CN106310385A - Preparation method and application of egg shell membrane composite nano-silver thin film used for artificial skin - Google Patents

Abstract

The invention discloses a preparation method and application of an egg film composite nano-silver film for artificial skin. The preparation method includes: discarding the egg white and egg yolk of fresh eggs, cleaning the egg shell with deionized water, separating the egg film, Then wash it with deionized water, cut it into a size of 1-3cm×1-3cm, put it in a hydrochloric acid solution with a concentration of 1-100mM and pH=1-3, soak it at room temperature for 1-4 days, and then wash it with deionized water. Then put it in a dopamine solution with a concentration of 2-4mg/mL and pH=8.5-9.5, soak it for 6-20 hours at room temperature, wash it with deionized water, and then put it in a silver nitrate solution with a concentration of 10-50nM. After soaking in light for 6-20 hours, it is washed with deionized water and dried naturally at normal temperature to obtain an egg membrane composite nano-silver thin film for artificial skin. The prepared egg membrane composite nano-silver thin film is used to manufacture artificial skin, has good antibacterial properties, has no obvious biological toxicity, and has a simple preparation process, less time-consuming and low cost.

Description

A preparation method of egg membrane composite nano-silver thin film for artificial skin and its application

technical field

The invention belongs to the technical field of material application, and in particular relates to a preparation method and application of an egg membrane composite nano-silver thin film for artificial skin.

Background technique

As the largest organ of the human body, the skin can protect internal organs from damage and maintain water and electrolyte balance. Skin defects caused by burns, trauma, diabetic foot ulcers, etc. will lead to serious infection, water and electrolyte imbalance, scar hyperplasia and other problems in the body, which will bring huge mental and physical pain and heavy medical burden to patients. Therefore, restoring the barrier function of the skin has become a top priority. However, some patients with large skin defects, such as patients with extensive burns, cannot effectively seal the wound in time due to their limited skin source. In order to solve this problem, it is very necessary to apply a suitable artificial skin to replace the self-skin source to seal the wound.

At present, many commercialized artificial skins such as Integra and AlloDerm have been used clinically abroad. Integra is an artificial skin developed by Burke and Yannas in 1980. It consists of a silicone rubber film to form the "epidermis", glutaraldehyde-crosslinked bovine type I collagen, chondroitin 6-sulfate and glucosamine to form the "dermis". Clinical application has found that it is beneficial to the infiltration and growth of wound tissue, easy to vascularize and not easy to degrade, and can effectively promote the healing of large area burn wounds. At the same time, it also has obvious effects on the prevention and treatment of keloids and hypertrophic scars. However, the artificial skin has high requirements on the wound surface, weak anti-infection ability, and poor mechanical strength and air permeability. At the same time, due to the complicated preparation process and high price, it is difficult to be widely promoted. AlloDerm is a commercial decellularized dermal matrix produced by American Life Science Company. It is an artificial skin made by removing the epidermis and cellular components of fresh cadaver skin. Because the cellular components are removed, AlloDerm reduces the immunogen However, it still retains the three-dimensional structure of the extracellular matrix scaffold and the complete basement membrane complex, which can guide the growth and expansion of newborn cells. However, due to the limited source of cadaver skin and the risk of virus transmission, AlloDerm is expensive and complicated in process, so its application is also limited.

Therefore, it is particularly important to prepare an artificial skin with low price, relatively simple operation process, good biocompatibility and good antibacterial effect.

The performance of scaffold materials is a key factor in the construction of artificial skin, and it is also a research hotspot in this field. Egg membrane is a fibrous mesh film located between egg shell and egg white, with good air permeability and mechanical properties. The egg membrane is mainly composed of proteins such as keratin and collagen, and also contains hyaluronic acid, chondroitin sulfate and other main components of the extracellular matrix. As a natural bioscaffold material, it has good biocompatibility, and is non-toxic and non-immunogenic. In addition, egg membranes are easy to obtain and cheap, and can be widely used in clinic simply and economically. At present, it has been used in the treatment of some superficial skin wounds.

Nano-silver is a widely used medical antibacterial agent, which has a good killing effect on bacteria and fungi. At the same time, nano-silver antibacterial is sustained and stable, with little drug resistance. Studies have confirmed that nano silver can effectively promote wound healing through antibacterial and anti-inflammatory. Therefore, nanosilver can effectively function as an antibacterial agent in artificial skin. However, nano-silver is easy to form silver oxide after contacting with oxygen in the air, which greatly reduces or even disappears its antibacterial effect. In recent years, studies have found that integrating nano-silver into biological scaffolds can effectively maintain its stability and antibacterial properties.

At present, there are many methods for preparing nano-silver and integrating it into biological scaffolds to prepare artificial skin, including electrospinning technology and application of reducing reagents. However, the operation of electrospinning technology is relatively complicated and the cost is high, and the commonly used reducing reagents take more time and are prone to produce environmental pollutants. In recent years, Messersmith discovered that dopamine can self-polymerize on the surface of various materials to form a layer of polymerized dopamine film, which can efficiently adhere to various molecules or cells. Polymerized dopamine has reducibility, and can reduce silver ions to nano silver particles in situ on the surface of the material. Studies have confirmed that this kind of nano-silver has good stability and antibacterial properties. In addition, dopamine is non-toxic, non-teratogenic, has good biocompatibility, and can be effectively used in the preparation of artificial skin.

In summary, it is a convenient, feasible, simple and practical method to prepare an antibacterial artificial skin made of egg membrane composited with nano-silver by using the egg membrane as a biological scaffold and utilizing the adhesion and reduction properties of dopamine.

Contents of the invention

The purpose of the present invention is to provide a method for preparing egg membrane composite nano-silver thin film for artificial skin. The egg membrane composite nano-silver thin film for artificial skin prepared by the invention has good antibacterial properties, no obvious biological toxicity, and can effectively promote the repair of full-thickness skin defect wounds. The preparation method has the advantages of simple process, less time-consuming and low cost. advantage.

The invention provides a method for preparing an egg membrane composite nano-silver film for artificial skin, comprising the following steps:

(1) Take fresh eggs, discard the egg whites and yolks, wash the egg shells in deionized water, separate the egg membranes, wash them with deionized water, and cut the egg membranes into 1-3cm×1-3cm sizes;

(2) Place the egg membrane prepared in step (1) in a hydrochloric acid solution with a concentration of 1-100mM and pH=1-3, soak it at room temperature for 1-4 days, and then wash it with deionized water for 3-5 times, 3-5 minutes each time;

(3) Dissolve dopamine in Tris-HCl buffer solution with a concentration of 10-100mM to prepare a dopamine solution with a concentration of 2-4mg/mL, and adjust the pH to 8.5-9.5;

(4) Put the egg membrane prepared in step (2) into the dopamine solution prepared in step (3), soak it at room temperature for 6-20 hours, and then wash it with deionized water for 3-5 times, each time for 3-5 minutes ;

(5) Place the egg membrane prepared in step (4) in a silver nitrate solution with a concentration of 10-50nM, soak it in the dark at room temperature for 6-20h, and then wash it with deionized water for 3-5 times, each time 3-5 Minutes, natural drying at room temperature, prepared egg membrane composite nano-silver thin film.

Further, the egg membranes prepared in the steps (1), (2) and (4) were washed with deionized water for 3 times, 5 minutes each time.

Further, the concentration of the hydrochloric acid solution in the step (2) is 1 mM, pH=3.

Further, the concentration of the Tris-HCl buffer in the step (3) is 10 mM.

Further, the dopamine solution prepared in the step (3) has a concentration of 2 mg/mL and a pH of 8.5.

Further, the concentration of the silver nitrate solution in the step (5) is 30nM.

The invention discloses a use of an egg membrane composite nano-silver film for artificial skin in the preparation of artificial skin or surgical dressing for treating skin defects.

Further, the skin defect is a full-thickness skin defect or a skin infection wound.

Carry out following performance test to egg membrane composite nano-silver thin film of the present invention.

1. Scanning electron microscope observation:

Referring to Fig. 1, the surface structure of the prepared egg membrane composite nano-silver thin film was observed under different magnifications by scanning electron microscope. Using Image-Pro Plus 6.0 software, measure the porosity of all electron microscope images of the film, export to an Excel table, calculate the average value and standard deviation of the porosity: the porosity of the film is 45±4.11%.

2. Transmission electron microscope observation:

Referring to Figure 2 and Figure 3, the prepared egg membrane composite nano-silver film was pulverized with an ultrasonic pulverizer, dissolved in deionized water, blown and beaten evenly to form a suspension, and a drop of the suspension was dropped onto a carbon-coated copper grid, and was analyzed by transmission. The shape and size of nano-silver in the egg membrane composite nano-silver thin film was observed by electron microscope. Using Image-J software, set the scale as the legend scale, select the scale fixedly, measure the diameters of all nano-silver particles, export them to an Excel table, calculate the size distribution map of nano-silver, see Figure 3, and obtain the average value: The diameter is 13.91nm.

The beneficial effects of the present invention are: compared with the prior art, the present invention uses the egg membrane as a biological scaffold, utilizes the adhesion and reduction properties of dopamine, and the antibacterial properties of nano silver to prepare an egg membrane composite nanometer for artificial skin. The silver thin film has good antibacterial performance and no obvious biological toxicity; the preparation method has low cost, less time consumption and simple operation process.

Description of drawings

Fig. 1 is a scanning electron micrograph of an egg membrane composite nano-silver thin film according to an embodiment of the present invention.

Fig. 2 is a transmission electron micrograph of an egg membrane composite nano-silver thin film according to an embodiment of the present invention.

Fig. 3 is a size distribution diagram of nano-silver in a transmission electron micrograph of an egg membrane composite nano-silver thin film according to an embodiment of the present invention.

Fig. 4 is a histogram of cell activity in the cytocompatibility experiment of the prepared egg membrane composite nano-silver thin film.

Figure 5 shows the antibacterial performance test results of the prepared egg membrane composite nano silver film (a—sterile filter paper sheet, b—egg membrane treated with hydrochloric acid, c—egg membrane treated with dopamine solution, d—egg membrane composite nano silver film).

Figure 6 shows the wound healing of mice on the third day and the seventh day after the wound was treated with the blank control group, Vaseline gauze, hydrochloric acid-treated egg membrane, dopamine solution-treated egg membrane, and egg membrane composite nano-silver film in animal experiments Comparison.

detailed description

Embodiment 1: a kind of preparation method of the egg membrane composite nano-silver thin film that is used for artificial skin, comprises the steps:

(1) Take a fresh egg, break it lightly, discard the egg white and yolk, wash the egg shell in deionized water, then carefully separate the egg membrane with tweezers, wash it again with deionized water, and cut the egg membrane into 2cm ×2cm size;

(2) Place the egg membrane prepared in step (1) in a hydrochloric acid solution, soak it at room temperature for 3 days, and then wash it with deionized water for 3 times, each time for 5 minutes. The concentration of the hydrochloric acid solution is 1mM, and the pH= 3;

(3) Dissolving dopamine into Tris-HCl solution, the concentration of the Tris-HCl solution is 10mM, the concentration of the prepared dopamine solution is 2mg/ml, and the pH is adjusted to 8.5;

(4) Place the egg membrane prepared in step (2) in the dopamine solution prepared in step (3), soak it at room temperature for 16 hours, and then wash it with deionized water for 3 times, each time for 5 minutes;

(5) Place the egg membrane prepared in step (4) in a silver nitrate solution, the concentration of the silver nitrate (5) solution is 30nM, soak for 18 hours in the dark at room temperature, and then wash it with deionized water 3 times, each Repeat for 5 minutes, and dry naturally at room temperature to prepare egg membrane composite nano-silver thin film.

Embodiment 2: a kind of preparation method of egg membrane composite nano-silver film for artificial skin comprises the following steps:

(1) Take a fresh egg, break it lightly, discard the egg white and yolk, wash the egg shell in deionized water, then carefully separate the egg membrane with tweezers, wash it again with deionized water, and cut the egg membrane into 1cm ×3cm size;

(2) Place the egg membrane prepared in step (1) in hydrochloric acid solution, soak it at room temperature for 1 day, and then wash it with deionized water for 3 times, each time for 5 minutes. The concentration of the hydrochloric acid solution is 10mM, and the pH= 1;

(3) Dissolving dopamine into Tris-HCl solution, the concentration of the Tris-HCl solution is 30mM, the concentration of the prepared dopamine solution is 2mg/ml, and the pH is adjusted to 9.0;

(4) Put the egg membrane prepared in step (2) into the dopamine solution prepared in step (3), soak it at room temperature for 6 hours, and then wash it with deionized water for 3 times, each time for 5 minutes;

(5) Place the egg membrane prepared in step (4) in a silver nitrate solution, the concentration of the silver nitrate (5) solution is 10nM, soak for 6 hours in the dark at room temperature, and then wash it with deionized water for 3 times. Repeat for 5 minutes, and dry naturally at room temperature to prepare egg membrane composite nano-silver thin film.

Embodiment 3: a kind of preparation method of egg membrane composite nano-silver thin film for artificial skin, comprises the steps:

(1) Take a fresh egg, break it lightly, discard the egg white and yolk, wash the egg shell in deionized water, then carefully separate the egg membrane with tweezers, wash it again with deionized water, and cut the egg membrane into 3cm ×3cm size;

(2) Place the egg membrane prepared in step (1) in a hydrochloric acid solution, soak it at room temperature for 4 days, and then wash it with deionized water for 5 times, each time for 3 minutes. The concentration of the hydrochloric acid solution is 100mM, and the pH= 3;

(3) Dissolving dopamine into Tris-HCl solution, the concentration of the Tris-HCl solution is 100mM, the concentration of the prepared dopamine solution is 4mg/ml, and the pH is adjusted to 9.5;

(4) Put the egg membrane prepared in step (2) into the dopamine solution prepared in step (3), soak it at room temperature for 20 hours, and then wash it with deionized water for 5 times, each time for 3 minutes;

(5) Place the egg membrane prepared in step (4) in a silver nitrate solution, the concentration of the silver nitrate (5) solution is 50nM, soak it in the dark at room temperature for 20h, and then wash it with deionized water for 5 times. Repeat for 3 minutes, and dry naturally at room temperature to prepare egg membrane composite nano-silver thin film.

Embodiment 4: a kind of preparation method of the egg membrane composite nano-silver thin film that is used for artificial skin, comprises the steps:

(1) Take a fresh egg, break it lightly, discard the egg white and yolk, wash the egg shell in deionized water, then carefully separate the egg membrane with tweezers, wash it again with deionized water, and cut the egg membrane into 2cm ×3cm size;

(2) Place the egg membrane prepared in step (1) in a hydrochloric acid solution, soak it at room temperature for 2 days, and then wash it with deionized water for 4 times, each time for 4 minutes. The concentration of the hydrochloric acid solution is 40mM, and the pH= 2;

(3) Dissolving dopamine into Tris-HCl solution, the concentration of the Tris-HCl solution is 50mM, the concentration of the prepared dopamine solution is 3mg/ml, and the pH is adjusted to 8.5;

(4) Put the egg membrane prepared in step (2) into the dopamine solution prepared in step (3), soak it at room temperature for 14 hours, and then wash it with deionized water 4 times, each time for 4 minutes;

(5) Place the egg membrane prepared in step (4) in a silver nitrate solution, the concentration of the silver nitrate (5) solution is 40nM, soak for 12 hours in the dark at room temperature, and then wash it with deionized water 4 times, every Repeat for 4 minutes, and dry naturally at room temperature to prepare egg membrane composite nano-silver thin film.

Embodiment 5: a kind of preparation method of the egg membrane composite nano-silver thin film that is used for artificial skin, comprises the steps:

(1) Take a fresh egg, break it lightly, discard the egg white and yolk, wash the egg shell in deionized water, then carefully separate the egg membrane with tweezers, wash it again with deionized water, and cut the egg membrane into 3cm ×2cm size;

(2) Place the egg membrane prepared in step (1) in a hydrochloric acid solution, soak it at room temperature for 3 days, and then wash it with deionized water for 5 times, each time for 4 minutes. The concentration of the hydrochloric acid solution is 80mM, and the pH= 3;

(3) Dissolving dopamine into Tris-HCl solution, the concentration of the Tris-HCl solution is 70mM, the concentration of the prepared dopamine solution is 4mg/ml, and the pH is adjusted to 9.5;

(4) Put the egg membrane prepared in step (2) into the dopamine solution prepared in step (3), soak it at room temperature for 10 hours, and then wash it with deionized water for 5 times, each time for 4 minutes;

(5) Place the egg membrane prepared in step (4) in a silver nitrate solution, the concentration of the silver nitrate (5) solution is 40nM, soak for 14 hours in the dark at room temperature, and then wash it with deionized water for 5 times. Repeat for 4 minutes, and dry naturally at room temperature to prepare egg membrane composite nano-silver thin film.

