Application of chiral gold nanomaterial in preparation of bactericidal drugs

Abstract

The application discloses application of chiral gold nanomaterial in preparation of sterilization drugs, and a preparation method of the chiral gold nanomaterial, which comprises the following steps: uniformly mixing a gold precursor aqueous solution and a chiral ligand solution containing a mercapto group, then dropwise adding a reducing agent solution, and obtaining the chiral gold nanomaterial after stirring reaction; and the chiral ligand containing the mercapto group is D-glutathione. The chiral gold nanomaterial has good biocompatibility, strong sterilization capacity and is not prone to cause drug resistance of bacteria.

Description

TECHNICAL FIELD

[0001] The application relates to the field of medicine, and in particular to application of chiral gold nanomaterials in preparation of bactericidal drugs. BACKGROUND

[0002] Bacterial infection has always been 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 a serious problem, leading to the emergence of pathogenic bacteria with multiple drug resistance. Due to the widespread occurrence and rapid spread of drug-resistant bacteria, the existing antibiotics against drug-resistant bacteria are extremely limited, and the development of new effective antibacterial agents is imminent.

[0003] Infections caused by Pseudomonas aeruginosa are very wide, and Pseudomonas aeruginosa is an important pathogen in many parts of the respiratory tract, blood stream, wound, central nervous system, abdominal cavity and the like, which is closely related to its perfect pathogenic mechanism. Another characteristic of Pseudomonas aeruginosa is its complex drug resistance mechanism, which also makes it often become a multi-drug resistant pathogen, increasing the difficulty of antibacterial treatment.

[0004] Pseudomonas aeruginosa has strong resistance to conventional antibacterial drugs and fast adaptability, and is listed by the World Health Organization (WHO) as a bacterium with great threat, and is also an important object of attention for the development of new antibiotics.

[0005] Unlike traditional antibiotics, gold nanoparticles have good biocompatibility, and also have biological inertness and unique optical properties, and are expected to become a traditional antibiotic substitute. 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 the surface chemical properties. Naked gold nanoparticles without coating will produce serious aggregation in main target organs such as liver and spleen, and these accumulations can induce gene mutation and liver necrosis and the like.

[0006] Chirality is a basic phenomenon in biological systems, and most biological molecules have chiral enantiomers, which play a role in a highly stable and ordered manner. Chiral nanomaterials will exhibit different bactericidal effects according to their chirality, and nanomaterials with a specific chiral configuration have certain advantages in the field of antibacterial. SUMMARY

[0007] The application provides application of chiral gold nanomaterials in preparation of bactericidal drugs, and the chiral gold nanomaterials have good biocompatibility, strong bactericidal capacity and are not easy to cause drug resistance of bacteria.

[0008] The technical scheme of the application is as follows:

[0009] The application of a chiral gold nanomaterial in the preparation of a sterilization drug, wherein the preparation method of the chiral gold nanomaterial comprises the following steps: uniformly mixing a gold precursor aqueous solution and a chiral ligand solution containing a mercapto group, then dropwise adding a reducing agent solution, and obtaining the chiral gold nanomaterial after stirring reaction; and the chiral ligand containing a mercapto group is D-glutathione.

[0010] The gold precursor is tetrachloroauric acid; and the reducing agent is sodium borohydride.

[0011] Preferably, the molar ratio of the gold precursor to the chiral ligand in the reaction system is 1:3-5.

[0012] Preferably, the molar ratio of the gold precursor to the reducing agent in the reaction system is 1:20-30 by dropwise adding the reducing agent solution.

[0013] Further preferably, the preparation method of the chiral gold nanomaterial comprises the following steps:

[0014] uniformly mixing a gold precursor aqueous solution and a chiral ligand solution containing a mercapto group, stirring reaction at room temperature for 10-60 min;

[0015] dropwise adding a reducing agent solution into the reaction system, and stirring reaction at room temperature for 60-180 min.

[0016] When the stirring speed is too low, the particle size of the obtained chiral gold nanomaterial is not uniform, and the product with a particle size that is too large is mixed. Preferably, the stirring speed is 400-600 rpm during the stirring reaction.

[0017] Preferably, the particle size of the chiral gold nanomaterial is 2-5 nm.

[0018] Preferably, the sterilization drug is a drug for killing drug-resistant bacteria.

