CN114732941A

CN114732941A - Preparation method of cleaning and antibacterial gel

Info

Publication number: CN114732941A
Application number: CN202210284568.5A
Authority: CN
Inventors: 张幼芃; 黄振伟; 黄海东
Original assignee: Anhui Huike Bio Engineering Technology Co ltd
Current assignee: Anhui Huike Bio Engineering Technology Co ltd
Priority date: 2022-03-22
Filing date: 2022-03-22
Publication date: 2022-07-12
Anticipated expiration: 2042-03-22
Also published as: CN114732941B

Abstract

The invention belongs to the technical field of hydrogel, in particular to a preparation method of clean antibacterial gel, which utilizes the combination of bacteriocin and hydrogel through cyanoacrylate load and is assisted by chitosan enzyme and the like to form the clean antibacterial gel, has good compliance of moisture retention, can be cut, can absorb seepage and permeate gas, has the advantages of promoting the moist healing of wounds, having no adhesion, enabling patients to feel comfortable, obviously relieving the pain feeling and the like, has activity or can promote the release of active substances, thereby accelerating the healing speed of wound surfaces, and the clean antibacterial hydrogel disclosed by the invention shows stable coverage, small displacement, lasting antibacterial property and rapid wound healing capability.

Description

Preparation method of cleaning and antibacterial gel

Technical Field

The invention belongs to the technical field of hydrogel materials, and particularly relates to a preparation method of a cleaning and antibacterial gel.

Background

The hydrogel is also called water-containing hydrogel, and is a hydrophilic net-shaped polymer swelling body which can swell in water, absorb and retain a large amount of water and is insoluble in water. Because of high water content, the hydrogel can be randomly shaped, is suitable for wound surfaces of any position, provides necessary conditions for wound healing, and has biocompatibility superior to fiber and film dressings. In addition, the hydrogel is very soft due to a large amount of moisture and is similar to organism tissues, so that adverse reactions can be reduced when the hydrogel is used as a human implant. Therefore, the hydrogel is widely used as an excellent biomedical material, such as a drug carrier, a tissue scaffold, a wound dressing and the like, and can prevent further wound injury and bacterial invasion. The antibacterial hydrogel can meet the requirements of both hydrogel and antibacterial function, and is mainly divided into antibacterial hydrogel containing inorganic nanoparticles, antibacterial hydrogel containing organic antibacterial agents and the like according to the difference between a matrix and the antibacterial agents. For example, silver particles, the conventional method mostly compounds nano silver particles with the implant directly, thereby endowing the implant with an antibacterial function. Taking hydrogel material as an example, after the nano silver ions are directly compounded with the hydrogel material, the nano silver particles have interaction with tissues or cells, but when the concentration of the nano silver particles exceeds a certain range, certain toxicity is generated on the cells and the tissues, and the nano silver particles penetrate or deposit on cell walls, so that the transportation of electrolytes and metabolites is influenced, the interaction with membrane proteins is caused, signal channels are activated, and the proliferation and the contraction of the cells are inhibited. Chinese patent CN2018115319408 discloses an antibacterial hydrogel capable of controllably releasing nano silver ions and a preparation method thereof, and aims to realize long-term slow release of nano silver particles in an implant; for example, chinese patent CN2016104668764 discloses a marine phycocolloid hydrogel and its preparation method, which mainly uses artificially synthesized antibacterial agents, such as antibacterial agents benzisothiazolinone, methylparaben, benzalkonium chloride, cetrimide, etc., however, the hydrogel using the synthesized antibacterial agents has the disadvantages and defects of poor biocompatibility and antibacterial property, and easy skin allergy. From the current research results, in the development of the antibacterial hydrogel, people have great efforts to utilize macromolecules such as polysaccharides and polyethers to prepare the antibacterial hydrogel with complicated structure, complicated method, complicated components and mixing with various antibacterial substances, but neglect the most basic biocompatibility and irritation, for example, although inorganic antibacterial agents such as Ag nanoparticles have good antibacterial property, the biocompatibility limits the wide application of the antibacterial hydrogel on the surface of human skin. In addition, long-term use and abuse of traditional antibiotic antibacterials, such as penicillins and the like, can lead to the development of resistant bacteria. Therefore, those skilled in the art are in need of developing a preparation method of a cleansing antibacterial gel, specifically, a bacteriocin hydrogel is formed by chemically or physically combining bacteriocin and hydrogel.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a preparation method of a clean bacteriostatic gel, and specifically, a bacteriocin hydrogel is formed by chemically or physically combining bacteriocin and hydrogel.

