WO2017201801A1

WO2017201801A1 - Antibacterial agent based on gold nanoparticle surface modified by nitrogen heterocyclic small molecule

Info

Publication number: WO2017201801A1
Application number: PCT/CN2016/087344
Authority: WO
Inventors: 蒋兴宇; 冯艳
Original assignee: 国家纳米科学中心
Priority date: 2016-05-23
Filing date: 2016-06-27
Publication date: 2017-11-30
Also published as: CN107412780B; CN107412780A

Abstract

Disclosed is a gold nanoparticle which is modified on its surface by a nitrogen heterocyclic small molecule that is preferably one or more molecules selected from a triazole with a mercapto, imidazole with a mercapto, purine with a mercapto and benzothiazole with a mercapto. The gold nanoparticle prepared exhibits good antibacterial properties against gram-negative bacteria, gram-positive bacteria and/or clinical bacteria isolates with multidrug resistance, and may have good biocompatibility. Also provided are a method for preparing the gold nanoparticle, an antibacterial composition and a kit comprising the gold nanoparticle, and a use of the gold nanoparticle and the antibacterial composition in the preparation of antibacterial products or medicines.

Description

Antibacterial agent based on gold nanoparticle surface modified nitrogen heterocyclic small molecule

Cross-reference to related applications

The present application claims priority to Chinese Patent Application No. CN201610345068.2, filed on May 23, 2016, which is hereby incorporated by reference.

Technical field

The present invention relates to a gold nanoparticle, in particular to a gold nanoparticle having a surface modified nitrogen heterocyclic small molecule, and a method for preparing the gold nanoparticle, an antibacterial composition containing the gold nanoparticle, a kit, and the gold nanoparticle and The use of an antimicrobial composition for the preparation of an antimicrobial product or medicament.

Background technique

Bacterial infection is one of the most important causes of threat to human health, but the abuse of traditional antibiotics based on small organic molecules has caused the emergence of super-resistant bacteria. Bacterial resistance has led to serious morbidity and mortality, especially in hospital-related infections, and at the same time increased public health costs. Thus, it is urgent to invent new effective antibacterial agents, especially for super-resistant bacteria.

Nanomaterials exhibit their potential antimicrobial performance due to their large specific surface area, flexible surface functionalization and inherent physicochemical properties. Nanosilver, nano-copper, nano-zinc oxide and carbon nanotubes all show certain antibacterial activity, but these materials are not used as ideal antibacterial agents due to their toxicity and poor antibacterial properties. Because of its good biocompatibility and mature synthetic methods, gold nanoparticles have attracted more and more attention as an antibacterial agent. Many studies have shown that gold nanoparticles have no antibacterial activity per se, but gold nanoparticles modified with thiols, amines and phosphorus compounds can be used as antibacterial agents. Compared with the antibiotic itself, the antibacterial activity of the gold nanoparticles modified with antibiotics is significantly enhanced, but the gold nanoparticles still have no effect on the resistant bacteria.

In order to synthesize effective antibacterially active gold nanoparticles, Applicants have invented a series of antibacterially active gold nanoparticles modified with mercaptopyrimidine against Gram-negative bacteria, Gram-positive bacteria and super Resistant bacteria have good antibacterial effects. However, these gold nanoparticles have some serious deficiencies. For example, the need for two small molecule ligands to simultaneously modify gold nanoparticles and the functional effects of the two ligands seriously affect the experiment. Repeatability and antibacterial effect.

Therefore, it is necessary to discover and study new gold nanoparticles with good reproducibility, high antibacterial activity and simple modification of ligands.

Summary of the invention

Therefore, in order to overcome the defects of the prior art, the present invention provides a gold nanoparticle having a surface-modified nitrogen heterocyclic small molecule, a method for preparing gold nanoparticles, an antibacterial composition containing the gold nanoparticle, and a kit. And the use of the gold nanoparticles and the antibacterial composition in the preparation of an antibacterial product or a medicament.

In order to achieve the above object, a first aspect of the present invention provides a gold nanoparticle having a surface modified with a nitrogen heterocyclic small molecule. Preferably, the nitrogen heterocyclic small molecule has a fluorenyl group. More preferably, the nitrogen heterocyclic small molecule is selected from one or more of a triazole having a fluorenyl group, an imidazole having a fluorenyl group, a fluorenyl group having a fluorenyl group, and a benzothiazole having a fluorenyl group. Further preferably, the nitrogen heterocyclic small molecule is selected from the group consisting of 3-amino-5-mercapto-1,2,4-triazole (ATT), 4-amino-3-linked ammonia-5-mercapto-1,2 , 4-triazole (AHMT), 2-mercaptoimidazole (MI), methimazole (MTM), 2-amino-6-mercaptopurine (AMP), and 6-amino-2-mercaptobenzothiazole (AMBT) One or more of them.

Different from the pyrimidine molecules used in the prior art, the nitrogen heterocyclic small molecule of the present invention has a five-membered ring structure, has certain biological activity, and is positive for Gram-negative bacteria and Gram-like after modification of gold nanoparticles. The bacteria have antibacterial effects and the antibacterial effect is good.