[0019] Further, the drug-resistant bacteria are pseudomonas aeruginosa.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] The chiral gold nanomaterial has high stability, controllable particle size and shape, good biocompatibility, and strong antibacterial ability, and is not easy to make bacteria resistant, can be used as a new drug for effectively resisting drug-resistant bacteria, reduces the risk of spread of drug-resistant bacteria, and provides a new choice for efficient treatment of bacterial infection wounds. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The TEM image of the chiral gold nanocluster, wherein, Figure 1 Figure (a) corresponds to comparative example 1, Figure 1 Figure (b) corresponds to comparative example 2,Figure 1 Example 1 (c);

[0023] Figure 2 TEM images of chiral gold nanoclusters, wherein, Figure 2 Example 1 (a), Figure 2 Example 1 (b), Figure 2 Example 1 (c);

[0024] Figure 3 Circular dichroism spectra of L-AuNCs, D-AuNCs and DL-AuNCs in Examples 1-3;

[0025] Figure 4 TEM images of chiral gold nanoclusters, wherein, Figure 4 Example 3 (a), Figure 4 Example 3 (b);

[0026] Figure 5 Antibacterial activity of L-AuNPs, D-AuNPs in Comparative Examples 3-4;

[0027] Figure 6 Antibacterial activity of L-AuNPs, D-AuNPs in Comparative Examples 3-4;

[0028] Figure 7 Bacterial resistance of D-AuNCs in Example 2;

[0029] Figure 8 Survival rate of human bronchial epithelial cells (BEAS-2B) treated with D-AuNCs in Example 2;

[0030] Figure 9 Survival rate of human umbilical vein endothelial cells (HUVEC) treated with D-AuNCs in Example 2;

[0031] Figure 10 Treatment scheme of D-AuNCs for recovery of wound injury in mice in Example 2;

[0032] Figure 11 Comparison of wound size of mice after treatment with D-AuNCs in Example 2. DETAILED DESCRIPTION

[0033] The application will be further described in conjunction with the accompanying drawings and examples. It should be noted that the following examples are intended to facilitate the understanding of the application and do not limit the application in any way.

[0034] Comparative Example 1: Synthesis of chiral gold nanomaterials

[0035] The main synthesis process of chiral gold nanomaterials is as follows:

[0036] A chiral ligand reaction solution was prepared by mixing 80 μL of tetrachloroauric acid trihydrate solution (477 mM) with 50 mL of L-GSH solution (0.15 mmol). After reacting at room temperature for 30 minutes (without stirring), 2 mL of sodium borohydride solution (1 mmol) was added dropwise, and the reaction was continued for 120 minutes (without stirring). The material was collected by centrifugation at 10000 g for 25 minutes using a 3 kDa ultrafiltration tube. The material was then washed three times, and the precipitate was dispersed in an aqueous solution and stored at 4 °C for later use. After the reaction, the particle size was analyzed using high-resolution transmission electron microscopy. The results are shown below. Figure 1 As shown in (a).

[0037] Comparative Example 2: Synthesis of Chiral Gold Nanomaterials

[0038] The main synthesis process of chiral gold nanomaterials is as follows:

[0039] A chiral ligand reaction solution was prepared by mixing 80 μL of tetrachloroauric acid trihydrate solution (477 mM) with 50 mL of L-GSH solution (0.15 mmol). The mixture was stirred at 50 rpm for 30 minutes at room temperature, followed by dropwise addition of 2 mL of sodium borohydride solution (1 mmol), and then stirred at 50 rpm for 120 minutes. The material was collected by centrifugation at 10000 g for 25 minutes using a 3 kDa ultrafiltration tube. The material was then washed three times, and the precipitate was dispersed in an aqueous solution and stored at 4 °C for later use. After the reaction, the particle size was analyzed using high-resolution transmission electron microscopy. The results are shown below. Figure 1 As shown in (b).

[0040] Example 1: Synthesis of chiral gold nanomaterials

[0041] This embodiment employs a one-step synthesis method to prepare chiral gold nanoclusters. Using chiral glutathione (L-GSH and D-GSH) as thiol groups, and by adding the 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 the chiral gold nanomaterials is as follows:

[0042] A chiral ligand reaction solution was prepared by mixing 80 μL of a solution of tetrachloroauric acid trihydrate (477 mM) with 50 mL of a solution of L-GSH (0.15 mmol). After stirring at 500 rpm for 30 minutes at room temperature, 2 mL of a solution of sodium borohydride (1 mmol) was added dropwise, and then stirring was continued at 500 rpm for 120 minutes. The material (L-AuNCs) was collected by centrifugation at 10000 g for 25 minutes using a 3 KDa ultrafiltration tube, and then the material was washed three times, dispersed in an aqueous solution, and stored at 4 °C for later use. After the reaction was completed, the particle size of the material was analyzed by high-resolution transmission electron microscopy, and the results are shown in FIG. 1. Figure 1 (c).

