Nanometer gold particle, its preparation method and application

Abstract

This invention discloses a gold nanoparticle, its preparation method, and its application, relating to the field of biomaterials technology. The gold nanoparticle is prepared by a method comprising the following steps: preparing a first mixed solution containing a gold salt, a ligand, and a stabilizer; performing a first reaction to obtain a reaction solution; preparing a second mixed solution containing a reducing agent and the reaction solution; and performing a second reaction to obtain the gold nanoparticle. The ligand has the structure shown in the following general formula: R1 is selected from Cl, Br, C1-C4 alkane groups or hydroxyl groups; R2 is selected from Cl, Br, C1-C4 alkane groups or hydroxyl groups. The prepared gold nanoparticle exhibits good biocompatibility and antibacterial properties, demonstrating significant social, economic, and technological benefits, as well as high practical application value and promising prospects for wider application.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a gold nanoparticle, its preparation method, and its application. Background Technology

[0002] Bacteria, as pathogens, can invade host cells, produce pathogenic substances, and cause infection. Statistics show that, out of a random sample of 100,000 deaths each year, approximately 33,000 are directly caused by bacterial infections. Antibiotics are used to treat bacterial infections and reduce their harm. However, while antibiotics have been very successful in treating bacterial infections, their overuse has led to significant waste of medical supplies and exacerbated problems such as antibiotic resistance and adverse reactions. Furthermore, because the resistance genes of superbugs can spread rapidly between different bacteria, antibiotics previously used to treat bacterial infections are becoming ineffective.

[0003] Therefore, it is important to provide new antibacterial substances. Summary of the Invention

[0004] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a method for preparing gold nanoparticles, which is simple in process and can prepare gold nanoparticles with good biocompatibility and antibacterial properties.

[0005] The present invention also provides gold nanoparticles prepared by the above preparation method.

[0006] The present invention also provides an antibacterial composition comprising gold nanoparticles.

[0007] The present invention also provides the application of the above preparation method, the above gold nanoparticles, or the above antibacterial composition.

[0008] A method for preparing gold nanoparticles according to a first aspect of the present invention includes the following steps:

[0009] A first mixed solution of gold salt, ligand, and stabilizer is prepared, and a first reaction is carried out to obtain a reaction solution; a second mixed solution containing a reducing agent and the reaction solution is prepared, and a second reaction is carried out to obtain gold nanoparticles;

[0010] The ligand has a structure represented by the following general formula:

[0011]

[0012] R1 is selected from Cl, Br, C1-C4 alkane groups or hydroxyl groups;

[0013] R2 is selected from Cl, Br, C1-C4 alkane groups or hydroxyl groups.

[0014] According to some embodiments of the present invention, the reaction temperature of the preparation method is 0 to 4°C.

[0015] According to some embodiments of the present invention, the alkane groups of C1 to C4 are -CH3, -CH2CH3, -CH2CH2CH3 or -CH2CH2CH2CH3.

[0016] According to some embodiments of the present invention, the gold salt is a soluble gold salt. The gold salt includes at least one selected from chloroauric acid, tetrachloroauric acid trihydrate, and tetrachloroauric acid tetrahydrate.

[0017] According to some embodiments of the present invention, the stabilizer includes at least one of mercaptoPEG, Tween80, and PVP. The stabilizer makes the synthesized gold nanoparticles more stable; otherwise, the synthesized gold nanoparticles would quickly precipitate in water.

[0018] According to some embodiments of the present invention, the number-average molecular weight of the thiol PEG is 500 to 5000. This better ensures the colloidal stability of the gold nanoparticles.

[0019] According to some embodiments of the present invention, the ligand includes at least one of the compounds shown in Formula I, Formula II, Formula III, Formula IV, and Formula V.

[0020]

[0021] According to some embodiments of the present invention, the molar ratio of the gold salt to the ligand is 1:0.1 to 2.5. For example, it can be 1:0.1, 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, 1:2.4 or 1:2.5.

[0022] According to some embodiments of the present invention, the molar ratio of the ligand to the stabilizer is 1:0.005 to 0.25. For example, it can be 1:0.005, 1:0.01, 1:0.02, 1:0.04, 1:0.06, 1:0.08, 1:0.1, 1:0.12, 1:0.14, 1:0.16, 1:0.18, 1:0.2, or 1:0.25.

[0023] According to some embodiments of the present invention, when the ligand is a compound of Formula I: the molar ratio of the gold salt to the ligand is 1:1.8 to 2.2, and the molar ratio of the ligand to the stabilizer is 1:0.08 to 0.22. Alternatively, the molar ratio of the gold salt to the ligand is 1:0.8 to 1.2, and the molar ratio of the ligand to the stabilizer is 1:0.08 to 0.22. Another option is that the molar ratio of the gold salt to the ligand is 1:0.4 to 0.6, and the molar ratio of the ligand to the stabilizer is 1:0.03 to 0.22 (e.g., 1:0.08 to 0.22). Finally, the molar ratio of the gold salt to the ligand is 1:0.2 to 0.3, and the molar ratio of the ligand to the stabilizer is 1:0.03 to 0.22 (e.g., 1:0.08 to 0.22 or 1:0.18 to 0.22). The molar ratio of the gold salt to the ligand is 1:0.1 to 0.15, and the molar ratio of the ligand to the stabilizer is 1:0.18 to 0.22.

[0024] According to some embodiments of the present invention, when the ligand is a compound of formula II: the molar ratio of the gold salt to the ligand is 1:1.8 to 2.2, and the molar ratio of the ligand to the stabilizer is 1:0.03 to 0.22 (for example, it can be 1:0.08 to 0.12). Alternatively, the molar ratio of the gold salt to the ligand is 1:0.8 to 1.2, and the molar ratio of the ligand to the stabilizer is 1:0.018 to 0.22 (for example, it can be 1:0.08 to 0.22). Or, the molar ratio of the gold salt to the ligand is 1:0.4 to 0.6, and the molar ratio of the ligand to the stabilizer is 1:0.03 to 0.22 (for example, it can be 1:0.08 to 0.22 or 1:0.08 to 0.12). The molar ratio of the gold salt to the ligand is 1:0.1 to 0.15, and the molar ratio of the ligand to the stabilizer is 1:0.08 to 0.22 (for example, it can be 1:0.18 to 0.22).