In one embodiment, the surface of the gold nanoparticles is modified with only one nitrogen-heterocyclic small molecule having a thiol group. The present invention provides six preferred nitrogen-heterocyclic small molecules having a fluorenyl group, two triazoles (ATT, AHMT), two imidazoles (MI, MTM), one hydrazine (AMP) and one benzothiazole. (AMBT), each of the above nitrogen heterocyclic small molecules can be modified with gold nanoparticles alone, and good antibacterial activity can be obtained.

Among them, the chemical structural formula of 3-amino-5-mercapto-1,2,4-triazole (ATT) is:

The chemical structural formula of 4-amino-3-linked ammonia-5-mercapto-1,2,4-triazole (AHMT) is:

The chemical structural formula of 2-mercaptoimidazole (MI) is:

The chemical structural formula of methimazole (MTM) is:

The chemical structural formula of 2-amino-6-mercaptopurine (AMP) is:

The chemical structural formula of 6-amino-2-mercaptobenzothiazole (AMBT) is:

The gold nanoparticle according to the first aspect of the present invention, wherein a molar ratio of the nitrogen heterocyclic small molecule to the gold element in the gold nanoparticle is from 0.1 to 0.99:1, preferably from 0.22 to 0.92:1; and / Or the gold nanoparticles have an average particle diameter of 1 to 35 nm, preferably 2 to 10 nm.

A second aspect of the invention provides a preparation method for preparing the gold nanoparticles of the first aspect of the invention, the preparation method comprising:

(1) dissolving the nitrogen heterocyclic small molecule in methanol (MeOH) and mixing with chloroauric acid (HAuCl4) to form a mixed solution;

(2) adding a reducing agent to the mixed liquid in the step (1), and fully reacting to obtain a crude gold nanoparticle;

(3) Purifying the crude gold nanoparticles in the step (2) to obtain the gold nanoparticles.

Preferably:

The molar ratio of the nitrogen heterocyclic small molecule to the chloroauric acid in the step (1) is from 1 to 10:1, most preferably 10:1;

The reducing agent in the step (2) is selected from one or more of sodium borohydride, sodium ascorbate and sodium citrate, most preferably sodium borohydride;

The molar ratio of the reducing agent to the chloroauric acid in the step (2) is from 1 to 10:1, preferably from 2 to 8:1, most preferably 3:1; and/or

The reaction in the step (2) is carried out under ice bath and stirring, and the reaction time is preferably from 30 minutes to 2 hours, most preferably 1 hour.

The production method according to the second aspect of the present invention, wherein, after the mixing of the step (1), the mixture is stirred under ice bath conditions and a nonionic surfactant is added to make it uniformly mixed. Wherein: the nonionic surfactant is preferably selected from one or more of Triton X-100, Tween or polyethylene glycol, most preferably Tween 80. And/or the molar ratio of the nonionic surfactant to the chloroauric acid is preferably from 0.1 to 2:1, more preferably from 0.5 to 1.5:1. Among them, it can be stirred using a magnetic stirrer.

The production method according to the second aspect of the present invention, wherein the purification in the step (3) comprises:

Remove methanol. The removal of methanol is preferably carried out by means of a rotary evaporator. and / or

Dialysis. The dialysis is preferably carried out using a 14 KD dialysis bag for 48 hours.

Preferably, distilled water is added to the gold nanoparticles after the methanol removal to dissolve, and then dialyzed.

In one embodiment, the above gold nanoparticles are prepared by using a sodium borohydride reduction method to synthesize different nitrogen heterocycles by adjusting the molar ratio of the nitrogen heterocyclic small molecule to the chloroauric acid. The modified gold nanoparticle, wherein the molar ratio of the nitrogen heterocyclic small molecule to the gold element in the gold nanoparticle is from 0.1 to 0.99:1, preferably from 0.22 to 0.92:1.

Specifically, the method for preparing the above gold nanoparticles may include:

(1) completely dissolving different nitrogen heterocyclic small molecules in methanol and then mixing with chloroauric acid to form a mixed liquid, the molar ratio of the nitrogen heterocyclic small molecule to the chloroauric acid is 1 to 10:1, the most Preferably 10:1;

(2) stirring the mixture in the step (1) under an ice bath magnetic stirrer, adding Tween 80 and thoroughly mixing and dissolving, adding sodium borohydride, and the molar ratio of the sodium borohydride to the chloroauric acid is 1 to 10:1, preferably 2 to 8:1, and most preferably 3:1, stirring is continued for 1 hour to obtain gold nanoparticles (crude);

(3) removing methanol by a rotary evaporator and adding a certain amount of distilled water to redissolve the gold Nanoparticles, after dialysis of the gold nanoparticles for 48 hours with a 14 KD dialysis bag, the gold nanoparticles were collected and stored for later use.

A third aspect of the invention provides an antibacterial composition comprising the gold nanoparticles of the first aspect of the invention or the gold nanoparticles prepared by the preparation method of the second aspect of the invention. Preferably, the antimicrobial composition is an antibacterial or antibacterial pharmaceutical composition. More preferably, the antibacterial or antibacterial pharmaceutical composition is one or more of the following species: Gram-negative bacteria, Gram-positive bacteria, and clinical isolates having multidrug resistance. Further preferably, the Gram-negative bacteria are selected from one or more of E. coli, P. aeruginosa, and K. pneumoniae. The Gram-positive bacteria are selected from the group consisting of S. aureus, and/or the multi-drug resistant clinical isolate is selected from the group consisting of multi-drug resistant Escherichia coli, multidrug resistant One or more of Pseudomonas aeruginosa and multidrug resistant Staphylococcus aureus. In one embodiment, the antibacterial pharmaceutical composition comprises a pharmaceutically acceptable carrier, and the gold nanoparticles of the first aspect of the invention or the gold nanoparticles prepared by the preparation method of the second aspect of the invention.

