Application of chiral gold nanomaterial in preparation of bactericidal drugs

Abstract

The invention discloses an application of a chiral gold nano-material in preparation of bactericidal drugs, and a preparation method of the chiral gold nano-material comprises the following steps: uniformly mixing a gold precursor aqueous solution with a sulfydryl-containing chiral ligand solution, dropwise adding a reducing agent solution, and carrying out a stirring reaction to obtain the chiral gold nano-material, the chiral ligand containing the sulfydryl group is D-glutathione. The chiral gold nanomaterial disclosed by the invention has good biocompatibility and strong bactericidal ability, and is not easy to enable bacteria to generate drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to the application of a chiral gold nanomaterial in the preparation of a bactericidal drug. Background Art

[0002] Bacterial infections have always posed a serious threat to human health. Since the discovery of penicillin in 1928, antibiotics have been widely used in clinical treatment and have achieved great success in treating bacterial infections. However, the abuse of antibiotics has become an increasingly serious problem, leading to the emergence of multi-drug resistant pathogenic bacteria. Due to the widespread occurrence and rapid spread of drug-resistant bacteria, the existing types of antibiotics that can combat drug-resistant bacteria are extremely limited, and the development of new and effective antibacterial agents is extremely urgent.

[0003] Infections caused by Pseudomonas aeruginosa are very extensive. In multiple parts such as the respiratory tract, bloodstream, wound surface, central nervous system, and abdominal cavity, Pseudomonas aeruginosa is an important pathogen, which is closely related to its perfect pathogenic mechanism. Another characteristic of Pseudomonas aeruginosa is its complex drug resistance mechanism, which also often makes it a multi-drug resistant pathogen and increases the difficulty of antibacterial treatment.

[0004] Pseudomonas aeruginosa has a particularly strong resistance to conventional antibacterial drugs and a fast adaptability, and is listed as a highly threatening bacterium by the World Health Organization (WHO), and is also a key focus for the research and development of new antibiotics.

[0005] Different from traditional antibiotics, gold nanoparticles have good biocompatibility, and also have biological inertness and unique optical properties, and are expected to become an alternative to traditional antibiotics. Gold nanomaterials have good application prospects in the fields of anti-inflammatory and antibacterial, but in actual in vivo experiments, the metabolism of gold nanomaterials is closely related to their surface chemical properties. Naked gold nanoparticles without a coating will cause serious aggregation in major target organs such as the liver and spleen, and these accumulations can induce gene mutations and liver necrosis, etc.

[0006] Chirality is a basic phenomenon in biological systems. Most biomolecules have chiral enantiomers, which function in a highly stable and orderly manner. Chiral nanomaterials will exhibit different bactericidal effects according to their chirality, and nanomaterials with specific chiral configurations show certain advantages in the antibacterial field. Summary of the Invention

[0007] The present invention provides the application of a chiral gold nanomaterial in the preparation of a bactericidal drug. The chiral gold nanomaterial has good biocompatibility, strong bactericidal ability and is not likely to cause bacteria to develop drug resistance.

[0008] The technical solution of the present invention is as follows: Application of a chiral gold nanomaterial in the preparation of a bactericidal drug. The preparation method of the chiral gold nanomaterial includes: mixing an aqueous solution of a gold precursor with a solution of a chiral ligand containing a mercapto group uniformly, and then dropwise adding a reducing agent solution. After stirring and reacting, the chiral gold nanomaterial is obtained. The chiral ligand containing a mercapto group is D-glutathione.

[0009] The gold precursor is chloroauric acid; the reducing agent is sodium borohydride.

[0010] Preferably, the molar ratio of the gold precursor to the chiral ligand in the reaction system is 1:3 - 5.

[0011] Preferably, the reducing agent solution is added dropwise so that the molar ratio of the gold precursor to the reducing agent in the reaction system is 1:20 - 30.

[0012] More preferably, the preparation method of the chiral gold nanomaterial includes: Mixing an aqueous solution of a gold precursor with a solution of a chiral ligand containing a mercapto group uniformly, and stirring and reacting at room temperature for 10 - 60 min; Adding the reducing agent solution dropwise to the reaction system, and stirring and reacting at room temperature for 60 - 180 min to obtain the product.

