CN110743035A

CN110743035A - Preparation method and application of intelligent antibacterial hydrogel

Info

Publication number: CN110743035A
Application number: CN201911074386.XA
Authority: CN
Inventors: 刘海波; 周倩; 王静; 李青云; 刘幽燕; 廖丹葵
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2019-11-06
Filing date: 2019-11-06
Publication date: 2020-02-04

Abstract

The invention discloses a preparation method and application of intelligent antibacterial hydrogel. This method is achieved by using vesicles loaded with self-extinguishing fluorescent dyes. The vesicle is sensitive to a cell virulence factor generated by the thallus and used for regulating the density of a key population, and the generated thallus toxin can cause the vesicle to break under the condition that a wound is infected by bacteria, so that the dye is released from the vesicle and emits strong fluorescence. Therefore, whether the wound is infected or not can be judged according to the fluorescence intensity, and the risk of infection is further reduced. In addition, synchronous intelligent response to pathogen detection and sterilization can be realized by loading a photosensitive antibacterial agent in the vesicle. Compared with the traditional pathogenic bacteria detection method, the method has the characteristics of simple preparation process, convenient operation, high efficiency, rapidness, accuracy and the like.

Description

Preparation method and application of intelligent antibacterial hydrogel

Technical Field

The invention belongs to the technical field of biosensing, and particularly relates to a preparation method and application of an intelligent antibacterial hydrogel.

Background

With the wide clinical application of antibiotic drugs, the problem of drug resistance of bacteria is getting worse, the drug resistance is getting worse, the separation rate of clinical multi-drug resistant strains is quite high, the drug resistance mechanism is complex and changeable, and great challenges are brought to clinical treatment. At present, the detection of pathogenic microorganisms and the drug resistance thereof are mainly completed by analysis means such as bacterial culture, biochemical identification, polymerase chain reaction amplification, real-time fluorescence quantitative gene amplification technology, enzyme-linked immunosorbent assay, chip technology and the like. The detection means has the problems of long detection period, high cost, complex operation and the like, and is difficult to meet the requirement of clinical real-time detection. Therefore, the development of a rapid and sensitive detection method has important practical significance for the prevention and control of pathogenic microorganisms and the like.

A wound dressing is a material that is applied to a skin wound to promote wound healing. The hydrogel dressing is a novel dressing invented in recent years, and is widely researched and applied in the field of medical dressings. There are studies on the drug release and antibacterial effects of PEO/PVA graft copolymer hydrogels prepared by irradiating a mixture of polyethylene oxide and polyvinyl alcohol with electron beams and incorporating gentamicin. Currently, antibacterial agents loaded on hydrogel finished products mainly comprise nano silver, sulfapyridine salt, curcumin and tetracycline drugs. Wherein the market share of the nano-silver antibacterial hydrogel is more than 60%. Research shows that the nano silver has great toxicity and is not suitable for wound surfaces. Therefore, antibiotic antibacterial agents are widely used, the hydrogel can achieve the purpose of resisting infection by releasing antibiotics and directly killing or inhibiting the growth of bacteria by interfering the important life process of the bacteria with the antibiotics, but the bacteria have fast adaptability and evolutionary capability, and are extremely easy to generate negative effects such as drug resistance, microbial flora imbalance and the like. There is therefore a need for an effective and less drug resistant alternative or adjunct to antibiotic therapy. Photodynamic antibacterial therapy (PACT) is one of the most promising therapies that have emerged in recent years. The PACT principle of antibacterial action is that photosensitizer selectively gathers on bacterial cell wall and cell membrane, under the excitation of appropriate wavelength light source, relies on the participation of oxygen, through active oxygen substances such as I type, II type reaction production singlet oxygen, directly breaks bacterial cell wall and membrane system, influences its metabolism, and then leads to the bacterium death. The PACT antibacterial effect can effectively kill pathogenic bacteria and inactivate bacterial toxin factors, and has the unique advantage of effectively killing pathogenic bacteria and not easily generating tolerance.

Based on the above problems, we have devised a hydrogel dressing having the ability to detect bacterial microorganisms and kill bacteria intelligently, in which intelligent detection of pathogenic bacteria is achieved by using vesicles loaded with self-extinguishing fluorescent dyes, and in addition, synchronous intelligent response to pathogenic bacteria detection and killing can be achieved by loading photosensitive antibacterial agents in the vesicles.

Disclosure of Invention

The invention aims to provide a preparation method and application of an intelligent antibacterial hydrogel.

The technical scheme for solving the technical problems is as follows:

1. a preparation method of intelligent antibacterial hydrogel comprises the following operation steps:

1) 73.4mg of phospholipid 1, 2-dipalmitoyl-Sn-glycero-3-phosphocholine (DPPC), 38.6mg of cholesterol and 34.6mg of 10, 12-tricosane-dienoic acid (TCDA) were weighed out and dissolved in 1mL of chloroform at a concentration of 100mmol/dm³. 660. mu.L of DPPC, 240. mu.L of cholesterol and 300. mu.L of TCDA were mixed in a nitrogen stream and then dried, freed of impurities and dehydrated.

2) Will contain 50mmol/dm³10mL buffer and lipid of fluorescent agentMixing, freezing, extruding the lipid mixture through polycarbonate membrane with vesicle extruder, and storing the vesicle at 4 deg.C.

3) The size of the product is 90X 1mm³The hydrogel dressing is characterized in that 4-methyl umbelliferyl- α -D-Mannoside (MUD) is grafted on the hydrogel, an enzyme released by pathogenic bacteria can cut MUD fluorescent agent, a fluorophore 4-methyl umbelliferone (4-MU) is released into the hydrogel, and blue light is shown under an ultraviolet lamp (lambda & gt 365 nm).

4) The filling materials in the sac are the sac body, a pathogen detection fluorescent agent and chitosan, the fluorescent agent not only can detect pathogens, but also can play a role in sterilization under the condition of natural illumination, and the main antibacterial photosensitizer is natural photosensitive antibacterial agents such as hypericin and the like.

2. The application of the intelligent antibacterial hydrogel comprises the following steps: the method is applied to detection of pathogenic bacteria, intelligent antibacterial experiments and influence of an intelligent photosensitive antibacterial agent on wound healing of mice.

Due to the adoption of the technical scheme, the preparation method of the intelligent adhesive bandage dressing with the detection and antibacterial effects has the beneficial technical effects that:

1. the raw material for preparing the antibacterial hydrogel dressing mainly comprises chitosan. The structure and properties of chitosan are similar to those of extracellular matrix; the biocompatibility is good, and the rejection reaction with human tissues is avoided; the chitosan has good biological activity, has the effects of inhibiting bacteria, reducing cholesterol and the like, and can also promote the growth of epithelial cells; the chitosan can be degraded in the presence of lysozyme, the degradation process is mild and safe, and the degradation product is glucosamine which can directly enter a metabolic pathway.

2. The vesicle filler mainly comprises the vesicle, the pathogen detection fluorescent agent and chitosan, the fluorescent agent in the vesicle is mainly a natural extract, the fluorescent agent not only can detect the pathogen but also can play a role in sterilization under natural light, and the detection and treatment of the pathogenic bacteria of the wound are synchronously realized.

3. The reagents used in the preparation of the invention have no toxic or side effect, and the prepared hydrogel dressing has good biocompatibility.

Drawings

FIG. 1 is a graph showing the effect of enzymatic reactions in solution.

A, B, C are Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus, respectively.

FIG. 2 is a graph showing the effect of enzymatic reactions on a hydrogel dressing.

Fig. 3 is a graph of the effect of the intelligent hydrogel wound dressing on detection of pathogens under an ultraviolet lamp.

In the figure, the left figure is a figure when not applied; the right image is the effect image after application.

FIG. 4 is a graph showing the antibacterial effect of hypericin.

In the figure, A, B, C, D photodynamic therapy group, photosensitizer only group, xenon lamp only irradiated group and blank control group.

FIG. 5 is a comparative graph of epidermal histology of mice in the experimental group and the control group of the animal of the present invention.

In the figure, A, B, C represents a normal mouse, an illuminated group and an unlit group.

Detailed description of the preferred embodiments

The present invention will be described in further detail with reference to examples. The following are examples of the present invention, and are not intended to limit the present invention, and any modifications, substitutions, improvements, etc. made on the basis of the present invention are included in the scope of the present invention.

Example 1 Intelligent antimicrobial hydrogel

The application of the intelligent antibacterial hydrogel in the detection of pathogenic bacteria comprises the following operation steps:

1. enzymatic reaction in solution

For the enzymatic reaction in the solution, 50. mu.L of MUD solution was added to a 96-well plate containing 50. mu.L of the bacterial solution, the 96-well plate was sealed with a sealing tape, and the plate was placed in a fluorescence cell to observe the fluorescence intensity as shown in FIG. 1.

2. Enzymatic reaction of hydrogel dressing on culture dish

50 mu L of MUD solution is grafted on the surface of the prepared hydrogel dressing, then the hydrogel dressing is placed on a culture dish coated with 50 mu L of bacterial liquid, and the luminous intensity of the hydrogel dressing is observed as shown in figure 2.

3. Preparation of hypericin clathrate

Adding β -CD (β -cyclodextrin) 5.0g into 30 ml of NaOH solution 30%, stirring at room temperature to dissolve β -CD, adding EP 2.41ml into the mixed solution at 30 ℃, stirring for 24h, cooling to room temperature, desalting by a dialysis method, evaporating the solution in a dialysis bag to be viscous, adding absolute ethyl alcohol, precipitating white solid, filtering the white solid, carrying out vacuum freeze drying to obtain β -CDP (β -cyclodextrin polymer), dissolving β -CDP 80mg and Hyp (hypericin) 10mg in a proper amount of water, fully stirring for at least 24h at room temperature, filtering the mixed solution after the reaction is finished, removing the residual Hyp to obtain a dark purple solution containing an inclusion complex, and carrying out vacuum freeze drying to obtain a dark purple product Hyp- β -CDP (hypericin inclusion complex).

4. Hypericin clathrate is filled into vesicle

Uniformly preparing a plurality of round holes in the hydrogel, adding vesicles and chitosan into the holes, wherein the fillers in the vesicles are hypericin clathrate or other natural photosensitive antibacterial agents.

Results of the experiment

A, B, C in the figure 1 is enzymatic reaction of pseudomonas aeruginosa, escherichia coli and staphylococcus aureus in a 96-well plate respectively, the enzymatic reaction lasts for 1h, and the fluorescent substrate MUD can fluoresce the three bacteria according to the judgment of the fluorescence intensity, which indicates that the fluorescent substrate MUD has a certain detection effect on the three bacteria; in FIG. 2, A, B, C culture dishes are respectively provided with Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus, the hydrogel dressing is cut into a square with the size of 1.5cm multiplied by 1.5cm, a fluorescent substrate is grafted on the hydrogel dressing, the dressing is placed on the culture dish, an enzymatic reaction is carried out for 1h, and the dressing has a detection effect on the three bacteria from the fluorescence intensity in the drawing; fig. 3 is a comparison of before and after the auxiliary materials are activated under an ultraviolet lamp, and it can be seen from the figure that the vesicles are cleaved by toxins released by pathogenic bacteria, hypericin is released, and red fluorescence is exhibited under the ultraviolet lamp (λ 365 nm).

Example 2

The application of the intelligent antibacterial hydrogel in an intelligent antibacterial experiment comprises the following operation steps:

1. bacterial culture

The bacteria were picked from the LB plate and placed in a 50ml centrifuge tube, 10ml of LB medium was added to the centrifuge tube, and the tube was incubated for 15 hours at 37 ℃ on a shaker.

2. Preparation of solid Medium

Taking a 1000ml beaker, adding 500ml of distilled water, weighing 5g of beef extract, 10g of peptone, 5g of agar and 5g of sodium chloride, dissolving in water, adjusting the pH to about 7.2 by using a 10% NaOH solution, fixing the volume to 1000ml, wrapping by using tin foil paper, and sterilizing in a sterilizing box for later use.

3. Antibacterial experiments

Diluting cultured strain with LB solution 10 times, and measuring OD₆₀₀And (4) diluting the strain to 0.01 for later use. Taking out the 96-well plate, adding bacterial liquid and photosensitive antibacterial agent, incubating in an incubator at 37 deg.C for 1h, irradiating one group for 30min, and treating the other group in dark for 30 min; after illumination, the solution in the 96-well plate is coated in a solid culture dish and then cultured for 12 hours at 37 ℃; the culture dish was removed and the growth of the bacteria in the culture dish was observed.

Results of the experiment

In FIG. 4A, B shows the growth of bacteria in the presence or absence of light, with a light sensitive antimicrobial agent added to the vesicles. It is clear from the figure that in the presence of light, no bacteria have grown; in the absence of light, the bacteria had grown over the entire dish.

C, D in FIG. 4 shows the growth of bacteria in the absence of light, without the addition of a photosensitive antimicrobial agent to the vesicles. It can be observed from the figure that the culture dish is full of bacteria regardless of whether it is illuminated or not.

Example 3

The application of the intelligent antibacterial hydrogel in the influence of the intelligent photosensitive antibacterial agent on the mouse wound healing comprises the following operation steps:

1 materials of the experiment

1.1 Experimental reagents

Hypericin or natural photosensitive antibacterial agent extract, medical alcohol, and diluted bacteria solution

1.2 Experimental animals

Kunming mouse

2 method of experiment

2.1 mouse wound model fabrication

Taking a healthy mouse, anesthetizing the mouse by using urethane, taking clean scissors, removing the hair of the mouse, disinfecting the hair-removed part by using medical alcohol, and then manufacturing an 2 wound model with the area of about 1cm on the back of the mouse by using the disinfected scissors and tweezers.

2.2 grouping and administration

The mice are divided into an experimental group and a control group, wherein each group comprises 10 mice, the experimental group is coated with bacteria and placed with intelligent hydrogel containing photosensitive antibacterial agent at the wounds of the mice, and the control group is coated with bacteria only at the wounds without other treatment. The experiment is to irradiate the wound of the mouse for 1h, and then observe and record the healing condition of the wound of the mouse. Mice were examined histologically on wound skin on day 7 of the experiment.

2.3 histological analysis

Soaking the skin in 4% formalin for 24 hr, rinsing skin tissue, dehydrating, transparentizing, soaking in wax, embedding, making wax block slice, and slicing with slicer (thickness of 3-5 μm); drying for 2-3h in an oven, dewaxing with xylene for 5min (4 times in total), and washing off xylene; scraping magnesium oxide on the surface of the hematoxylin, and dyeing the hematoxylin; staining the dewaxed slices in hematoxylin for 5min, washing with tap water for three times, differentiating with 1% hydrochloric acid alcohol differentiation solution, soaking the slices in water for 5-10min, soaking the slices in eosin staining solution for 30s, observing the color under a microscope, prolonging the soaking time if the color is light, soaking the slices in 95% alcohol once, drying in an oven, dropping 2 drops of neutral gum, covering with a cover glass, and drying overnight.

2.4 results of the experiment

FIG. 5 is a histological section of a wound of a mouse, wherein A is a section of a normal mouse not infected with bacteria, B is an illuminated group infected with a bacterial solution and then added with a photosensitive antibacterial agent, and C is a non-illuminated group infected with a bacterial solution and then added with a photosensitive antibacterial agent. Observed under a microscope: b contains a small amount of lymphocytes, and C has a large amount of lymphocytes, so that the photosensitive antibacterial agent can play a role in sterilization under the condition of illumination.

In conclusion, the invention provides the wound dressing with detection and antibacterial effects, and the three common pathogenic bacteria, namely pseudomonas aeruginosa, escherichia coli and staphylococcus aureus, are subjected to enzymatic reaction, and the three common pathogenic bacteria are efficiently and sensitively detected in real time according to the fact that whether fluorescence is emitted and the strength of a fluorescence signal is detected. Natural photosensitive antibacterial agents such as hypericin and the like are filled into the vesicles, cytotoxin generated by pathogenic bacteria can cause the response of vesicle cracking, so that the photosensitive antibacterial agents are released from the vesicles, and the intelligent detection and treatment of the pathogenic bacteria are synchronously carried out.

Claims

1. A preparation method of intelligent antibacterial hydrogel is characterized by comprising the following operation steps: 1) 73.4mg of phospholipid 1, 2-dipalmitoyl-Sn-glycero-3-phosphocholine (DPPC), 38.6mg of cholesterol and 34.6mg of 10, 12-tricosane-dienoic acid (TCDA) were weighed out and dissolved in 1mL of chloroform at a concentration of 100mmol/dm³Mixing 660 mu L of DPPC, 240 mu L of cholesterol and 300 mu L of TCDA in nitrogen flow, and then drying, removing impurities and dehydrating;

2) will contain 50mmol/dm³Mixing 10mL buffer solution of fluorescent agent with lipid, freezing for circulation, extruding the lipid mixture through a polycarbonate membrane by a vesicle extruder, and storing the vesicle at 4 ℃ for later use;

3) the size of the product is 90X 1mm³The hydrogel dressing is characterized in that 4-methyl umbelliferyl- α -D-Mannoside (MUD) is grafted on the hydrogel, an enzyme released by pathogenic bacteria can cut MUD fluorescer, a fluorophore 4-methyl umbelliferone (4-MU) is released into the hydrogel, blue light is shown under an ultraviolet lamp (lambda is 365nm), cylindrical pores with uniform intervals are carved on the prepared hydrogel dressing, and each pore has uniform diameter and depth;

2. An application of intelligent antibacterial hydrogel in the detection of pathogenic bacteria is characterized in that,

1) enzymatic reaction in solution

For the enzymatic reaction in the solution, 50 mul of MUD solution is added into a 96-well plate filled with 50 mul of bacterial liquid, the 96-well plate is sealed by a sealing adhesive tape, and the 96-well plate is placed into a fluorescence pool to observe the fluorescence intensity;

2) enzymatic reaction of hydrogel dressing on culture dish

Grafting 50 mu L of MUD solution on the surface of the prepared hydrogel dressing, then placing the hydrogel dressing on a culture dish coated with 50 mu L of bacterial liquid, and observing the luminous intensity of the hydrogel dressing;

3) preparation of hypericin clathrate

Adding β -CD (β -cyclodextrin) 5.0g into 30% NaOH solution 10ml, stirring at room temperature to dissolve β -CD, adding EP 2.41ml into the mixed solution at 30 ℃, stirring for 24h, cooling to room temperature, desalting by a dialysis method, evaporating the solution in a dialysis bag to be viscous, adding absolute ethyl alcohol, precipitating white solid, filtering the white solid, and carrying out vacuum freeze drying to obtain β -CDP (β -cyclodextrin polymer), dissolving β -CDP 80mg and Hyp (hypericin) 10mg in a proper amount of water, fully stirring for at least 24h at room temperature, filtering the mixed solution after the reaction is finished, removing the residual Hyp to obtain a deep purple solution containing an inclusion compound, and carrying out vacuum freeze drying to obtain a deep purple product Hyp- β -CDP (hypericin inclusion compound);

4) hypericin clathrate is filled into vesicle

3. The application of the intelligent antibacterial hydrogel in an intelligent antibacterial experiment is characterized by comprising the following steps of;

1) bacterial culture

Picking bacteria from an LB flat plate, putting the bacteria into a 50ml centrifuge tube, adding 10ml of LB culture solution into the centrifuge tube, and putting the centrifuge tube into a shaker at 37 ℃ for culturing for 15 hours for later use;

2) preparation of solid Medium

Taking a 1000ml beaker, adding 500ml of distilled water, weighing 5g of beef extract, 10g of peptone, 5g of agar and 5g of sodium chloride, dissolving in water, adjusting the pH to about 7.2 by using a 10% NaOH solution, fixing the volume to 1000ml, wrapping by using tin foil paper, and sterilizing in a sterilizing box for later use;

3) antibacterial experiments

Diluting cultured strain with LB solution 10 times, and measuring OD₆₀₀Diluting the strain to 0.01 for later use, taking out a 96-well plate, adding a bacterial liquid and a photosensitive antibacterial agent, incubating for 1h in an incubator at 37 ℃, and irradiating one group for 30min while keeping the other group away from light for 30 min; after illumination, the solution in the 96-well plate is coated in a solid culture dish and then cultured for 12 hours at 37 ℃; the culture dish was removed and the growth of the bacteria in the culture dish was observed.

4. The application of the intelligent antibacterial hydrogel in the influence of the intelligent photosensitive antibacterial agent on the healing of the mouse wound is characterized by comprising the following steps of;

1) experimental Material

1.1) test reagents

Hypericin or natural photosensitive antibacterial agent extract, medical alcohol, and diluted bacteria solution;

1.2) mouse of Experimental animal Kunming

2) Experimental methods

2.1) mouse wound model preparation

Anaesthetizing a healthy mouse by using urethane, removing hair of the mouse by using clean scissors, disinfecting the hair-removed part by using medical alcohol, and manufacturing an 2 wound model with the area of about 1cm on the back of the mouse by using the disinfected scissors and tweezers;

2.2) grouping and administration

Dividing the mice into an experimental group and a control group, wherein each group comprises 10 mice, the experimental group is coated with bacteria and placed with intelligent hydrogel containing photosensitive antibacterial agent at the wounds of the mice, and the control group is coated with bacteria only at the wounds without other treatment; the wound of the mouse is illuminated for 1h in the experiment, then the healing condition of the wound of the mouse is observed and recorded, and the wound skin of the mouse is subjected to histological examination on the 7 th day of the experiment;

2.3) histological analysis