Preparation method and application of pyrimidine-coated gold nanoparticle antibacterial material

Abstract

A preparation method and application of a pyrimidine-coated gold nanoparticle antibacterial material, the invention belongs to the technical field of antibacterial nanomaterials. The invention aims to solve the problem that the existing gold nanoparticle antibacterial material has a complicated synthesis process and cannot have an antibacterial effect on both Gram-negative bacteria and Gram-positive bacteria. Method: 1. Prepare a CMTPC solution; 2. Mix the CMTPC solution with a chloroauric acid solution, and then drop a NaOH solution; 3. Heat the reaction. The invention is used for the preparation and application of a pyrimidine-coated gold nanoparticle antibacterial material.

Description

Technical Field

[0001] The invention belongs to the technical field of antibacterial nano materials. Background Art

[0002] As we all know, bacteria are one of the most numerous organisms with extremely high adaptability and reproduction rate, especially pathogenic bacteria. Even a small amount can indirectly cause human infection by contaminating water sources, food, medical equipment, etc., or directly infect the human body, and severe infection can be life-threatening. Antibiotics are an effective method for treating such diseases. However, the long-term and excessive use of antibiotics by people has prompted the evolution of bacteria, causing bacteria to overcome the effects of antibiotics through a series of ways such as mutations and develop drug resistance, resulting in unsatisfactory antibiotic treatment effects. Faced with this dilemma, exploring new antibacterial materials has also become an important means to combat bacterial infections.

[0003] Gold nanomaterials have attracted extensive attention in the field of biomedical research because they are easy to functionalize on the surface and have unique physical and chemical properties, optical characteristics, and excellent biocompatibility. However, the following problems still exist with regard to antibacterial gold nanoparticles:

[0004] (1) In addition to stabilizers, most chemical synthesis methods of gold nanoparticles require additional chemical reducing agents, which are dangerous. In addition, their synthesis requires high reaction temperatures, high equipment requirements, and certain safety hazards.

[0005] (2) Most of the antibacterial materials based on gold nanoparticles need to modify the surface ligands of antibiotics and organic small molecules on the basis of the synthesized gold nanoparticles, and the synthesis steps are complicated.

[0006] (3) Currently, the antibacterial effect of gold nanoparticles is not ideal. Some gold nanoparticles can only have antibacterial effects on Gram-negative bacteria or Gram-positive bacteria alone and do not have broad-spectrum antibacterial properties. Summary of the invention

[0007] The present invention aims to solve the problem that the existing gold nanoparticle antibacterial materials have complex synthesis processes and cannot have antibacterial effects on both Gram-negative bacteria and Gram-positive bacteria at the same time, and further provides a preparation method and application of pyrimidine-coated gold nanoparticle antibacterial materials.

[0008] A method for preparing a pyrimidine-coated gold nanoparticle antibacterial material is carried out according to the following steps:

[0009] 1. Adding NaOH solution dropwise into the dispersed solution of 2-methylthio-5-chloropyrimidine-4-carboxylic acid to obtain a CMTPC solution;

[0010] 2. Mix the CMTPC solution with the chloroauric acid solution, then dropwise add the NaOH solution and mix well to obtain a mixed system;

[0011] 3. The mixed system is heated to react to obtain a colloidal solution, the colloidal solution is centrifuged to remove the supernatant, and the nanoparticles are separated and finally the nanoparticles are dispersed in sterile ultrapure water to obtain a pyrimidine-coated gold nanoparticle antibacterial material.

[0012] The invention discloses an application of a pyrimidine-coated gold nanoparticle antibacterial material, wherein the pyrimidine-coated gold nanoparticle antibacterial material is used for inhibiting and sterilizing gram-negative bacteria and gram-positive bacteria.

[0013] The beneficial effects of the present invention are:

[0014] (1) The present invention realizes for the first time the synthesis of gold nanoparticles by a one-step reaction using 2-methylthio-5-chloropyrimidine-4-carboxylic acid as a protective ligand and a reducing agent.