[0013] When the stirring speed is too low, the obtained chiral gold nanomaterial has uneven particle size and is mixed with products having too large particle size. Preferably, during the stirring reaction, the stirring speed is 400 - 600 rpm.

[0014] Preferably, the particle size of the chiral gold nanomaterial is 2 - 5 nm.

[0015] Preferably, the bactericidal drug is a drug for killing drug-resistant bacteria.

[0016] Furthermore, the drug-resistant bacteria are Pseudomonas aeruginosa.

[0017] Compared with the prior art, the beneficial effects of the present invention are: The chiral gold nanomaterial of the present invention has high stability, controllable particle size and shape, good biocompatibility, strong antibacterial ability, and is not likely to cause bacteria to develop drug resistance. It can be used as a new drug to effectively combat drug-resistant bacteria, reduce the transmission risk of drug-resistant bacteria, and provide a new option for the efficient treatment of bacterial infection wounds. Description of the Drawings

[0018] Figure 1 It is a TEM image of chiral gold nanoclusters. Among them, Figure 1 (a) corresponds to Comparative Example 1, Figure 1 (b) corresponds to Comparative Example 2, Figure 1 and (c) corresponds to Example 1; Figure 2 TEM images of chiral gold nanoclusters, where Figure 2 in (a) corresponds to Example 1, Figure 2 in (b) corresponds to Example 2, Figure 2 in (c) corresponds to Example 3; Figure 3 are the circular dichroism spectra of L-AuNCs, D-AuNCs, and DL-AuNCs in Examples 1-3; Figure 4 TEM images of chiral gold nanoclusters, where Figure 4 in (a) corresponds to Comparative Example 3, Figure 4 in (b) corresponds to Comparative Example 4; Figure 5 are the antibacterial activity images of L-AuNCs, D-AuNCs, and DL-AuNCs in Examples 1-3; Figure 6 are the antibacterial activity images of L-AuNPs and D-AuNPs in Comparative Examples 3-4; Figure 7 is the bacterial drug resistance image of D-AuNCs in Example 2; Figure 8 is the survival rate image of human bronchial epithelial cells (BEAS-2B) treated with D-AuNCs in Example 2; Figure 9 is the survival rate image of human umbilical vein endothelial cells (HUVEC) treated with D-AuNCs in Example 2; Figure 10 is the flowchart of the treatment plan for the recovery of mouse wound injury with D-AuNCs in Example 2; Figure 11 is the comparison chart of the wound size of mice after treatment with D-AuNCs in Example 2. Detailed implementation manners

[0019] The present invention will be further described in detail below in conjunction with the drawings and examples. It should be noted that the following examples are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0020] Comparative Example 1: Synthesis of chiral gold nanomaterials The main synthesis process of chiral gold nanomaterials is as follows: 80 μL of tetrachloroauric acid trihydrate solution (477 mM) was mixed with 50 mL of L-GSH (0.15 mmol) solution to obtain a chiral ligand reaction solution. After reacting for 30 minutes at room temperature (without stirring), 2 mL of sodium borohydride solution (1 mmol) was added dropwise, and then the reaction was carried out for 120 minutes (without stirring). The material was collected by centrifugation at 10,000 g for 25 minutes using a 3 KDa ultrafiltration tube, and then the material was washed 3 times. The precipitate was dispersed in an aqueous solution and stored at 4 °C for later use. After the reaction, the particle size of the material was analyzed by high-resolution transmission electron microscopy, and the results are as shown in Figure 1 Figure (a).

