Preparation method and application of Pickering emulsion

Abstract

The invention relates to the technical field of preparation of Pickering emulsion, in particular to a preparation method and application of Pickering emulsion, comprising the following steps: A, dissolving giant salamander skin collagen in acetic acid to obtain a giant salamander skin collagen solution, and dialyzing out the acetic acid to obtain a giant salamander skin collagen gel for standby use; B, respectively adding giant salamander bone polypeptide and giant salamander liver oil to the giant salamander skin collagen gel obtained in step A for homogenization to obtain a dispersed Pickering emulsion; the application is used for burns and scalds.

Description

Technical Field

[0001] The invention relates to the technical field of Pickering emulsion preparation, and in particular to a preparation method and application of Pickering emulsion. Background Art

[0002] Burns and scalds are the fourth largest type of human injuries. Due to their high morbidity and mortality, they have become a major public health problem worldwide. At present, burns and scalds are generally treated with anti-inflammatory and moisturizing burn ointments to help recovery, or with drugs that promote the growth of new skin tissue, which will help recovery. For example, erythromycin ointment and mupirocin ointment are applied for treatment, but their efficacy is poor and the wounds recover slowly. Summary of the invention

[0003] In order to solve the above technical problems, an embodiment of the present invention provides a preparation method and application of a Pickering emulsion.

[0004] To achieve the above object, the embodiments of the present invention adopt the following technical solutions:

[0005] On the one hand, the present invention provides a method for preparing a Pickering emulsion, comprising the following steps:

[0006] A. Dissolving giant salamander skin collagen in acetic acid to obtain a giant salamander skin collagen solution, and dialyzing out the acetic acid to obtain a giant salamander skin collagen gel for later use;

[0007] B. Add the giant salamander bone polypeptide and giant salamander liver oil to the giant salamander skin collagen gel obtained in step A and homogenize to obtain a dispersed Pickering emulsion.

[0008] The Pickering emulsion prepared in the examples of the present application has suitable viscosity, is easy to adhere to the wound, can avoid the toxic side effects such as hemolysis and allergies caused by traditional emulsion surfactants, and at the same time has antibacterial and anti-inflammatory effects, removes reactive oxygen species, and promotes wound healing. Among them, the fatty acids that are beneficial to wound healing in giant salamander liver oil are rich and can reduce the inflammatory response of the wound; the giant salamander skin collagen as solid stable particles avoids the toxic side effects of traditional emulsion surfactants and can promote cell proliferation; the giant salamander bone antioxidant polypeptide can remove excess reactive oxygen species produced by oxidative stress during wound healing.

[0009] In some embodiments, in step A, the concentration of the giant salamander skin collagen solution is 70-100 mg / mL.

[0010] In some embodiments, in step A, the method for preparing giant salamander skin collagen comprises the following steps:

[0011] The fat and pigment are removed from the giant salamander skin, and then 0.5 mol / L acetic acid is added to the giant salamander skin according to a solid-liquid ratio of 1:10-30, and then pepsin with a mass fraction of 0.2-2% is added for extraction, filtration and centrifugation, and the supernatant is taken, and 5 mol / L NaCl solution is added to the supernatant to make the concentration of NaCl 0.9 mol / L, and the mixture is allowed to stand for 24 hours to separate layers, and then centrifuged at 8000 r / min for 15 minutes to recover the flocs, and 0.5 mol / L acid is added to the flocs to dissolve them, and the supernatant is taken, and 5 mol / L NaCl is added to the supernatant to make the concentration of NaCl 0.9 mol / L, and the mixture is allowed to stand for 24 hours to separate layers, and then centrifuged at 10000 r / min for 20 minutes to recover the flocs, and the flocs are dialyzed and freeze-dried to obtain giant salamander skin collagen.

[0012] In some embodiments, in step B, the method for preparing giant salamander liver oil comprises the following steps:

[0013] Neutral protease was added to the giant salamander liver at 11000 U / g, and the enzymatic hydrolysis temperature was controlled at 50°C, the enzymatic hydrolysis time was 3 h and the solid-liquid ratio was 1:1.5. Then the pH value was adjusted, the enzyme was inactivated and the upper oil was recovered by centrifugation to obtain crude oil, which needed to be degummed, deacidified and decolorized to obtain giant salamander liver oil.

[0014] In some embodiments, in step B, the method for preparing the giant salamander bone polypeptide comprises the following steps:

[0015] The giant salamander bones were cleaned and pre-treated with decalcification, and the pre-treated giant salamander bones were enzymatically hydrolyzed with alkaline protease at 55°C, 9000 U / g enzyme addition, and pH 12 for 3 h at a solid-liquid ratio of 5 mg / mL. The pH value was adjusted, the enzyme was inactivated, the supernatant was centrifuged, and the filtrate was collected by ultrafiltration membrane interception. After freeze-drying, giant salamander bone polypeptides were obtained.