[0015] (2) The synthesis process of the gold nanoparticles prepared by the present invention is simple, the reaction temperature is mild, the cost is low, and the environment is friendly. 2-Methylthio-5-chloropyrimidine-4-carboxylic acid is non-toxic as an organic ligand and is often used in pharmaceutical synthesis. Its methylthio group can form a specific bonding reaction with the Au core to prevent further aggregation of the gold nanoparticles in the solution.

[0016] (3) The gold nanoparticles synthesized by the present invention can not only effectively inhibit the growth of bacteria, but also kill 99% of the bacteria in a short period of time, kill 8 orders of magnitude of Pseudomonas aeruginosa (P.aeruginosa) and 6 orders of magnitude of Staphylococcus aureus (S.aureus) within 2 hours, and have good antibacterial effects on both Gram-negative and Gram-positive bacteria, reaching the national Class I antibacterial standard.

[0017] (4) The gold nanoparticles synthesized by the present invention can be stably stored for 2 days in various environments (water, PBS buffer, 0.9% saline) at 37°C and for 1 month at 4°C (pure water), which is beneficial to the practical application of nanomaterials.

[0018] The invention is used for a preparation method and application of a pyrimidine-coated gold nanoparticle antibacterial material. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a flow chart of the preparation of the pyrimidine-coated gold nanoparticle antibacterial material of Example 1;

[0020] Figure 2 The transmission electron microscopy (TEM) images of the MP-AuNPs prepared in Example 1, A is a transmission electron microscopy image, B is a transmission electron microscopy image at high magnification, C is an electron diffraction image, and D is a transmission electron element mapping image;

[0021] Figure 3 is a UV-visible absorption spectrum, 1 is MP-AuNPs in Example 1, 2 is CMTPC, and 3 is HAuCl4;

[0022] Figure 4 The stability test of MP-AuNPs in different solutions at 37°C, A is 0.9% saline, B is water, C is PBS buffer (pH=5), and D is PBS buffer (pH=9);

[0023] Figure 5 is the absorbance at 529 nm of MP-AuNPs in 4°C water for 3, 6, 9, 15, and 30 days;

[0024] Figure 6 The antibacterial effect diagram of Pseudomonas aeruginosa (A) and Staphylococcus aureus (B), 1 is MP-AuNPs solution (concentration 1pM), 2 is CMTPC solution (0.2wt%), 3 is blank;

[0025] Figure 7 The diagram shows the sterilization process of MP-AuNPs;

[0026] Figure 8 The graph shows the bactericidal effect on Pseudomonas aeruginosa (A) and Staphylococcus aureus (B), 1 is the MP-AuNPs solution (concentration 1 pM), 2 is the CMTPC solution (0.2 wt%), and 3 is the blank. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.

[0028] Specific implementation method 1: This implementation method is a method for preparing a pyrimidine-coated gold nanoparticle antibacterial material, which is carried out according to the following steps:

[0029] 1. Adding NaOH solution dropwise into the dispersed solution of 2-methylthio-5-chloropyrimidine-4-carboxylic acid to obtain a CMTPC solution;

[0030] 2. Mix the CMTPC solution with the chloroauric acid solution, then dropwise add the NaOH solution and mix well to obtain a mixed system;

[0031] 3. The mixed system is heated to react to obtain a colloidal solution, the colloidal solution is centrifuged to remove the supernatant, and the nanoparticles are separated and finally the nanoparticles are dispersed in sterile ultrapure water to obtain a pyrimidine-coated gold nanoparticle antibacterial material.