[0021] Comparative Example 2: Synthesis of Chiral Gold Nanomaterials The main synthesis process of chiral gold nanomaterials is as follows: 80 μL of tetrachloroauric acid trihydrate solution (477 mM) was mixed with 50 mL of L-GSH (0.15 mmol) solution to obtain a chiral ligand reaction solution. After stirring at 50 rpm for 30 minutes at room temperature, 2 mL of sodium borohydride solution (1 mmol) was added dropwise, and then the mixture was stirred at 50 rpm for 120 minutes. The material was collected by centrifugation at 10,000 g for 25 minutes using a 3 KDa ultrafiltration tube, and then the material was washed 3 times. The precipitate was dispersed in an aqueous solution and stored at 4 °C for later use. After the reaction, the particle size of the material was analyzed by high-resolution transmission electron microscopy, and the results are as shown in Figure 1 Figure (b).

[0022] Example 1: Synthesis of Chiral Gold Nanomaterials In this example, a one-step synthesis method was used to prepare chiral gold nanoclusters. The material uses chiral glutathione (L-GSH and D-GSH) as the thiol group, and by adding a strong reducing agent sodium borohydride, chiral gold nanoclusters with uniform particle size, good water solubility, and stable optical properties were prepared. The main synthesis process of chiral gold nanomaterials is as follows: 80 μL of tetrachloroauric acid trihydrate solution (477 mM) was mixed with 50 mL of L-GSH (0.15 mmol) solution to obtain a chiral ligand reaction solution. After stirring at 500 rpm for 30 minutes at room temperature, 2 mL of sodium borohydride solution (1 mmol) was added dropwise, and then the mixture was stirred at 500 rpm for 120 minutes. The material (L-AuNCs) was collected by centrifugation at 10,000 g for 25 minutes using a 3 KDa ultrafiltration tube, and then the material was washed 3 times. The precipitate was dispersed in an aqueous solution and stored at 4 °C for later use. After the reaction, the particle size of the material was analyzed by high-resolution transmission electron microscopy, and the results are as shown in Figure 1 Figure (c).

[0023] By comparing the particle sizes of the synthetic materials under the three reaction conditions of Comparative Example 1, Comparative Example 2, and Example 1, it was found that without stirring (Comparative Example 1), the material properties were unstable and prone to aggregation; under low-speed stirring (Comparative Example 2), the material particle sizes were non-uniform, with large particles of about 9 nm doped; while under high-speed stirring (Example 1), the particle sizes were synthesized uniformly, and the material particle sizes were all about 3 nm.

[0024] Example 2: Synthesis of chiral gold nanomaterials The main synthesis process of chiral gold nanomaterials is as follows: Mix 80 μL of chloroauric acid trihydrate solution (477 mM) with 50 mL of D-GSH (0.15 mmol) solution to obtain a chiral ligand reaction solution. Under room temperature conditions, stir at 500 rpm for 30 minutes, then gradually add 2 mL of sodium borohydride solution (1 mmol), and then stir at 500 rpm for 120 minutes. Collect the material (D-AuNCs) by centrifugation at 10000g for 25 minutes using a 3 KDa ultrafiltration tube, then wash the material 3 times, disperse the precipitate in an aqueous solution, and store it at 4 °C for later use.

[0025] Example 3: Synthesis of chiral gold nanomaterials The main synthesis process of racemic gold nanomaterials is as follows: Mix 80 μL of chloroauric acid trihydrate solution (477 mM) with 50 mL of a mixed solution of D-GSH and L-GSH (0.075 mmol of D-GSH and 0.075 mmol of L-GSH) to obtain a chiral ligand reaction solution. Under room temperature conditions, stir at 500 rpm for 30 minutes, then gradually add 2 mL of sodium borohydride solution (1 mmol), and then stir at 500 rpm for 120 minutes. Collect the material (DL-AuNCs) by centrifugation at 10000g for 25 minutes using a 3 KDa ultrafiltration tube, then wash the material 3 times, disperse the precipitate in an aqueous solution, and store it at 4 °C for later use.

[0026] Figure 2 For the transmission electron microscope images of the chiral gold nanoclusters prepared in Examples 1-3, Figure 2 in (a) is L-AuNCs; Figure 2 in (b) is D-AuNCs; Figure 2 in (c) is DL-AuNCs. The material morphology is stable and the size distribution is uniform.