A fourth aspect of the invention provides a kit comprising the gold nanoparticles of the first aspect of the invention or the gold nanoparticles prepared by the method of the second aspect of the invention.

A fifth aspect of the invention provides an application of the gold nanoparticle of the first aspect of the invention, the gold nanoparticle prepared by the preparation method of the second aspect of the invention or the antimicrobial composition of the third aspect of the invention Use in the preparation of an antimicrobial product or in the preparation of a medicament for the prevention and/or treatment of a condition caused by a microorganism.

Preferably:

The antibacterial product or drug is one or more of one or more of the following species: Gram-negative bacteria, Gram-positive bacteria, and clinical isolates having multidrug resistance. Wherein the Gram-negative bacteria are preferably selected from one or more of the group consisting of Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae, and the Gram-positive bacteria are preferably selected from the group consisting of Staphylococcus aureus. The multidrug resistant clinical isolate is preferably selected from one or more of multidrug resistant Escherichia coli, multidrug resistant Pseudomonas aeruginosa, and multidrug resistant Staphylococcus aureus. . and / or

The condition caused by the microorganism is a condition caused by one or more of Gram-negative bacteria, Gram-positive bacteria, and clinical isolates having multidrug resistance. The condition is preferably selected from one or more of the following: skin and subcutaneous tissue infections, wound infections, otitis media, meningitis, peritonitis, Enteritis, bronchitis, pneumonia, respiratory infections, urinary tract infections, sepsis or sepsis.

A sixth aspect of the invention provides a method of preventing and/or treating a condition caused by a microorganism, the method comprising administering to a subject a therapeutically effective amount of the gold nanoparticle of the first aspect of the invention, using the invention The gold nanoparticle prepared by the preparation method of the second aspect or the antimicrobial composition of the third aspect of the invention.

Preferably:

The condition caused by the microorganism may be a condition caused by Gram bacteria and/or a clinical isolate having multidrug resistance. The condition may preferably be selected from one or more of the following: skin and subcutaneous tissue infections, wound infections, otitis media, meningitis, peritonitis, enteritis, bronchitis, pneumonia, respiratory infections, urinary tract infections, sepsis, pus Toxic and other diseases. The Gram-negative bacteria may be Gram-negative bacteria and Gram-positive bacteria. Wherein the Gram-negative bacteria is preferably selected from one or more of the group consisting of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae, and the Gram-positive bacteria are preferably selected from the group consisting of Staphylococcus aureus. The multi-drug resistant clinical isolate is preferably selected from one or more of multidrug resistant Escherichia coli, multidrug resistant Pseudomonas aeruginosa, and multidrug resistant Staphylococcus aureus. Kind. and / or

The administration can be delivered to the subject by one or more of oral, injection, patch, spray, and other known techniques. The subject can be any animal, such as a human, having the above conditions. The effective amount can include an amount effective to treat, reduce, alleviate, alleviate, eliminate, or avoid one or more symptoms of the condition, the condition seeking to be treated, or alternatively, the condition seeking to be avoided, or In addition, a clinically identifiable favorable change is produced in the condition or its effect.

The invention also provides a preferred method of treatment comprising one or more of the following steps: (1) determining whether the disease species is a condition caused by the microorganism; (2) directly employing the gold nanoparticles of the invention or employing The gold nanoparticles prepared by the method of the present invention; (3) administering a therapeutically effective amount of the gold nanoparticles to a subject in need thereof; (4) detecting a therapeutic effect of the disease. The preferred method of treatment may further comprise: (5) detecting the gold nanoparticles in serum in the subject; (6) detecting the gold nanoparticles at the lesion of the condition.

A seventh aspect of the invention provides a gold nanoparticle for preventing and/or treating a condition caused by a microorganism, the gold nanoparticle being the gold nanoparticle of the first aspect of the invention or the preparation method according to the second aspect of the invention The gold nanoparticles were prepared.

Compared with the prior art, the gold nanoparticles prepared by the invention are against Gram-negative bacteria, Gram Positive bacteria and/or multi-drug resistant clinical isolates exhibit good antibacterial properties, and some of the prepared gold nanoparticles have good biocompatibility.

The invention utilizes a reduction method to synthesize a series of gold nanoparticles with antibacterial effect, which is used for sterilizing Gram-negative bacteria and Gram-positive bacteria and clinical isolates with multi-drug resistance, thereby realizing gold The potential of nanoparticles as an antibacterial agent. The small molecule ligand (ie, the nitrogen heterocyclic small molecule) used in the present invention does not have any antibacterial activity per se, and is modified to a gold nanoparticle by a reduction method, and even has a multidrug for Gram-negative bacteria and Gram-positive bacteria. The drug-resistant clinical isolates showed strong antibacterial activity, and the gold nanoparticles modified by the imidazole small molecule had very good biocompatibility. Based on the present invention, new antibacterial studies of similar molecularly modified gold nanoparticles can be further explored to explore the regularity and mechanism of gold nanoparticles as an antibacterial agent, and to avoid the study of some molecules with poor biocompatibility.