The invention is realized by the following technical scheme:

a preparation method of a cleaning bacteriostatic gel comprises the following operation steps:

(1) adding 45-50 parts by weight of deionized water and 16-18 parts by weight of carbomer into a reaction bottle, magnetically stirring for dissolving, then adjusting the pH of the system to 3.5-4 by using a hydrochloric acid solution with the mass fraction of 5%, slowly dropwise adding 12-15 parts of cyanoacrylate monomer at the stirring speed of 800r/min, and adjusting the pH of the system to 6.8-7.0 by using 0.1mol/L potassium carbonate aqueous solution after 2-3 h to obtain a gel solution;

since the cyano group in the alpha position is a strong electron-withdrawing group, the beta carbon atom is rendered more electropositive, so that it has a great tendency to polymerize, and upon contact with an anion, it is converted from a monomer to a polymer. And then the cyanoacrylate microspheres are introduced into the hydrogel, and the organic rigid particles of the cyanoacrylate microspheres toughen to form a cold-drawing effect, so that the physical and mechanical properties of the hydrogel are further improved. (2) Uniformly mixing the gel solution and the bacteriocin according to the mass ratio of 7-9: 1, and then placing the mixture in a constant-temperature oscillator for oscillation for 2.4 hours; filtering out the hydrogel, cleaning, washing, and drying in an oven at 60 ℃ for 0.5-1 h to obtain the bacteriocin-loaded carbomer gel; the constant temperature oscillator has the advantages of high temperature control precision, uniform temperature distribution, high heating speed and low noise, and can ensure that the bacteriocin is uniformly and rapidly loaded on the carbomer gel.

(3) According to the weight parts, sequentially adding 8-13 parts of glycerol polyethylene glycol hydrogenated castor oil, 36-43 parts of polyvinyl alcohol, 56-61 parts of bacteriocin-loaded carbomer gel, 8-12 parts of polyvidone and 6-8 parts of chitosan enzyme into 690-740 parts of distilled water, stirring, performing ultrasonic defoaming treatment for 40-50 min, and performing freezing-thawing cycle treatment for 5-7 times to obtain the clean antibacterial gel.

Further, in the step (1), the polyvinyl alcohol in the step (3) is one of PVA05-88 or PVA 17-88.

Further, in the step (1), the carbomer is one of carbomer 934P, carbomer 974P and carbomer 1342.

Further, in the step (3), the povidone is one or a mixture of PVPK30 and PVPK 90.

Further, in the step (2), the bacteriocin is one of colicin and nisin or a mixture of the colicin and the nisin.

Bacteriocins are a class of antibacterial substances produced by certain bacteria, and have certain antibacterial action although the action range is narrow. The name of bacteriocin is named according to the species produced, e.g.the bacteriocin produced by E.coli is called colicin.

Further, in the step (3), the cyanoacrylate is one of α -n-butyl cyanoacrylate and α -heptyl cyanoacrylate, or a mixture of both.

Cyanoacrylate, commonly used as an adhesive, was first developed and applied by kodak companies, such as n-butyl α -cyanoacrylate to prepare 504 medical adhesive, which can strongly adhere to body tissues and has a high adhesion speed. Has no toxicity, little reaction to tissues, no thrombosis and simple sterilization.

Further, in the step (3), the operation of the freezing-thawing cycle treatment is to place the mixture into a refrigerator with the temperature of-26 ℃ for cold 24 hours, take the mixture out, thaw the mixture with tap water at room temperature until the temperature reaches 0-2 ℃, and complete the freezing-thawing process.

