阅读说明：本技术 一种万古霉素修饰的黑磷量子点抗菌剂、制备方法及应用 (Vancomycin-modified black phosphorus quantum dot antibacterial agent, and preparation method and application thereof ) 是由 杨燕美 支晓钰 田晓涵 张晔 牛伟华 李然 于 2020-06-11 设计创作，主要内容包括：本发明具体涉及一种万古霉素修饰的黑磷量子点抗菌剂、制备方法及应用。抗菌药物的开发具有周期长、费用昂贵的缺点,并且菌株耐药性的形成速度超过药物的研发速度。为了提供具有更好抗菌效果、降低耐药性产生的药物,本发明设计采用万古霉素修饰黑磷量子点作为抗菌活性成分,所述万古霉素通过共价键结合于黑磷量子点表面。经本发明研究证实,经万古霉素修饰的黑磷量子点抗菌性能具有显著的提升,有望成为临床抗菌药物一线活性成分,具有良好的开发意义。(The invention particularly relates to a vancomycin-modified black phosphorus quantum dot antibacterial agent, and a preparation method and application thereof. The development of antibacterial drugs has the disadvantages of long period and high cost, and the formation speed of the drug resistance of the bacterial strains exceeds the development speed of the drugs. In order to provide a medicament with better antibacterial effect and reduced drug resistance, the invention adopts vancomycin modified black phosphorus quantum dots as an antibacterial active ingredient, and the vancomycin is bonded on the surfaces of the black phosphorus quantum dots through covalent bonds. The research of the invention proves that the antibacterial performance of the vancomycin-modified black phosphorus quantum dot is remarkably improved, and the black phosphorus quantum dot is expected to become a first-line active ingredient of clinical antibacterial drugs and has good development significance.)

1. The antibacterial agent is characterized by being vancomycin-modified black phosphorus quantum dots.

2. The antimicrobial agent of claim 1, wherein the vancomycin is bound to the black phosphorus quantum dots by a covalent bond.

3. The process for producing the antibacterial agent according to claim 1 or 2, which comprises dissolving vancomycin hydrochloride and N-hydroxysuccinimide in a mixture, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-Boc- (ethylenedioxy) diethylamine and N, N-diisopropylethylamine, mixing, centrifuging, drying the precipitate obtained by the centrifugation, treating the precipitate with dichloromethane, trifluoroacetic acid and diethyl ether, and adding black phosphorus quantum dots to obtain the antibacterial agent.

4. The method for preparing the antibacterial agent according to claim 3, which comprises the following specific steps: dissolving vancomycin hydrochloride and N-hydroxysuccinimide in DMSO, cooling the solvent, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), mixing, putting into an ice bath, continuously adding N-BoC- (ethylenedioxy) diethylamine and N, N-Diisopropylethylamine (DIPEA), stirring overnight, adding acetone, and centrifuging to obtain a precipitate; separating the precipitate part by a liquid phase to obtain a sample, and treating the sample by a mixed solution of dichloromethane and trifluoroacetic acid and ether in turn; adding a black phosphorus quantum dot solution, and adjusting the pH value to 7-8 to obtain the vancomycin-modified black phosphorus quantum dot.

5. The method for producing an antibacterial agent according to claim 4, wherein the sample is obtained by liquid phase separation under methanol/water (v: 90: 10); and (4) retaining the amino deprotection product, and drying to obtain the sample amino- (ethylenedioxy) diethylamine-vancomycin.

6. The method for preparing the antibacterial agent according to claim 4, wherein in the mixed solution of dichloromethane and trifluoroacetic acid, the volume ratio of dichloromethane to trifluoroacetic acid is 3: 1.5-2.5;

or the sample is sequentially treated by a mixed solution of dichloromethane and trifluoroacetic acid and diethyl ether as follows: and (3) placing the sample into a mixed solution of dichloromethane and trifluoroacetic acid, stirring for 0.8-1.2 h under an ice-bath condition, removing a solvent part, slowly adding a cold ether solution in times, removing ether, and drying.

7. The preparation method of the antibacterial agent according to claim 4, wherein the pH of the sample is adjusted to 7-8 by NaOH solution after the black phosphorus quantum dot solution is added to the sample, a supernatant part is obtained by centrifugation, and the vancomycin-modified black phosphorus quantum dot is obtained by washing and dialysis.