[0032] In this embodiment, the pyrimidine small molecule 2-methylthio-5-chloro-4-pyrimidinecarboxylic acid is used as an intermediate for synthesizing a variety of drugs, which is safe and non-toxic to humans. The pyrimidine small molecule 2-methylthio-5-chloro-4-pyrimidinecarboxylic acid is used as a reducing agent and a stabilizing ligand, a part of which can reduce the trivalent gold in chloroauric acid to zero-valent gold, and the other part is successfully modified on the surface of gold nanoparticles to prevent further aggregation of gold nanoparticles without the need for additional reducing agents. The prepared gold nanoparticles were added to the bacterial liquid of Pseudomonas aeruginosa and Staphylococcus aureus, and their broad-spectrum antibacterial effect was observed by the growth curve method and the plate coating method.

[0033] The beneficial effects of this embodiment are:

[0034] (1) This embodiment realizes for the first time the synthesis of gold nanoparticles by a one-step reaction using 2-methylthio-5-chloropyrimidine-4-carboxylic acid as a protective ligand and a reducing agent.

[0035] (2) The synthesis process of the gold nanoparticles prepared in this embodiment is simple, the reaction temperature is mild, the cost is low, and the environment is friendly. 2-Methylthio-5-chloropyrimidine-4-carboxylic acid is non-toxic as an organic ligand and is often used in pharmaceutical synthesis. Its methylthio group can form a specific bonding reaction with the Au core to prevent further aggregation of the gold nanoparticles in the solution.

[0036] (3) The gold nanoparticles synthesized by this embodiment can not only effectively inhibit the growth of bacteria, but also kill 99% of bacteria in a short period of time, killing 8 orders of magnitude of Pseudomonas aeruginosa (P.aeruginosa) and 6 orders of magnitude of Staphylococcus aureus (S.aureus) within 2 hours, and have good antibacterial effects on both Gram-negative and Gram-positive bacteria, reaching the national Class I antibacterial standard.

[0037] (4) The gold nanoparticles synthesized in this embodiment can stably exist for 2 days in various environments at 37°C (water, PBS buffer, 0.9% saline) and can be stored for 1 month at 4°C (pure water), which is beneficial to the practical application of nanomaterials.

[0038] Specific implementation method 2: This implementation method is different from the specific implementation method 1 in that the concentration of NaOH in the CMTPC solution described in step 1 is 9.6 mmol / L to 12 mmol / L. The rest is the same as the specific implementation method 1.

[0039] Specific embodiment 3: This embodiment is different from specific embodiment 1 or 2 in that the concentration of 2-methylthio-5-chloropyrimidine-4-carboxylic acid in the CMTPC solution in step 1 is 1 mmol / L to 30 mmol / L. Others are the same as specific embodiment 1 or 2.

[0040] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the concentration of 2-methylthio-5-chloropyrimidine-4-carboxylic acid in the mixed system described in step 2 is 0.83 mmol / L to 25 mmol / L. Other aspects are the same as specific embodiments 1 to 3.

[0041] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that the concentration of chloroauric acid in the mixed system described in step 2 is 0.16 mmol / L to 4 mmol / L. The rest is the same as specific embodiments 1 to 4.

[0042] Specific implementation example 6: This implementation example is different from specific implementation examples 1 to 5 in that the concentration of NaOH in the mixed system described in step 2 is 13 mmol / L to 25 mmol / L. The rest is the same as specific implementation examples 1 to 5.

[0043] Specific embodiment 7: This embodiment differs from Specific embodiments 1 to 6 in that the heating reaction in step 3 is carried out at a reaction temperature of 40° C. to 70° C. for 1 h to 2.5 h. The rest is the same as Specific embodiments 1 to 6.

[0044] Specific embodiment 8: This embodiment differs from specific embodiments 1 to 7 in that the centrifugation in step 3 is carried out at a temperature of 4°C to 10°C and a rotation speed of 10000 rpm to 11000 rpm for 40 min to 60 min. The rest is the same as specific embodiments 1 to 7.

[0045] Specific implementation method 9: This implementation method provides an application of a pyrimidine-coated gold nanoparticle antibacterial material. The pyrimidine-coated gold nanoparticle antibacterial material is used for inhibiting and sterilizing Gram-negative bacteria and Gram-positive bacteria.