[0027] Figure 3 For the circular dichroism spectrum of the chiral gold nanoclusters prepared by the present invention. The material has excellent optical activity, and the circular dichroism spectrum signal is mirror symmetric.

[0028] Comparative Example 3: Synthesis of Chiral Gold Nanomaterials Mix 125 μL of chloroauric acid trihydrate solution (477 mM) with 100 mL of L-GSH solution (0.15 mM). Under room temperature conditions, after stirring at 240 rpm for 30 minutes, gradually add 146 μL of sodium hydroxide solution (2 M) to adjust the pH of the system to 11, and then stir for another 5 minutes. Subsequently, place it in a water bath at 37 °C for 11 hours. Centrifuge at 13000g for 25 minutes to remove free GSH molecules in the solution, and then wash the material 3 times to obtain left-handed gold nanoparticles (L-AuNPs). Disperse the precipitate in an aqueous solution and store it at 4 °C for standby.

[0029] After the reaction, use high-resolution transmission electron microscopy (TEM) to examine the particle size of the material. The results are as Figure 4 shown in (a) below. The prepared gold nanomaterials have a uniform size distribution, and the particle size is about 7 nm.

[0030] Comparative Example 4: Synthesis of Chiral Gold Nanomaterials Mix 125 μL of chloroauric acid trihydrate solution (477 mM) with 100 mL of D-GSH solution (0.15 mM). Under room temperature conditions, after stirring at 240 rpm for 30 minutes, gradually add 146 μL of sodium hydroxide solution (2 M) to adjust the pH of the system to 11, and then stir for another 5 minutes. Subsequently, place it in a water bath at 37 °C for 11 hours. Centrifuge at 13000g for 25 minutes to remove free GSH molecules in the solution, and then wash the material 3 times to obtain right-handed gold nanoparticles (D-AuNPs). Disperse the precipitate in an aqueous solution and store it at 4 °C for standby.

[0031] After the reaction, use high-resolution transmission electron microscopy (TEM) to examine the particle size of the material. The results are as Figure 4 shown in (b) below. The prepared gold nanomaterials have a uniform size distribution, and the particle size is about 7 nm.

[0032] Example 4: Antibacterial Activity and Bacterial Drug Resistance Test of Chiral Gold Nanomaterials In this invention, Pseudomonas aeruginosa is used as the experimental bacterial strain, and the turbidimetry method is adopted to investigate the effect of chiral gold nanoclusters on bacterial survival rate. The specific scheme is as follows: Cultivate the bacteria to the logarithmic phase, and then gradually dilute the bacterial solution with the culture medium until its density is 1×10 5CFU / mL, and the diluted bacterial solution was used as the standby bacterial solution. The materials prepared in Examples 1-3, namely L-AuNCs, D-AuNCs, and DL-AuNCs, and the materials prepared in Comparative Examples 3-4, namely L-AuNPs and D-AuNPs, were formulated into different concentrations (0.2, 1, 5, 10, 20, 40 μg / mL) with MH liquid medium. Then, the materials were mixed with the standby bacterial solution and co-incubated for 12 h under the culture conditions of 37 °C and 200 rpm. After the exposure, 100 μL of the mixed solution was taken from each treatment group and placed in a 96-well plate. Finally, the OD value of each sample was measured at 600 nm with a microplate reader. Taking the bacterial solution diluted with MH liquid medium growing normally for 12 hours as the blank control, the bacterial survival rate = [OD600 (bacterial solution exposed to materials with different concentrations) - OD600 (material background)] / OD600 (blank control) × 100%.

[0033] Figure 5 Effects of L-AuNCs, D-AuNCs, and DL-AuNCs prepared in Examples 1-3 on the survival rate of Pseudomonas aeruginosa. The results showed that all three materials exhibited concentration-dependent antibacterial activity, that is, with the increase in the material concentration, the antibacterial efficiency increased accordingly. However, there were significant differences in the antibacterial effects of different chiral materials at the same concentration. The antibacterial activities of L-AuNCs and DL-AuNCs were relatively weak. Even at a concentration of 40 μg / mL, the antibacterial rate was only about 80%. While the antibacterial property of D-AuNCs was significantly better than that of L-AuNCs and DL-AuNCs. At a concentration of 10 μg / mL, the antibacterial rate exceeded 80%, and at 20 μg / mL, the antibacterial rate could reach more than 95%. It indicated that the antibacterial property of the material had obvious chirality dependence, and the D-type material had the strongest antibacterial effect.