[0016] In some embodiments, in step B, the mass ratio of the giant salamander skin collagen, giant salamander bone polypeptide and giant salamander liver oil is (0.4-2):0.4:(10-50).

[0017] On the other hand, the Pickering emulsion prepared by the preparation method provided in the examples of the present application is used in the treatment of burns and scalds. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A This is the surface of the scanning electron microscope image of the collagen of the skin of the giant salamander in the embodiment of the present application;

[0019] Figure 1B This is the side view of the scanning electron microscope image of the collagen in the skin of the giant salamander in the example of the present application;

[0020] Figure 2This is a state diagram of the Pickering emulsion of the present application example after storage for 30 days;

[0021] Figure 3 This is a diagram showing the changing state of the Pickering emulsion before and after centrifugation in an embodiment of the present application;

[0022] Figure 4A This is a curve diagram showing the effect of the concentration of collagen from the skin of giant salamander on the particle size of Pickering emulsion in the embodiment of the present application;

[0023] Figure 4B This is a bar graph showing the effect of the concentration of collagen from the skin of giant salamander on the average particle size of Pickering emulsion in the examples of the present application;

[0024] Figure 4C This is a graph showing the effect of different oil-water ratios on the particle size of the Pickering emulsion in the examples of the present application;

[0025] Figure 4D This is a bar graph showing the effect of different oil-water ratios on the average particle size of the Pickering emulsion in the examples of the present application;

[0026] Figure 5A This is a line graph showing the effect of the concentration of collagen in the skin of giant salamander on the ζ-potential of Pickering emulsion in the embodiment of the present application;

[0027] Figure 5B This is a line graph showing the effect of the oil-water phase ratio on the ζ-potential of the Pickering emulsion in the examples of the present application;

[0028] Figure 5C Schematic diagram of Pickering emulsion prepared with different concentrations of giant salamander skin collagen in the examples of the present application;

[0029] Figure 5D Schematic diagram of Pickering emulsions prepared with different oil-water phase ratios in the examples of the present application;

[0030] Fig. 6A This is a scatter diagram of the effect of different concentrations of giant salamander skin collagen on the rheological properties of Pickering emulsion in the examples of this application;

[0031] Figure 6B This is a scatter diagram showing the effect of different oil-water phase ratios on the process performance of Pickering emulsion in the examples of this application;

[0032] Fig. 7A Laser confocal images of Pickering emulsions prepared with different concentrations of giant salamander skin collagen in the examples of this application; giant salamander skin collagen and giant salamander liver oil were stained with Nile blue (green) and Nile red (red), respectively, scale: 50 μm;

[0033] Figure 7BLaser confocal images of Pickering emulsions prepared with different oil-water ratios in the examples of this application; the giant salamander skin collagen and giant salamander liver oil were stained with Nile blue (green) and Nile red (red), respectively, scale: 50 μm;

[0034] Figure 8 This is a bar graph showing the effects of the Pickering emulsion and different components on the viability of L929 cells in the examples of this application (p<0.05);

[0035] Fig. 9A These are the wound appearance pictures of rats scalded on days 0, 7, 14, and 19 in this application example;

[0036] Fig. 9B This is a bar graph of wound healing rates of rat wounds on days 0, 7, 14, and 19 in the examples of this application (p<0.05);

[0037] Fig. 9C This is a diagram of the contraction trajectory of the rat wound at different time periods in the embodiment of the present application;

[0038] Fig. 10A These are H&E staining images of wound tissues of rats at 7, 14, and 19 days after scalding in the examples of this application;

[0039] Fig. 10B The Masson staining images of the wound tissues of rats at 7, 14, and 19 days after scalding in the present application example;

[0040] Fig.11A These are images of TNF-α and IL-6 immunofluorescence staining and H&E staining of rat wounds after 5 days of treatment in the present application example;

[0041] Fig. 11B The fluorescence intensity of TNF-α (B) and IL-6 (p<0.05) of the rat wound surface after 5 days of treatment in the present application example;

[0042] Fig. 11C The fluorescence intensity of IL-6 on the 5th day after wound treatment in rats in the present application example (p<0.05);

[0043] Fig.12 This is a bar graph of the ROS content in the wound tissue of rats on the 5th day after injury in the example of this application (p<0.05);

[0044] Fig.13 This is a bar graph of EGF content in the wound surface of rats scalded on days 7, 14, and 19 in the example of this application (p<0.05);

[0045] Fig.14 These are CD31 immunohistochemical staining images of wound sections of rats scalded on days 7, 14, and 19 in the examples of this application. DETAILED DESCRIPTION

[0046] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in some embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0047] Example

[0048] Burns and scalds are the fourth largest type of human injuries. Due to their high morbidity and mortality, they have become a major public health problem worldwide. At present, burns and scalds are generally treated with anti-inflammatory and moisturizing burn ointments to help recovery, or with drugs that promote the growth of new skin tissue, which will help recovery. For example, erythromycin ointment and mupirocin ointment are applied for treatment, but their efficacy is poor and the wounds recover slowly.