[0046] Specific embodiment 10: This embodiment is different from specific embodiment 9 in that: the Gram-negative bacteria is Pseudomonas aeruginosa, and the Gram-positive bacteria is Staphylococcus aureus. Other aspects are the same as specific embodiment 9.

[0047] The following examples are used to verify the beneficial effects of the present invention:

[0048] Embodiment 1, combined with Figure 1 Specific instructions:

[0049] A method for preparing a pyrimidine-coated gold nanoparticle antibacterial material is carried out according to the following steps:

[0050] 1. Add NaOH solution dropwise into the dispersed solution of 2-methylthio-5-chloropyrimidine-4-carboxylic acid, and dissolve it by ultrasonication for 1 min at an ultrasonic power of 100 W to obtain a CMTPC solution;

[0051] The concentration of NaOH in the CMTPC solution is 9.6 mmol / L; the concentration of 2-methylthio-5-chloropyrimidine-4-carboxylic acid in the CMTPC solution is 10 mmol / L;

[0052] 2. Mix the CMTPC solution with chloroauric acid solution (HAuCl4), then dropwise add NaOH solution, and shake and mix for 1 min to obtain a mixed system;

[0053] The concentration of 2-methylthio-5-chloropyrimidine-4-carboxylic acid in the mixed system is 8.3 mmol / L;

[0054] The concentration of chloroauric acid in the mixed system is 1.7 mmol / L;

[0055] The concentration of NaOH in the mixed system is 13.2 mmol / L;

[0056] 3. Under the condition of reaction temperature of 55°C, the mixed system was heated to react for 1.5h to obtain a colloidal solution. The colloidal solution was centrifuged to remove the supernatant, and the nanoparticles were separated. The nanoparticles were dispersed in sterile ultrapure water. The centrifugation and dispersion were repeated 3 times to obtain a solution containing pyrimidine-coated gold nanoparticle antibacterial material, namely, MP-AuNPs solution.

[0057] The centrifugation in step 3 is specifically carried out at a temperature of 4° C. and a rotation speed of 11000 rpm for 40 minutes.

[0058] The 2-methylthio-5-chloropyrimidine-4-carboxylic acid (CMTPC abbreviated as MP, CAS: 61727-33-1) used in this example was purchased from Aladdin Reagent (Shanghai) Co., Ltd., and the molecular formula is as follows:

[0059]

[0060] The MP-AuNPs solution prepared in Example 1 was dropped on a copper mesh and then dried for transmission electron microscopy; Figure 2 The transmission electron microscope (TEM) image of MP-AuNPs prepared in Example 1, A is a transmission electron microscope image, B is a transmission electron microscope image at high magnification, C is an electron diffraction image, and D is a transmission electron element mapping image; it can be seen from the figure that the shape of the synthesized gold nanoparticles is spherical, with good dispersibility in water, no agglomeration, and an average diameter of about 13nm. The high-resolution TEM image shows a set of lattice planes with an inter-plane d spacing of 0.22nm, corresponding to the (111) plane of Au. In addition, the TEM-mapping image verifies the uniform dispersion of S and Au elements, indicating that CMTPC (MP) is successfully modified on the surface of the nanoparticles as a protective ligand.

[0061] Figure 3 The UV-visible absorption spectrum, 1 is the MP-AuNPs of Example 1, 2 is CMTPC, and 3 is HAuCl4; it can be seen from the figure that CMTPC and HAuCl4 have almost no absorption peaks at 500-600nm, while the synthesized MP-AuNPs have a broad absorption band at 529nm, which is the surface plasmon resonance (SPR) peak, indicating that the synthesized product is gold nanoparticles. In addition, the absorption of CMTPC near 263nm and 306nm belongs to the carboxyl group and pyrimidine ring.