8. The method of claim 4, wherein the method of preparing the black phosphorus quantum dot comprises the steps of: putting black phosphorus in an N-methyl pyrrolidone reagent, uniformly dispersing, and heating and reacting under an alkaline condition to obtain a black phosphorus quantum dot antibacterial agent in a supernatant part;

Preferably, the preparation method of the black phosphorus quantum dot comprises the following specific steps: adding the black phosphorus crystals into NMP under the protection of inert gas for uniform dispersion, and reacting the NMP solution of the black phosphorus with alkali at 120-160 ℃ for 4-8 h under the protection of the inert gas; after the reaction is finished, centrifuging to obtain supernatant black phosphorus, and drying to obtain the black phosphorus quantum dots;

further, the black phosphorus crystals were ground into powder and then added to NMP, and uniformly dispersed by ultrasonic.

Furthermore, the alkali is sodium hydroxide, and the mass ratio of the black phosphorus to the sodium hydroxide is 1: 9-11;

further, the inert gas is nitrogen;

further, the centrifugation is as follows: and centrifuging the reacted solution at 6000-8000 r to obtain a supernatant part, continuously centrifuging the supernatant part at the rotating speed of 15000-16000 r to obtain supernatant black phosphorus, and washing and drying to obtain the black phosphorus quantum dots.

9. Use of the antibacterial agent of claim 1 or 2 for the preparation of an antibacterial product.

10. Use of an antimicrobial agent according to claim 9 in the manufacture of an antimicrobial product, wherein the antimicrobial product comprises an antimicrobial medicament or a wash-and-care product;

preferably, the antibacterial agent is used for inhibiting gram-positive bacteria and gram-negative bacteria;

Preferably, the antibacterial agent comprises the antibacterial agent of claim 1 or 2, and further comprises the necessary auxiliary materials in pharmacy;

preferably, the antibacterial agent comprises the antibacterial agent of claim 1 or 2, and further comprises other antibacterial active ingredients.

Technical Field

The invention belongs to the technical field of antibiosis, and particularly relates to a vancomycin-modified black phosphorus quantum dot antibacterial agent, a preparation method of the antibacterial agent and application of the antibacterial agent in preparation of an antibacterial product.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Various pathogenic bacteria are widely distributed in nature, and the spread and infection of the pathogenic bacteria can cause various diseases, thereby threatening the health of human beings. Killing bacteria or preventing the proliferation of bacteria is currently the main idea of antibacterium. Among the traditional multiple antibacterial drugs, antibiotics are a large class of drugs with the widest clinical application range and the largest variety, and have better bactericidal effect. However, excessive use of antibiotic resistance presents a serious problem: drug resistance, toxic and side effects, dysbacteriosis and the like. However, in recent years, the development of new antibiotics has been a difficult dilemma. On one hand, the over-developed soil resources lead to the fact that new antibiotics cannot be screened from soil microorganisms, and synthetic chemistry is only improved on a certain type of antibiotics, so that the type is single; on the other hand, the development of the antibacterial agent needs a long period (more than 10 years) and is expensive (more than 10 hundred million dollars), while the generation of a generation of the drug-resistant bacteria only needs two years, and the development speed of the antibacterial agent is far lower than that of the drug-resistant bacteria. Therefore, while the next generation of antibiotics is sought, the idea is converted to find a new antibacterial mechanism, and the research and development of new generation, green, broad-spectrum and economic antibacterial drugs and materials are very urgent.

In recent years, the research on the antibacterial function of nano materials has become a new front-edge hotspot of the interdisciplinary discipline in the research field of nano medicine. Besides good biocompatibility, the two-dimensional nano material also has very good repairability. With the development of the preparation and characterization technology of nano materials, people have prepared more and more novel nano materials, such as phospholene. The novel two-dimensional nanomaterial graphene has a two-dimensional structure of graphene, and has oxidation capacity due to the special electrical properties of the novel two-dimensional nanomaterial graphene, so that the antibacterial capacity of the novel two-dimensional nanomaterial graphene can be remarkably enhanced. Meanwhile, phosphorus is an essential element in a living body, so that the phosphorus has incomparable advantages in the application of the phosphorus in the field of biomedicine.

Vancomycin belongs to tricyclic glycopeptide antibiotics, and at present, the recent research on vancomycin shows that the vancomycin combination has a certain inhibition effect on the drug resistance of certain bacteria, so that the research on the vancomycin modified black phosphorus quantum dot antibacterial agent has a good prospect.

Disclosure of Invention

Based on the research background, the invention designs the mode of combining the antibacterial drug and the two-dimensional nano material to obtain the active ingredient with improved antibacterial effect. According to the research of the invention, vancomycin is modified on the surface of the black phosphorus quantum dot, so that the in-vitro antibacterial activity of the medicine can be obviously improved, the antibacterial range of the medicine is expanded, the dosage of the medicine is expected to be reduced, and the generation of drug-resistant strains is reduced.