Embodiment 6: Carry out cytocompatibility experiment to the prepared egg membrane composite nano-silver film For the cytocompatibility of the prepared egg membrane composite nano-silver film for artificial skin, the following experiment is carried out:

(1) Experimental grouping: experimental group: egg membrane composited with nano-silver film; control group: egg membrane treated with hydrochloric acid and egg membrane treated with dopamine;

(2) The samples of the experimental group and the control group prepared in Example 1 were respectively made into film discs with a diameter of 6 mm by a puncher, soaked in 75% ethanol solution for disinfection, and then rinsed with phosphate buffer for 3 times;

(3) Place the sterile discs in 96-well plates, and replant 6 wells in each group;

(4) Take GFP mouse fibroblasts in the logarithmic growth phase, inoculate 8×10 ³ cells per well on a film disc, add DMEM medium containing 10% fetal bovine serum to 200 μL, and grow cells adherently for 24 hours change fluid;

(5) On the 3rd day after culture, take the films of 3 wells in each group and observe the cell adhesion and growth morphology under a fluorescence microscope; the remaining 3 wells are used for cell proliferation detection experiments, the original medium is discarded, and 100 μL of new medium is added to each well. culture medium and 10 μL MTT reagent, incubate at 37°C for 4 hours, then discard the medium, add 100 μl dimethyl sulfoxide to dissolve the purple crystals on the film, shake on the shaker for 10 minutes, use a microplate reader at 570nm The optical density of each well was detected at the wavelength, and a histogram was drawn to detect the effect of egg membrane composite nano-silver on cell activity.

Results: See Figure 4. Under the microscope, it can be seen that the cells in the three groups all grew well, showing a typical spindle-shaped expansion, and the cell viability test showed that there was no significant difference between the egg membrane composite nano-silver group and the dopamine-treated egg membrane group, suggesting the addition of nano-silver It did not cause obvious cytotoxicity and was relatively safe. Egg membranes treated with dopamine significantly promoted cell growth compared with egg membranes not soaked in dopamine, indicating that dopamine has good biocompatibility.

Embodiment 7: Carrying out the antibacterial performance detection experiment of the egg membrane composite nano-silver thin film that makes

In order to test the antibacterial performance of the prepared egg membrane composite nano-silver film for artificial skin, the following experiments were carried out by using the disc diffusion method:

(1) Experimental grouping: experimental group: egg membrane composite nano-silver film prepared in Example 1; control group: egg membrane treated with sterile filter paper hydrochloric acid, egg membrane treated with dopamine solution;

(2) Make each group of films into sterile discs according to the method in Example 6;

(3) Dilute the bacterial suspension of Escherichia coli and Staphylococcus aureus to 0.5 standard McFarland units (106cfu/ml), and then dip the bacterial solution with a sterile cotton swab and spread it evenly on the MH agar culture dish , each strain was repeated 3 times;

(4) Carefully place each group of film discs on the MH agar culture dish, and place the culture dish in a 37°C incubator;

(5) Take out the petri dish after 24 hours, take pictures and record.

Results: See Figure 5, in the culture dishes of Escherichia coli and Staphylococcus aureus, obvious antibacterial rings appeared around the egg membrane composite nano-silver film, indicating that it has good antibacterial effect on both bacteria.

Example 8: Carry out full-thickness skin defect wound repair experiment on the prepared egg membrane composite nano-silver film

(1) Preparation of full-thickness skin defect wound model in BALB/C mice (purchased from the Experimental Animal Center of Third Military Medical University): The experimental animals were intraperitoneally injected with 1% pentobarbital sodium (100mg/kg, 0.01ml/g) anaesthetization. The mice were fixed in the prone position, and after the skin preparation of the back operation area, routine disinfection with 75% ethanol was performed. A full-thickness skin defect wound with a diameter of 4 mm was created on both sides of the back with a punch. Take 35 BALB/C mice, 7 mice in each of the experimental group and the control group, and prepare wound models according to the above method.