[0062] The stability of the MP-AuNPs prepared in Example 1 in different solutions at 37° C. was tested: specifically, the UV spectra of the MP-AuNPs in 0.9% by mass saline, water, PBS buffer (pH=5) and PBS buffer (pH=9) were tested for 12 h, 24 h, 36 h and 48 h; Figure 4 The stability test of MP-AuNPs in different solutions at 37°C, A is 0.9% saline, B is water, C is PBS buffer (pH=5), and D is PBS buffer (pH=9). After being placed at 37°C for two days, the surface plasmon resonance absorption peak in its UV spectrum did not undergo red shift, indicating that MP-AuNPs can exist stably without agglomeration.

[0063] Testing the stability of the MP-AuNPs prepared in Example 1 in water at 4°C: Testing the absorbance of the MP-AuNPs at 529 nm in water for 3 days, 6 days, 9 days, 15 days and 30 days; Figure 5 This is the absorbance of MP-AuNPs at 529nm in 4°C water for 3 days, 6 days, 9 days, 15 days and 30 days. It can be seen that there is no obvious change in the absorbance, proving that MP-AuNPs have good stability.

[0064] Figure 6 The antibacterial effect diagram of Pseudomonas aeruginosa (A) and Staphylococcus aureus (B), 1 is MP-AuNPs solution (concentration 1pM), 2 is CMTPC solution (0.2wt%), and 3 is blank. The test process is as follows:

[0065] Bacterial activation: Take out the preserved P. aeruginosa and S. aureus from the 4°C refrigerator and put them in the clean bench. Use an inoculation loop to take a colony and add it to LB medium (ingredients: NaCl is 10g / L, yeast extract powder is 5g / L, tryptone is 10g / L), and culture it in a constant temperature shaker at 130r / min and 37°C for 12h.

[0066] Add materials: Prepare a 96-well plate, take 100 μL LB medium (bacterial concentration is 10 6 CFU / mL), 100 μL of MP-AuNPs solution (concentration 1 pM) or CMTPC solution (0.2 wt%) was added to each well of the experimental group, and 100 μL of sterile water was added to the positive control group.

[0067] The well plate was placed in a fully automatic curve growth instrument and cultured for 16 hours at 130 r / min and 37°C. All the above experiments were performed in a sterile operating table.

[0068] The optical density (OD) at 600 nm 600 ) reflects the concentration of bacteria in the culture medium. The sterile LB liquid culture medium is clear and transparent. As the number of bacteria in the culture medium increases, the solution gradually becomes turbid, accompanied by a decrease in the transmittance of the solution. Figure 6 It can be seen that the bacterial growth of the control group and the group with CMTPC was almost not inhibited, while the group containing MP-AuNPs completely inhibited the growth of P. aeruginosa ( Figure 6 A) and S. aureus( Figure 6 B), which proves that MP-AuNPs effectively slowed down the growth of Gram-negative and Gram-positive bacteria and had a broad-spectrum antibacterial activity.

[0069] Figure 7 The diagram shows the sterilization process of MP-AuNPs; Figure 8 The sterilization effect diagram of Pseudomonas aeruginosa (A) and Staphylococcus aureus (B) is shown in Figure 1. 1 is MP-AuNPs solution (concentration 1pM), 2 is CMTPC solution (0.2wt%), and 3 is blank. The test process is as follows:

[0070] Bacterial activation and treatment: Take out the preserved P.aeruginosa and S.aureus from the 4℃ refrigerator and put them in the clean bench. Use an inoculation loop to take a colony and add it to LB medium (ingredients: NaCl is 10g / L, yeast extract powder is 5g / L, tryptone is 10g / L), and culture it in a constant temperature shaker at 130r / min and 37℃ for 12h. Centrifuge the obtained bacterial solution (6000rpm, 5min) and resuspend it in PBS buffer (0.01M, pH=7), and adjust the bacterial solution concentration to OD 600 =0.2.