[0025] According to some embodiments of the present invention, when the ligand is a compound of formula III: the molar ratio of the gold salt to the ligand is 1:1.8 to 2.2, and the molar ratio of the ligand to the stabilizer is 1:0.08 to 0.22. Alternatively, the molar ratio of the gold salt to the ligand is 1:0.8 to 1.2, and the molar ratio of the ligand to the stabilizer is 1:0.03 to 0.22.

[0026] According to some embodiments of the present invention, when the ligand is a compound of formula IV: the molar ratio of the gold salt to the ligand is 1:1.8 to 2.2, and the molar ratio of the ligand to the stabilizer is 1:0.08 to 0.22 (for example, 1:0.18 to 0.22). Alternatively, the molar ratio of the gold salt to the ligand is 1:0.8 to 1.2, and the molar ratio of the ligand to the stabilizer is 1:0.08 to 0.22 (for example, 1:0.08 to 0.12). Or, the molar ratio of the gold salt to the ligand is 1:0.4 to 0.6, and the molar ratio of the ligand to the stabilizer is 1:0.03 to 0.22 (for example, 1:0.08 to 0.22 or 1:0.18 to 0.22). Or, the molar ratio of the gold salt to the ligand is 1:0.1 to 0.15, and the molar ratio of the ligand to the stabilizer is 1:0.18 to 0.22.

[0027] According to some embodiments of the present invention, when the ligand is a compound of formula V: the molar ratio of the gold salt to the ligand is 1:1.8 to 2.2, and the molar ratio of the ligand to the stabilizer is 1:0.03 to 0.22 (for example, it can be 1:0.08 to 0.22). Alternatively, the molar ratio of the gold salt to the ligand is 1:0.8 to 1.2, and the molar ratio of the ligand to the stabilizer is 1:0.03 to 0.22 (for example, it can be 1:0.08 to 0.22 or 1:0.18 to 0.22).

[0028] According to some embodiments of the present invention, the concentration of the gold salt in the first mixed solution is 30-50 mg / mL. For example, it can be 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL or 50 mg / mL.

[0029] According to some embodiments of the present invention, the reaction solvent for the first reaction and / or the second reaction includes at least one of methanol and deionized water.

[0030] According to some embodiments of the present invention, the preparation of the first mixed solution of gold salt, ligand, and stabilizer comprises: adding a ligand solution and a stabilizer solution dropwise to a gold salt solution. The volume ratio of the gold salt solution to the ligand solution and the stabilizer solution is 1:0.4-0.6:0.4-0.6.

[0031] According to some embodiments of the present invention, the reaction time of the first reaction is 10 to 30 minutes. For example, it can be 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes.

[0032] According to some embodiments of the present invention, the reducing agent includes sodium borohydride (NaBH4).

[0033] According to some embodiments of the present invention, the molar ratio of the gold salt to the reducing agent is 1:2 to 4. For example, it can be 1:2, 1:2.3, 1:2.5, 1:2.7, 1:3, 1:3.3, 1:3.5, 1:3.8 or 1:4.

[0034] According to some embodiments of the present invention, the concentration of the reducing agent in the second mixed solution is 0.3 to 0.6 mg / mL. For example, it can be 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, or 0.6 mg / mL.

[0035] According to some embodiments of the present invention, preparing a second mixed solution containing a reducing agent and a reaction solution comprises: adding a reducing agent solution dropwise to the reaction solution. The concentration of the reducing agent solution is 2-3 mg / mL. For example, it can be 2 mg / mL, 2.1 mg / mL, 2.2 mg / mL, 2.3 mg / mL, 2.4 mg / mL, 2.5 mg / mL, 2.6 mg / mL, 2.7 mg / mL, 2.8 mg / mL, 2.9 mg / mL, or 3 mg / mL. By adding the reducing agent dropwise and stirring the reaction, the reducing agent can be more fully reduced to obtain gold nanoparticles.

[0036] According to some embodiments of the present invention, the reaction time of the second reaction is 70 to 100 minutes. For example, it can be 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, or 100 minutes.

[0037] According to some embodiments of the present invention, the second reaction includes reacting sequentially at a first rotational speed for 10 min to 20 min and at a second rotational speed for 50 min to 80 min; the first rotational speed is greater than the second rotational speed.

[0038] According to some embodiments of the present invention, the first rotational speed is 1000 to 2000 rpm. For example, it can be 1000 rpm, 1100 rpm, 1200 rpm, 1300 rpm, 1400 rpm, 1500 rpm, 1600 rpm, 1700 rpm, 1800 rpm, 1900 rpm or 2000 rpm.

[0039] According to some embodiments of the present invention, the second rotational speed is 400 rpm to 800 rpm. For example, it can be 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm, 750 rpm or 800 rpm.

[0040] According to some embodiments of the present invention, the preparation method further includes post-reaction processing. The post-reaction processing includes purification.

[0041] According to some embodiments of the present invention, the purification process includes dialysis. The molecular weight cutoff for dialysis is 12–15 kDa (e.g., 14 kDa). The dialysis time is 48–72 hours.

[0042] According to a second aspect of the present invention, gold nanoparticles are prepared by the above-described preparation method.

[0043] According to some embodiments of the present invention, the average hydrated particle size of the gold nanoparticles is 8 nm to 50 nm. For example, it can be 8 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm or 50 nm.

[0044] According to some embodiments of the present invention, the potential of the gold nanoparticles is -25mV to -5mV. For example, it can be -25mV, -23mV, -20mV, -18mV, -15mV, -13mV, -10mV, -8mV, or -5mV.