[0034] Figure 6 Effects of L-AuNPs and D-AuNPs prepared in Comparative Examples 3-4 on the survival rate of Pseudomonas aeruginosa. The results showed that the antibacterial properties of L-AuNPs and D-AuNPs were significantly weaker than those of L-AuNCs and D-AuNCs. At a concentration of 20 μg / mL, the antibacterial rate was only about 50%.

[0035] Since D-AuNCs prepared in Example 2 had excellent antibacterial activity, further investigation was carried out on whether bacteria would develop drug resistance after being treated with it. The specific scheme was as follows: First, the first-generation Pseudomonas aeruginosa was cultured to the logarithmic phase, and then the bacterial solution was serially diluted with MH medium until its density was 1×10 5CFU / mL. This bacterial solution was used as the standby bacterial solution. The concentration of D-AuNCs prepared in Example 2 was formulated to 20 μg / mL with the culture medium, and then the material and the bacterial solution were mixed and treated under the culture conditions of 37 °C and 200 rpm for 12 h. After the exposure ended, 100 μL of the mixed solution was taken and placed in a 96-well plate, and finally the OD value of each sample was measured at 600 nm with a microplate reader. A 1 μL inoculation loop was used to dip the exposed bacterial solution, and the bacterial solution was inoculated on the solid medium. After sealing the film, it was inverted and placed in an incubator at 37 °C for about 20 hours. At this time, single colonies of Pseudomonas aeruginosa after the first exposure were obtained. Then, an inoculation loop was used to pick a single colony and place it in a liquid medium for amplification culture. The bacterial solution obtained at this time was the second-generation Pseudomonas aeruginosa. The above operations were repeated to obtain highly passaged exposed bacteria by repeating the exposure.

[0036] Figure 7 It is a graph showing the change in bacterial drug resistance in the passage experiment. The results showed that under the condition that Pseudomonas aeruginosa was continuously exposed to D-AuNCs for 20 generations, its sensitivity to the material did not decrease significantly, and the antibacterial efficiency remained at about 95% all the time, indicating that the treatment with D-AuNCs material had high antibacterial properties and did not cause bacteria to develop drug resistance, and could be used as a new drug to effectively combat drug-resistant bacteria and reduce the transmission risk of drug-resistant bacteria. The bacteria did not develop drug resistance.

[0037] Example 5: Cytotoxicity test of D-AuNCs The present invention adopted the CCK8 method to evaluate the effect of D-AuNCs prepared in Example 2 on the cell viability of human bronchial epithelial cells (BEAS-2B) and human umbilical vein endothelial cells (HUVEC), and to investigate the biocompatibility and toxicity of the material, providing a basis for in vitro experiments for subsequent experiments on treating mouse wounds with D-AuNCs.

[0038] The specific scheme was as follows: BEAS-2B and HUVEC cells were seeded at 1×10 per well 5The cells were seeded into a 96-well cell culture plate at a certain number and cultured overnight at 37 °C under 5% CO₂. The next day, when the cell density reached approximately 80%, the cells were rinsed once with PBS, and then treated with D-AuNCs at different concentrations (0, 5, 10, 20, 40, 80 μg / mL) prepared in complete medium containing 10% FBS for 24 h. The medium was aspirated, the cells were gently rinsed once with PBS, 100 μL of CCK8 working solution was added, and the cells were incubated in the cell culture incubator for another 2 h. Finally, the OD values of each sample were measured at 450 nm and 690 nm using a microplate reader, and the absorbance at 690 nm was used as a correction. After normalizing to the blank group, the cell viability was calculated using the following formula: Cell viability = (OD450 - OD690) (experimental group) / (OD450 - OD690) (blank group) × 100%.