According to the technical effects, the invention provides the following technical scheme:

in a first aspect of the invention, an antibacterial agent is provided, wherein the antibacterial agent is vancomycin-modified black phosphorus quantum dots.

As is known in the art, vancomycin is a glycopeptide antibiotic, is used for treating serious infection which is not effective in other antibiotic treatment, has high nephrotoxicity and narrow antibacterial spectrum, and is only used for gram-positive bacteria. According to the research result of the invention, the bacteriostatic effect of the black phosphorus quantum dot is not obvious when the black phosphorus quantum dot is applied to bacteria alone, but the bacteriostatic effect is improved by four times after the black phosphorus quantum dot is modified by vancomycin, and the black phosphorus quantum dot modified by the vancomycin not only has a suppression effect on gram-positive bacteria, but also has a significant suppression effect on gram-negative bacteria. The modified black phosphorus quantum dots expand the antibacterial spectrum of vancomycin, reduce the dosage of vancomycin, reduce the occurrence of drug resistance, and develop the black phosphorus quantum dots as antibacterial active ingredients into antibacterial drugs to be combined with other antibacterial active ingredients to prepare the antibacterial drugs, or combine the antibacterial drugs with surfactant active ingredients to prepare washing and care products with antibacterial effects.

In a second aspect of the present invention, there is provided a method for producing the antibacterial agent according to the first aspect, wherein the method comprises mixing and dissolving vancomycin hydrochloride and N-hydroxysuccinimide, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-BoC- (ethylenedioxy) diethylamine and N, N-diisopropylethylamine, mixing and centrifuging, drying the precipitate obtained by centrifuging, treating the precipitate with dichloromethane, trifluoroacetic acid and diethyl ether, and then adding black phosphorus quantum dots to obtain the antibacterial agent.

In a third aspect of the invention, there is provided the use of an antibacterial agent according to the first aspect in the manufacture of an antibacterial product.

The beneficial effects of one or more technical schemes are as follows:

the invention relates to a vancomycin-modified black phosphorus quantum dot antibacterial agent and a preparation method thereof. The black phosphorus quantum dots are only single atom layer thick, the specific surface area is very large, and the sharp boundary of the atom thickness can directly damage the cell membrane of bacteria; moreover, the black phosphorus quantum dots have excellent optical performance, can induce the generation of active oxygen and cause the peroxidation of a thallus biological system; the modified antibacterial material still adsorbs biological macromolecules such as protein, nucleic acid, phospholipid and the like when entering a biological microenvironment, and a two-dimensional nano material-biological macromolecule compound is formed on the surface of the modified antibacterial material, so that the contact between the material and bacteria can be prevented, and the antibacterial effect of the antibacterial material is improved. The vancomycin-modified black phosphorus quantum dot antibacterial agent prepared by the invention has good biocompatibility and strong antibacterial effect.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

Fig. 1 is a TEM image of the black phosphorus quantum dots described in example 1.

FIG. 2 is a photograph of an experiment of inhibiting bacteria by black phosphorus quantum dots as described in example 2;

wherein, fig. 2A is a blank control group cultured for 2h, fig. 2B is an experimental group added with 10 microliter of quantum dot black phosphorus, and fig. 2C is an experimental group added with 20 microliter of quantum dot black phosphorus.

FIG. 3 is a mass spectrum of vancomycin hydrochloride described in example 2.

FIG. 4 is a mass spectrum of the intermediate product described in example 2.

FIG. 5 is a mass spectrum of vancomycin-modified black phosphorus quantum dots in example 2.

Fig. 6 is an ultraviolet fluorescence spectrum of vancomycin hydrochloride, the black phosphorus quantum dots and the vancomycin-modified black phosphorus quantum dots in example 2.

FIG. 7 is a photograph showing the bactericidal effect of the vancomycin-modified black phosphorus quantum dots in example 2;

wherein, fig. 7A is a blank control group cultured for 1h, fig. 7B is 10 microliters of the black phosphorus quantum dots modified by adding vancomycin, and fig. 7C is 20 microliters of the black phosphorus quantum dots modified by adding vancomycin.

FIG. 8 is a quantum dot black phosphorus bactericidal effect histogram after quantum dot black phosphorus and vancomycin are modified.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As introduced in the background art, in order to further develop the antibacterial active ingredient, the invention provides a vancomycin-modified black phosphorus quantum dot antibacterial agent as the antibacterial active ingredient.