The technical solution of the present invention will be further described in detail below through the accompanying drawings and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

1 is a schematic diagram of a preparation method and antibacterial of gold nanoparticles provided by the present invention;

2 is a cytotoxicity evaluation of human umbilical vein endothelial cells at different concentrations of gold nanoparticles prepared in the second, fourth, sixth, eighth, tenth and twelfth embodiments;

3 to 14 are transmission electron microscope scans and particle size distribution diagrams of the gold nanoparticles prepared in the first to twelfth embodiments, respectively.

The best way to implement the invention

The invention is further illustrated by the following examples, which are intended to be in no way intended to

This section provides a general description of the materials used in the tests of the present invention and the test methods. While many of the materials and methods of operation used to accomplish the objectives of the present invention are well known in the art, the present invention is still described in detail herein. It will be apparent to those skilled in the art that, in the context, the materials and methods of operation of the present invention are well known in the art unless otherwise specified.

The reagents and instruments used in the following examples are as follows:

Reagents:

Chloroauric acid, purchased from Sinopharm Chemical Reagent Co., Ltd.; 3-amino-5-mercapto-1,2,4-triazole (ATT) was purchased from Beijing Bailingwei Technology Co., Ltd., 4-amino-3- hydrazine- 5-Mercapto-1,2,4-triazole (AHMT) was purchased from Saen Chemical Technology (Shanghai) Co., Ltd., 2-mercaptoimidazole (MI) was purchased from Beijing Bailingwei Technology Co., Ltd., and methimazole (MTM) Purchased from Saan Chemical Technology (Shanghai) Ltd., 2-amino-6-mercaptopurine (AMP) was purchased from Beijing Bailingwei Technology Co., Ltd., and 6-amino-2-mercaptobenzothiazole (AMBT) was purchased from Beijing Enoch Technology Co., Ltd.

instrument:

Transmission Electron Microscopy (TEM), model Tecnai G2 20 S-TWIN, FEI, USA.

Microplate reader, model Tecan infinite M200, TECAN.

First embodiment

This example is intended to illustrate the surface-modified 3-amino-5-mercapto-1,2,4-triazole gold nanoparticles (ATT-Au) of the present invention and a process for the preparation thereof.

For the preparation method adopted in this embodiment, reference may be made to the schematic diagram in FIG. 1 , specifically:

(1) 3-amino-5-mercapto-1,2,4-triazole is completely dissolved in methanol and then mixed with chloroauric acid to form a mixed solution, the 3-amino-5-mercapto-1, 2, The molar ratio of 4-triazole and the chloroauric acid is shown in Table 1.

(2) stirring the mixture in the step (1) under an ice bath magnetic stirrer, adding Tween 80 and thoroughly mixing and dissolving, adding sodium borohydride, the molar ratio of the Tween 80 to the chloroauric acid and The molar ratio of the sodium borohydride to the chloroauric acid was as shown in Table 1, and stirring was continued for 1 hour to obtain a crude gold nanoparticle.

(3) The methanol was removed by a rotary evaporator, the gold nanoparticles were redissolved by adding a certain amount of distilled water, and the gold nanoparticles were dialyzed for 48 hours with a 14 KD dialysis bag, and the gold nanoparticles were collected and stored for use.

The particle size of the gold nanoparticles was about 1 to 15 nm as determined by TEM, and the measurement results are shown in FIG. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticles was 0.36:1 as determined by an X-ray energy spectrometer.

Second embodiment

The preparation method of this embodiment is basically the same as that of Example 1, except that the nitrogen heterocyclic small molecule specified in Table 1 below, the molar ratio of the nitrogen heterocyclic small molecule to the chloroauric acid, and the Tween are the same. The molar ratio of 80 to the chloroauric acid, and the molar ratio of the sodium borohydride to the chloroauric acid are prepared.

The particle size of the gold nanoparticles was about 1 to 5 nm as determined by TEM, and the measurement results are shown in Fig. 4. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticles was 0.53:1 as determined by an X-ray energy spectrometer.

Third embodiment

This example is intended to illustrate the surface-modified 4-amino-3-linked ammonia-5-mercapto-1,2,4-triazole gold nanoparticles (AHMT-Au) of the present invention and a process for the preparation thereof.

The particle size of the gold nanoparticles was about 2 to 15 nm as determined by TEM, and the measurement results are shown in Fig. 5. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticles was 0.31:1 as determined by an X-ray energy spectrometer.

Fourth embodiment

The particle size of the gold nanoparticles was about 1 to 3 nm as determined by TEM, and the measurement results are shown in Fig. 6. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticles was 0.56:1 as determined by an X-ray energy spectrometer.

Fifth embodiment

This example is intended to illustrate the surface-modified 2-mercaptoimidazole gold nanoparticles (MI-Au) of the present invention and a process for the preparation thereof.

The particle size of the gold nanoparticles was about 2 to 35 nm as determined by TEM, and the measurement results are shown in Fig. 7. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticles was 0.34:1 as determined by an X-ray energy spectrometer.

Sixth embodiment

The particle size of the gold nanoparticles was about 2 to 10 nm as determined by TEM, and the measurement results are shown in Fig. 8. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticles was determined by an X-ray energy spectrometer to be 0.46:1.

Seventh embodiment

This example is intended to illustrate the surface-modified methimazole-based gold nanoparticles (MTM-Au) of the present invention and a process for the preparation thereof.

The particle size of the gold nanoparticles was about 2 to 15 nm as determined by TEM, and the measurement results are shown in Fig. 9. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticles was 0.22:1 as determined by an X-ray energy spectrometer.

Eighth embodiment

The particle size of the gold nanoparticles was about 1 to 10 nm as determined by TEM, and the measurement results are shown in FIG. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticles was 0.25:1 as determined by an X-ray energy spectrometer.

Ninth embodiment

This example is intended to illustrate the surface-modified 2-amino-6-mercaptopurine gold nanoparticles (AMP-Au) of the present invention and a process for the preparation thereof.

The particle size of the gold nanoparticles was about 2 to 10 nm as determined by TEM, and the measurement results are shown in FIG. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticle was 0.30:1 as determined by an X-ray energy spectrometer.

Tenth embodiment

The particle size of the gold nanoparticles was about 1 to 5 nm as determined by TEM, and the measurement results are shown in FIG. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticle was 0.40:1 as determined by an X-ray energy spectrometer.

Eleventh embodiment

This example is intended to illustrate the surface-modified 6-amino-2-mercaptobenzothiazole gold nanoparticles (AMBT-Au) of the present invention and a process for the preparation thereof.

The particle size of the gold nanoparticles was about 1 to 12 nm as determined by TEM, and the measurement results are shown in FIG. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticle was 0.40:1 as determined by an X-ray energy spectrometer.

Twelfth embodiment

The particle size of the gold nanoparticles was about 1 to 8 nm as determined by TEM, and the measurement results are shown in Fig. 14. The molar ratio of the nitrogen heterocyclic small molecule to gold in the finally obtained gold nanoparticles was 0.92:1 as determined by an X-ray energy spectrometer.

Table 1

The types of nitrogen heterocyclic small molecules of the first to twelfth embodiments, the nitrogen heterocyclic small molecule and chloroauric acid, Tween 80 and chloroauric acid, and the molar ratio of sodium borohydride to chloroauric acid

First test case

The antibacterial effect of the gold nanoparticles prepared in the second, fourth, sixth, eighth, tenth and twelfth embodiments was tested, indicating that the gold nanoparticles have a good antibacterial effect.

The comparison method is as follows:

Using the minimum inhibitory concentration, Gram-negative bacteria and Gram-positive bacteria and their related multi-drug resistant clinical isolates were cultured by broth method to the logarithmic phase, and then diluted to ensure The number of bacteria used in the experiment was 10 ⁴ to 10 ⁵ , and different strains were inoculated into 96-well plates, each of which was 100 μl. The synthesized different gold nanoparticles were added to the first well of each strain from the original concentration, the volume was added to 100 μl, and then the dilution was carried out step by step. The last well was the only strain as a blank control, and all operations were super clean. Taichung. Thereafter, the 96-well plate was placed at 37 ° C for constant incubation for 24 hours, and the minimum inhibitory concentration of each of the different gold nanoparticles was evaluated by observing the transparency of the well plate and the blank control. The test results are shown in Table 2.

It can be seen from the test results that the gold nanoparticles prepared in the second, fourth, sixth, eighth, tenth and twelfth embodiments have good antibacterial effects against Gram-negative bacteria and resistant Gram-negative bacteria, and most of them It also has good antibacterial effects against Gram-positive bacteria and resistant Gram-positive bacteria.

Table 2 The detection of antibacterial effects of gold nanoparticles prepared in the second, fourth, sixth, eighth, tenth and twelfth embodiments

Second test case

Cytotoxicity experiments were performed on the gold nanoparticles prepared in the second, fourth, sixth, eighth, tenth and twelfth embodiments to evaluate the cytotoxicity of the gold nanoparticles.

The assessment method is as follows:

Human umbilical vein endothelial cells in good condition were inoculated in 96-well plates, with 4 to 50,000 cells per well, ensuring the same order of magnitude of cells per well. Incubate in 37 ° C culture until the cells adhered to the cells, and add different gold nanoparticles. And the dilution was carried out step by step. The surrounding of the 96-well plate was not subjected to experimental treatment, and only a blank control was used. After incubating for 24 hours in a 37 ° C incubator, the liquid per well was gently aspirated, 10 to 20 μl of cck-8 reagent dye was added, and cultured in an incubator for 2 to 4 hours until the color of the well plate changed significantly. The absorbance at 450 nm of each well in a 96-well plate was measured with a microplate reader. Then, the cell viability of each well was calculated by the calculation formula of the reagent method, thereby determining the toxicity of the corresponding gold nanoparticles to human umbilical vein endothelial cells. The test results are shown in Figure 2.

It can be seen from the test results that even if the concentration of the gold nanoparticles prepared in the sixth and eighth embodiments is as high as 100 μg/mL, the activity of the corresponding human umbilical vein endothelial cells is almost 100%, indicating that the two gold nanoparticles have a concentration of Good biocompatibility. Further, the gold nanoparticles prepared in the twelfth embodiment have a cell activity higher than that of the gold nanoparticles prepared in the other examples to be evaluated except the sixth and eighth embodiments at a concentration of 50 μg/mL or more. .

Kit

The present invention also provides a kit comprising the gold nanoparticles of the first aspect of the invention or the gold nanoparticles prepared by the preparation method of the second aspect of the invention.

In addition, instructions for use and/or use/analysis software may also be included in the kit.

