereby has all the beneficial effects of the gold nanoclusters.

[0111] The technical solutions of the present application can be better understood in combination with the specific embodiments below.

[0112] In the following examples:

[0113] Chloroauric acid, sodium borohydride were purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.

[0114] Methanol was purchased from sigma company.

[0115] Sodium borohydride was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0116] Dialysis bag, 2kDa MW cut off, purchased from Solarbio company.

[0117] Imipenem, meropenem were purchased from Solarbio company.

[0118] Ertapenem, purchased from aladdin company.

[0119] Carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant Enterobacteriaceae bacteria, carbapenem-resistant Acinetobacter baumannii were from the Third Affiliated Hospital of Guangzhou Medical University.

[0120] Human umbilical vein endothelial cells (HUVEC) were purchased from Beijing Dingguochangsheng Biotechnology Co., Ltd.

[0121] Nanoparticle size and zeta potential analyzer (NZS), model Zetasizer Nano ZS, purchased from Malvern Instruments Ltd., UK; ultraviolet-visible spectrophotometer, model UV-2600i, purchased from SHIMADZU.

[0122] Example: Preparation of tiopronin modified gold nanoclusters

[0123] Example 1

[0124] The prepared gold nanoclusters are as shown in Figure 1 This embodiment is used to illustrate the preparation method and performance characterization of tiopronin activated gold nanoclusters. The gold nanoclusters are modified with tiopronin, and the molar ratio of chloroauric acid trihydrate to tiopronin is 1:10. The gold nanoclusters are denoted as Au-TTP NCs.

[0125] (1) In a round-bottom flask, 1 millimole of chloroauric acid trihydrate and 10 millimoles of tiopronin were dissolved in 10 milliliters of methanol solution, and mixed in an ice water bath for 20 minutes (800 revolutions per minute) until the molecules were completely dissolved.

[0126] (2) 2.4 mg / mL sodium borohydride was dissolved in 2.5 mL of methanol and 2.5 mL of pure water, and added dropwise into a round-bottom flask under vigorous stirring (1500 rpm). The color of the solution in the flask turned yellow, and the reaction was continued for 1 hour under the same conditions.

[0127] (3) The obtained tiopronin-modified gold nanoclusters were dialyzed in a dialysis bag (2 kDa MW cut-off, Solarbio) for 48 hours to remove untreated chemicals. The gold nanoclusters were sterilized by filtering through a 0.22-micron filter (Millipore) and stored in a 4-degree Celsius refrigerator for future use.

[0128] Example 2

[0129] This example provides a gold nanocluster, which differs from Example 1 only in that the molar ratio of chloroauric acid trihydrate to tiopronin is 1:0.5. This gold nanocluster is denoted as Au-PTP NCs.

[0130] Example 3

[0131] This example provides a gold nanocluster, which differs from Example 1 only in that the molar ratio of chloroauric acid trihydrate to tiopronin is 1:1. This gold nanocluster is denoted as Au-OTP NCs.

[0132] Example 4

[0133] This example provides a gold nanocluster, which differs from Example 1 only in that the molar ratio of chloroauric acid trihydrate to tiopronin is 1:2. This gold nanocluster is denoted as Au-WTP NCs.

[0134] Example 5

[0135] This example provides a gold nanocluster, which differs from Example 1 only in that the molar ratio of chloroauric acid trihydrate to tiopronin is 1:4. This gold nanocluster is denoted as Au-FTP NCs.

[0136] Materials characterization of gold nanoclusters

[0137] The relevant characterization is used for the study of the physicochemical properties of gold nanoclusters with different ligands.

[0138] 1. The ultraviolet-visible spectrophotometer (UV-2600i, SHIMADZU) was used to test the ultraviolet-visible spectrum of the gold nanoclusters.

[0139] 2. The size and potential of the material were determined using a nanoparticle size and Zeta potential analyzer (NZS) (instrument model Zetasizer Nano ZS, purchased from Malvern Instruments Ltd., UK).