[0045] According to some embodiments of the present invention, the MIC value of the gold nanoparticles against MRSA is 0.5 to 32 μg / mL.

[0046] An antibacterial composition according to a third aspect of the present invention comprises gold nanoparticles as described in the second aspect of the present invention.

[0047] According to some embodiments of the present invention, the antibacterial composition may further include other components with antibacterial activity, preferably without affecting the function of the gold nanoparticles.

[0048] According to some embodiments of the present invention, the antibacterial composition further includes excipients or carriers.

[0049] The application of the preparation method as described in the first aspect embodiment of the present invention, or the gold nanoparticles as described in the second aspect embodiment, or the antibacterial composition as described in the third aspect embodiment, in the preparation of antibacterial products according to the fourth aspect embodiment of the present invention.

[0050] According to some embodiments of the present invention, the gold nanoparticles are used to inhibit bacterial cell wall synthesis.

[0051] According to some embodiments of the present invention, the antimicrobial product is selected from pharmaceuticals, formulations, reagent kits, packaging materials, medical supplies, or fabrics.

[0052] According to some embodiments of the present invention, the antibacterial product is used to fight bacteria.

[0053] According to some embodiments of the present invention, the bacteria include Gram-positive bacteria. The Gram-positive bacteria include clinical isolates resistant to antibiotics.

[0054] According to some embodiments of the present invention, the Gram-positive bacteria include at least one of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, methicillin-resistant Staphylococcus epidermidis, Enterococcus faecalis, multidrug-resistant Enterococcus faecalis, hemolytic Staphylococcus, and methicillin-resistant hemolytic Staphylococcus.

[0055] The present invention has at least the following beneficial effects:

[0056] The gold nanoparticles of this invention exhibit broad-spectrum antibacterial activity against Gram-positive bacteria, demonstrating long-lasting antibacterial and highly effective bactericidal properties. Compared with vancomycin, a commonly used antibiotic in clinical practice, their antibacterial performance is superior. Furthermore, these antibacterial gold nanoparticles possess good biocompatibility, making them promising candidate drugs for combating Gram-positive bacterial infections and holding promise for controlling bacterial infections and their spread. The preparation method in this embodiment employs a one-pot process, which is simple, and the resulting antibacterial material exhibits excellent colloidal stability and superior antibacterial performance. In conclusion, this invention contributes to controlling drug-resistant bacterial infections and their spread, and is of great significance for improving people's quality of life and promoting social health. It possesses significant social, economic, and technological benefits, as well as high practical application value and promising prospects for wider application.

[0057] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0058] Figure 1 The results of detecting the MIC values ​​of gold nanoparticles prepared with the compound shown in Formula I against different Staphylococcus aureus strains; where Figure A represents Staphylococcus aureus ATCC 29213 and Figure B represents MRSA.

[0059] Figure 2 The results of detecting the MIC values ​​of gold nanoparticles prepared with the compound shown in Formula II against different Staphylococcus aureus strains; where Figure A represents Staphylococcus aureus ATCC 29213 and Figure B represents MRSA.

[0060] Figure 3 The results of detecting the MIC values ​​of gold nanoparticles prepared with the compound shown in Formula III against different Staphylococcus aureus strains; where Figure A represents Staphylococcus aureus ATCC 29213 and Figure B represents MRSA.

[0061] Figure 4The results of detecting the MIC values ​​of gold nanoparticles prepared with the compound shown in Formula IV against different Staphylococcus aureus strains; where Figure A represents Staphylococcus aureus ATCC 29213 and Figure B represents MRSA.

[0062] Figure 5 The detection results of MIC values ​​of gold nanoparticles prepared with the compound shown in Formula V against different Staphylococcus aureus strains; where Figure A is Staphylococcus aureus ATCC 29213 and Figure B is MRSA.

[0063] Figure 6 The average particle size of the gold nanoparticles in Examples 1 and 2 are as follows;

[0064] Figure 7 The potential values ​​of the gold nanoparticles in Examples 1 and 2 are the detection results.

[0065] Figure 8 The UV-Vis absorption spectra of the gold nanoparticles in Examples 1 and 2 are shown.

[0066] Figure 9 Time-based sterilization curves for low-concentration gold nanoparticles in Examples 1 and 2;

[0067] Figure 10 Time-based sterilization curves for high-concentration gold nanoparticles in Examples 1 and 2;

[0068] Figure 11 The effects of gold nanoparticles in Examples 1 and 2 on the growth curves of Staphylococcus aureus ATCC 29213 are shown in Figure A, which is the growth curve at a concentration of 1 μg / mL, and Figure B is the growth curve at a concentration of 2 μg / mL.

[0069] Figure 12 The effects of gold nanoparticles in Examples 1 and 2 on the growth curves of MRSA are shown in Figure A, which is the growth curve at an effective concentration of 1 μg / mL, and Figure B is the growth curve at an effective concentration of 2 μg / mL.

[0070] Figure 13 The effects of gold nanoparticles in Examples 1 and 2 on routine blood parameters in mice;

[0071] Figure 14 The effects of gold nanoparticles in Examples 1 and 2 on blood biochemical parameters in mice;

[0072] Figure 15 This is a TEM image showing the effect of gold nanoparticles on bacteria in Example 2;

[0073] Figure 16 This is to demonstrate the in vivo antibacterial effect of the gold nanoparticles in Example 2. Detailed Implementation

[0074] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0075] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0076] In the description of this invention, the use of terms such as "first," "second," etc., is for the purpose of distinguishing technical features only and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0077] In the description of this invention, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0078] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0079] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, or article that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, or article.

[0080] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0081] In the embodiments of the present invention, the number average molecular weight of the thiol PEG used is 2000, purchased from SIGMA-ALDRICH, catalog number 729140-1G, trade name polyethylene glycol methyl ether thiol.

[0082] In an embodiment of the present invention, the MIC value of gold nanoparticles is detected by the following method. The steps are as follows:

[0083] 1) Preparation of bacterial culture: Staphylococcus aureus ATCC 29213 (hereinafter referred to as Sa), MRSA, Staphylococcus epidermidis (hereinafter referred to as SE), methicillin-resistant Staphylococcus epidermidis (MRSE), Enterococcus faecalis (hereinafter referred to as EF), multidrug-resistant Enterococcus faecalis (MREF), hemolytic Staphylococcus (hereinafter referred to as SH), and methicillin-resistant hemolytic Staphylococcus faecalis (MRSH) were inoculated into LB solid culture plates and cultured overnight at 37°C; single colonies were then picked and inoculated into LB liquid medium and cultured overnight at 37°C; an appropriate amount of bacterial suspension was taken and diluted with LB liquid medium to a bacterial concentration of 10. 6 CFU / mL was used to obtain a diluted bacterial suspension.

[0084] 2) Preparation of drug solution: Dilute the gold nanoparticles to a certain concentration using LB liquid culture medium to obtain a series of particle solutions with different concentration gradients.

[0085] 3) MIC determination: Add equal volumes of diluted bacterial suspension and drug solution to each well of a 96-well plate. Each treatment group was repeated three times. After incubation at 37°C for 24 hours, observe the colony growth and turbidity in each well to determine the minimum drug concentration required to inhibit colony proliferation. Compared with the blank control group, the lowest drug concentration that keeps the culture medium clear, from high to low concentration, is the MIC.

[0086] Effect of compound (ligand 23) shown in Formula I on the antibacterial properties of gold nanoparticles

[0087] Gold nanoparticles were prepared using the compound shown in Formula I (CAS No. 51618-29-2) as a ligand, and the effect of synthesis conditions on the antibacterial properties of the gold nanoparticles was investigated by orthogonal method.

[0088]

[0089] The preparation of gold nanoparticles was carried out under ice-water bath conditions. The specific steps are as follows:

[0090] 10 mL of methanol was stirred in an ice-water bath for 10 min. Then, HAuCl4·3H2O solution (41 mg, 0.1 mmol, dissolved in 1 mL of methanol) was added and stirred for another 10 min to obtain a pale yellow, transparent solution. 5 mL of ligand solution (dissolved in methanol) and 5 mL of mercaptoPEG solution (dissolved in methanol) were then slowly added dropwise. The solution immediately turned turbid with pale yellow flocculent matter. After stirring for 20 min, NaBH4 (12 mg, 0.3 mmol, freshly dissolved in 5 mL of methanol) was slowly added dropwise while stirring vigorously (1500 rpm). The solution immediately turned black, then dark brown, and stirring continued for another 15 min. The stirring speed was then reduced (600 rpm), and stirring continued in an ice-water bath for one hour. The reaction solution was dialyzed against deionized water (14 kDa MW cut-toff, Millipore) for 72 h and sterilized using a 0.22 μm filter (Millipore) to obtain gold nanoparticles.

[0091] Different amounts of ligand were added, with the ligand solutions containing 0.2 mmol, 0.1 mmol, 0.05 mmol, 0.025 mmol, and 0.0125 mmol of the compound shown in Formula I, corresponding to molar ratios of gold to ligand (Au:ligand) of 1:2, 1:1, 2:1, 4:1, and 8:1, respectively. Different amounts of thiol PEG were also added, resulting in molar ratios of thiol PEG to ligand (PEG:ligand) of 0.01:1, 0.02:1, 0.05:1, 0.1:1, and 0.2:1, corresponding to 1%, 2%, 5%, 10%, and 20%, respectively. For example, when the ligand solution contained 0.2 mmol of the compound shown in Formula I and the thiol PEG solution contained 0.04 mmol of thiol PEG, the PEG:ligand ratio was 20%.

[0092] The MIC value test results of the gold nanoparticles are as follows: Figure 1 As shown.

[0093] Effect of compound (ligand 34) of Formula II on the antibacterial properties of gold nanoparticles

[0094] Gold nanoparticles were prepared using the compound shown in Formula II (CAS No. 5331-91-9) as a ligand, and the effect of synthesis conditions on the antibacterial properties of the gold nanoparticles was investigated by orthogonal method.

[0095]

[0096] The preparation of gold nanoparticles was carried out under ice-water bath conditions. The specific steps are as follows:

[0097] 10 mL of methanol was stirred in an ice-water bath for 10 min. Then, HAuCl4·3H2O solution (41 mg, 0.1 mmol, dissolved in 1 mL of methanol) was added and stirred for another 10 min to obtain a pale yellow, transparent solution. 5 mL of ligand solution (dissolved in methanol) and 5 mL of mercaptoPEG solution (dissolved in methanol) were then slowly added dropwise. The solution immediately turned turbid with pale yellow flocculent matter. After stirring for 20 min, NaBH4 (12 mg, 0.3 mmol, freshly dissolved in 5 mL of methanol) was slowly added dropwise while stirring vigorously (1500 rpm). The solution immediately turned black, then dark brown, and stirring continued for another 15 min. The stirring speed was then reduced (600 rpm), and stirring continued in an ice-water bath for one hour. The reaction solution was dialyzed against deionized water (14 kDa MW cut-toff, Millipore) for 72 h and sterilized using a 0.22 μm filter (Millipore) to obtain gold nanoparticles.

[0098] Different amounts of ligand were added, with the ligand solutions containing 0.2 mmol, 0.1 mmol, 0.05 mmol, 0.025 mmol, and 0.0125 mmol of the compound shown in Formula II, corresponding to molar ratios of gold to ligand (Au:ligand) of 1:2, 1:1, 2:1, 4:1, and 8:1, respectively. Different amounts of thiol PEG were also added, resulting in molar ratios of thiol PEG to ligand (PEG:ligand) of 0.01:1, 0.02:1, 0.05:1, 0.1:1, and 0.2:1, corresponding to 1%, 2%, 5%, 10%, and 20%, respectively. For example, when the ligand solution contained 0.2 mmol of the compound shown in Formula II and the thiol PEG solution contained 0.04 mmol of thiol PEG, the PEG:ligand ratio was 20%.

[0099] The MIC value test results of the gold nanoparticles are as follows: Figure 2 As shown.

[0100] Effect of compound (ligand 39) of formula III on the antibacterial properties of gold nanoparticles

[0101] Gold nanoparticles were prepared using the compound shown in Formula III (CAS No. 155559-81-2), and the effects of synthesis conditions on the antibacterial properties of the gold nanoparticles were investigated by orthogonal method.

[0102]

[0103] The preparation of gold nanoparticles was carried out under ice-water bath conditions. The specific steps are as follows:

[0104] 10 mL of methanol was stirred in an ice-water bath for 10 min. Then, HAuCl4·3H2O solution (41 mg, 0.1 mmol, dissolved in 1 mL of methanol) was added and stirred for another 10 min to obtain a pale yellow, transparent solution. 5 mL of ligand solution (dissolved in methanol) and 5 mL of mercaptoPEG solution (dissolved in methanol) were then slowly added dropwise. The solution immediately turned turbid with pale yellow flocculent matter. After stirring for 20 min, NaBH4 (12 mg, 0.3 mmol, freshly dissolved in 5 mL of methanol) was slowly added dropwise while stirring vigorously (1500 rpm). The solution immediately turned black, then dark brown, and stirring continued for another 15 min. The stirring speed was then reduced (600 rpm), and stirring continued in an ice-water bath for one hour. The reaction solution was dialyzed against deionized water (14 kDa MW cut-toff, Millipore) for 72 h and sterilized using a 0.22 μm filter (Millipore) to obtain gold nanoparticles.

[0105] Different amounts of ligand were added, with the ligand solutions containing 0.2 mmol, 0.1 mmol, 0.05 mmol, 0.025 mmol, and 0.0125 mmol of the compound shown in Formula III, corresponding to molar ratios of gold to ligand (Au:ligand) of 1:2, 1:1, 2:1, 4:1, and 8:1, respectively. Different amounts of thiol PEG were also added, resulting in molar ratios of thiol PEG to ligand (PEG:ligand) of 0.01:1, 0.02:1, 0.05:1, 0.1:1, and 0.2:1, corresponding to 1%, 2%, 5%, 10%, and 20%, respectively. For example, when the ligand solution contained 0.2 mmol of the compound shown in Formula III and the thiol PEG solution contained 0.04 mmol of thiol PEG, the PEG:ligand ratio was 20%.

[0106] The MIC value test results of the gold nanoparticles are as follows: Figure 3 As shown.

[0107] Effect of compound (ligand 27) of formula IV on the antibacterial properties of gold nanoparticles

[0108] Gold nanoparticles were prepared using the compound shown in Formula IV (CAS No. 2268-79-3) as a ligand, and the effect of synthesis conditions on the antibacterial properties of the gold nanoparticles was investigated by orthogonal method.

[0109]

[0110] The preparation of gold nanoparticles was carried out under ice-water bath conditions. The specific steps are as follows:

[0111] 10 mL of methanol was stirred in an ice-water bath for 10 min. Then, HAuCl4·3H2O solution (41 mg, 0.1 mmol, dissolved in 1 mL of methanol) was added and stirred for another 10 min to obtain a pale yellow, transparent solution. 5 mL of ligand solution (dissolved in methanol) and 5 mL of mercaptoPEG solution (dissolved in methanol) were then slowly added dropwise. The solution immediately turned turbid with pale yellow flocculent matter. After stirring for 20 min, NaBH4 (12 mg, 0.3 mmol, freshly dissolved in 5 mL of methanol) was slowly added dropwise while stirring vigorously (1500 rpm). The solution immediately turned black, then dark brown, and stirring continued for another 15 min. The stirring speed was then reduced (600 rpm), and stirring continued in an ice-water bath for one hour. The reaction solution was dialyzed against deionized water (14 kDa MW cut-toff, Millipore) for 72 h and sterilized using a 0.22 μm filter (Millipore) to obtain gold nanoparticles.

[0112] Different amounts of ligand were added, with the ligand solutions containing 0.2 mmol, 0.1 mmol, 0.05 mmol, 0.025 mmol, and 0.0125 mmol of the compound shown in Formula IV, corresponding to molar ratios of gold to ligand (Au:ligand) of 1:2, 1:1, 2:1, 4:1, and 8:1, respectively. Different amounts of thiol PEG were also added, resulting in molar ratios of thiol PEG to ligand (PEG:ligand) of 0.01:1, 0.02:1, 0.05:1, 0.1:1, and 0.2:1, corresponding to 1%, 2%, 5%, 10%, and 20%, respectively. For example, when the ligand solution contained 0.2 mmol of the compound shown in Formula IV and the thiol PEG solution contained 0.04 mmol of thiol PEG, the PEG:ligand ratio was 20%.

[0113] The MIC value test results of the gold nanoparticles are as follows: Figure 4 As shown.

[0114] Effect of compound V (ligand 38) on the antibacterial properties of gold nanoparticles

[0115] Gold nanoparticles were prepared using the compound shown in Formula V as a ligand (CAS No. 80087-71-4), and the effects of synthesis conditions on the antibacterial properties of the gold nanoparticles were investigated by orthogonal method.

[0116]

[0117] The preparation of gold nanoparticles was carried out under ice-water bath conditions. The specific steps are as follows:

[0118] 10 mL of methanol was stirred in an ice-water bath for 10 min. Then, HAuCl4·3H2O solution (41 mg, 0.1 mmol, dissolved in 1 mL of methanol) was added and stirred for another 10 min to obtain a pale yellow, transparent solution. 5 mL of ligand solution (dissolved in methanol) and 5 mL of mercaptoPEG solution (dissolved in methanol) were then slowly added dropwise. The solution immediately turned turbid with pale yellow flocculent matter. After stirring for 20 min, NaBH4 (12 mg, 0.3 mmol, freshly dissolved in 5 mL of methanol) was slowly added dropwise while stirring vigorously (1500 rpm). The solution immediately turned black, then dark brown, and stirring continued for another 15 min. The stirring speed was then reduced (600 rpm), and stirring continued in an ice-water bath for one hour. The reaction solution was dialyzed against deionized water (14 kDa MW cut-toff, Millipore) for 72 h and sterilized using a 0.22 μm filter (Millipore) to obtain gold nanoparticles.

[0119] Different amounts of ligand were added, with the ligand solutions containing 0.2 mmol, 0.1 mmol, 0.05 mmol, 0.025 mmol, and 0.0125 mmol of the compound shown in Formula V, corresponding to molar ratios of gold to ligand (Au:ligand) of 1:2, 1:1, 2:1, 4:1, and 8:1, respectively. Different amounts of thiol PEG were also added, resulting in molar ratios of thiol PEG to ligand (PEG:ligand) of 0.01:1, 0.02:1, 0.05:1, 0.1:1, and 0.2:1, corresponding to 1%, 2%, 5%, 10%, and 20%, respectively. For example, if the ligand solution contained 0.2 mmol of the compound shown in Formula V and the thiol PEG solution contained 0.04 mmol of thiol PEG, then the PEG:ligand ratio would be 20%.

[0120] The MIC value test results of the gold nanoparticles are as follows: Figure 5 As shown.

[0121] A suitable ratio of high PEG concentration and high ligand concentration can make the synthesized gold nanoparticles more stable and have better antibacterial properties.

[0122] Example 1

[0123] This example provides a gold nanoparticle (hereinafter referred to as: 6-chloro-2-mercaptobenzothiazole). It is prepared under ice-water bath conditions. The specific preparation method is as follows:

[0124] 10 mL of methanol was stirred in an ice-water bath for 10 min. Then, HAuCl4·3H2O solution (41 mg, 0.1 mmol, dissolved in 1 mL of methanol) was added and stirred for another 10 min to obtain a pale yellow, transparent solution. Then, 5 mL of ligand solution (containing 0.2 mmol of the compound shown in Formula I, dissolved in methanol) and 5 mL of mercaptoPEG solution (containing 0.04 mmol of mercaptoPEG, dissolved in methanol) were slowly added dropwise. The solution immediately turned turbid and contained pale yellow flocculent matter. After stirring for 20 min, NaBH4 (12 mg, 0.3 mmol, freshly dissolved in 5 mL of methanol) was slowly added dropwise while stirring vigorously (1500 rpm). The solution immediately turned black, then dark brown, and stirring continued for 15 min. Then, the stirring speed was reduced (600 rpm), and stirring continued in an ice-water bath for one hour. The reaction solution was dialyzed with deionized water (14kDa MW cut-toff, Millipore) for 72 hours and then sterilized with a 0.22μm filter (Millipore) to obtain gold nanoparticles.

[0125] Example 2

[0126] This example provides a gold nanoparticle (hereinafter referred to as: 5-chloro-2-mercaptobenzothiazole). It is prepared under ice-water bath conditions. The specific preparation method is as follows:

[0127] 10 mL of methanol was stirred in an ice-water bath for 10 min. Then, HAuCl4·3H2O solution (41 mg, 0.1 mmol, dissolved in 1 mL of methanol) was added and stirred for another 10 min to obtain a pale yellow, transparent solution. Then, 5 mL of ligand solution (containing 0.2 mmol of the compound shown in Formula II, dissolved in methanol) and 5 mL of mercaptoPEG solution (containing 0.04 mmol of mercaptoPEG, dissolved in methanol) were slowly added dropwise. The solution immediately turned turbid and contained pale yellow flocculent matter. After stirring for 20 min, NaBH4 (12 mg, 0.3 mmol, freshly dissolved in 5 mL of methanol) was slowly added dropwise while stirring vigorously (1500 rpm). The solution immediately turned black, then dark brown, and stirring continued for 15 min. Then, the stirring speed was reduced (600 rpm), and stirring continued in an ice-water bath for one hour. The reaction solution was dialyzed with deionized water (14kDa MW cut-toff, Millipore) for 72 hours and then sterilized with a 0.22μm filter (Millipore) to obtain gold nanoparticles.

[0128] Detection Example 1

[0129] The gold nanoparticles were diluted to 128 μg / mL with ultrapure water. The average particle size and particle size distribution of the gold nanoparticles were measured and recorded using a particle size analyzer. The potential values ​​of the gold nanoparticles were measured and recorded using a Malvern potentiometer. The UV-Vis absorption curves were measured using a UV-Vis spectrophotometer, and the absorption peak positions and intensities of the gold nanoparticles were recorded.

[0130] The results are as follows Figures 6-8 As shown.

[0131] The average hydrated particle size of the gold nanoparticles in Example 1 was 15.63±6.18 nm, and the potential value was -7.58±1.50 mV; the average hydrated particle size of the gold nanoparticles in Example 2 was 11.58±1.36 nm, and the potential value was -16.03±3.56 mV.

[0132] Detection Example 2

[0133] This example demonstrates the testing of the antibacterial properties of gold nanoparticles.

[0134] (1) The detection results of the MIC values ​​of the gold nanoparticles in Examples 1 and 2 for SE, MRSE, EF, MREF, SH and MRSH are shown in Table 1.

[0135] Table 1

[0136]

[0137] (2) Time-based sterilization curve test: The experimental method is as follows:

[0138] 1) Preparation of bacterial culture medium: Staphylococcus aureus ATCC 29213 and MRSA were inoculated into LB agar plates and cultured overnight at 37°C; single colonies were picked and inoculated into LB liquid medium and cultured overnight at 37°C; an appropriate amount of bacterial suspension was taken and diluted with LB liquid medium to a suitable bacterial concentration to obtain a diluted bacterial suspension.

[0139] 2) Preparation of drug solution: Dilute the gold nanoparticles to a certain concentration using LB liquid culture medium to obtain a drug solution.

[0140] 3) Plot the time-killing curve: Add equal volumes of diluted bacterial suspension and drug solution to 96-well plates. Each treatment group was repeated 3 times; the control group had the drug solution replaced with an equal volume of LB liquid medium, and samples were taken every 3 hours to measure bacterial growth.

[0141] For Staphylococcus aureus ATCC 29213: The initial bacterial concentration of Staphylococcus aureus ATCC 29213 is 1×10⁻⁶. 6At CFU / mL, the effective concentrations of the gold nanoparticles in Examples 1 and 2 were set to 1 and 2 μg / mL, respectively. The initial bacterial concentration of Staphylococcus aureus ATCC 29213 was 1 × 10⁻⁶. 10 At CFU / mL, the effective concentration of the gold nanoparticles in Examples 1 and 2 was set to 8 μg / mL, respectively.

[0142] For MRSA: The initial bacterial concentration of MRSA is 5 × 10⁻⁶. 5 At CFU / mL, the effective concentrations of gold nanoparticles in Example 1 were set to 2 and 4 μg / mL, respectively, and the effective concentrations of gold nanoparticles in Example 2 were set to 1 and 2 μg / mL, respectively. The initial bacterial concentration of MRSA was 1 × 10⁻⁶. 9 At CFU / mL, the effective concentration of the gold nanoparticles in Examples 1 and 2 was set to 8 μg / mL, respectively.

[0143] The results are as follows Figures 9-10 As shown.

[0144] The antibacterial effects of the gold nanoparticles in Examples 1 and 2 were both concentration- and time-dependent.

[0145] (3) Bacterial growth curve test:

[0146] 1) Preparation of bacterial culture: Staphylococcus aureus ATCC 29213 and MRSA were inoculated into LB agar plates and incubated overnight at 37°C; single colonies were then picked and inoculated into LB liquid medium and incubated overnight at 37°C; an appropriate amount of bacterial suspension was taken and diluted with LB liquid medium to a suitable bacterial concentration to obtain a diluted bacterial suspension (concentration of 2×10⁻⁶). 5 (CFU / mL)

[0147] 2) Preparation of drug solutions: The gold nanoparticles and vancomycin were diluted to 2 and 4 μg / mL, respectively, using LB liquid medium to obtain drug solutions.

[0148] 3) Plotting bacterial growth curves: Add equal volumes of diluted bacterial suspension and drug solution to 96-well plates. Each treatment group was repeated three times. In the control group, the drug solution was replaced with an equal volume of LB liquid medium, and bacterial growth was measured over 36 hours. Bacterial growth was expressed as absorbance (OD) at 600 nm.

[0149] The results are as follows Figures 11-12 As shown.

[0150] The gold nanoparticles in Examples 1 and 2 can effectively inhibit the growth of Staphylococcus aureus and MRSA.

[0151] Detection Example 3

[0152] This study tested the effects of gold nanoparticles on blood parameters in mice to assess their biological effects and safety.

[0153] Nine Balb / c mice (purchased from Guangdong Provincial Animal Center, 6-8 weeks old, 6-18g, 9 females) were randomly divided into a control group, a Example 1 group (6-chloro-2-mercaptobenzothiazole), and a Example 2 group (5-chloro-2-mercaptobenzothiazole), with 3 mice in each group. They were raised and treated according to laboratory animal management regulations. The control group was administered 200 μL of physiological saline via tail vein injection; the Example 1 group was administered 50 mg / kg bw of Example 1 gold nanoparticles (200 μL) via tail vein injection; and the Example 2 group was administered 50 mg / kg bw of Example 2 gold nanoparticles (200 μL) via tail vein injection. Two days later, blood samples were collected from the mice via orbital blood collection. Blood samples were subjected to routine blood tests using equipment such as a blood cell analyzer (including indicators such as white blood cell count (WBC), red blood cell count (RBC), hemoglobin concentration, and platelet count (PLT)); and blood samples were subjected to biochemical tests using equipment such as a biochemical analyzer (including serum protein (ALB), liver function indicators (aspartate aminotransferase AST, alanine aminotransferase ALT, etc.), kidney function indicators (creatinine CR, blood urea nitrogen Urea, etc.), serum alkaline phosphatase (ALP), serum uric acid (UA), and total protein (TP), etc.).

[0154] The results are as follows Figures 13-14 As shown.

[0155] The gold nanoparticles in Examples 1 and 2 had little effect on various indicators in mouse blood. This suggests that the gold nanoparticles prepared in the examples of this invention have good biosafety.

[0156] Detection Example 4

[0157] This example demonstrates the antibacterial mechanism of gold nanoparticles.

[0158] The concentrations of 1×10⁻⁶ gold nanoparticles were treated with 8 μg / mL of gold nanoparticles from Example 2. 8CFU / mL *Staphylococcus aureus* ATCC 29213 or MRSA were treated for 6 h as the treatment group (NP Treatment); untreated *Staphylococcus aureus* or MRSA served as the control group (Control). Bacteria were fixed with 2.5% glutaraldehyde in 0.1M carboxylate buffer (pH 7.4) for 3 h, washed twice with PBS, then fixed with 0.1% osmotic acid for 30 min, washed three times with PBS, and subjected to a gradient dehydration process using different concentrations (v / v) of ethanol-water solution (ethanol concentrations of 30%, 50%, 70%, 90%, 95%, and 100%, respectively), each dehydration lasting 10 min. The dehydrated samples were embedded in Epon812 and polymerized at 60°C for 24 h. The samples were then cut into ultrathin slices with a thickness of 60–70 nm and placed in… On the mesh of the coating. Adjust the TEM's accelerating voltage, contrast, aperture size, and other parameters according to the sample type and the structural features to be observed to obtain optimal imaging results. Use the TEM's focusing system to focus the electron beam onto the sample, ensuring a clear image. Select the area and acquire the image.

[0159] The results are as follows Figure 15 As shown.

[0160] Gold nanoparticles did not affect the integrity of bacterial cell membranes, but they did cause abnormal cell wall synthesis. This indicates that gold nanoparticles exert their antibacterial effect by inhibiting cell wall synthesis.

[0161] Case 5

[0162] This example demonstrates the in vivo antibacterial effect of gold nanoparticles.

[0163] Twenty-eight female Balb / c mice (purchased from Guangdong Provincial Animal Center, 6-8 weeks old, 16-18g) were used to establish a neutrophil-deficient thigh infection model in mice. On day 1, mice were injected intraperitoneally with cyclophosphamide solution (physiological saline) at a dose of 200 mg / kg body weight (bw). On day 4, mice were injected intraperitoneally with cyclophosphamide solution at a dose of 150 mg / kg body weight (bw), with an injection volume of 0.2 mL. The thigh infection model mice were randomly divided into 7 groups of 4 mice each. After disinfecting the dorsal skin of the thigh with 75% alcohol, MRSA was inoculated into the thigh muscles. 0.2 mL of bacterial suspension (bacterial concentration 1×10⁻⁶) was inoculated into each thigh. 9 (CFU / mL). Two legs were taken from each mouse for counting, resulting in 8 data points per group. The treatment methods for each group of mice are as follows:

[0164] (1) Baseline: 2 hours after MRSA inoculation, the bacterial content at the infection site on the thigh of mice in the baseline group was measured.

[0165] (2) Blank control group (saline): 2 hours after MRSA inoculation, 100 μL of physiological saline was administered to the thigh infection model mice via subcutaneous injection, once.

[0166] (3) Positive control group (vanco 20mg / kg): 2 hours after MRSA inoculation, the mice with thigh infection model were given 20mg / kg vancomycin (100μL, in physiological saline) by subcutaneous injection, once.

[0167] (4) 2.5 mg / kg group: 2 h after MRSA inoculation, mice with thigh infection were given 2.5 mg / kg of gold nanoparticles from Example 2 (100 μL, in physiological saline) by subcutaneous injection. The administration was repeated once.

[0168] (5) 5mg / kg group: 2 hours after MRSA inoculation, mice with thigh infection were given 5mg / kg of gold nanoparticles from Example 2 (100μL, in physiological saline) via subcutaneous injection. The administration was repeated once.

[0169] (6) 10 mg / kg group: 2 h after MRSA inoculation, mice with thigh infection were given 10 mg / kg of gold nanoparticles from Example 2 (100 μL, in physiological saline) via subcutaneous injection. The administration was repeated once.

[0170] (7) 20 mg / kg group: 2 h after MRSA inoculation, mice with thigh infection were given 20 mg / kg of gold nanoparticles from Example 2 (100 μL, in physiological saline) via subcutaneous injection. The administration was repeated once.

[0171] Mice in the blank control group, positive control group, 2.5 mg / kg group, 5 mg / kg group, 10 mg / kg group, and 20 mg / kg group were injected with the drug 24 hours later. The bacterial content at the infection site in the thigh of the mice was measured. The method for measuring the bacterial content at the infection site in the thigh of the mice was as follows: the mice were sacrificed, the thigh muscles were isolated under sterile conditions, weighed, and homogenized in 1.5 mL of sterile PBS solution in an ice bath. After serial 10-fold dilution, the homogenate was spread on LB solid medium and incubated at 35°C for 18 hours. After that, the colony count was performed.

[0172] The results are as follows Figure 16 As shown.

[0173] The gold nanoparticles of Example 2 demonstrated good antibacterial activity in a mouse thigh infection model with neutrophils. Compared with the blank control group, the bacterial content in the leg was reduced by orders of magnitude 1.39, 1.61, 1.24, and 1.96 at doses of 2.5 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg of gold nanoparticles, respectively, while the reduction was only by orders of magnitude 0.78 at a dose of 20 mg / kg vancomycin. The gold nanoparticles of Example 2 exhibit superior in vivo antibacterial activity compared to vancomycin.

[0174] In summary, the gold nanoparticles prepared in this application have excellent antibacterial effects.

[0175] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A method for preparing gold nanoparticles, characterized in that, Includes the following steps: A first mixed solution of gold salt, ligand, and stabilizer is prepared, and a first reaction is carried out to obtain a reaction solution; a second mixed solution containing a reducing agent and the reaction solution is prepared, and a second reaction is carried out to obtain gold nanoparticles; The ligand has a structure represented by the following general formula: ； R1 is selected from Cl, Br, C1-C4 alkane groups or hydroxyl groups; R2 is selected from Cl, Br, C1-C4 alkane groups or hydroxyl groups; The stabilizer includes at least one of mercaptoPEG, Tween80, and PVP; The molar ratio of the gold salt to the ligand is 1:0.1~2.5; The molar ratio of the ligand to the stabilizer is 1:0.005~0.25; The molar ratio of the gold salt to the reducing agent is 1:2~4.

2. The preparation method according to claim 1, characterized in that, The gold salt includes at least one of chloroauric acid, tetrachloroauric acid trihydrate, and tetrachloroauric acid tetrahydrate.

3. The preparation method according to claim 1, characterized in that, The reducing agent includes sodium borohydride.

4. A type of gold nanoparticle, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 3.

5. The gold nanoparticles according to claim 4, characterized in that, The average hydrated particle size of the gold nanoparticles is 8 nm to 50 nm.

6. An antibacterial composition, characterized in that, Including the gold nanoparticles as described in claim 4 or 5.

7. The antibacterial composition according to claim 6, characterized in that, The antibacterial composition also includes excipients or carriers.

8. The use of the gold nanoparticles of claim 4 or 5, or the antibacterial composition of claim 6 or 7, in the preparation of antibacterial products.

Images