Pharmaceutical composition

The present invention also relates to an antibacterial pharmaceutical composition comprising a pharmaceutically acceptable carrier, and the gold nanoparticles of the first aspect of the invention or the gold nanoparticles prepared by the production method of the second aspect of the invention. The gold nanoparticles can be in an effective amount or in a therapeutically effective amount in the pharmaceutical composition.

As used herein, "effective amount" refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.

As used herein, a "pharmaceutically acceptable" ingredient is suitable for use in humans and/or animals (eg, mammals and birds) without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, has reasonable benefits / risk ratio substance. "Pharmaceutically acceptable carrier" means a carrier for administration, and may include various excipients, diluents and the like.

The pharmaceutical composition of the present invention may contain a safe and effective amount of the gold nanoparticles of the present invention as an active ingredient together with a pharmaceutically acceptable carrier. Such carriers can include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, and the dosage form of the pharmaceutical composition of the present invention can be prepared as an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, a diluent, and the like as needed. For example, physiological saline or an aqueous solution containing glucose and other excipients is usually prepared in a conventional manner. The pharmaceutical composition is more suitably manufactured under sterile conditions. The pharmaceutically acceptable carrier of the present invention includes, but is not limited to, water, physiological saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or combination. The choice of carrier should generally be matched to the mode of administration, as is well known to those of ordinary skill in the art.

For example, for oral administration in the form of a tablet or capsule, the gold nanoparticles can be combined with an oral, non-toxic pharmaceutically acceptable carrier such as ethanol, glycerol, water, and the like. Dusts can be prepared by comminuting the mixture into suitable fine sizes and mixing with a similar mashed pharmaceutical carrier such as an edible sugar such as starch or mannitol. Flavoring agents, preservatives, dispersing agents, and coloring agents may also be included.

Capsules can be prepared by preparing a powder mixture as described above and filling into a shaped gelatin capsule. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture prior to the filling stage. Can also be added to disintegrants or solubilized Agents such as agar-agar, calcium carbonate or sodium carbonate improve the availability of the drug when ingesting the capsule.

Moreover, suitable binders, lubricants, disintegrants, and colorants can also be incorporated into the mixture when desired or necessary. Suitable binders include starch, gelatin, natural gums such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, poly Ethylene glycol, wax, etc. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like. Tablets are prepared by preparing a powder mixture, granulating, pre-pressing, adding a lubricant and a disintegrant, and compressing into tablets. The powder mixture is obtained by mixing the gold nanoparticles with a diluent or matrix as described above, optionally with a binder such as carboxymethylcellulose, alginate, gelatin or polyvinylpyrrolidone, with a retarder solution such as paraffin, An absorption enhancer such as a quaternary ammonium salt and/or an absorbent such as bentonite, kaolin or dicalcium phosphate is mixed and suitably mashed to prepare. The powder mixture can be prepared by wetting with a gum or solution of a binder such as syrup, starch paste, cellulosic or polymeric material and pressurizing the screen. As another granulation method, the powder mixture can be prepared by a tableting machine to obtain an incompletely formed pre-tablet which can be a granule. The granules can be lubricated by the addition of stearic acid, stearate, talc or mineral oil to prevent sticking of the tablet forming mold. The lubricated mixture is then compressed into tablets. The gold nanoparticles of the present invention can also be directly compressed into tablets by mixing with a free flowing inert carrier without the need for a granulation or pre-pressing stage. A clear or opaque protective coating consisting of shellac, a coating of sugar or polymeric material and a polishing coating of wax may be provided. Dyes can be added to these coatings to distinguish between different unit dosage forms.

Oral liquids such as solutions, syrups and elixirs can be prepared in dosage units such that a given dosage comprises a predetermined amount of gold nanoparticles. A syrup can be prepared by dissolving the gold nanoparticles in a suitable perfumed liquid solution, and the elixirs can be prepared by using non-toxic alcohol-containing excipients. Suspensions can be prepared by dispersing the gold nanoparticles in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitan esters, preservatives, flavoring additives such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like, may also be added.

Dosage unit formulations for oral administration can be microencapsulated, where appropriate. The formulations may also be prepared for delayed or sustained release, for example by coating or embedding particulate materials in polymers, waxes and the like.

Gold nanoparticles for use in accordance with the present invention may also be administered by liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be prepared from a variety of phospholipids, such as cholesterol, stearamide or phosphophosphatidylcholine.

Gold nanoparticles used in accordance with the present invention can also be delivered by using monoclonal antibodies as separate carriers for molecular coupling. The gold nanoparticles can also be coupled to a soluble polymer such as a targeted drug carrier. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl group Acrylamide-phenol, polyhydroxyethylaspartamide, or polyethyleneoxypolylysine substituted with palmitoyl residues. Moreover, the gold nanoparticles can be coupled to a class of biodegradable polymers for obtaining controlled release of a drug such as polylactic acid, polycaprolactone, polyhydroxybutyric acid, polyorthoesters, polycarboxylates. Crosslinked or amphiphilic block copolymers of aldehydes, polydihydropyrans, polycyanoacrylates and hydrogels.

A pharmaceutical composition suitable for transdermal administration can be a separate patch which is expected to remain in direct contact with the epidermis of the recipient for an extended period of time. For example, the active ingredient can be delivered by iontophoresis as generally described in Pharmaceutical Research, 3(6), (1986).