[0140] The experimental results are shown in Figure 6. Figure 2 It can be seen that each test curve shows a clear characteristic absorption peak of gold nanoclusters, proving that gold nanoclusters are successfully prepared. In addition, Figure 3 It can be seen that the five gold nanoclusters prepared have a size of 1-10 nm and are all negatively charged. Figure 4 It can be seen that all the gold nanoclusters prepared are negatively charged. The Zeta potential of the gold nanoclusters is related to the proportion of the thiol-containing negatively charged ligand. As the proportion of the negatively charged ligand increases, the Zeta potential of the gold nanoclusters shifts to the negative side, confirming that the ligand is effectively modified on the surface of the gold nanoclusters.

[0141] Evaluation of the synergistic antibacterial performance of gold nanoclusters

[0142] This test example is used to study the synergistic antibacterial performance of gold nanoclusters modified with different ligands synthesized in Example 1. The minimum inhibitory concentration (MIC) is determined by microdilution or well plate dilution method, and the specific test method is as follows:

[0143] 1. The carbapenem-resistant Klebsiella pneumoniae (CR K.p) was inoculated in liquid LB medium and cultured to the logarithmic phase.

[0144] 2. Using a sterile transparent 96-well plate, the common antibiotics meropenem (Mer), ertapenem (Ert), and imipenem (Imi) were first grouped. The first well of each group was the highest concentration of the sample to be tested, with a volume of 200 μL and a concentration of 256 μg / mL.

[0145] 3. The remaining wells were each added with 100 μL of culture medium, and then 100 μL of the first well liquid was taken out and added to the second well for mixing. The concentration of the sample to be tested was 128 μg / mL. The subsequent wells were diluted by the same method in a ratio of 2, and after mixing, 100 μL was directly taken out and discarded. The last well was the negative control group, and the concentration of the sample to be tested was 0 μg / mL.

[0146] 4. The bacteria cultured to the logarithmic phase were adjusted to a concentration of 1×10 5 CFU / mL with culture medium, and 100 μL of the above bacterial solution was added to each well. Then the plate was placed in a constant temperature incubator at 37°C and cultured for 24 h. Three parallel samples were prepared for each concentration.

[0147] 5. The turbidity of each well was observed, and the lowest concentration of the sample to be tested corresponding to the well with a transparent and non-turbid solution was the minimum inhibitory concentration.

[0148] The synergistic antibacterial performance of gold nanoclusters was determined by chessboard dilution method, and the specific test method is as follows:

[0149] 1. Carbapenem-resistant Klebsiella pneumoniae (CR K.p), carbapenem-resistant Pseudomonas aeruginosa (CR P.a), carbapenem-resistant Acinetobacter baumannii (CR A.b), carbapenem-resistant Enterobacteriaceae (CR E.coli) and methicillin-resistant Staphylococcus aureus (MRSA) were inoculated in liquid LB medium and cultured to logarithmic phase.

[0150] 2. Different concentrations of antibiotic imipenem and gold nanocluster solution were obtained by continuous half dilution, so that the concentration was 2 times the final concentration.

[0151] 3. Then in the 96-well plate, gold nanocluster solution was added in the order of decreasing concentration in the x-axis direction, and the highest final concentration was 64 μg / mL. Similarly, the same volume of antibiotic imipenem solution was added in the order of decreasing concentration in the y-axis direction, and the highest final concentration was 64 μg / mL.

[0152] 4. The bacteria cultured to logarithmic phase were adjusted to a concentration of 1×10 5 CFU / mL, 100 μL of the above bacterial solution was added to each well, and then the plate was placed in a constant temperature incubator at 37°C and cultured for 24 h.

[0153] 5. Then the 96-well plate was placed in a 37°C shaking bed for 12 h, and the absorbance at 600 nm was determined using a microplate reader.