[0039] Figure 8 and Figure 9 show the effects of D-AuNCs on the viability of BEAS-2B cells and HUVEC cells, respectively. When the concentration reached 80 μg / mL, the cells still maintained good viability, indicating that D-AuNCs had low toxicity and good biocompatibility.

[0040] Example 6: Treatment of wounds in mice infected with bacteria using D-AuNCs In this invention, 8-week-old male C57BL / 6 mice were used as experimental subjects. After shaving the hair on the back, a circular wound with a diameter of approximately 10 - 12 mm was cut on the back of each mouse using surgical scissors, and 20 μL of Pseudomonas aeruginosa was inoculated at the wound site to establish a bacterial infection model. After 24 hours of infection, the mice were randomly divided into 3 groups (3 mice in each group). The first group was the PBS treatment group, that is, a sterile PBS solution was applied to the wound site during each treatment. The second group was the levofloxacin treatment group (levofloxacin is a commonly used antibiotic for treating Pseudomonas aeruginosa infection), that is, a 200 μg / mL levofloxacin solution was applied to the wound site during each treatment. The third group was the D-AuNCs treatment group, that is, a 200 μg / mL D-AuNCs solution prepared in Example 2 was applied to the wound site during each treatment. The treatment was continued for three days, and then the mice were normally fed for another four days, and the changes in the size of the wound were observed and recorded.

[0041] Figure 10 and Figure 11 are the mouse treatment protocol and the change diagram of wound healing. Compared with the PBS treatment group and the levofloxacin treatment group, the wounds of the mice in the D-AuNCs treatment group healed significantly faster. On the seventh day, the wounds in the D-AuNCs treatment group were basically healed, showing a better treatment effect than traditional antibiotics, indicating its significant advantage in promoting the healing of wounds infected with bacteria.

[0042] The embodiments described above have elaborated in detail on the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of a chiral gold nanomaterial in the preparation of a bactericidal drug, characterized in that, The preparation method of the chiral gold nanomaterial includes: uniformly mixing an aqueous solution of a gold precursor with a solution of a chiral ligand containing a mercapto group, and then dropwise adding a reducing agent solution, and obtaining the chiral gold nanomaterial after stirring reaction; the chiral ligand containing a mercapto group is D-glutathione.

2. Use of the chiral gold nanomaterial according to claim 1 in the preparation of a bactericidal drug, characterized in that, The gold precursor is chloroauric acid; the reducing agent is sodium borohydride.

3. Use of the chiral gold nanomaterial according to claim 1 in the preparation of a bactericidal drug, characterized in that, The molar ratio of the gold precursor to the chiral ligand in the reaction system is 1:3-5.

4. Use of the chiral gold nanomaterial according to claim 1 in the preparation of a bactericidal drug, characterized in that, The reducing agent solution is dropwise added so that the molar ratio of the gold precursor to the reducing agent in the reaction system is 1:20-30.

5. Use of the chiral gold nanomaterial according to claim 1 in the preparation of a bactericidal drug, characterized in that, The preparation method of the chiral gold nanomaterial includes: uniformly mixing an aqueous solution of a gold precursor with a solution of a chiral ligand containing a mercapto group, and stirring and reacting at room temperature for 10-60 min; dropwise adding a reducing agent solution to the reaction system, and stirring and reacting at room temperature for 60-180 min to obtain the product.

6. Use of the chiral gold nanomaterial according to claim 5 in the preparation of a bactericidal drug, characterized in that, When stirring and reacting, the stirring speed is 400-600 rpm.

7. Use of the chiral gold nanomaterial according to claim 1 in the preparation of a bactericidal drug, characterized in that, The particle size of the chiral gold nanomaterial is 2-5 nm.

8. Use of the chiral gold nanomaterial according to claim 1 in the preparation of a bactericidal drug, characterized in that, The bactericidal drug is a drug for killing drug-resistant bacteria.

9. Use of the chiral gold nanomaterial according to claim 8 in the preparation of a bactericidal drug, characterized in that, The drug-resistant bacteria are Pseudomonas aeruginosa.

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