Pharmaceutical compositions suitable for topical administration may be formulated as ointments, creams, suspensions, powders, solutions, pastes, gels, sprays, aerosols or oils.

For the treatment of the eye or other external tissues such as the mouth and skin, the formulation is preferably applied as a topical ointment or cream. When formulated as an ointment, the active ingredient can be applied with a paraffin or water miscible cream base. Further, the active ingredient may be formulated into a cream containing a water-in-oil type cream base or an oil-in-water type base.

Pharmaceutical compositions suitable for topical administration to the eye comprise eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solution.

Pharmaceutical compositions adapted for topical administration to the mouth include lozenges, pastilles, and mouth lotions.

Pharmaceutical compositions adapted for rectal administration may be presented as a suppository or enemas.

A pharmaceutical composition suitable for nasal administration wherein the carrier is a solid comprises a coarse powder having a particle size of from 20 to 500 microns which is administered in the form of ingesting an olfactory agent, i.e., from a powder-containing container adjacent to the nose. Rapid inhalation through the nasal cavity. Suitable formulations for administration in a spray or nasal drops wherein the carrier is a liquid include aqueous or oil solutions of the active ingredient gold nanoparticles.

Pharmaceutical compositions suitable for administration by inhalation include fine particle powders or mists which can be produced by various types of metered doses of pressurized aerosols, nebulizers or inhalers.

The pharmaceutical composition suitable for vaginal administration may be a pessary, tampons, cream, gel, paste, foam or spray formulation.

Pharmaceutical compositions adapted for parenteral administration include aqueous or non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes which render the formulation isotonic to the intended recipient; Aqueous suspensions of aqueous use may contain suspending and thickening agents. The formulation can be enclosed in unit or multi-dose containers, such as sealed ampoules and vials, can be stored under lyophilization (lyophilization), and requires only the addition of sterile liquid carriers, such as water for injection, prior to immediate use. . Extemporaneous injection solutions or suspensions can be prepared from sterile powders, granules and tablets.

It will be understood that in addition to the above ingredients, the formulations may contain other agents conventional in the art in connection with the type of formulation discussed above, for example those suitable for oral administration may include flavoring agents.

The effective amount of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, The route of medicine, etc. In general, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg to 50 mg/kg of animal body weight per day (preferably 0.0001 mg to 10 mg/kg of animal body weight), a satisfactory effect can be obtained. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.

The invention also provides the use of the pharmaceutical composition for the preparation of an antimicrobial product or for the preparation of a medicament for the prevention and/or treatment of a condition caused by a microorganism.

Prevention and/or treatment

The invention also provides a method of preventing and/or treating a condition caused by a microorganism, the method comprising administering to a subject a therapeutically effective amount of the gold nanoparticle of the first aspect of the invention, employing the second aspect of the invention The gold nanoparticle prepared by the preparation method or the antimicrobial composition of the third aspect of the invention.

The gold nanoparticle or pharmaceutical composition of the present invention can pass through the gastrointestinal tract, nasal cavity, trachea, lung, non-lesion site vein or epidermis, intradermal, subcutaneous, intracardiac, muscle, bone marrow, abdominal cavity, epidural, Oral, sublingual, ocular, rectal, vaginal, urethra, ear canal and other routes of administration. Preferred modes of administration or modes of administration include oral, respiratory, injection, transdermal, mucosal, or intraluminal administration.

Wherein, the oral administration means includes swallowing, incorporation, and the like. The method of administration of the respiratory tract includes an inhalation method such as ultrasonic atomization inhalation, oxygen atomization inhalation, hand pressure atomization inhalation, and the like. The administration mode of injection includes arterial injection, intravenous injection, intramuscular injection, intracardiac injection, intradermal injection, and the like. The transdermal or transdermal administration methods include iontophoresis, electroporation, and the like. The mucosal administration forms include nasal mucosa administration, oral mucosal administration, ocular mucosal administration, rectal mucosal administration, uterine administration, and vaginal mucosal administration. The method of administration of the lumen includes rectal administration, vaginal administration, urethral administration, nasal administration, ear canal administration, and the like.

All documents (including patent documents or non-patent documents) mentioned in the present invention are incorporated herein by reference in their entirety as if they are individually incorporated by reference.

While the invention has been described in detail, it is obvious that various changes in the various conditions can be made without departing from the spirit and scope of the invention. It is to be understood that the invention is not limited to the embodiments, but is intended to be included within the scope of the appended claims.

Claims

a gold nanoparticle, characterized in that the surface of the gold nanoparticle is modified with a nitrogen heterocyclic small molecule;

Preferably, the nitrogen heterocyclic small molecule has a sulfhydryl group;

More preferably, the nitrogen heterocyclic small molecule is selected from one or more of a triazole having a fluorenyl group, an imidazole having a fluorenyl group, a fluorenyl group having a fluorenyl group, and a benzothiazole having a fluorenyl group;

Further preferably, the nitrogen heterocyclic small molecule is selected from the group consisting of 3-amino-5-mercapto-1,2,4-triazole, 4-amino-3-linked ammonia-5-mercapto-1,2,4- One or more of triazole, 2-mercaptoimidazole, methimazole, 2-amino-6-mercaptopurine, and 6-amino-2-mercaptobenzothiazole.
The gold nanoparticle according to claim 1, wherein:

a molar ratio of the nitrogen heterocyclic small molecule to the gold element in the gold nanoparticle is from 0.1 to 0.99:1, preferably from 0.22 to 0.92:1; and/or

The gold nanoparticles have an average particle diameter of from 1 to 35 nm, preferably from 2 to 10 nm.
The method for preparing a gold nanoparticle according to claim 1 or 2, wherein the preparation method comprises:

(1) dissolving the nitrogen heterocyclic small molecule in methanol and mixing with chloroauric acid to form a mixed solution.