[0154] The interaction between gold nanoclusters and antibiotics was evaluated by fractional inhibitory concentration index (FICI), and the calculation formula was:

[0155] FICI = FIC a + FIC b

[0156] FIC a = MIC of gold nanoclusters when used in combination / MIC of gold nanoclusters when used alone

[0157] FICb = MIC of antibiotic in combination / MIC of antibiotic alone

[0158] Wherein, when FICI≤0.5 is defined as synergistic effect between a and b, 0.5<FICI≤1 is defined as additive effect between a and b; when FICI>1 is defined as no relationship between the two.

[0159] The antibacterial results of three antibiotics meropenem, imipenem, and ertapenem against carbapenem-resistant Klebsiella pneumoniae (CR K.p) are shown in Table 1. Among the three antibiotics, meropenem and imipenem showed resistance to CR K.p bacteria, and ertapenem did not show resistance to CR K.p bacteria. Among the four bacteria, CR K.p 3 and CR K.p 4 exhibited the inhibitory effect of antibiotics on them. The results of the checkerboard dilution method are shown in Table 1. Figure 5 As shown in Table 1, the FICI of Au-PTP NCs C (1:0.5), Au-OTP NCs (1:1) and imipenem were 0.75, 0.625, respectively, showing antagonistic effect on imipenem. The FICI of Au-WTP NCs (1:2), Au-FTP NCs (1:4), Au-TTP NCs (1:10) and imipenem were 0.188, 0.375 and 0.047, respectively, showing synergistic effect with imipenem, among which Au-TTP NCs (1:10) showed the most significant antibacterial effect. Therefore, Au NC (1:10) was selected as the best combination with imipenem.

[0160] To test whether the gold nanoclusters and imipenem have synergistic effect on different strains, we selected CR K.p, CR A.b, CR P.a, CR E.coli and MRSA as model strains. As shown in Table 2, the combination of gold nanoclusters and imipenem was effective on gram-negative bacteria, including CR K.p, CR A.b, CR P.a, CR E.coli, and the FICI was 0.047, 0.313, 0.094, 0.5, respectively. Gold nanoclusters had no synergistic antibacterial effect on gram-positive bacteria MRSA. The combination of imipenem and gold nanoclusters can inhibit the growth of gram-negative bacteria and restore the effect of imipenem. Figure 6

[0161] Table 1 Minimum inhibitory concentration MIC (μg / mL)

[0162]

[0163] Kinetics of gold nanoclusters synergistic antibacterial effect

[0164] ​The experiment will use the gold nanoclusters synthesized in Example 1, imipenem and their combination to incubate bacteria for bactericidal kinetics study, the specific test method is as follows:

[0165] 1. Carbapenem-resistant Klebsiella pneumoniae (CR K.p), carbapenem-resistant Acinetobacter baumannii (CR A.b) and carbapenem-resistant Enterobacteriaceae (CR E.coli) were inoculated in LB liquid medium and cultured at 37℃ to logarithmic phase.

[0166] 2. Phosphate buffered saline solution, gold nanoclusters (10 μg / mL), imipenem (2 μg / mL) and their combination were added to the test tube respectively. Among them, for the CR K.p group, the combination of gold nanoclusters and imipenem was 20 μg / mL+2 μg / mL and 10 μg / mL+8 μg / mL respectively; for the CR E.coli group, the combination of gold nanoclusters and imipenem was 10 μg / mL+2 μg / mL and 10 μg / mL+4 μg / mL respectively; for the CR A.b group, the combination of gold nanoclusters and imipenem was 16 μg / mL+8 μg / mL and 16 μg / mL+16 μg / mL respectively.

[0167] 10μg / mL+2μg / mL and 10μg / mL+4μg / m; for the CR A.b group, the combination of gold nanoclusters and imipenem was 16μg / mL+8μg / mL and 16μg / mL+16μg / mL respectively.

[0168] 3. The bacteria cultured to logarithmic phase were added to the test tube with culture medium to make the final concentration 1×10 5 CFU / mL.

[0169] 4. The bacterial suspension in the test tube was taken at 0h, 1h, 3h, 6h, 9h, 24h and plated on LB agar plate to determine the number of bacteria. At 24h, the liquid was taken out and the OD 600 value was read.

[0170] The results of bactericidal kinetics are shown in Figure 7 20μg / mL gold nanoclusters combined with 2μg / mL imipenem and 10μg / mL gold nanoclusters combined with 8μg / mL imipenem can rapidly kill CR K.p bacteria within 9 hours. Compared with phosphate buffered saline solution, gold nanoclusters and imipenem single drug treatment group, the combination of gold nanoclusters and imipenem shows excellent antibacterial ability, OD600 is 0.04, the difference is extremely significant (P<0.01). In CR E.coli and CR A.b groups, the synergistic bactericidal effect of gold nanoclusters and imipenem is also obtained, and the bacteria can be completely killed in 12 hours.

[0171] Effect of gold nanoclusters combined with antibiotics on the microstructure of bacteria

[0172] In this experiment, the effects of different ligand-modified gold nanoclusters synthesized in Example 1 combined with imipenem on the structure of bacteria will be observed using a scanning electron microscope (SEM).

[0173] 1. Carbapenem-resistant Klebsiella pneumoniae was inoculated in liquid LB medium and cultured to the logarithmic phase.

[0174] 2. The cultured bacteria were centrifuged at 6000 rpm for 3 min to collect the bacterial cells, which were then washed once with PBS buffer. Phosphate buffered saline (control group), gold nanoclusters (10 μg / mL), and imipenem (8 μg / mL) were added, respectively, as well as gold nanoclusters combined with imipenem (20 μg / mL + 2 μg / mL) and gold nanoclusters combined with imipenem (10 μg / mL + 8 μg / mL). The bacteria were adjusted to a final concentration of 1 x 10 5 CFU / mL with culture medium, and the final volume of the five solutions was 2 mL, which was incubated at 37°C for 9 hours.

[0175] 3. After 9 hours, the bacterial cells were collected by centrifugation, and the five solutions were washed once with phosphate buffered saline. 2.5% glutaraldehyde fixing solution was added to the bacterial cells and soaked for 24 h.

[0176] 4. The bacterial cells were washed three times with PBS buffer, and then dehydrated with 30%, 50%, 70%, 90%, 95%, and 100% ethanol for 15 min each. After removing the 100% ethanol, the bacterial cells were transferred to a silicon wafer, air-dried, and observed for morphological changes using SEM.

[0177] The results of the scanning electron microscope are shown in Figure 8 As shown, the CR K.p bacteria in the control group without material treatment were short and thick rods with smooth surfaces and intact cell walls. The cell walls of the bacteria treated with gold nanoclusters and imipenem were relatively intact. However, the CR K.p bacteria treated with gold nanoclusters and imipenem combination showed damage to the cell walls and surface collapse and shrinkage, demonstrating that the combination of gold nanoclusters and imipenem has antibacterial effects and causes damage to the bacterial cell walls.

[0178] Evaluation of the cytotoxicity of gold nanoclusters

[0179] In this experiment, the cytotoxicity of different ligand-modified gold nanoclusters synthesized in Example 1 will be tested.

[0180] 1. In 96-well plates, 8000 HUVEC cells (human umbilical vascular endothelial cells) and 3T3 cells (mouse embryonic fibroblasts) were inoculated in each well, and 100 μL of culture medium was added to each well. The cells were incubated overnight at 37°C in an incubator containing 5% CO2 to allow the cells to adhere completely.

[0181] 2. The culture medium was discarded, and the gold nanoclusters were diluted from a concentration of 400 μg / mL by 2-fold stepwise. 100 μL of gold nanoclusters at different concentrations were added to the 96-well plates and incubated with the cells for 24 h. Control wells (100 μL of culture medium) were set up, and six replicates were set up for each concentration.

[0182] 3. The culture medium in each well was discarded, and CCK8 reagent (purchased from Biyun Tian Biotechnology Co., Ltd.) was added to the 96-well plates of step two as the experimental group. A 96-well plate of the same specification was taken and added with culture medium and CCK8 reagent as the blank group. A 96-well plate of the same specification was taken and added with cells and CCK8 reagent as the negative control group.