The invention has the beneficial effects that:

the invention introduces cyanoacrylate microspheres into hydrogel, rigid particles of the cyanoacrylate microspheres toughen to form a cold drawing effect, spherical particles are dispersed in a matrix before drawing, brittle particles yield and deform in the same size with the matrix during drawing to absorb considerable energy, so that the toughness of a blend is improved, the mechanical property of the hydrogel can be enhanced, high tensile strength and better compression property are realized, meanwhile, the loaded bacteriocin provides lasting antibacterial property for the hydrogel and forms a synergistic antibacterial effect with chitosan enzyme, the main antibacterial active ingredients of the clean antibacterial gel prepared by the preparation method disclosed by the invention are bacteriocin and chitosan enzyme, and the gel has multiple medical health-care effects of resisting bacteria, diminishing inflammation, increasing immunity, promoting wound healing, nourishing and repairing mucous membranes and the like, and also has good biocompatibility and biodegradability, is safe and nontoxic, wherein chitosan is a polymer formed by connecting glucosamine through beta-1, 4-bonds, is rich in active groups, can be adsorbed with pathogenic cell membranes and can be coagulated, or penetrate into pathogenic bacteria to disturb metabolism of pathogenic bacteria, thereby exerting broad-spectrum antibacterial effect, not easily generating drug resistance, and having lasting effect, has obvious inhibiting and killing effect on mycoplasma or chlamydia infection, high cyanoacrylate polymerization activity, alkali and very weak water capable of initiating polymerization and being wetted by blood, can be subjected to nontoxic and biochemical depolymerization, has good biodegradability and biocompatibility, can be used as a carrier material to prepare the loaded bacteriocin, and improves the action efficiency of the bacteriocin by means of degradation performance and drug release performance, the cleaning gel with uniform bacteriostatic action is prepared by simple preparation steps through processes of freezing, thawing, defoaming and the like.

Compared with the prior art, the invention has the following advantages:

the invention combines bacteriocin and hydrogel through cyanoacrylate load, and is assisted by chitosan enzyme and the like to form clean antibacterial gel, which has the advantages of good compliance of moisture retention, tailorability, capability of absorbing seepage, permeability of gas, promotion of moist healing of wounds, no adhesion, comfortable feeling of patients, obvious pain relief and the like, and the hydrogel has activity or can promote the release of active substances, so that the healing speed of wound surfaces is accelerated.

Detailed Description

The invention is illustrated by the following specific examples, which are not intended to be limiting.

Example 1

Firstly, adding 50 parts by weight of deionized water and 18 parts by weight of carbomer 974P into a reaction bottle, magnetically stirring for dissolution, then adjusting the pH of the system to 4 by using a hydrochloric acid solution with the mass fraction of 5%, slowly dropwise adding 15 parts of alpha-heptyl cyanoacrylate at the stirring speed of 800r/min, and after 3 hours, adjusting the pH of the system to 7.0 by using 0.1mol/L potassium carbonate aqueous solution to obtain a gel solution;

step two, uniformly mixing the gel solution and bacteriocin colicin according to the mass ratio of 9:1, and then placing the mixture in an SHZ-88 water bath constant temperature oscillator at 30 ℃ for oscillation for 2.4 hours with the oscillation frequency of 100 times; filtering out the hydrogel, cleaning, washing, and drying in an oven at 60 ℃ for 1h to obtain the bacteriocin-loaded carbomer gel;

thirdly, adding 13 parts of glycerol polyethylene glycol hydrogenated castor oil, 43 parts of polyvinyl alcohol PVA05-88, 61 parts of bacteriocin-loaded carbomer gel, 12 parts of povidone PVPK90 and 8 parts of chitosan enzyme into 740 parts of distilled water in sequence by weight, stirring, performing ultrasonic defoaming treatment for 50min, and performing freezing-unfreezing circulation treatment for 7 times to obtain the clean antibacterial gel, wherein the longitudinal tensile strength of the gel is 1.4MPa, the transverse tensile strength of the gel is 1.3MPa, the elongation at break of the gel is 654.3%, the peel strength of the gel at a width of 1cm is 1.2N/cm, and the skin sensitization of the gel is as follows: post-challenge rating of 0, skin irritation: the response stimulation index is 0, and the water vapor transmission rate is 112.5g/m²24h, bacteriostatic activity: staphylococcus aureus (ATCC6538) 93.0%, Escherichia coli (ATCC25922) 98.4%, Candida albicans (ATCC10231) 93.0%.

Coli (ATCC25922) MIC colicin 2.69 × 10^-4Coli (ATCC25922) MIC chitosanase 2.8X 10^-3、FIC＝0.36

Example 2

Firstly, adding 45 parts of deionized water and 16 parts of carbomer 934P into a reaction bottle in parts by weight, magnetically stirring for dissolution, then adjusting the pH of the system to 3.5 by using a hydrochloric acid solution with the mass fraction of 5%, slowly dropwise adding 12 parts of alpha-n-butyl cyanoacrylate at the stirring speed of 800r/min, and adjusting the pH of the system to 6.8 by using 0.1mol/L potassium carbonate aqueous solution after 2 hours to obtain a gel solution;

step two, uniformly mixing the gel solution and bacteriocin nisin according to the mass ratio of 7:1, and then placing the mixture into an SHZ-88 water bath constant-temperature oscillator for oscillation for 2.4 hours with the oscillation frequency of 100 times; filtering out the hydrogel, cleaning, washing, and drying in an oven at 60 ℃ for 0.5h to obtain the carbomer gel loaded with the bacteriocin;

thirdly, sequentially adding 8 parts of glycerol polyethylene glycol hydrogenated castor oil, 36 parts of polyvinyl alcohol PVA05-88, 56 parts of bacteriocin-loaded carbomer gel, 8 parts of povidone PVPK30 and 6 parts of chitosan enzyme into 690 parts of distilled water by weight, stirring, performing ultrasonic defoaming treatment for 40min, and performing freezing-unfreezing cycle treatment for 5 times to obtain the clean antibacterial gel, wherein the longitudinal tensile strength of the clean antibacterial gel is 1.1MPa, the transverse tensile strength of the clean antibacterial gel is 1.0MPa, the elongation at break is 612.7%, the peel strength of the clean antibacterial gel with the width of 1cm is 1.2N/cm, and the skin sensitization is as follows: post-challenge rating of 0, skin irritation: the response stimulation index is 0, and the water vapor transmission rate is 112.5g/m²24h, bacteriostatic activity: 95.2% of staphylococcus aureus (ATCC6538), 93.8% of escherichia coli (ATCC25922) and 94.1% of candida albicans (ATCC 10231).

Note: the detection was performed with reference to the following criteria: the bacteriostasis rate test is carried out according to the method in the GB15979-2002 hygienic standard of disposable sanitary products; irritation and skin sensitization test: removing back hair of 10cm × 15cm from two sides of spinal column of animal 24 hr before test, and dripping 0.5ml of test sample leaching solution and control solution onto 2.5cm × 2.5cm absorbent gauze pieces respectively. The gauze pieces soaked with the sample leaching solution and the solvent contrast solution are respectively directly contacted with the skins on both sides of the spine of the rabbit, and then are fixedly attached for 4 hours by using bandages. And removing the patch after the contact period is finished. The tensile strength and elongation at break were determined according to GB/T1040.3-2006. The test sample is a strip with the length of 150mm and the width of 15mm, the distance between the clamps is 100mm, and the test speed is 250 mm/min.