(2) adding a reducing agent to the mixed liquid in the step (1), and sufficiently reacting to obtain a crude gold nanoparticle.

(3) purifying the crude gold nanoparticles in the step (2), that is, obtaining the gold nanoparticles;

Preferably:

The molar ratio of the nitrogen heterocyclic small molecule to the chloroauric acid in the step (1) is from 1 to 10:1, most preferably 10:1;

The reducing agent in the step (2) is selected from one or more of sodium borohydride, sodium ascorbate and sodium citrate, most preferably sodium borohydride;

The molar ratio of the reducing agent to the chloroauric acid in the step (2) is from 1 to 10:1, preferably from 2 to 8:1, most preferably 3:1; and/or

The reaction in the step (2) is carried out under ice bath and stirring, and the reaction time is preferably from 30 minutes to 2 hours, most preferably 1 hour.
The method for preparing a gold nanoparticle according to claim 3, wherein after the mixing in the step (1), the mixture is stirred and added with a nonionic surfactant under ice bath conditions. well mixed;

among them:

The nonionic surfactant is preferably selected from one or more of Triton X-100, Tween or polyethylene glycol, most preferably Tween 80; and/or

The molar ratio of the nonionic surfactant to the chloroauric acid is preferably from 0.1 to 2:1, more preferably from 0.5 to 1.5:1.
The method for preparing a gold nanoparticle according to claim 3 or 4, wherein the purifying in the step (3) comprises:

Methanol is removed, the removal of methanol is preferably carried out by a rotary evaporator; and/or

Dialysis, the dialysis preferably uses a 14KD dialysis bag for 48 hours;

Preferably, distilled water is added to the gold nanoparticles after the methanol removal to dissolve, and then dialyzed.
An antibacterial composition comprising the gold nanoparticles of claim 1 or 2 or gold nanoparticles prepared by the method of any one of claims 3 to 5;

Preferably, the antibacterial composition is an antibacterial agent or an antibacterial pharmaceutical composition;

More preferably, the antibacterial or antibacterial composition is one or more of the following strains: Gram-negative bacteria, Gram-positive bacteria, and clinical isolates having multidrug resistance;

Further preferably:

The Gram-negative bacteria are selected from one or more of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae;

The Gram-positive bacteria are selected from the group consisting of Staphylococcus aureus; and/or

The multi-drug resistant clinical isolate is selected from one or more of multidrug resistant Escherichia coli, multidrug resistant P. aeruginosa, and multidrug resistant S. aureus.
A kit comprising the gold nanoparticles of claim 1 or 2 or gold nanoparticles prepared by the method of any one of claims 3 to 5.
The gold nanoparticle according to claim 1 or 2, the gold nanoparticle prepared by the method according to any one of claims 3 to 5 or the antibacterial composition according to claim 6 in the preparation of an antibacterial product or in preparation for use in preparation Use in a medicament for preventing and/or treating a condition caused by a microorganism;

Preferably:

The antibacterial product or drug is one or more of the following strains: Gram-negative bacteria, Gram-positive bacteria, and multi-drug resistant clinical isolates; wherein the Gram-negative bacteria Preferred choice From one or more of Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae, the Gram-positive bacteria are preferably selected from the group consisting of Staphylococcus aureus, the multi-drug resistant clinical isolate Preferably one or more selected from the group consisting of multidrug resistant Escherichia coli, multidrug resistant P. aeruginosa, and multidrug resistant S. aureus; and/or

The condition caused by the microorganism is a condition caused by one or more of Gram-negative bacteria, Gram-positive bacteria, and multi-drug resistant clinical isolates; the condition is preferably selected from the following one Species or more: skin and subcutaneous tissue infections, traumatic infections, otitis media, meningitis, peritonitis, enteritis, bronchitis, pneumonia, respiratory infections, urinary tract infections, sepsis or sepsis.
A method for preventing and/or treating a condition caused by a microorganism, the method comprising administering to a subject a therapeutically effective amount of the gold nanoparticle according to claim 1 or 2, using the claim 3 The gold nanoparticle prepared by the method of any of the above 5 or the antimicrobial composition of claim 6;

Preferably:

The condition caused by the microorganism is a condition caused by one or more of Gram-negative bacteria, Gram-positive bacteria, and multi-drug resistant clinical isolates; the condition is preferably selected from the following one Species or types: skin and subcutaneous tissue infections, traumatic infections, otitis media, meningitis, peritonitis, enteritis, bronchitis, pneumonia, respiratory infections, urinary tract infections, sepsis or sepsis; and/or

The mode of administration is selected from one or more of oral, injection, patch and spray.
A gold nanoparticle for preventing and/or treating a condition caused by a microorganism, characterized in that the gold nanoparticle is the gold nanoparticle of claim 1 or 2 or the method of any one of claims 3 to 5 Gold nanoparticles prepared by the method.