[0183] 4. The OD450 value of the test cell culture solution was tested. 450 The OD450 value of the gold nanoclusters was negligible.

[0184] Cell viability = (experimental group - blank) / (control group - blank) * 100%.

[0185] The results are shown in Figure 9 Table 1, which shows that the gold nanoclusters of the present application have no toxicity to the HUVEC and 3T3 cells, and the cell survival rate is between 95-105% at 12 h, 24 h and 48 h.

[0186] Example 8: Evaluation of hemolytic performance of gold nanoclusters

[0187] This test example is used to test the hemolysis test of the tiopronin-modified gold nanoclusters synthesized in Example 1.

[0188] 1. Preparation of blood cell suspension: Mouse whole blood was taken and mixed with about 10 times the amount of 0.9% sodium chloride solution. Centrifugation was performed at 1200 r / min for 15 minutes, and the supernatant was removed to obtain the precipitated red blood cells. The red blood cells were washed with 0.9% sodium chloride solution until the supernatant was not red. The obtained red blood cells were prepared into a 2% suspension with 0.9% sodium chloride solution for use.

[0189] 2. Experimental Procedure: Take nine clean centrifuge tubes, numbered 1 to 7. Tube 1 is the positive control (water), tube 2 is the negative control, and tubes 3-9 contain different drug concentrations: 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively. Add 2% red blood cell suspension to each tube, mix well, and immediately incubate at 37°C. Observe every 15 minutes, then every hour after 1 hour, and perform testing after 3 hours.

[0190] 3. Results observation: After 3 hours of incubation, centrifuge at 1200 r / min for 15 minutes, record the image, and measure the absorbance at 540 nm using a UV-Vis spectrophotometer.

[0191] The results of this experiment are as follows: Figure 10 As shown, the gold nanoclusters do not cause blood clotting.

[0192] In vivo biosafety assessment of gold nanoclusters

[0193] This test case was used to evaluate the biosafety of the thiopronine-modified gold nanoclusters synthesized in Example 1.

[0194] 1. Each mouse was injected intraperitoneally with physiological saline solution (control group) and gold nanoclusters (experimental group), with the injection dose of gold nanoclusters being greater than 50 mg / kg body weight: 50 mg / kg.

[0195] 2. Observe the survival rate of mice at 12, 24, and 48 hours, and record biochemical indicators, including complete blood count and liver and kidney function indicators. Three mice were used in each group, and the experiment was repeated three times.

[0196] The results of this experiment are shown in Table 2. At 12, 24, and 48 hours after treatment with high-concentration (50 mg / kg) gold nanoclusters, the biochemical indicators of the two groups with different administration routes, including complete blood count (RBC: red blood cell count, WBC: white blood cell count, and PLT: platelet count), liver function indicators (ALT: alanine aminotransferase, AST: aspartate aminotransferase, ALB: albumin), and kidney function indicators (ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; CR: creatinine), showed no significant differences compared to the control group (mice were injected with the same volume of saline without gold nanoclusters at the same site), and all were within the normal range. These results indicate that the gold nanoclusters in this invention have high biosafety and can be safely used in various biological applications.

[0197] Table 2. In vivo biosafety assessment results of the gold nanoclusters prepared in the examples.

[0198]

[0199] Effect of gold nanoclusters combined with antibiotics in treatment of bacterial infection in animal models

[0200] Establishment of mouse peritoneal bacterial infection model: CR K.p, CR A.b and CR E.coli were selected as model strains.

[0201] 1. BALB / c female mice were randomly divided into 5 groups, n = 3 in each group, and placed in drug or control cages. Cyclophosphamide was injected intraperitoneally 4 days (150 mg / kg) and 1 day (100 mg / kg) before bacterial infection, respectively.

[0202] 2. The bacteria were cultured to the logarithmic phase, the bacterial bodies were collected by centrifugation, and washed twice with phosphate buffered saline.

[0203] 3. CR K.p, CR A.b and CR E.coli strains were suspended in 1 x 10 5 CFU / mL and intranasally injected, respectively.

