CN110743035A - Preparation method and application of intelligent antibacterial hydrogel - Google Patents

Abstract

The invention discloses a preparation method and application of an intelligent antibacterial hydrogel. This approach is achieved by using vesicles loaded with self-extinguishing fluorescent dyes. The vesicles are sensitive to the cytotoxicity factor regulated by the key population density produced by the bacteria. In the case of bacterial infection in the wound, the generated bacterial toxins can cause the rupture of the vesicles, so that the dyes are released from the vesicles and emitted. Strong fluorescence. Therefore, according to the magnitude of the fluorescence intensity, it can be determined whether the wound is infected, thereby reducing the risk of infection. In addition, the synchronous intelligent response to pathogen detection and sterilization can be achieved by loading photosensitive antimicrobial agents in vesicles. Compared with the traditional pathogenic bacteria detection method, the invention has the characteristics of simple preparation process, convenient operation, high efficiency, rapidity, accuracy and the like.

Description

A kind of preparation method and application of intelligent antibacterial hydrogel

technical field

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

Background technique

With the wide application of antibiotics in clinical practice, the problem of bacterial drug resistance is becoming more and more serious, and its drug resistance continues to intensify. The isolation rate of clinical multidrug-resistant strains is quite high. bring great challenges. At present, the detection and drug resistance of pathogenic microorganisms are mainly completed by bacterial culture, biochemical identification, polymerase chain reaction amplification, real-time fluorescent quantitative gene amplification technology, enzyme-linked immunosorbent assay and chip technology. The above detection methods have problems such as long detection period, high cost, and complicated operation, which are difficult to meet the needs of clinical real-time detection. Therefore, the development of rapid and sensitive detection methods has important practical significance for the prevention and control of pathogenic microorganisms.

A wound dressing is a material that covers a wound on the skin to promote wound healing. Hydrogel dressing is a new type of dressing invented in recent years, which has been widely researched and applied in the field of medical dressing. Some studies have prepared PEO/PVA graft copolymer hydrogel by irradiating the mixture of polyethylene oxide and polyvinyl alcohol with electron beam, and incorporated gentamicin to study the drug release and antibacterial effect of the hydrogel. At present, the antibacterial agents loaded in the finished hydrogel products mainly include nano-silver, sulfapyridine salts, curcumin and tetracyclines. Among them, the market share of nano-silver antibacterial hydrogel is more than 60%. Studies have shown that nano-silver has great toxicity and is not suitable for wounds. Therefore, antibiotic antibacterial agents are widely used. Hydrogels achieve the purpose of anti-infection by releasing antibiotics, interfering with the important life process of bacteria with antibiotics, directly killing them or inhibiting the growth of bacteria. However, due to the rapid adaptability of bacteria themselves and evolutionary ability, it is extremely prone to negative effects such as drug resistance and microbial dysbiosis. Therefore, it is necessary to use an effective and less resistant method to replace or supplement antibiotic therapy. Photonamic antimicrobial chemotherapy (PACT) is one of the most promising treatments in recent years. The antibacterial action principle of PACT is that the photosensitizer selectively accumulates on the bacterial cell wall and cell membrane. Under the excitation of the appropriate wavelength light source, relying on the participation of oxygen, reactive oxygen species such as singlet oxygen are generated through the type I and type II reactions, which directly destroy the bacterial cell wall. and membrane system, affecting its metabolism, which in turn leads to bacterial death. The antibacterial effect of PACT can not only effectively kill pathogenic bacteria, but also inactivate bacterial toxin factors, which has the unique advantage of effectively killing pathogenic bacteria without easily developing tolerance.

Based on the above problems, we designed a hydrogel dressing capable of detecting bacterial microorganisms and intelligently sterilizing, in which the intelligent detection of pathogenic bacteria is realized by using vesicles loaded with self-extinguishing fluorescent dyes, and the Loaded with photosensitive antibacterial agents to achieve simultaneous intelligent response to pathogen detection and sterilization.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a preparation method and application of an intelligent antibacterial hydrogel.

The technical scheme that the present invention solves the above-mentioned technical problems is as follows:

1. a preparation method of an intelligent antibacterial hydrogel, the operation steps are as follows:

1) Weigh 73.4 mg of phospholipid 1,2-dipalmitoyl-Sn-glycero-3-phosphocholine (DPPC), 38.6 mg of cholesterol and 34.6 mg of 10,12-tricosadadienoic acid (TCDA) and dissolved in In 1 mL of chloroform, the concentration was 100 mmol/dm ³ . 660 μL of DPPC, 240 μL of cholesterol and 300 μL of TCDA were taken and mixed in nitrogen flow, followed by drying, impurity removal and dehydration.

2) Mix 10 mL of buffer containing 50 mmol/dm ³ fluorescent agent and lipid, freeze and circulate, and finally extrude the lipid mixture through a polycarbonate membrane with a vesicle extruder, and store the vesicles at 4°C for later use.

³ ) A hydrogel dressing with a size of 90 × 90 × 1 mm is made, and 4-methylumbelliferyl-α-D-mannoside (MUD) is grafted on the hydrogel, and the enzymes released by pathogenic bacteria can cut The MUD fluorophore, which releases the fluorophore 4-methylumbelliferone (4-MU) into the hydrogel, exhibits blue light under UV light (λ=365 nm). The prepared hydrogel dressing is engraved with evenly spaced cylindrical pores, each of which has a uniform diameter and depth.

4) The fillings in the capsule are vesicles, pathogenic bacteria detection fluorescent agent and chitosan. This fluorescent agent can not only detect pathogenic bacteria but also play a role in sterilization under natural light conditions. The main antibacterial photosensitizer There are natural photosensitive antibacterial agents such as hypericin.

2. Application of an intelligent antibacterial hydrogel: applied to the detection of pathogenic bacteria, intelligent antibacterial experiments and the effect of intelligent photosensitizing antibacterial agents on wound healing in mice.

Due to the adoption of the above technical solutions, the method for preparing an intelligent Band-Aid dressing with detection and antibacterial effect of the present invention has the following advantageous technical effects:

1. The raw material of the present invention to prepare the antibacterial hydrogel dressing is mainly chitosan. The structure and properties of chitosan are similar to the extracellular matrix; it has good biocompatibility and does not react with human tissues; chitosan also has good biological activity, with antibacterial, cholesterol-lowering and other effects, and can also promote epithelial Cell growth; chitosan can be degraded in the presence of lysozyme, the degradation process is smooth and safe, and the degradation product is glucosamine, which can directly enter the metabolic pathway.

2. The fillings in the capsules of the present invention are mainly vesicles, pathogenic bacteria detection fluorescent agents and chitosan, and the fluorescent agents in the capsules are mainly natural extracts, which can not only detect pathogenic bacteria but also detect pathogenic bacteria in natural light. It can play the role of sterilization, and the invention realizes the simultaneous detection and treatment of the pathogenic bacteria in the wound.

3. The reagents used in the preparation of the present invention are all non-toxic and side effects, and the prepared hydrogel dressing has good biocompatibility.

Description of drawings

Figure 1 is a diagram of the effect of the enzymatic reaction in solution.

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

Figure 2 is a diagram showing the effect of enzymatic reaction on the hydrogel dressing.

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

Figure 3 shows the effect of the smart hydrogel wound dressing on the detection of pathogenic bacteria under UV light.

In the figure, the left picture is the picture before application; the right picture is the effect picture after application.

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

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

Figure 5 is a histological comparison diagram of the mouse epidermis between the experimental group of the animal experiment of the present invention and the control group.

In the figure, A, B, and C are normal mice, light group, and no light group, respectively.

specific implementation

The present invention will be further described in detail below in conjunction with the examples. The following are examples of implementations of the present invention, which are not intended to limit the present invention, and any modifications, substitutions and improvements made on the basis of the present invention are included within the protection scope of the present invention.

Embodiment 1. A kind of intelligent antibacterial hydrogel

The application of intelligent antibacterial hydrogel in the detection of pathogenic bacteria, the operation steps are as follows:

1. Enzymatic reaction in solution

For the enzymatic reaction in solution, add 50 μL of MUD solution to a 96-well plate containing 50 μL of bacterial solution, seal the 96-well plate with sealing tape, and place it in a fluorescent cell to observe the fluorescence intensity as shown in Figure 1.

2. Enzymatic Reaction of Hydrogel Dressing on Petri Dish

50 μL of MUD solution was grafted on the surface of the prepared hydrogel dressing, and then placed on a petri dish coated with 50 μL of bacterial solution, and the luminescence intensity of the hydrogel dressing was observed as shown in Figure 2.

3. Preparation of Hypericin Inclusion Complex

5.0 g of β-CD (β-cyclodextrin) was added to 10 ml of 30% NaOH solution, and the β-CD was dissolved by stirring at room temperature. 2.41ml of EP was added to the above mixed solution at 30°C, stirred for 24h, and cooled to room temperature. Desalting by dialysis, steaming the solution in the dialysis bag to a viscous state, adding absolute ethanol, a white solid is precipitated, filtering the white solid, and vacuum freeze drying to obtain β-CDP (β-cyclodextrin polymer) . Dissolve 80 mg of β-CDP and 10 mg of Hyp (hypericin) in an appropriate amount of water, and stir well at room temperature for at least 24 h. After the reaction, the mixed solution was filtered to remove the remaining Hyp to obtain a dark purple solution containing an inclusion complex, which was vacuum freeze-dried to obtain a dark purple product Hyp-β-CDP (hypericin inclusion complex).

4. Inclusion of hypericin into vesicles

Several round holes are uniformly prepared in the hydrogel, vesicles and chitosan are added in the holes, and the filling in the vesicles is hypericin inclusion complex or other natural photosensitive antibacterial agent.

Experimental results

A, B, and C in Figure 1 are the enzymatic reactions of Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus in a 96-well plate respectively. The enzymatic reaction is 1 h. Judging from the fluorescence intensity, the fluorescent substrate MUD has These three bacteria will fluoresce, indicating that the fluorescent substrate MUD has a certain detection effect on these three bacteria; in Figure 2, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus are on the culture dishes A, B, and C, respectively. The hydrogel dressing was cut into squares with a size of 1.5cm×1.5cm, and the fluorescent substrate was grafted onto the hydrogel dressing, and then the dressing was placed on a petri dish, and the enzymatic reaction was carried out for 1h. From the fluorescence intensity in the figure , this dressing has a detection effect on these three kinds of bacteria; Figure 3 is the comparison picture before and after the excipients are activated under the ultraviolet lamp. It can be seen from the picture that the toxin released by the pathogenic bacteria causes the vesicles to crack, and the hypericin is released. , showing red fluorescence under UV lamp (λ=365nm).

Example 2

The application of intelligent antibacterial hydrogel in intelligent antibacterial experiment, the operation steps are as follows:

1. Bacterial culture

Pick bacteria from the LB plate, put them in a 50ml centrifuge tube, add 10ml of LB culture medium to the centrifuge tube, and culture in a shaker at 37°C for 15h until use.

2. Preparation of solid medium

Take a 1000ml beaker, put it in 500ml of distilled water, weigh 5g of beef extract, 10g of peptone, 5g of agar, and 5g of sodium chloride and dissolve it in water, adjust the pH to about 7.2 with 10% NaOH solution, and set the volume to 1000ml. Packed and sterilized in a sterilization box for later use.

3. Antibacterial test

Dilute the cultured strains 10 times with LB solution, measure the OD ₆₀₀ value, and then dilute the strains to 0.01 for use. Take out the 96-well plate, add bacterial solution and photosensitizing antibacterial agent, incubate for 1 h in a 37°C incubator, expose one group to light for 30 minutes, and treat the other group to avoid light for 30 minutes; after lighting, apply the solution in the 96-well plate to In a solid petri dish, then cultured at 37°C for 12 h; take out the petri dish and observe the growth of bacteria in the petri dish.

Experimental results

In Figure 4, A and B are the growth of bacteria in the presence or absence of light when photosensitive antimicrobial agents are added to the vesicles. It is obvious from the figure that in the case of light, the bacteria did not grow; in the absence of light, the bacteria have overgrown the entire petri dish.

C and D in Figure 4 show the growth of bacteria in the presence or absence of light without adding photosensitizing antibacterial agents to the vesicles. From the picture, it can be observed that the petri dishes are covered with bacteria with or without light.

Example 3

The application of smart antibacterial hydrogel in the effect of smart photosensitizing antibacterial agent on wound healing in mice, the operation steps are as follows:

1 Experimental material

1.1 Experimental reagents

Hypericin or natural photosensitive antibacterial agent extract, medical alcohol, diluted bacterial liquid

1.2 Experimental animals

Kunming mice

2 Experimental methods

2.1 Making a mouse wound model

Take a healthy mouse, anesthetize it with urethane, take clean scissors, remove the mouse hair, sterilize the hair removal area with medical alcohol, and then use the sterilized scissors and tweezers to make an area on the back of the mouse about approx. For the 1cm2 wound model.

2.2 Grouping and Administration

The mice were divided into an experimental group and a control group, with 10 mice in each group. The experimental group applied bacteria to the wounds of the mice and placed smart hydrogels containing photosensitizing antibacterial agents, while the control group only applied bacteria to the wounds. Do not do other processing. The wounds of the mice were illuminated for 1 h, and then the wound healing of the mice was observed and recorded. On the seventh day of the experiment, the mouse wound skin was histologically examined.

2.3 Histological analysis

The taken skin was soaked in 4% formalin for 24 hours, and the skin tissue was routinely rinsed, dehydrated, transparent, dipped in wax, and embedded to make a wax block section, which was sliced with a microtome (thickness of 3-5 μm). Use an oven to dry for 2-3h, dewax with xylene for 5min (4 times in total), wash away xylene; scrape off the magnesia on the surface of hematoxylin, and stain it; put the above-mentioned dewaxed section in hematoxylin Stain for 5 min, wash three times with tap water, differentiate with 1% hydrochloric acid alcohol differentiation solution, soak the sections in water for 5-10 min, soak the sections in eosin staining solution for 30 s, observe the color under a microscope, if the color is light, then prolong the soaking time, Soak the sections in 95% alcohol, put them in an oven to dry, add 2 drops of neutral gum, cover with a cover glass, and let dry overnight.

2.4 Experimental results

Figure 5 shows the histological sections of mouse wounds, A is a normal mouse section that was not infected with bacteria, B was a light group with photosensitizing antibacterial agents added after being infected with bacterial liquid, and C was a non-illuminated group with photosensitizing antibacterial agents added after being infected with bacterial liquid . Observed from the microscope: B contains a small amount of lymphocytes, C has a large number of lymphocytes, which indicates that the photosensitizing antibacterial agent can play a bactericidal effect under light conditions.

To sum up, the present invention is a wound dressing with detection and antibacterial effect, by carrying out enzymatic reaction on three common pathogenic bacteria of Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus, according to whether the The intensity of fluorescence and its fluorescence signal enables real-time, efficient and sensitive detection of these three common pathogenic bacteria. When natural photosensitive antibacterial agents such as hypericin are filled into the vesicles, the cytotoxins produced by pathogenic bacteria will cause the response of vesicle lysis, so that the photosensitive antibacterial agents are released from the vesicles, realizing intelligent detection and detection of pathogenic bacteria. Synchronization of treatment.

Claims (4)

1. a preparation method of an intelligent antibacterial hydrogel, is characterized in that, operation steps are as follows: 1) take by weighing 73.4mg phospholipid 1,2-dipalmitoyl-Sn-glycerol-3-phosphocholine (DPPC), 38.6 mg of cholesterol and 34.6 mg of 10,12-tricosadadienoic acid (TCDA) were dissolved in 1 mL of chloroform, respectively, at a concentration of 100 mmol/dm ³ , 660 μL of DPPC, 240 μL of cholesterol and 300 μL of TCDA were mixed in nitrogen flow, and then drying, impurity removal and dehydration; 2) Mix 10 mL of buffer containing 50 mmol/dm ³ fluorescent agent and lipid, freeze and circulate, and finally extrude the lipid mixture through a polycarbonate membrane with a vesicle extruder, and store the vesicles at 4°C for later use; ³ ) A hydrogel dressing with a size of 90 × 90 × 1 mm is made, and 4-methylumbelliferyl-α-D-mannoside (MUD) is grafted on the hydrogel, and the enzymes released by pathogenic bacteria can cut MUD fluorescent agent, the fluorophore 4-methylumbelliferone (4-MU) is released into the hydrogel, showing blue light under the ultraviolet lamp (λ=365nm), and the prepared hydrogel dressing is engraved with uniform Spaced cylindrical apertures, each of uniform diameter and depth; 4) The fillings in the capsule are vesicles, pathogenic bacteria detection fluorescent agent and chitosan. This fluorescent agent can not only detect pathogenic bacteria but also play a role in sterilization under natural light conditions. The main antibacterial photosensitizer There are natural photosensitive antibacterial agents such as hypericin. 2. the detection application of a kind of intelligent antibacterial hydrogel in pathogenic bacteria, is characterized in that, 1) Enzymatic reaction in solution For the enzymatic reaction in solution, add 50 μL of MUD solution to a 96-well plate containing 50 μL of bacterial solution, seal the 96-well plate with sealing tape, and place it in a fluorescence cell to observe the fluorescence intensity; 2) Enzymatic reaction of hydrogel dressings on petri dishes Graft 50 μL of MUD solution on the surface of the prepared hydrogel dressing, and then put it on a petri dish coated with 50 μL of bacterial liquid to observe the luminescence intensity of the hydrogel dressing; 3) Preparation of Hypericin Inclusion Complex Add 5.0g β-CD (β-cyclodextrin) to 10ml of 30% NaOH solution, stir at room temperature to dissolve β-CD, add 2.41ml of EP to the above mixed solution at 30°C, stir for 24h, and cool to room temperature; dialyze Desalting method, steam the solution in the dialysis bag to a viscous state, add absolute ethanol, a white solid is precipitated, the white solid is filtered, and vacuum freeze-dried to obtain β-CDP (β-cyclodextrin polymer); Dissolve 80 mg of β-CDP and 10 mg of Hyp (hypericin) in an appropriate amount of water, and fully stir at room temperature for at least 24 hours; after the reaction, filter the mixed solution to remove the remaining Hyp to obtain an inclusion complex. The dark purple solution of the substance was vacuum freeze-dried to obtain the dark purple product Hyp-β-CDP (hypericin inclusion complex); 4) Hypericin inclusion complex is filled into vesicles Several round holes are uniformly prepared in the hydrogel, vesicles and chitosan are added in the holes, and the filling in the vesicles is hypericin inclusion complex or other natural photosensitive antibacterial agent. 3. an intelligent antibacterial hydrogel is applied in an intelligent antibacterial experiment, wherein the steps are as follows; 1) Bacterial culture Pick bacteria from the LB plate, put them in a 50ml centrifuge tube, add 10ml of LB culture medium to the centrifuge tube, and culture in a shaker at 37°C for 15h until use; 2) Preparation of solid medium Take a 1000ml beaker, put it in 500ml of distilled water, weigh 5g of beef extract, 10g of peptone, 5g of agar, and 5g of sodium chloride and dissolve it in water, adjust the pH to about 7.2 with 10% NaOH solution, and set the volume to 1000ml. Pack it well, put it in a sterilization box and sterilize it for later use; 3) Antibacterial test Dilute the cultured strain 10 times with LB solution, measure the OD ₆₀₀ value, then dilute the strain to 0.01, set aside for use, take out the 96-well plate, add bacterial liquid and photosensitizing antibacterial agent, and incubate in an incubator at 37°C For 1 h, one group was exposed to light for 30 min, and the other group was treated in the dark for 30 min; after lighting, the solution in the 96-well plate was coated on a solid petri dish, and then incubated at 37 °C for 12 h; the petri dish was taken out, and the petri dish was observed. growth of bacteria. 4. the application of a kind of intelligent antibacterial hydrogel in the influence of intelligent photosensitizing antibacterial agent on mouse wound healing, it is characterised in that the steps are as follows; 1) Experimental materials 1.1) Experimental reagents Hypericin or natural photosensitive antibacterial agent extract, medical alcohol, diluted bacterial liquid; 1.2) Experimental animal Kunming mice 2) Experimental method 2.1) Creation of mouse wound model Take a healthy mouse, anesthetize it with urethane, take clean scissors, remove the mouse hair, sterilize the hair removal area with medical alcohol, and then use the sterilized scissors and tweezers to make an area on the back of the mouse about approx. It is a 1cm2 wound model; 2.2) Grouping and administration The mice were divided into an experimental group and a control group, with 10 mice in each group. The experimental group applied bacteria to the wounds of the mice and placed smart hydrogels containing photosensitizing antibacterial agents, while the control group only applied bacteria to the wounds. No other treatment was performed; the wounds of the mice were illuminated for 1 hour, and then the wound healing of the mice was observed and recorded, and the wound skin of the mice was histologically examined on the 7th day of the experiment; 2.3) Histological analysis The taken skin was soaked in 4% formalin for 24 hours, and the skin tissue was routinely rinsed, dehydrated, transparent, dipped in wax, and embedded to make a wax block section, which was sliced with a microtome (thickness of 3-5 μm). Use an oven to dry for 2-3h, dewax with xylene for 5min (4 times in total), wash away xylene; scrape off the magnesia on the surface of hematoxylin, and stain it; put the above-mentioned dewaxed section in hematoxylin Stain for 5 min, wash three times with tap water, differentiate with 1% hydrochloric acid alcohol differentiation solution, soak the sections in water for 5-10 min, soak the sections in eosin staining solution for 30 s, observe the color under a microscope, if the color is light, then prolong the soaking time, Soak the sections in 95% alcohol, put them in an oven to dry, add 2 drops of neutral gum, cover with a cover glass, and let dry overnight.

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