CN111012798B

CN111012798B - Nano antibacterial agent for killing drug-resistant bacteria and preparation method thereof

Info

Publication number: CN111012798B
Application number: CN202010039409.XA
Authority: CN
Inventors: 汪联辉; 宇文力辉; 单京阳; 杨凯丽
Original assignee: Nanjing University of Posts and Telecommunications
Current assignee: Nanjing University of Posts and Telecommunications
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2022-03-18
Anticipated expiration: 2040-01-15
Also published as: CN111012798A

Abstract

The invention discloses a nano antibacterial agent for quickly and efficiently killing drug-resistant bacteria and a preparation method thereof, wherein the main component of the nano antibacterial agent is cuprous thiomolybdate (Cu) prepared by a mechanical stripping method₂MoS₄) The nano material is simple to prepare and suitable for mass production. Irradiating with laser in near infrared region for 2-20 min, Cu₂MoS₄The nano material shows high antibacterial performance to drug-resistant escherichia coli and drug-resistant staphylococcus aureus; the nano antibacterial agent not only has good biocompatibility and extremely low cytotoxicity, but also can effectively treat drug-resistant bacterial infection under the skin of a living body in a short time.

Description

Nano antibacterial agent for killing drug-resistant bacteria and preparation method thereof

Technical Field

The invention relates to the fields of nano antibacterial technology and drug-resistant bacteria infection, in particular to a nano antibacterial agent capable of quickly and efficiently killing drug-resistant bacteria and a preparation method thereof.

Background

Antibiotics refer to a class of substances produced by bacteria, molds, or other microorganisms during life that have anti-pathogenic or other activities. Antibiotics have developed rapidly since the clinical application of penicillin in 1940, and the types of antibiotics today have reached thousands. However, a very obvious side effect of the widely used antibiotics is that the microorganisms can generate drug resistance against the antibiotics after the antibiotics are used, for example, the antibiotics are abused, so that the pathogenic microorganisms existing in the environment are resistant, if the pathogenic infection of the human body becomes resistant bacteria, the effective drugs are difficult to perform targeted treatment, and the continuous appearance and rapid spread of the antibiotic-resistant strains seriously threaten the health of the human body. At present, 70 million people die annually from antimicrobial resistance, if not contained in an effective manner, and by 2050, the number of deaths annually will rise to 1000 million, and the cost of treatment and development will rise dramatically to $ 100 trillion (https:// www.wired.com/2015/02/oneill-amr-2 /). The development of new antibiotics is one of the approaches to solve the problem, but the method has long cost period and high cost, and the use of the new antibiotics can also lead to the generation of new drug-resistant bacteria. For this reason, related researchers have been working on developing antibacterial compounds and nanomaterials that can replace antibiotics to solve this problem.

With the continuous development of nano biomedicine, novel antibacterial nano materials are produced at the same time, and the drug resistance of bacteria can be greatly weakened. In recent years, many nanomaterials (such as silver, carbon-based nanomaterials and metallic oxygen/sulfides, etc.) have been applied in the antibacterial field, and their strategies leading to bacterial inactivation mainly include: (1) the inherent antibacterial activity of the nano material destroys protein and DNA in bacteria by releasing metal ions; (2) killing bacteria by producing excessive heat based on the photothermal conversion properties of the nanomaterial; (3) based on the catalytic properties of the nanomaterials, the catalytic generation of active oxygen destroys lipids and DNA.

However, the nano antibacterial materials disclosed in the prior art still have the disadvantages, which are mainly reflected in the following aspects: the existing antibacterial nano material has low antibacterial activity; the laser power density is unsafe; the preparation process of the nano material is complex. Therefore, there is a need to develop an antibacterial nanomaterial with low cost, high efficacy and high safety to solve the related dilemma faced by the current medical industry.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a nano antibacterial agent for quickly and efficiently killing drug-resistant bacteria and a preparation method thereof, and Cu prepared by a simple mechanical stripping method₂MoS₄The nanometer material can be used for effectively treating drug-resistant bacterial infection.

The technical scheme of the invention is as follows: a nano-class antibacterial agent for quickly and efficiently killing the medicines-resistant bacteria contains Cu (0.5-5 wt.%)₂MoS₄Nano material and 5-50 parts of surfactant.

Further, Cu₂MoS₄The nano material is nano sheet, nano granule, nano film,One of the nanodot forms.

Further, Cu₂MoS₄The particle size of the nano material is 5-100 nm.

Further, the surfactant comprises one or more of polyether F127, hydroxymethyl cellulose, polyvinylpyrrolidone, hexadecyltriethyl ammonium bromide and hexadecyltrimethyl ammonium chloride.