[0204] 4. Half an hour after infection, treatment was performed by intranasal administration. The first, second and third groups of mice were treated with phosphate buffered saline, gold nanoclusters (10 mg / kg) and imipenem (8 mg / kg), respectively, and the fourth and fifth groups were treated with gold nanoclusters and imipenem (10 mg / kg + 8 mg / kg). Three hours later, the fifth group was injected with a second dose of gold nanoclusters and imipenem (10 mg / kg + 8 mg / kg).

[0205] 5. The mice were euthanized 24 hours after infection. The mouse organ tissues (heart, liver, spleen, lung and kidney) were collected, homogenized and plated on LB agar plates, and the number of bacterial colonies was counted.

[0206] The experimental results are shown in Figure 11 In the CR K.p bacterial infection model, the lungs of mice injected with phosphate buffered saline showed obvious congestion and hemorrhage, and the lesions were often scattered in the lobes of both lungs. The lungs of mice treated with gold nanoclusters (alone) or imipenem (alone) showed a slight decrease in congestion points. The lungs treated with gold nanoclusters combined with imipenem showed a significant reduction in congestion and hemorrhage.

[0207] After homogenizing the lung tissue and performing standard plate colony counting, it was observed that the lungs of the mice treated with PBS contained a large amount of CR K. p bacteria. The number of bacteria in the lungs of the mice treated with GNC alone or imipenem alone was reduced, but the antibacterial effect was not obvious. The antibacterial effect of the lungs of the mice treated with GNC and imipenem was significantly improved, and the colony forming units were reduced by three orders of magnitude. The antibacterial effect of GNC and imipenem on bacteria was also verified in the hearts, livers, spleens and kidneys of the mice.

[0208] To evaluate the potential of the synergistic regimen and the effect of personalized treatment, we also selected CR E. coli and CR A. b as experimental objects, as shown in Figure 12 , Figure 13 According to the results of lung imaging, the lungs of the mice treated with PBS contained a large amount of congestion, and the congestion area was almost distributed in the entire lung lobe, and the bacterial load was high. Compared with the single drug group, the congestion of the lungs of the mice treated with GNC and imipenem was significantly reduced, and the amount of bacteria was also significantly reduced, which showed a good effect of clearing bacteria. The results of GNC and imipenem in enhancing the antibacterial ability were also exhibited in the hearts, livers, spleens and kidneys of the mice. In general, the in vivo antibacterial study not only proved that GNC restored the antibacterial effect of imipenem, but also verified the broad-spectrum effect of GNC and imipenem against gram-negative pathogens.

[0209] The above embodiments are combined to illustrate the present application in detail, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the present application.

Claims

1. The application of gold nanoclusters in the preparation of antibacterial drugs, characterized in that, The antibacterial drug includes gold nanoclusters modified with thiopronine, wherein the gold nanoclusters have gold atoms and sulfur atoms, and the gold nanoclusters are used in combination with carbapenem antibiotics to synergistically inhibit carbapenem-resistant Gram-negative pathogens, wherein the carbapenem antibiotics are meropenem, imipenem or ertapenem. The gold atoms and the sulfur atoms are bonded by covalent bonds; The particle size range of the gold nanoclusters is 1 nm to 20 nm.

2. The application according to claim 1, characterized in that, The gold nanoclusters are prepared by mixing a gold source, thioproline, and a reducing agent and reacting them to obtain the gold nanoclusters.

3. The application according to claim 2, characterized in that, The gold source includes chloroauric acid.

4. The application according to claim 2, characterized in that, The reducing agent includes sodium borohydride.

5. The application according to claim 2, characterized in that, The molar ratio of the gold source and thiopronin is 1:0.5~10.

6. The application according to claim 2, characterized in that, Includes the following steps: S1: Mix and dissolve the gold source and thioproline in a solvent to obtain a mixture; S2: Under stirring conditions, a reducing agent is added to the mixture, and after reaction, a crude product is obtained; S3: Purify the crude product to obtain the gold nanoclusters.

Images