[0049] In order to solve the above technical problems, the present application provides a method for preparing a Pickering emulsion, comprising the following steps:

[0050] A. Dissolve the giant salamander skin collagen in 5 mol / mL acetic acid to obtain a giant salamander skin collagen solution, then dialyze the solution in ultrapure water for three days, changing the water every 6 hours, and after the acetic acid is dialyzed out, the collagen is in a gel state and the acetic acid is dialyzed out to obtain a giant salamander skin collagen gel for later use;

[0051] B. Add the giant salamander bone polypeptide and giant salamander liver oil to the giant salamander skin collagen gel obtained in step A, and homogenize at 10,000 rpm for 5 min using a homogenizer to obtain a dispersed Pickering emulsion.

[0052] The Pickering emulsion prepared in the examples of the present application has suitable viscosity, is easy to adhere to the wound, can avoid the toxic side effects such as hemolysis and allergies caused by traditional emulsion surfactants, and at the same time has antibacterial and anti-inflammatory effects, removes reactive oxygen species, and promotes wound healing. Among them, the fatty acids that are beneficial to wound healing in giant salamander liver oil are rich and can reduce the inflammatory response of the wound; the giant salamander skin collagen as solid stable particles avoids the toxic side effects of traditional emulsion surfactants and can promote cell proliferation; the giant salamander bone antioxidant polypeptide can remove excess reactive oxygen species produced by oxidative stress during wound healing.

[0053] In some embodiments, in step A, the concentration of the giant salamander skin collagen solution is 70-100 mg / mL. Exemplarily, the concentration may be 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL, which is not specifically limited in the embodiments of the present application.

[0054] In some embodiments, in step A, the method for preparing giant salamander skin collagen comprises the following steps:

[0055] Collection of giant salamander skin: Kill healthy second-generation giant salamanders, remove residual meat, and cut into 5mm×5mm pieces for later use.

[0056] Removing fat: After removing the residual meat, put the skin of the giant salamander into 10% by volume isopropanol (solid-liquid ratio is 1:10), soak for 24 hours, change the liquid every 12 hours, and then wash it with tap water five times, and then wash it with ultrapure water five times to remove fat.

[0057] Removing pigments: Soak the giant salamander skin, from which the fat has been removed, in a 0.1 mol / L sodium hydroxide solution, changing the solution every 12 hours for 24 hours. After soaking, wash it five times with tap water and then five times with ultrapure water to remove the pigments.

[0058] Enzymatic extraction of collagen from giant salamander skin: add 0.5 mol / L acetic acid to the giant salamander skin from which fat and pigment have been removed, at a solid-liquid ratio of 1:10-30, and then add 0.2-2% of the weight of giant salamander skin pepsin to assist in extraction, then filter with gauze, adjust the pH of the filtrate to 8, let stand for 10 minutes (to inactivate the enzyme), and then adjust the pH to the original pH with 0.5 mol / L acid. Centrifuge at 8000r / min for 15min, take the supernatant, add 5mol / L NaCl solution to the supernatant to make the NaCl concentration of 0.9mol / L, let stand for 24h to separate layers, centrifuge at 8000r / min for 15min, recover the flocs, add 0.5mol / L acid to the flocs to dissolve them, take the supernatant, add 5mol / L NaCl to the supernatant to make the concentration of 0.9mol / L, let stand for 24h to separate layers, centrifuge at 10000r / min for 20min to recover the flocs, (if the flocs clump, add acetic acid to dissolve them and then dialyze them) dialyze them in distilled water for 3d, change the solution every 24h, put them in a -80℃ refrigerator for 1d after dialysis, and then freeze-dry them to obtain giant salamander skin collagen.

[0059] In some embodiments, in step B, the method for preparing giant salamander liver oil comprises the following steps:

[0060] Preparation method of giant salamander liver oil: The giant salamander liver was transported to the laboratory at -20℃, thawed in tap water, the fascia was removed, cut into small pieces and mixed, and then packaged in 50g portions and stored at -40℃ for later use.

[0061] After the packaged giant salamander liver was thawed, it was broken in a homogenizer and treated with neutral protease at an enzyme addition amount of 11000U / g, an enzymatic hydrolysis temperature of 50℃, an enzymatic hydrolysis time of 3 h and a solid-liquid ratio of 1:1.5. During the process, the pH value was adjusted with 0.5 mol / L acetic acid and 0.5 mol / L sodium hydroxide. After the end, the liver was treated with boiling water for 10 min to inactivate the enzyme. The liver was centrifuged at 10000 r / min for 10 min, and the upper oil was recovered to obtain crude oil.