[0043] By comparing the particle sizes of the materials synthesized under the three reaction conditions of Comparative Example 1, Comparative Example 2, and Example 1, it was found that in the case of no stirring (Comparative Example 1), the material was unstable and easy to aggregate; in the case of low-speed stirring (Comparative Example 2), the particle size of the material was not uniform, and there were about 9 nm large particles doped; and in the case of high-speed stirring (Example 1), the particle size was uniform, and the particle size of the material was about 3 nm.

[0044] Example 2: Synthesis of chiral gold nanomaterials

[0045] The main synthesis process of the chiral gold nanomaterials is as follows:

[0046] A chiral ligand reaction solution was prepared by mixing 80 μL of a solution of tetrachloroauric acid trihydrate (477 mM) with 50 mL of a solution of D-GSH (0.15 mmol). After stirring at 500 rpm for 30 minutes at room temperature, 2 mL of a solution of sodium borohydride (1 mmol) was added dropwise, and then stirring was continued at 500 rpm for 120 minutes. The material (D-AuNCs) was collected by centrifugation at 10000 g for 25 minutes using a 3 KDa ultrafiltration tube, and then the material was washed three times, dispersed in an aqueous solution, and stored at 4 °C for later use.

[0047] Example 3: Synthesis of chiral gold nanomaterials

[0048] The main synthesis process of the racemic gold nanomaterials is as follows:

[0049] A chiral ligand reaction solution was prepared by mixing 80 μL of a tetrachloroauric acid trihydrate solution (477 mM) with a mixed solution of 50 mL of D-GSH and L-GSH (D-GSH: 0.075 mmol, L-GSH: 0.075 mmol). After stirring at 500 rpm for 30 minutes at room temperature, 2 mL of a sodium borohydride solution (1 mmol) was added dropwise, and then stirring was continued at 500 rpm for 120 minutes. The material (DL-AuNCs) was collected by centrifugation at 10000 g for 25 minutes using a 3 KDa ultrafiltration tube, and then the material was washed three times, the precipitate was dispersed in an aqueous solution, and the material was stored at 4 ℃ for later use.

[0050] Figure 2 Transmission electron microscopy images of the chiral gold nanoclusters prepared in Examples 1-3, Figure 2 (a) is L-AuNCs; Figure 2 (b) is D-AuNCs; Figure 2 (c) is DL-AuNCs. The material has a stable morphology and a uniform size distribution.

[0051] Figure 3 Circular dichroism spectra of the chiral gold nanoclusters prepared in the present application. The material has excellent optical activity, and the circular dichroism spectrum signal is mirror-symmetric.

[0052] Comparative Example 3: Synthesis of chiral gold nanomaterials

[0053] A 125 μL tetrachloroauric acid trihydrate solution (477 mM) was mixed with 100 mL of an L-GSH solution (0.15 mM). After stirring at 240 rpm for 30 minutes at room temperature, 146 μL of a sodium hydroxide solution (2 M) was added dropwise, the pH of the system was adjusted to 11, and then stirring was continued for 5 minutes. Subsequently, the system was placed in a 37 ℃ water bath for 11 hours. The free GSH molecules in the solution were removed by centrifugation at 13000 g for 25 minutes, and then the material was washed three times to obtain left-handed gold nanoparticles (L-AuNPs). The precipitate was dispersed in an aqueous solution and stored at 4 ℃ for later use.

[0054] After the reaction was completed, the particle size of the material was investigated using high-resolution transmission electron microscopy (TEM), and the results are shown in Figure 4 (a) in the figure. The gold nanomaterials prepared have a uniform size distribution, and the particle size is about 7 nm.

[0055] Comparative Example 4: Synthesis of chiral gold nanomaterials

[0056] The 125 μL tetrachloroauric acid trihydrate solution (477 mM) was mixed with 100 mL D-GSH solution (0.15 mM). After stirring at room temperature for 30 minutes at 240 rpm, 146 μL sodium hydroxide solution (2 M) was added dropwise to adjust the pH of the system to 11, and then stirred for another 5 minutes. Subsequently, it was placed in a 37°C water bath for 11 hours. 13000g centrifugation was performed for 25 minutes to remove free GSH molecules in the solution, and then the material was washed 3 times to obtain dextrorotatory gold nanoparticles (D-AuNPs), which were dispersed in an aqueous solution and stored at 4 ℃ for standby.