Staphylococcus aureus (ATCC6538) MIC nisin 1.63X 10^-5Staphylococcus aureus (ATCC6538) MIC Chitosan enzyme 3.14X 10^-3、FIC＝0.27；

And (3) culturing bacteria: transforming the activated bacteriaInoculating to liquid culture medium, culturing at 37 deg.C for 12 hr, centrifuging at 3000r min for 5min, discarding supernatant, and diluting the precipitate with sterilized liquid culture medium to 10%⁶CFU/mL is used as a test bacterial liquid (beef extract peptone culture medium), the cultured fungal cells are diluted to 2X 10' CFUmL by using a liquid culture medium, 100/AL is taken and put into each experimental hole of a 96-well plate, then 100 mu L of antibacterial peptide with certain concentration is respectively added into each experimental hole, and two groups are set as a control group: one group was 100 μ L of culture medium; the other group is 100 mu L of bacterial liquid, and the bacterial liquid is simultaneously put into an incubator at 28 ℃ for 24 hours. On the basis of small inhibitory concentration (MIC), 4 XMIC antibacterial peptide and 4 XMIC antibiotic are respectively added into each 75/L of experiment holes of a 96-hole micro-culture plate, dilution is carried out in a multiple ratio mode continuously, each original concentration hole is diluted into 20 experiment holes, and then 100/L of bacteria liquid to be detected (10) is added into each experiment hole⁶CFU/mL), together with two controls: one group is 250/culture solution; the other group is 250L of bacterial liquid, and then the bacterial liquid is put into a constant temperature incubator at 37 ℃ for 12 hours. The Minimum Inhibitory Concentration (MIC) of the antimicrobial peptide and the antibiotic at the time of co-culture was calculated.

Claims

1. The preparation method of the cleaning and bacteriostasis gel is characterized by comprising the following operation steps:

(1) adding 45-50 parts by weight of deionized water and 16-18 parts by weight of carbomer into a reaction bottle, dissolving by magnetic stirring, adjusting the pH of the system to 3.5-4 by using a hydrochloric acid solution with the mass fraction of 5%, slowly dripping 12-15 parts of cyanoacrylate monomer at the stirring speed of 600-800 r/min, and adjusting the pH of the system to 6.8-7.0 by using 0.1mol/L potassium carbonate aqueous solution after 2-3 h to obtain a gel solution;

(2) uniformly mixing the gel solution and the bacteriocin according to the mass ratio of 7-9: 1, and then placing the mixture in a constant-temperature oscillator for oscillation for 2-2.4 hours; filtering out the gel, cleaning, washing, and drying in an oven at 50-60 ℃ for 0.5-1 h to obtain the carbomer gel loaded with the bacteriocin;

(3) according to the weight parts, 8-13 parts of glycerol polyethylene glycol hydrogenated castor oil, 36-43 parts of polyvinyl alcohol, 56-61 parts of bacteriocin-loaded carbomer gel, 8-12 parts of polyvidone and 6-8 parts of chitosan enzyme are sequentially added into 690-740 parts of distilled water, stirred, subjected to ultrasonic defoaming treatment for 40-50 min, subjected to freezing-unfreezing cycle treatment for 5-7 times, and then the clean antibacterial gel is obtained.

2. The method for preparing a cleansing bacteriostatic gel according to claim 1, wherein in the step (3), the polyvinyl alcohol is one of PVA05-88 or PVA17-88, and the povidone is one of PVPK30 or PVPK90 or a mixture of both.

3. The method for preparing a cleansing bacteriostatic gel according to claim 1, characterized in that in the step (1), the carbomer is one of carbomer 934P, carbomer 974P and carbomer 1342.

4. The method for preparing a cleansing bacteriostatic gel according to claim 1, wherein in the step (1), the cyanoacrylate is one of α -n-butyl cyanoacrylate and α -heptyl cyanoacrylate or a mixture of the two.

5. The method for preparing a cleansing bacteriostatic gel according to claim 1, wherein in the step (2), the bacteriocin is one or a mixture of colicin and nisin.

6. The preparation method of a cleaning bacteriostatic gel according to claim 1, characterized in that in the step (3), the freezing-thawing cycle treatment is carried out by putting the gel into a refrigerator of-20 ℃ for cooling for 24h, taking out the gel, thawing the gel with tap water at room temperature until the temperature reaches 0-2 ℃, and then completing a freezing-thawing process.