In a first aspect of the invention, an antibacterial agent is provided, wherein the antibacterial agent is vancomycin-modified black phosphorus quantum dots.

Preferably, the vancomycin is bound to the black phosphorus quantum dots through a covalent bond.

Preferably, the size of the black phosphorus quantum dot is 1.5-2 nm.

Preferably, the mass ratio of the black phosphorus quantum dots to the vancomycin is 1: 18 to 22.

In a second aspect of the present invention, there is provided a method for producing the antibacterial agent according to the first aspect, wherein the method comprises mixing and dissolving vancomycin hydrochloride and N-hydroxysuccinimide, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-BoC- (ethylenedioxy) diethylamine and N, N-diisopropylethylamine, mixing and centrifuging, drying the precipitate obtained by centrifuging, treating the precipitate with dichloromethane, trifluoroacetic acid and diethyl ether, and adding black phosphorus quantum dots after HPLC separation to obtain the antibacterial agent.

Preferably, the preparation method comprises the following specific steps: dissolving vancomycin hydrochloride and N-hydroxysuccinimide in DMSO, cooling the solvent, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), mixing, putting into an ice bath, continuously adding N-BoC- (ethylenedioxy) diethylamine and N, N-Diisopropylethylamine (DIPEA), stirring overnight, adding acetone, and centrifuging to obtain a precipitate; obtaining the sample through liquid phase separation, and treating the sample through mixed solution of dichloromethane and trifluoroacetic acid and ether in turn; adding a black phosphorus quantum dot solution, and adjusting the pH value to 7-8 to obtain the vancomycin-modified black phosphorus quantum dot.

Preferably, the precipitated fraction is treated sequentially with a mixture of dichloromethane and trifluoroacetic acid and diethyl ether as follows: and (3) placing the sample into a mixed solution of dichloromethane and trifluoroacetic acid, stirring for 0.8-1.2 h under an ice-bath condition, removing a solvent part, slowly adding a cold ether solution in times, removing ether, and drying.

Preferably, the sample is obtained by liquid phase separation under methanol/water (v: v ═ 90: 10); and (4) retaining the amino deprotection product, and drying to obtain the sample amino- (ethylenedioxy) diethylamine-vancomycin.

More preferably, in the mixed solution of dichloromethane and trifluoroacetic acid, the volume ratio of dichloromethane to trifluoroacetic acid is 3: 1.5-2.5.

Preferably, after the black phosphorus quantum dot solution is added into the sample, the pH value is adjusted to 7-8 by adopting a NaOH solution, a supernatant part is obtained by centrifugation, and the vancomycin-modified black phosphorus quantum dot is obtained by washing and dialyzing.

Preferably, the preparation method of the black phosphorus quantum dot comprises the following steps: putting black phosphorus in an N-methyl pyrrolidone (NMP) reagent for uniform dispersion, and heating and reacting under an alkaline condition to obtain the black phosphorus quantum dot antibacterial agent in a supernatant part.

Further preferably, the preparation method of the black phosphorus quantum dot comprises the following specific steps: adding the black phosphorus crystals into NMP under the protection of inert gas for uniform dispersion, and reacting the NMP solution of the black phosphorus with alkali at 120-160 ℃ for 4-8 h under the protection of the inert gas; and after the reaction is finished, centrifuging to obtain supernatant black phosphorus, and drying to obtain the black phosphorus quantum dots.

Further, the black phosphorus crystals were ground into powder and then added to NMP, and uniformly dispersed by ultrasonic.

Furthermore, the alkali is sodium hydroxide, and the mass ratio of the black phosphorus to the sodium hydroxide is 1: 9-11.

Further, the inert gas is nitrogen.

Further, the centrifugation is as follows: and centrifuging the reacted solution at 6000-8000 r to obtain a supernatant part, continuously centrifuging the supernatant part at the rotating speed of 15000-16000 r to obtain supernatant black phosphorus, and washing and drying to obtain the black phosphorus quantum dots.

In a third aspect of the invention, there is provided the use of an antibacterial agent according to the first aspect in the manufacture of an antibacterial product.

Preferably, the antimicrobial product comprises an antimicrobial medicament or a wash care product.

Further preferably, the antibacterial agent is used for inhibiting gram-positive bacteria and gram-negative bacteria.

Further preferably, the antibacterial agent comprises the antibacterial agent of the first aspect, and further comprises pharmaceutically necessary auxiliary materials.

It is further preferred that the antimicrobial medicament comprises the antimicrobial agent of the first aspect, and further comprises other antimicrobial active ingredients.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.