Furthermore, the nano antibacterial agent with the concentration of 5-100 mug/mL can effectively kill drug-resistant bacteria such as escherichia coli with resistance to chloramphenicol, streptomycin and tetracycline and staphylococcus aureus with resistance to methicillin in the near-infrared two-zone laser irradiation for 2-20 min.

The preparation method of the nano antibacterial agent specifically comprises the following steps:

1) cu production using mechanical stripping₂MoS₄Nano materials: putting bulk Cu₂MoS₄Placing the materials and the surfactant in a ball milling tank for ball milling;

2) after the ball milling is finished, carrying out gradient centrifugation on the ball-milled mixed material to purify the nano material;

3) dispersing purified nanomaterial in ultrapure water, Cu₂MoS₄The concentration of the nano material water solution is 5-100 mug/mL, and the nano material water solution is stored at 4 ℃ for standby.

Further, the ball milling conditions in step 1 are: the ball milling speed is 400-700 rpm, and the ball milling time is 12-48 h.

Further, the conditions for performing gradient centrifugation in step 2 are: the centrifugal speed is 5000-.

The invention has the beneficial effects that:

1. the main component of the nano antibacterial agent prepared by the invention is Cu₂MoS₄The nano material avoids the problem of drug-resistant bacteria caused by using antibiotics;

2. the nano antibacterial agent prepared by the invention has broad-spectrum antibacterial performance and can be used for inactivating drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria;

3. Cu₂MoS₄use of nanomaterials forThe treatment of the subcutaneous drug-resistant bacteria infection of the living model shows high-efficiency treatment effect under low dosage, and the biological safety can be improved by low dosage;

4. when in specific use, the laser is near-infrared two-zone laser (1064 nm), and the power density is 1W/cm²The safety is higher;

5. the antibacterial action time of the nano antibacterial agent is less than 0.5 h, so that the treatment period is greatly shortened;

6. cu disclosed in the invention₂MoS₄The preparation process of the nano material is simple, the operability is strong, and the method is suitable for industrial batch production and has wide market application prospect.

Drawings

FIG. 1 is a graph showing Cu verification in example 2₂MoS₄Effect graph of blood compatibility of nanometer material;

FIG. 2 is a graph showing Cu verification of example 2₂MoS₄Effect graph of nano material cytotoxicity;

FIG. 3 is a graph showing Cu verification of example 3₂MoS₄Effect diagram of in vitro antibacterial performance of the nano material;

FIG. 4 is a graph showing Cu verification in example 4₂MoS₄Effect diagram of the antibacterial performance of the nanometer material living body;

wherein CMS is Cu₂MoS₄Nanomaterials, MDRE. coliIs multidrug resistant Escherichia coli, MDRS. aureusIs multi-drug resistant staphylococcus aureus, and NIR-II is near infrared two-zone laser.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit of the invention.

Example 1, Cu₂MoS₄Preparation of nanomaterials

Weighing 0.5-5 g of bulk Cu₂MoS₄The material and 5-50 g polyvinylpyrrolidone (molecular weight 8-360 k) are put in a ball milling tank, the ball milling speed is set to be 500 rpm, and the ball milling time is set to beAnd (4) 12 h. After the ball milling is finished, the nano material is purified by selecting a gradient centrifugation condition with the centrifugation speed of 12000 rpm and the centrifugation time of 1 h, and finally dispersed in ultrapure water and stored in a refrigerator at 4 ℃ for later use.

Example 2 Cu₂MoS₄Biological safety testing of nanomaterials

1. Study of Cu Using fresh mouse blood₂MoS₄Blood compatibility of the nanomaterial.

Adding 3-10 mL of physiological saline into a centrifuge tube filled with fresh blood, uniformly mixing, placing the centrifuge tube into a centrifuge, and purifying and extracting red blood cells by using the physiological saline.

And then dissolving pure 0.1-1 mL of red blood cells into 5-10 mL of physiological saline and uniformly mixing. Multiple tubes were filled with 1 mL of Cu at various concentrations₂MoS₄0.01-0.8 mL of red blood cells are respectively added into the physiological saline solution of the nano material, and after uniform mixing, incubation is carried out for 0.5-10 h at 37 ℃ and the rotating speed of 50-200 rpm.

As can be seen from FIG. 1, Cu₂MoS₄The nano material has good blood compatibility.

2. Study of Cu Using HeLa cells₂MoS₄Cytotoxicity of nanomaterials.

HeLa cells (1-6X 10)⁴) With different concentrations of Cu₂MoS₄The nanomaterials were incubated at 37 ℃ for 24 h, followed by validation of Cu using LDH, MTT or live/dead cell staining experiments₂MoS₄Cytotoxicity of nanomaterials.

As can be seen from FIG. 2, Cu₂MoS₄Nanomaterials have very low cytotoxicity.

Example 3, Cu₂MoS₄In-vitro antibacterial performance test of nano material

Escherichia coli (gram-negative bacteria) resistant to chloramphenicol, streptomycin and tetracycline and Staphylococcus aureus (gram-positive bacteria) resistant to methicillin are respectively stored in 5-30% of glycerol and stored in a refrigerator at-80 ℃.

A part of the frozen stock solution is picked up by using a sterile pipette tip, respectively scraped on LB plates, and cultured overnight at 37 ℃ to form colonies visible to the naked eye.

Individual colonies were picked using a pipette tip in LB (1-20 mL) medium and shaken overnight (220 rpm, 37 ℃).

And (3) putting 1 mL of overnight bacterial suspension into a 1.5 mL centrifuge tube, centrifuging at 5000-12000 rpm for 1-5 min, and adding physiological saline to wash twice. The final centrifugation product was resuspended by adding 1 mL of physiological saline and pipetting.

200. mu.L of the suspension was placed in a 96-well plate and the optical density value (OD 600 nm) was measured with a microplate reader to determine the concentration of the bacteria.

Respectively taking equal volume of bacterial suspension and Cu₂MoS₄Aqueous nanomaterial solutions were placed in triplicate in centrifuge tubes using saline as a control.

Laser in near infrared two regions (wavelength is 1064 nm, power density is 1W/cm)²(the safe power density is high enough to be,Small Methods 2017, 1, 1600032)) for 2-20 min.

Evaluation of Cu by plate count method₂MoS₄Antibacterial property of the nano material.

As can be seen from the results shown in FIG. 2, the Cu-transition time was compared with that of the control group₂MoS₄The growth amount of the bacterial strains on the culture dish plate of the drug-resistant escherichia coli (gram-negative bacteria) and the drug-resistant staphylococcus aureus (gram-positive bacteria) treated by the nano material is very small, which indicates that the Cu₂MoS₄The nanometer material has broad spectrum, high efficiency and fast antibacterial performance.

Example 4 Cu₂MoS₄Nano material for treating subcutaneous drug-resistant bacterial infection of living body

To study Cu₂MoS₄The antibacterial activity of the nanometer material on living bodies, and a drug-resistant bacteria infection model is established on the right side of the back vertebra of the mouse.

Balb/c mice (18-22 g) were cleaned of their back hairs using a mouse hair pusher and injected subcutaneously with a 100 μ L volume of 1 x 10 into the right side of the dorsal spine of the mice using a syringe⁶~1 * 10¹⁰Drug-resistant bacteria of CFU/mL, mice freely moving24 h。

The mice were divided into 4 groups (physiological saline group, physiological saline + laser irradiation group, Cu)₂MoS₄Nanomaterial group and Cu₂MoS₄Nano material + laser irradiation group), 10-100 mul of normal saline (control group) and equal volume of Cu are injected in situ by using an injector respectively₂MoS₄The nanometer material is applied to the affected area with physiological saline solution (treatment group), and near infrared two-zone laser (wavelength of 1064 nm, power density of 1W/cm) is used²) Irradiating for 2-20 min.

Changes in the area of the infected area were recorded using a vernier caliper, and recovery of the infected area was recorded by photographing.

From FIG. 3, it can be seen that Cu is present in comparison with the control group₂MoS₄The nano material shows excellent treatment effect on drug-resistant bacterial infection and has obvious living antibacterial activity.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. However, the above description is only an example of the present invention, the technical features of the present invention are not limited thereto, and any other embodiments that can be obtained by those skilled in the art without departing from the technical solution of the present invention should be covered by the claims of the present invention.

Claims

1. A preparation method of a nano antibacterial agent for killing drug-resistant bacteria is characterized by comprising the following steps:

1) cu production using mechanical stripping₂MoS₄Nano materials: 0.5-5 parts of bulk Cu₂MoS₄Placing the material and 5-50 parts of polyvinylpyrrolidone into a ball milling tank for ball milling, wherein the ball milling rotation speed is 400-700 rpm, and the ball milling time is 12-48 h;

3) dispersing purified nanomaterial in ultrapure water, Cu₂MoS₄The concentration of the nano material water solution is 5-100 mug/mL, and the nano material water solution is stored at 4 ℃ for standby;

the nano antibacterial agent with the concentration of 5-100 mug/mL can effectively kill drug-resistant bacteria such as Escherichia coli with resistance to chloramphenicol, streptomycin and tetracycline and Staphylococcus aureus with resistance to methicillin in the near-infrared region for 2-20 min of laser irradiation.

2. The method for preparing nano antibacterial agent for killing drug-resistant bacteria according to claim 1, wherein the Cu is₂MoS₄The particle size of the nano material is 5-100 nm.

3. The method for preparing a nano antibacterial agent for killing drug-resistant bacteria according to claim 1, wherein the conditions for performing gradient centrifugation in the step 2) are as follows: the centrifugal speed is 5000-.