[0071] Material addition: prepare 2mL sterile centrifuge tubes, add 500μL of the above bacterial solution to each centrifuge tube, take 500μL MP-AuNPs solution (concentration 1pM) or 500mL CMTPC solution (0.2wt%) in the experimental group, add them to the bacterial solution of P.aeruginosa and S.aureus, respectively, add 500μL sterile water to the blank control group, place in a 37℃ constant temperature shaker, and incubate at 130r / min for 1h, 2h, 3h, 4h, and 5h, respectively.

[0072] Plate coating: Place the sterile culture dish prepared in advance into the clean bench, take 100 μL of the bacterial solution of the experimental group and the control group cultured at different times, spread it evenly on the solid culture dish with a sterile coating rod, and place it in a 37°C constant temperature incubator for 24 hours.

[0073] Plate count: Calculate the bactericidal efficiency of MP-AuNPs after incubation with bacteria for different times.

[0074] from Figure 8 It can be seen that the prepared MP-AuNPs can effectively kill 99.99% of P. aeruginosa ( Figure 8 A) and 99% of S. aureus ( Figure 8 B). With the increase of incubation time, the colony count of P. aeruginosa decreased by 8 orders of magnitude in only 2 hours, and the colony count of S. aureus decreased by 6 orders of magnitude. In addition, CMTPC alone cannot kill bacteria directly, indicating that its antibacterial activity is caused by the newly generated gold nanoparticles. The experimental results show that the gold nanoparticles synthesized by this method have high bactericidal efficiency against both Gram-negative and Gram-positive bacteria and have broad-spectrum bactericidal activity.

Claims

1. A method for preparing a pyrimidine-coated gold nanoparticle antibacterial material, characterized in that It is carried out in the following steps:

1. Adding NaOH solution dropwise into the dispersed solution of 2-methylthio-5-chloropyrimidine-4-carboxylic acid to obtain a CMTPC solution; 2. Mix the CMTPC solution with the chloroauric acid solution, then dropwise add the NaOH solution and mix well to obtain a mixed system; 3. heating the mixed system to react to obtain a colloidal solution, centrifuging the colloidal solution to remove the supernatant, separating and obtaining nanoparticles, and finally dispersing the nanoparticles in sterile ultrapure water to obtain a pyrimidine-coated gold nanoparticle antibacterial material; The concentration of NaOH in the CMTPC solution described in step 1 is 9.6 mmol / L to 12 mmol / L; The concentration of 2-methylthio-5-chloropyrimidine-4-carboxylic acid in the CMTPC solution described in step 1 is 1 mmol / L to 30 mmol / L; The concentration of 2-methylthio-5-chloropyrimidine-4-carboxylic acid in the mixed system described in step 2 is 0.83 mmol / L to 25 mmol / L; The concentration of chloroauric acid in the mixed system described in step 2 is 0.16 mmol / L to 4 mmol / L; The concentration of NaOH in the mixed system described in step 2 is 13 mmol / L to 25 mmol / L.

2. The method for preparing a pyrimidine-coated gold nanoparticle antibacterial material according to claim 1, characterized in that The heating reaction described in step 3 is specifically carried out at a reaction temperature of 40° C. to 70° C. for 1 h to 2.5 h.

3. The method for preparing a pyrimidine-coated gold nanoparticle antibacterial material according to claim 1, characterized in that The centrifugation described in step 3 is specifically carried out at a temperature of 4°C to 10°C and a rotation speed of 10000 rpm to 11000 rpm for 40 min to 60 min.

4. The use of a pyrimidine-coated gold nanoparticle antibacterial material prepared as claimed in claim 1, characterized in that Pyrimidine-coated gold nanoparticle antibacterial materials are used for the inhibition and sterilization of Gram-negative and Gram-positive bacteria.

5. The use of a pyrimidine-coated gold nanoparticle antibacterial material according to claim 4, characterized in that The Gram-negative bacteria is Pseudomonas aeruginosa; the Gram-positive bacteria is Staphylococcus aureus.

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