(2) Experimental group: the egg membrane composite nano-silver film sample prepared in Example 1 was cut into a size of 6×6 mm, washed with sterile saline, covered on the wound on the back of the mouse, and then fixed on the back with a negative pressure suction film Skin; control group: Vaseline gauze, egg membrane treated with hydrochloric acid, and egg membrane treated with dopamine were used to cover the wound, and then fixed with negative pressure suction membrane, while the blank control group was fixed only with negative pressure suction membrane.

(3) Immediately after the operation, take pictures and record them as the original wound area on day 0, then open the wounds of mice in each group on days 3 and 7 to check the healing status, and take pictures and record them. After the photographs were taken on the 7th day after the operation, the mice were sacrificed by neck breaking.

(4) Image Pro Plus 6.0 was used to analyze the wound photos of each group on the 3rd and 7th days, and the residual wound area (the area surrounded by the wound margin) was calculated, and the wound healing rate was calculated according to the following formula:

Wound healing rate = (original wound area - residual wound area) / original wound area.

Results: See Figure 6. There was no redness, swelling and allergies on the wound surface of the egg membrane composite nano-silver film group, no exudate, pus and abscess formation, and no hyperemia, edema and necrosis in the surgical area. On the 3rd day after operation, the wound healing rate of the egg membrane composite nano-silver film group was higher than that of the other groups, and there was a statistical difference compared with the blank control group and the Vaseline gauze group; on the 7th day after the operation, the egg membrane composite nano-silver film group The healing rate of the group was higher than that of the other four groups, and the difference was statistically significant. The results indicate that the prepared egg membrane composite nano-silver film for artificial skin can significantly promote wound healing.

Claims (8)

1. the preparation method for the eggshell membrane composite Nano Ag films of artificial skin, it is characterised in that include walking as follows Rapid:

(1) take Fresh Egg, discard Ovum Gallus domesticus album and egg yolk, Ovum crusta Gallus domesticus is placed in deionized water cleaning, separate eggshell membrane, then spend After ionized water cleans, eggshell membrane is cut into 1 3cm × 1 3cm size；

(2) eggshell membrane that step (1) prepares being placed in concentration is 1 100mM, in the hydrochloric acid solution of pH=1 3, soaks under room temperature 14 days, then clean 35 times with deionized water, every all over 35 minutes；

(3) being dissolved in by dopamine in the Tris HCl buffer that concentration is 10 100mM, prepared concentration is that 2 4mg/mL are many Bar amine aqueous solution, by pH regulator to 8.5 9.5；

(4) eggshell membrane that step (2) prepares is placed in the dopamine solution that step (3) prepares, soaks 6 20h under room temperature, so Clean 35 times with deionized water afterwards, every all over 35 minutes；

(5) eggshell membrane that step (4) prepares being placed in concentration is in 10 50nM silver nitrate solutions, and under room temperature, lucifuge soaks 6 20h, then cleans 35 times with deionized water, every all over 35 minutes, and natural drying under room temperature prepares eggshell membrane composite nano silver Thin film.

The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its It is characterised by that the eggshell membrane deionized water that described step (1), step (2) and step (4) prepare cleans 3 times, every all over 5 minutes.

The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its The concentration being characterised by the hydrochloric acid solution of described step (2) is 1mM, pH=3.

The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its The Tris HCl buffer concentration being characterised by described step (3) is 10mM.

The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its It is characterised by that the dopamine solution concentration that described step (3) prepares is 2mg/mL, pH=8.5.

The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its The concentration being characterised by the silver nitrate solution of described step (5) is 30nM.

7. the eggshell membrane composite Nano Ag films for artificial skin prepared by the method described in claim 16 any bar In preparation purposes in the artificial skin treating the skin injury of people or surgical dressing.

Purposes the most according to claim 7, described skin injury is skin full-thickness defects or skin infection wound surface.