[0062] Crude oil needs to be degummed, deacidified, decolorized and refined to improve the quality of liver oil.

[0063] Degumming: Weigh 50 g of crude oil into a conical flask, place it in a magnetic stirring bath and heat it to 60°C, add 60% phosphoric acid (2% of the oil weight), and stir at 60°C for 60 min. Then centrifuge at 10,000 rpm for 15 min at room temperature, and the upper layer is the degummed oil.

[0064] Deacidification: weigh 50 g of degummed oil into a conical flask, the amount of alkali added = 7.13 × 10 4 × oil weight × oil acid value + oil weight × 4%, add 20% NaOH solution by mass, place in a heat-collecting constant temperature magnetic stirring bath, and stir at 40°C for 40 min. Centrifuge at 10,000 rpm for 15 min at room temperature, take the upper layer of oil, add 85°C, 10% oil weight deionized water for washing, until the waste liquid after washing is neutral. Then centrifuge at 10,000 rpm for 15 min at room temperature to remove residual water, and the upper layer is the deacidified oil.

[0065] Decolorization: Weigh 50 g of deacidified oil into a conical flask, add activated clay (10% of the oil weight), and stir at 60°C for 30 minutes. Then centrifuge at 10,000 rpm for 15 minutes at room temperature. The upper layer of oil is the decolorized oil.

[0066] In some embodiments, in step B, the method for preparing the giant salamander bone polypeptide comprises the following steps:

[0067] Pretreatment of giant salamander bones: Collect bones and remove residual meat from the surface, chop them into small pieces and grind them in a grinder, place them in a 10-fold volume of 0.5 mol / L sodium hydroxide solution, stir them at 4°C for 24 h, and replace the solution every 12 h. After treatment, wash them in 10-fold volume of ultrapure water, decalcify them in a 20-fold volume of 0.5 mol / L EDTA-2Na (pH=8.0) solution, stir them at 4°C for 24 h, and replace the solution every 12 h.

[0068] Preparation of giant salamander bone polypeptides: The giant salamander bone polypeptides were prepared by enzymatic hydrolysis. The pretreated giant salamander bones were hydrolyzed for 3 h at a solid-liquid ratio of 5 mg / mL using alkaline protease at 55°C, an enzyme addition of 9000 U / g, and a pH value of 12. During the period, the pH value was adjusted using 0.5 mol / L acetic acid and 0.5 mol / L sodium hydroxide solution. After the enzymatic hydrolysis was completed, the enzyme was inactivated by boiling water for 10 min, and the supernatant was taken after centrifugation at 10000 rpm for 10 min.

[0069] Ultrafiltration separation of giant salamander bone polypeptide:

[0070] After enzymatic hydrolysis of giant salamander bones, ultrafiltration membranes with different molecular weights (300D, 1kD, 3kD, 5kD) were used to intercept, and the filtrate was collected and freeze-dried to obtain giant salamander bone polypeptides with molecular weights ranging from 300 to 1kD, 1kD to 3kD, 3kD to 5kD, and greater than 5kD. 300-1000D giant salamander bone polypeptides were selected to prepare Pickering emulsions.

[0071] In some embodiments, the mass ratio of the giant salamander skin collagen, giant salamander bone polypeptide and giant salamander liver oil is (0.4-2):0.4:(10-50). For example, the mass ratio may be 0.4:0.4:10, 1:0.4:30, 2:0.4:50, 1.5:0.4:40, 1.8:0.4:45, which is not specifically limited in the embodiments of the present application.

[0072] 1.1. Screening of Giant Salamander Skin Collagen Concentration (i.e., studying the effect of the amount of Giant Salamander Skin Collagen Gel on Pickering Emulsion in step B)

[0073] The mass ratio of giant salamander skin collagen, giant salamander bone polypeptide and giant salamander liver oil is (0.4~2):0.4:20. The effect of different amounts of giant salamander skin collagen on Pickering emulsion was studied, and its particle size, rheological properties, ζ-potential and stability were determined. The single variable was the amount of giant salamander skin collagen. Therefore, ultrapure water was added to the giant salamander skin collagen gel in step B, and the concentrations of giant salamander skin collagen were respectively configured to be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL and 25 mg / mL. The mass of the fixed oil-water phase was 2:8, and the concentration of giant salamander bone polypeptide was 5 mg / mL to prepare Pickering emulsion.

[0074] 1.2 Screening of Giant Salamander Liver Oil Concentration

[0075] The concentration of giant salamander skin collagen was fixed at 20 mg / mL, and the concentration of giant salamander bone polypeptide was fixed at 5 mg / mL. The mass ratio of giant salamander liver oil: water was set to 1:9, 2:8, 3:7, 4:6, and 5:5, that is, the mass ratio of giant salamander skin collagen, giant salamander bone polypeptide and giant salamander liver oil was 1.6:0.4:(10-50) to prepare Pickering emulsion, and the effect of giant salamander liver oil concentration (single variable, i.e., the amount of giant salamander liver oil) on the properties of Pickering emulsion was studied.

[0076] (1) Pickering emulsion particle size

[0077] The Pickering emulsion was diluted 30 times with ultrapure water, and the particles were dispersed by ultrasound with an ultrasonic power of 150 W for 5 min. The volume average particle size of the emulsion was measured by a Malvern laser particle size analyzer with a measurement range of 0.1-300 μm. Each sample was tested three times and the average value was taken.

[0078] (2) Pickering emulsion ζ-potential

[0079] The Pickering emulsion was diluted 30 times and the ζ-potential was measured using a nanoparticle size potentiometer. Each sample was tested three times and the average value was taken.

[0080] (3) Pickering emulsion stability

[0081] Storage stability: The prepared Pickering emulsion was placed in a 5 mL glass bottle and observed for any oil / water phase separation during the 30-day storage period.

[0082] Centrifugal stability: The prepared Pickering emulsion was placed in a centrifuge tube and centrifuged at 3500 rpm for 5 min to observe whether there was any oil-water phase separation.

[0083] (4) Rheological properties of Pickering emulsion

[0084] The specific operation is as follows: the Pickering emulsion is taken out of the refrigerator at 4 °C and placed for 3 h, the sample is placed on the Malvern rheometer plate, and the shear rate is set from 0.01 to 100 S -1 Gradually increase the shear rate and record the change in the viscosity of the Pickering emulsion as the shear rate increases.

[0085] (5) Laser confocal scanning microscopy

[0086] Laser confocal scanning microscopy was used to locate and visualize the skin collagen of giant salamander and liver oil of giant salamander in Pickering emulsion. 20 μL of Nile red and Nile blue A dye (1 mg / mL) were added to 1 mL of emulsion, incubated overnight in the dark at room temperature, placed on a concave laser confocal microscope slide, and observed with a 400x magnifying glass. Nile red was excited by 405 nm argon laser to observe the skin collagen of giant salamander, and Nile blue A was excited by 633 nm helium-neon (He-Ne) laser to observe the liver oil of giant salamander.

[0087] (6) Data analysis

[0088] Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test using SPSS 25.0. All experiments were repeated three times and the results are presented as mean ± SD.

[0089] 1.3 Results and Analysis

[0090] (1) Characterization of collagen in giant salamander skin

[0091] The formation of Pickering emulsions is affected by factors such as particle type, concentration, size, density, and packaging form. Therefore, we used SEM to observe the microscopic morphology of collagen and investigate its potential for preparing Pickering emulsions. Figure 1A The collagen surface of the giant salamander skin Figure 1B The side of the giant salamander skin collagen protein shows that the surface and side of the giant salamander skin collagen protein have a porous structure, and the pores are distributed between 100 ± 60 μm. The pores are small, and have the characteristics of low density, high specific surface area, high permeability and high encapsulation capacity. Therefore, the giant salamander skin collagen protein has potential interfacial adsorption capacity and is a good material for preparing Pickering emulsions with controllable pore size and shape.

[0092] (2) Pickering emulsion stability

[0093] Storage stability is one of the important indicators to determine whether the Pickering emulsion can be used in practice. The prepared Pickering emulsion was stored at room temperature for 30 days to observe whether there was stratification and oil-water phase precipitation. The results are as follows: Figure 2 shown.

[0094] After the Pickering emulsion was placed for 30 days, a large amount of oil precipitated when the concentration of giant salamander skin collagen was 5 mg / mL. This was because the concentration of giant salamander skin collagen was low. When forming droplets with oil, a small amount of oil failed to combine with giant salamander skin collagen and was precipitated under long-term gravity. In addition, a small amount of oil will precipitate after the Pickering emulsion prepared under high oil-water ratio conditions for 30 days. This is because a large amount of oil is not attached to giant salamander skin collagen, and oil aggregation leads to precipitation. This is consistent with the results of Pickering emulsions prepared by starch and nanocellulose, that is, low concentrations of solid particles or high concentrations of oil phase will affect the stability of Pickering emulsions. Therefore, Pickering emulsions prepared with giant salamander skin collagen concentration greater than 5 mg / mL and oil-water ratio less than 5:5 have better storage stability.

[0095] The stability of Pickering emulsions prepared under different conditions was observed by centrifugation. Figure 3 As shown. When the concentration of giant salamander skin collagen is 5 mg / mL, the centrifugal force causes the upper layer of oil and the lower layer of giant salamander skin collagen to be clearly separated. Although there is no obvious stratification at 10 mg / mL, the color of the lower layer of the centrifuge tube is lighter. As the concentration of giant salamander skin collagen increases further, the Pickering emulsion has no stratification and color difference after centrifugation. In the Pickering emulsions prepared with different oil-water ratios, when the ratio is 1:9, obvious stratification occurs. This is because the oil phase is too small and cannot combine with a sufficient amount of giant salamander skin collagen, resulting in the centrifugal precipitation of giant salamander skin collagen. Similarly, when the oil phase is too high (ratio of 5:5), there is not enough giant salamander skin collagen to combine, and the oil will also be precipitated under centrifugation. Therefore, when the concentration of giant salamander skin collagen is greater than 10 mg / mL and the Pickering emulsion prepared with an oil-water ratio between 2:8 and 4:6 has better centrifugal stability.

[0096] (3) Pickering emulsion particle size

[0097] The droplet size is an important measurement parameter that has a great influence on the characteristics, stability and application of Pickering emulsions. Therefore, we investigated the effects of different concentrations of collagen from giant salamander skin and different mass ratios of oil and water on the particle size of Pickering emulsions.

[0098] When the oil-water ratio was fixed at 2:8, the effect of different concentrations of giant salamander skin collagen on the particle size of Pickering emulsion was as follows: Figure 4A It can be seen that at 5 mg / mL, the Pickering emulsion particle size is the largest. This is because the giant salamander skin collagen particles are not enough to cover the oil-water interface, thus forming larger droplets. As the concentration of giant salamander skin collagen increases, the Pickering emulsion droplets gradually decrease. Figure 4BAs shown in the figure, the minimum value is reached at 20 mg / mL, and the average particle size is 17.88 μm. This is because the increase in the concentration of giant salamander skin collagen can fully react with oil and promote the formation of small droplets. As the concentration of giant salamander skin collagen further increases to 25 mg / mL, the average particle size of Pickering emulsion increases to 41.57 μm. This is because the concentration of giant salamander skin collagen exceeds the maximum value of binding with oil, resulting in the formation of larger particles.

[0099] The concentration of collagen from the skin of giant salamander was fixed at 20 mg / mL. The effects of different oil / water ratios on the particle size of Pickering emulsion were as follows: Figure 4C As shown in Figure 2, when the oil-water ratio is 1:9 and 2:8, the particle size of the Pickering emulsion is small, but when the oil-water ratio continues to increase, the particle size of the Pickering emulsion gradually increases. Figure 4D As shown in the figure, when the oil-water ratio is 1:9 and 2:8, the average particle size of the Pickering emulsion is small, 18.89 μm and 17.88 μm, respectively, and then increases. In addition, most Pickering emulsions are formed by a high oil-water ratio, such as the emulsion formed by pea protein isolate and starch nanocrystals, whose oil phase exceeds 50%. However, Pickering emulsions with too high oil content will cause adverse reactions to the skin, limiting their application. Therefore, the optimal conditions for preparing giant salamander skin collagen-giant salamander liver oil Pickering emulsions are when the concentration of giant salamander skin collagen is 20 mg / mL and the oil-water ratio is 2:8.

[0100] (4) Pickering emulsion ζ-potential

[0101] The concentration of solid particles compared with the oil-water ratio is a key factor affecting the ζ-potential of Pickering emulsions. The ζ-potential can reflect the stability of Pickering emulsions, and its size determines whether the dispersed system can maintain good stability in the application environment. The effect of different concentrations of giant salamander skin collagen compared with different oil-water ratios on the ζ-potential in Pickering emulsions is shown in Figure 2. FIG. 5A to FIG. 5D shown.

[0102] like Figure 5A As shown in the figure, the absolute value of the ζ-potential of the prepared Pickering emulsion decreases with the increase of the concentration of giant salamander skin collagen. When it reaches 15 mg / mL, the potential value is about 5 mV. This is because the increase in protein concentration leads to insufficient electrostatic interaction between droplets to repel each other, thus forming a semi-solid emulsion gel with a certain viscosity. Figure 5C and Figure 5D It can also be seen that the low-concentration Pickering emulsion has a strong flow performance after inversion, while the high-concentration Pickering emulsion does not flow to the bottom of the bottle after inversion and has a certain viscosity. Figure 5BThe effect of different oil-water ratios on the ζ-potential of the Pickering emulsion is shown in the figure. When the ratio is 1:9, 2:8, and 3:7, the potential values ​​are 4.62 mV, 4.92 mV, and 2.18 mV, respectively. When the ratio is increased to 4:6 and 5:5, the potential values ​​are -18.3 and -11.34. This is because the electrostatic repulsion of the increased oil phase is enhanced after it combines with the protein to produce droplets. In addition, studies have also shown that the formation of semi-solid emulsion gel can improve the stability of Pickering emulsions, which can also be reflected in the previous stability test results.

[0103] (5) Rheological properties of Pickering emulsion

[0104] Giant salamander skin collagen is dispersed in the emulsion, forming a three-dimensional structure with oil and water, and has a certain viscosity. The effect of giant salamander skin collagen and oil concentration on the viscosity of Pickering emulsion is shown in Figure 2. Fig. 6A and Figure 6B shown.

[0105] The viscosity of the giant salamander skin collagen-liver oil Pickering emulsion decreases with the increase of shear rate, showing shear thinning. This is because the polymer liquid changes its conformation under the action of shear force during flow and is oriented along the direction of the external force. The relative flow resistance between the polymers after orientation becomes smaller, and the viscosity decreases accordingly. The shear thinning property of the Pickering emulsion is beneficial during smooth extrusion. Fig. 6A In the study, the viscosity of the Pickering emulsion increased with the increase of the concentration of giant salamander skin collagen. This is because the three-dimensional structure of giant salamander skin collagen may have a thickening effect, thus affecting the viscosity of the Pickering emulsion. Figure 6B In the experiment, the initial viscosity of Pickering emulsion increased with the increase of oil-water ratio, and the final viscosity difference became smaller under high shear. Viscosity is an important factor affecting whether the dressing can fit tightly and firmly on the wound. High-viscosity Pickering emulsion can better contact with the wound, while low-particle Pickering emulsion can better deliver drugs to the wound. After comprehensive consideration, the concentration of giant salamander skin collagen was 20 mg / mL and the oil-water ratio was 2:8 as the prescription for preparing burn healing drugs.

[0106] (6) Laser confocal scanning microscopy

[0107] Laser confocal microscopy was used to further study the Pickering emulsion type. Fig. 7A and Figure 7B The red giant salamander liver oil was covered by the green giant salamander skin collagen, indicating that the prepared Pickering emulsion was an oil-in-water type. Fig. 7A It is shown in the figure that as the concentration of collagen in giant salamander skin increases, the number of large droplets gradually decreases, causing the average particle size of the Pickering emulsion to decrease, confirming the results of the particle size experiment. Figure 7BIt shows that with the increase of the oil-water ratio, the binding of Pickering emulsion becomes tighter and agglomerates are formed at a ratio of 4:6. This may be because after the oil phase increases, there is not enough giant salamander skin collagen to adsorb on it, resulting in agglomeration between droplets. Finally, at a ratio of 5:5, larger droplets are formed and the particle size of Pickering emulsion increases.

[0108] In summary, the Pickering emulsion was prepared with giant salamander liver oil as the oil phase and giant salamander skin collagen as the stabilizer. The effects of different concentrations and different oil-water ratios on the particle size, ζ-potential, stability and rheological properties of the Pickering emulsion were studied, and the emulsion type was observed by laser confocal microscopy. The results are as follows:

[0109] (1) The concentration of collagen in giant salamander skin and the ratio of oil to water will affect the stability of the emulsion. At high protein concentration (greater than or equal to 15 mg / mL) and the oil-to-water ratio between 2:8 and 4:6, the Pickering emulsion will not stratify after long-term storage or centrifugation.

[0110] (2) The Pickering emulsion prepared under the conditions of giant salamander skin collagen concentration of 20 mg / mL and oil-water phase ratio of 2:8 had an average particle size of 17.88 μm and a ζ-potential of 4.92 (mV). The particle size was small and the Pickering emulsion was in the form of a semisolid gel.

[0111] (3) The Pickering emulsions prepared under different conditions all have the property of shear thinning. The concentration of collagen from the skin of giant salamander has a greater influence on its viscosity, which increases with the increase of the concentration of collagen from the skin of giant salamander. The oil-water ratio has a smaller influence on its viscosity, and its viscosity only increases slightly with the increase of the oil-water ratio.

[0112] (4) The results of laser confocal microscopy showed that the giant salamander liver oil-giant salamander skin collagen Pickering emulsion was an oil-in-water emulsion.

[0113] 2. Experimental example of repairing deep second degree burns in rats with giant salamander liver oil-giant salamander skin collagen-peptide Pickering emulsion

[0114] Wound healing is a complex biological process. Normal skin has the ability to regenerate itself after injury. Usually, wound healing goes through hemostasis, inflammation, cell proliferation and remodeling. A large amount of ROS will be produced in the early stage of inflammation. High concentrations of ROS-induced inflammation and bacterial infection are the main reasons for prolonged wound healing. Currently, hydrogels are mainly used to load antioxidant active substances to remove high concentrations of reactive oxygen species. These dressings have a multi-purpose effect because they can be applied to almost all areas of the body, cool and cover wounds, and remove heat from wounds through evaporation. However, hydrogel dressings usually have high viscosity. Changing dressings can cause new epithelial damage, delay healing, and cause pain to patients. At the same time, the process of changing hydrogel dressings is very long.

[0115] The micron or submicron particle size and high surface area of ​​the droplets in Pickering emulsion can enhance skin penetration, continuously release active ingredients, achieve targeted delivery, and improve the stability of active ingredients. In addition, it has the advantage of being prepared by biocompatible particle stabilizers, and has attracted attention in the field of local skin applications in recent years. In addition, Pickering emulsion has low viscosity and can reduce secondary damage when changing dressings. Therefore, it is feasible to use Pickering emulsion as a burn repair dressing.

[0116] In the experimental example of the present application, a rat deep second-degree burn model was constructed, and the wound was treated with Pickering emulsion prepared from giant salamander bone polypeptide, giant salamander skin collagen, and giant salamander liver oil, wherein the giant salamander skin collagen was 20 mg / mL, the oil-water ratio was 2:8, and the giant salamander bone polypeptide was 5 mg / mL. The wound healing rate was observed, and the healing mechanism was preliminarily explored through H&E staining of wound tissue, Masson staining, TNF-α, IL-6 immunofluorescence staining, CD31 immunohistochemical staining, ROS reactive oxygen species content determination, and EGF content detection, providing a theoretical basis for the treatment of deep second-degree burns with Pickering emulsion.

[0117] 2.1 Materials and methods

[0118] (1) Main materials

[0119] DAB colorimetric kit, bovine serum albumin, saline, phosphomolybdic acid solution, 5% BSA blocking solution, and TritonX-10 were purchased from Beijing Solebold Technology Co., Ltd. Immunohistochemistry kits were purchased from Wuhan Boste Bioengineering Co., Ltd. Citrate solution repair solution was purchased from Duolaimi Biotechnology Co., Ltd. Hematoxylin, eosin, and Ponceau red staining solutions were purchased from Shanghai Aladdin Reagent Co., Ltd. Anhydrous ethanol, H2O2, xylene, and other reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Cover glass and slides were purchased from Jiangsu Shitai Experimental Equipment Co., Ltd. Anti-fluorescence quenching sealing agent was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Jingwanhong was provided by Guizhou Chenchun Pharmaceutical Chain Co., Ltd.

[0120] (2) Equipment and instruments

[0121] The equipment and instruments used in this chapter include: dehydrator (Excelsior AS, Thermo Fisher Scientific, USA), paraffin embedding machine (YB-6LF, Xiaogan Yaguang Medical Electronic Technology Co., Ltd.), paraffin slicer (HM 340E, Thermo Fisher Scientific, USA), electric constant temperature incubator (DHP-9082, Ningguo Shaying Scientific Instrument Co., Ltd.), fluorescence inverted microscope (AxioVert.A1, Carl Zeiss Group, Germany), pathology scanner (Panoramic MIDI, Jinan Tangier Electronics Co., Ltd.), optical microscope (BH22, Olympus Corporation, Japan), immunohistochemistry pen (FPP06B, Shanghai Biyuntian Biotechnology Co., Ltd.) and microplate reader (MK3, Thermo Fisher Scientific, USA).

[0122] (3) Cytotoxicity

[0123] L929 mouse epithelial fibroblasts were used to detect the cytotoxicity of giant salamander bone polypeptide (BP), giant salamander liver oil (LO), giant salamander skin collagen gel (SC), and Pickering emulsion (PE). The specific operation was as follows: after L929 cells were revived, passaged, and counted, cells in the logarithmic growth phase were selected and cultured at 1×10 4 The cells were added into a 96-well plate at a density of 100 cells / well (100 μL). After culturing for 24 hours, the culture medium was aspirated and 100 μL each of culture medium (Control), BP (5 mg / mL polypeptide dissolved in culture medium), LO (10 mg / mL liver oil dissolved in DMSO), SC (skin collagen gel of giant salamander was immersed in culture medium at 37°C for 24 hours, and the culture medium after immersion was used for the test), and PE (10 mg / mL dissolved in culture medium) were added. After further culturing for 24 hours, cell viability was detected by CCK-8.

[0124] (4) Rat deep second-degree burn model

[0125] 72 SD rats (250 g) were housed in an animal room and fed freely. After 1 week of adaptation, they were weighed. The hair on the back of the rats was carefully shaved with an electric hair clipper, and the remaining hair on the back was gently wiped with a cotton ball moistened with warm water. 24 hours later, 10% chloral hydrate (0.1 mL / 20.0 g) was injected intraperitoneally for anesthesia. The rats were fixed on a wooden board, and a 1.2 cm diameter capillary tube with no plugs at both ends was used. One end was placed on the back of the rat's hind limbs, and the other end was injected with 97-100 ° C hot water. The hot water was poured out after 15 seconds, and one wound was made on each side, forming a circular wound with a diameter of 1.2 cm.

[0126] (5) Animal grouping and drug administration method

[0127] The scalded rats were randomly divided into 6 groups: negative control (normal saline), giant salamander bone polypeptide (BP), liver oil (LO), giant salamander skin collagen gel (SC), