WO2019169873A1 - Nano antibacterial gel for treating wound infection and promoting healing and preparation method therefor - Google Patents

Abstract

A nano antibacterial gel for treating wound infection and promoting healing, the components thereof being as follows: 0.5-5 parts of an aqueous solution of a Cu₂WS₄ nanomaterial, and 0.5-5 parts of an aqueous solution of a gelling agent. The Cu₂WS₄ nanomaterial has antibacterial properties against Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (E. coli) and resistant bacteria (methicillin-resistant Staphylococcus aureus (MRSA)) whether in the shade or in the presence of light, may treat an MRSA infection in a wound and has the ability to promote wound healing.

Description

Nano antibacterial gel for treating wound infection and promoting healing and preparation method thereof Technical field

The invention belongs to the field of nano antibacterial technology and trauma treatment, and particularly relates to a nano antibacterial gel for treating wound infection and promoting healing.

Background technique

Bacteria have always been the main cause of harm to public health. How to effectively control and prevent the growth and spread of bacteria has been a topic of great concern. Therefore, antibacterial preparations have always occupied a broad market in the medical field. Most antibacterial preparations are prepared by antibiotics or traditional Chinese medicines. Antibiotics are mainly used to treat various bacterial infections or pathogenic microbial infections. However, with the long-term and wide-ranging application of antibiotics, the drug resistance of bacteria has become stronger and stronger, and microbial infection has seriously threatened the health and safety of human beings. The clinical effect of antibacterial preparations prepared by using traditional Chinese medicines is not obvious.

It is worth noting that worldwide, the annual cost of treatment for infectious diseases is as high as hundreds of billions of dollars. Many pharmaceutical companies are willing to invest in the development of new drugs for a long time. However, the development of a new high-efficiency antibacterial preparation is not only extremely expensive, but also It takes a long time, and the produced preparation may also have a very short service life due to bacterial resistance. Therefore, the development of an antibacterial agent with excellent effects through a new type of technology is receiving more and more attention from pharmaceutical companies.

With the development of nanotechnology and nanobiomedicine, new ways to control microbial infections have emerged: the combination of biological methods and nanotechnology to produce nano-antibacterial new materials, and combined with gels to better affect the human body. The main ways in which nanomaterials cause bacteria to inactivate are: (1) the nanoparticles directly adhere to the bacterial membrane, or directly destroy the bacterial membrane; (2) the metal ions released by the nanoparticles destroy the protein or DNA in the bacteria; The production of reactive oxygen species destroys lipids and DNA. Here, nanomaterials exhibit characteristics that are not restricted by bacterial resistance, and more importantly, the production of active species can penetrate into bacteria more easily and destroy bacterial DNA and proteins. In recent years, many nanomaterials (such as silver, graphene, titanium dioxide, transition metal chalcogenides, etc.) have been used in the field of antibacterial; gel is a polymer or macromolecular aggregate that absorbs a large amount of solvent but is insoluble in solvents. They are rapidly swellable in water and maintain their shape and three-dimensional network structure, also known as "soft materials." Gels exhibit many of the properties of polymers that do not dissolve freely, and such materials can remain in place under physiological conditions while maintaining antimicrobial activity. These properties make them ideal for wound healing, implants, catheter coatings, skin infections, and even orifice barriers.

However, in the prior art, there are several problems: metal nanoparticles have certain toxicity, and excessive use may remain in the environment; the preparation process of the antibacterial nano material is complicated and cumbersome; the inhibition rate is low.

Summary of the invention

In view of the above problems, the present invention aims to solve the deficiencies in the prior art, and provides a nano antibacterial gel for treating wound infection and promoting healing, and selecting Cu ₂ WS ₄ nano material as an antibacterial agent and adding a corresponding gel agent. The Cu ₂ WS ₄ nano antibacterial gel is formed to treat wound infection and promote wound healing.

In order to achieve the above object, the technical solution adopted by the present invention is as follows: The nano antibacterial gel for treating wound infection and promoting healing according to the present invention is prepared by mixing an aqueous solution of Cu ₂ WS ₄ nano material and an aqueous solution of a gel. The mixture is prepared by mixing 0.5-5 parts of an aqueous solution of Cu ₂ WS ₄ nano material; 0.5-5 parts of a gel solution aqueous solution, and the volume ratio of the aqueous solution of the Cu ₂ WS ₄ nano material to the aqueous solution of the gel is 1:0.5. ～5, the concentration of the aqueous solution of the Cu ₂ WS ₄ nano material is 1 to 100 μg/mL, and the mass percentage of the aqueous solution of the gelling agent is 0.1% to 50%.

Further, the Cu ₂ WS ₄ nano material may be one of a nanosheet, a nanocube, and a nanofilm.

Further, the nanosheet has a particle diameter of 500 to 1000 nm, a nanocube size of 10 to 500 nm, and a nanofilm diameter of 100 to 300 nm.

Further, the gel is one or more of gelatin, peach gum, sodium alginate, agar, hydroxymethylcellulose, polyvinylpyrrolidone, and agarose.

Further, a method for preparing a nano antibacterial gel for treating wound infection and promoting healing, comprising the steps of:

Step 1: preparing an aqueous solution of Cu ₂ WS ₄ nano material;

Step 2: Dissolve the gel in water, heat at high temperature to form an aqueous solution, and wait until the gel aqueous solution is cooled to

35~55 ° C, adding an aqueous solution of Cu ₂ WS ₄ nano material, mixing until uniform;

Step 3: The above aqueous solution of Cu ₂ WS ₄ nano material is stored at room temperature to obtain a nano antibacterial gel.

The beneficial effects are:

(1) The present invention employs Cu ₂ WS ₄ nanomaterial as an antibacterial agent, combines a natural gelling agent, does not contain a traditional Chinese medicine component and an antibiotic, and thus has no side effects and problems caused by the resistant bacteria.

(2) A broad spectrum of antibacterial properties. It can be used in Gram-positive bacteria, Gram-negative bacteria and drug-resistant bacteria, and is not subject to external ambient light.

(3) The amount of product is extremely low, which greatly improves biosafety.

(4) Cu ₂ WS ₄ nano antibacterial gel is used for wound healing of wounds in living organisms and wound treatment of drug-resistant infections, exhibiting effective treatment and promoting wound healing at low doses.

(5) The preparation process of Cu ₂ WS ₄ nano antibacterial gel is simple.

(6) The method of using gel is easier to apply and act on the wound, which greatly improves the drug delivery rate.

DRAWINGS

1a is a schematic view showing the results of verifying the hemolysis effect of Cu ₂ WS ₄ nanomaterial according to Embodiment 1 of the present invention;

1b is a schematic diagram showing the results of verifying the biosafety of Cu ₂ WS ₄ nanomaterials according to Embodiment 1 of the present invention;

2 is a schematic view showing the results of in vitro antibacterial properties of Cu ₂ WS ₄ nanomaterials in Example 2 of the present invention;

Figure 3a is a photograph of a Cu ₂ WS ₄ nano antibacterial gel of the present invention;

3b is a schematic view showing the results of in vitro antibacterial properties of Cu ₂ WS ₄ nano antibacterial gel according to Example 3 of the present invention;

4 is a schematic view showing the effect of Cu ₂ WS ₄ nano antibacterial gel on wound healing in vivo according to Example 4 of the present invention;

Among them, Water: water; Saline: normal saline; CWS: Cu ₂ WS ₄ nanomaterials; Heart: heart; Liver: liver; Spleen: spleen; Lung: lung; kidney: kidney; E. coli: Escherichia coli Negative bacteria); S. aureus: Staphylococcus aureus (Gram-positive bacteria); Light no treatment: no treatment with Cu ₂ WS ₄ nanomaterials under illumination; Dark CWS: treatment with Cu ₂ WS ₄ nanomaterials under shading; Light CWS: Treatment with Cu ₂ WS ₄ nanomaterials under illumination.

Detailed ways

In order to enable a person skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention are further described below with reference to FIGS. 1-4 and the embodiments, but the scope of protection of the present invention is not limited thereto. .

The basic principles, main features and advantages of the present invention have been shown and described above. However, the above description is only a specific embodiment of the present invention, and the technical features of the present invention are not limited thereto, and any other embodiments obtained by those skilled in the art without departing from the technical solution of the present invention should be covered in the present invention. Within the scope of the invention patent.

Example 1

a) Referring to Figure 1a, the hemolysis effect of the nanomaterials was studied using fresh human blood.

First, 3-10 mL of physiological saline was added to a centrifuge tube containing fresh blood, and after mixing, the centrifuge tube was placed in a centrifuge. The mixture was centrifuged at 500 to 3000 rpm for 5 minutes at 4 ° C, and washed with physiological saline for 3 times under the same centrifugation conditions.

Then, 0.1-1 mL of pure red blood cells are dissolved in 5-10 mL of physiological saline and mixed. 0.01-0.8 mL of red blood cells were added to a physiological saline solution (0.8-34 μg/mL) of 1 mL of different concentrations of Cu ₂ WS ₄ nanomaterial, mixed, and placed in a small shaker.

Incubate for 0.5 to 10 h at a temperature of 37 ° C and a rotational speed of 50 to 200 rpm.

As can be seen from Figure 1a, the Cu ₂ WS ₄ nanomaterials have good biocompatibility.

b) Verification of biosafety of living organisms with reference to Figure 1b

BALA/c mice (18-22 g, female) were divided into 2 groups (6 in each group), and 100 μL of normal saline was injected into the tail vein as a control group; 100 μL of Cu ₂ WS ₄ nanomaterial aqueous dispersion was injected as an experimental group. Then, two groups of mice were placed in a cage.

Fourteen days after the mice were free to move, the mice were euthanized, and the main organs (heart, liver, spleen, lung, kidney) were removed and fixed in formalin, followed by sectioning, embedding, staining, and the like.

Finally, the changes of these organ tissues were observed by an Olympus inverted microscope;

From the results shown in Figure 1b, it can be seen that the organ tissue treated with Cu ₂ WS ₄ nanomaterials has no significant change compared with the control group, indicating that the Cu ₂ WS ₄ nanomaterial is non-toxic.

Example 2

Referring to Fig. 2, Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) were separately stored in 5 to 30% of glycerin, and stored in a -80 ° C refrigerator.

A portion of the cryopreservation solution was picked up using a sterile tip and scraped on LB plates and incubated at 37 ° C overnight to form macroscopic colonies.

Use a pipette tip to pick up a single colony in LB (peptone 10g·L ^-1 , yeast 5g·L ^-1 , sodium chloride 10g·L ^-1 ) (1-20mL) culture medium, shake overnight (220rpm, 37 ° C) .

Take 1 mL of the overnight bacterial suspension in a 1.5 mL centrifuge tube, centrifuge at 5000 to 12000 rpm for 1 to 5 minutes, and wash twice with physiological saline. The final centrifuged product was added to 1 mL of physiological saline and resuspended for use.

200 μL of the suspension was taken in a 96-well plate, and the optical density value (OD 600 nm) was measured by a microplate reader to determine the concentration of the bacteria.

An equal volume of bacterial suspension and Cu ₂ WS ₄ nanomaterial aqueous solution were taken in a centrifuge tube in triplicate, and deionized water was used instead of Cu ₂ WS ₄ nanomaterial.

It was shaken at 220 rpm for 0.5 to 12 h in a 37 ° C shaker.

The antibacterial properties of Cu ₂ WS ₄ nanomaterials were evaluated using a plate count method.

It can be seen from the results shown in Fig. 2 that CWS-treated inoculation with Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram), whether in shading or under light conditions, compared with the control group The growth of strains on the culture plate of the positive bacteria was extremely small, indicating that CWS has broad-spectrum antibacterial properties and is not subject to external ambient light.

Example 3

Referring to Figures 3a and 3b, an aqueous gelling solution having a mass percentage of 0.1% to 50% is first formulated and autoclaved at 121 °C for 20 minutes.

Then, an equal volume of aqueous gel solution (35-55 ° C) and saline (Saline) or Cu ₂ WS ₄ nanomaterial aqueous dispersion were weighed into a glass bottle, and then allowed to form a gel after standing at room temperature for a period of time. The gel imaging results are shown in Figure 3a.

Staphylococcus aureus is stored in glycerin and stored in a -80 ° C refrigerator. A portion of the cryopreservation solution was picked up on a LB agar plate using a sterile tip, placed in a 37 ° C incubator, and allowed to stand overnight. Subsequently, a single colony was picked up in a 50 mL centrifuge tube containing LB medium using a pipette tip, blown and mixed, and the tube was placed in a 37 ° C shaker and shaken (200 rpm) overnight.

200 μL of the bacterial suspension was taken in a 96-well plate, and the optical density value (OD 600 nm) was measured by a microplate reader to determine the bacterial concentration.

A series of dilutions of the bacterial suspension were carried out using physiological saline to finally obtain a bacterial suspension having a concentration of 1 × 10 ⁸ CFU/mL.

Take 10 to 200 μL of the bacterial suspension on LB solid agar plates, apply uniformity using a glass coater, and add a 2 to 50 μL volume of physiological saline gel (control group, Saline) and 2 to 50 μL Cu ₂ WS using a pipette. _{The 4} nanometer antibacterial gel (treatment group, CWS) was incubated on LB agar plates in duplicate and placed in an oven at 37 °C for 24 h. The growth of the strain was as shown in Fig. 3b, which can be seen from Fig. 3b. In comparison, a significant inhibition zone appeared around the site where the Cu ₂ WS ₄ nano antibacterial gel was inoculated, indicating that the Cu ₂ WS ₄ nano antibacterial gel has the ability to inhibit bacterial growth.

Example 4

a) To study the treatment of wound infection by the Cu ₂ WS ₄ nano-antibacterial gel, a wound model was established on the back of the mouse.

The hair on the back of BALA/c mice (18-22 g) was cleaned, and a wound of 2 to 8 mm in diameter was cut with a surgical scissors, and a volume of 100 μL of 1*10 ⁴ to 1*10 ⁹ CFU/mL was dropped using a pipetting gun. Bacteria or resistant bacteria on the wound.

Rats were divided into 2 groups (6 in each group), and every 5 hours, 5 to 50 μL saline gel (control group) and 5-50 μL Cu ₂ WS ₄ nano antibacterial gel (treatment group) were separately used with a pipette. On the wound, and photographed the wound healing.

b) To investigate the promotion of wound healing by the Cu ₂ WS ₄ nano antibacterial gel, a wound model was established on the back of the mouse.

The hair on the back of BALA/c mice (18-22 g) was cleaned and a wound of 2 to 10 mm in diameter was cut using surgical scissors.

The rats were divided into 2 groups (6 in each group), and every 5 hours, every 24 hours, 5 to 50 μL saline gel (control group) and 5 to 50 μL Cu ₂ WS ₄ nano antibacterial gel were respectively taken using a pipetting gun. (Therapeutic group) was placed on the wound and photographed to record the wound healing.

It can be seen from Fig. 4 that compared with the control group, the wound healing effect of the mice treated with Cu ₂ WS ₄ nano antibacterial gel is better, indicating that the Cu ₂ WS ₄ nano antibacterial gel has the treatment of drug resistant bacteria infection. The wound and the ability to promote wound healing.

Claims (6)

1. A nano antibacterial gel for treating wound infection and promoting healing, characterized in that the specific components are as follows:

   0.5-5 parts of an aqueous solution of Cu ₂ WS ₄ nano material;

   An aqueous solution of the gelling agent is 0.5 to 5 parts.
2. The nano antibacterial gel for treating wound infection and promoting healing according to claim 1, wherein the concentration of the aqueous solution of the Cu ₂ WS ₄ nano material is from 1 to 100 μg/mL, the gelling agent The mass percentage of the aqueous solution is from 0.1% to 50%.
3. The nano antibacterial gel for treating wound infection and promoting healing according to claim 1, wherein the Cu ₂ WS ₄ nano material is one of a nanosheet, a nanocube, and a nanofilm.
4. The nano antibacterial gel for treating wound infection and promoting healing according to claim 1, wherein the gelling agent is gelatin, peach gum, sodium alginate, agar, hydroxymethylcellulose, One or more of polyvinylpyrrolidone and agarose.
5. The nano antibacterial gel for treating wound infection and promoting healing according to claim 3, wherein the nanosheet has a particle diameter of 500 to 1000 nm, the nanocube size is 10 to 500 nm, and the nanofilm diameter is small. It is 100 to 300 nm.
6. The method for preparing a nano-antibacterial gel for treating wound infection and promoting healing according to any one of claims 1 to 4, comprising the steps of:

   Step 1: preparing an aqueous solution of Cu ₂ WS ₄ nano material;

   Step 2: Dissolve the gel in water, heat to form an aqueous solution at a high temperature, and cool the gel aqueous solution to 35-55 ° C, add an aqueous solution of Cu ₂ WS ₄ nano material, and mix until uniform;

   Step 3: The gel solution of the above Cu ₂ WS ₄ nano material is stored at room temperature to obtain a nano antibacterial gel.

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