[0057] After the reaction was completed, the particle size of the material was investigated by high-resolution transmission electron microscopy (TEM), and the results are shown in Fig. 2 (b). Figure 4 As shown in Fig. 2 (b), the gold nanomaterials prepared have uniform size distribution, and the particle size is about 7 nm.

[0058] Example 4: Bacteriostatic activity and bacterial drug resistance test of chiral gold nanomaterials

[0059] In the present application, Pseudomonas aeruginosa was used as the experimental strain, and the turbidity method was used to investigate the influence of chiral gold nanoclusters on the survival rate of bacteria. The specific scheme is as follows: the bacteria were cultured to the logarithmic phase, and then the bacterial solution was diluted with the culture medium to a density of 1×10 5 CFU / mL, and the diluted bacterial solution was used as the standby bacterial solution. The materials were prepared into different concentrations (0.2, 1, 5, 10, 20, 40 μg / mL) of L-AuNCs, D-AuNCs, DL-AuNCs prepared in Examples 1-3 and L-AuNPs, D-AuNPs prepared in Comparative Examples 3-4 by using MH liquid medium, and then the materials were mixed with the standby bacterial solution, and incubated at 37°C and 200 rpm for 12 h. After the exposure, 100 μL of the mixed solution was taken from each treatment group into a 96-well plate, and finally the OD value of each sample was measured at 600 nm by using a spectrometer. The bacterial solution diluted with the MH liquid medium was normally grown for 12 hours as the blank control, and the survival rate of the bacteria was calculated as [OD600 (bacterial solution exposed to different concentrations of materials)-OD600 (background of the materials)] / OD600 (blank control) × 100 %.

[0060] Figure 5The influence of L-AuNCs, D-AuNCs and DL-AuNCs prepared in Examples 1-3 on the survival rate of P. aeruginosa. The results show that the three materials all exhibit concentration-dependent antibacterial activity, that is, the antibacterial efficiency increases with the increase of the concentration of the material. However, the antibacterial effects of different chiral materials at the same concentration are significantly different. The antibacterial activity of L-AuNCs and DL-AuNCs is relatively weak, and even at a concentration of 40 μg / mL, the antibacterial rate is only about 80 %, while the antibacterial activity of D-AuNCs is significantly better than that of L-AuNCs and DL-AuNCs, and the antibacterial rate is more than 80 % at a concentration of 10 μg / mL, and more than 95 % at a concentration of 20 μg / mL. This shows that the antibacterial performance of the material has obvious chirality dependence, and the antibacterial effect of the D-type material is the strongest.

[0061] Figure 6 The influence of L-AuNPs and D-AuNPs prepared in Comparative Examples 3-4 on the survival rate of P. aeruginosa. The results show that the antibacterial activity of L-AuNPs and D-AuNPs is significantly weaker than that of L-AuNCs and D-AuNCs, and the antibacterial rate is only about 50 % at a concentration of 20 μg / mL.

[0062] Since D-AuNCs prepared in Example 2 have excellent antibacterial activity, it is further investigated whether the bacteria will develop drug resistance after treatment. The specific scheme is as follows: first, the first generation of P. aeruginosa is cultured to the logarithmic phase, and then the bacterial solution is diluted with MH medium to a density of 1×10 5 CFU / mL, and this bacterial solution is used as the bacterial solution to be used. The concentration of D-AuNCs prepared in Example 2 is prepared to be 20 μg / mL with the culture medium, and then the material and the bacterial solution are mixed, and treated at 37 °C, 200 rpm for 12 h. After exposure, 100 μL of the mixed solution is taken in a 96-well plate, and finally the OD value of each sample is measured at 600 nm with a spectrophotometer. 1 μL of the exposed bacterial solution is taken with a loop, and the bacterial solution is inoculated on a solid culture medium, and after sealing, it is placed in a 37 °C incubator for about 20 hours, at which time the single colony of the first exposed P. aeruginosa is obtained. Then a single colony is picked up with a loop and placed in a liquid culture medium for amplification culture, and the bacterial solution obtained at this time is the second generation of P. aeruginosa, and the above operation is repeated to obtain high-generation exposed bacteria.

[0063] Figure 7The results show that the sensitivity of P. aeruginosa to the material does not decrease significantly under the condition of being exposed to D-AuNCs for 20 generations in succession, and the bacteriostatic efficiency is always maintained at about 95%, indicating that the D-AuNCs material treatment has high antibacterial property and does not make the bacteria resistant, and can be used as a new drug to effectively resist drug-resistant bacteria and reduce the risk of spread of drug-resistant bacteria. The bacteria do not produce drug resistance.

[0064] Example 5: Cytotoxicity test of D-AuNCs

[0065] The present application uses the CCK8 method to evaluate the influence of D-AuNCs prepared in Example 2 on the cell activity of human bronchial epithelial cells (BEAS-2B) and human umbilical vein endothelial cells (HUVEC), and investigates the biocompatibility and toxicity of the material, providing a basis for in vitro experiments for subsequent experiments of treating mouse wounds with D-AuNCs.

[0066] The specific scheme is: BEAS-2B and HUVEC cells are inoculated into a 96-well cell culture plate at a number of 1×10 5 Each well, and cultured overnight at 37°C, 5% CO2. The next day, when the cell density was about 80%, the cells were washed once with PBS, and then treated with D-AuNCs at different concentrations (0, 5, 10, 20, 40, 80 μg / mL) containing 10% FBS for 24 h. The culture medium was aspirated, the cells were gently washed once with PBS, 100 μL of CCK8 working solution was added, and the cells were incubated in the cell incubator for 2 h. Finally, the OD value of each sample was measured at 450 nm and 690 nm by a microplate reader, and the absorbance at 690 nm was used as a correction. After normalization of the blank group, the cell survival rate was calculated, and the calculation formula was as follows: cell viability = (OD450-OD690) (experimental group) / (OD450-OD690) (blank group) x 100%.

[0067] Figure 8 and Figure 9 respectively, the activity of D-AuNCs on BEAS-2B cells and HUVEC cells. When the concentration reaches 80 μg / mL, the cells can still maintain good activity, indicating that the toxicity of D-AuNCs is low, and has good biocompatibility.

[0068] Example 6: Treatment of D-AuNCs on bacterial infected mouse wound surface

[0069] The present application takes 8-week-old male C57BL / 6 mice as experimental objects, after shaving the back, using surgical scissors to cut a circular wound with a diameter of about 10-12 mm on the back of each mouse, and inoculating 20 μL of Pseudomonas aeruginosa 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 per group). The first group is the PBS treatment group, that is, each time the wound is treated with sterile PBS solution. The second group is the levofloxacin treatment group (levofloxacin is a commonly used antibiotic for treating Pseudomonas aeruginosa infection), that is, each time the wound is treated with a 200 μg / mL solution of levofloxacin. The third group is the D-AuNCs treatment group, that is, each time the wound is treated with a 200 μg / mL solution of D-AuNCs prepared in Example 2. Continuous treatment for three days, and then the mice are normally raised for four days, and the size of the wound is observed and recorded.

[0070] Figure 10 and Figure 11 is a mouse treatment plan and wound healing condition change diagram. Compared with the PBS treatment group and the levofloxacin treatment group, the wound of the mice in the D-AuNCs treatment group heals significantly faster. On the seventh day, the wound of the D-AuNCs treatment group is basically healed, showing a better treatment effect than traditional antibiotics, indicating that it has a significant advantage in promoting the healing of bacterial infection wounds.

[0071] The above-described examples detail the technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the present application. Any modifications, supplements, and equivalent replacements made within the principle range of the present application shall be included in the protection scope of the present application.

Claims

1. Use of chiral gold nanomaterials in the preparation of bactericidal medicaments, characterized in that, The preparation method of the chiral gold nanomaterial comprises the following steps: uniformly mixing a gold precursor aqueous solution with a chiral ligand solution containing a mercapto group, then adding a reducing agent solution drop by drop, and obtaining the chiral gold nanomaterial after stirring reaction; the chiral ligand containing a mercapto group is D-glutathione; The gold precursor is tetrachloroauric acid; the reducing agent is sodium borohydride; the molar ratio of the gold precursor to the chiral ligand in the reaction system is 1:3-5; the molar ratio of the gold precursor to the reducing agent in the reaction system is 1:20-30; During the stirring reaction, the stirring speed is 400-600 rpm; The particle size of the chiral gold nanomaterial is 2-5 nm; The bactericidal drug is a drug for killing drug-resistant bacteria, and the drug-resistant bacteria are Pseudomonas aeruginosa.

2. Use of the chiral gold nanomaterial according to claim 1 for the preparation of an antiseptic drug, characterized in that, The preparation method of the chiral gold nanomaterial comprises: Uniformly mixing a gold precursor aqueous solution with a chiral ligand solution containing a mercapto group, and stirring reaction at room temperature for 10-60 min; Adding a reducing agent solution drop by drop into the reaction system, and stirring reaction at room temperature for 60-180 min.

Images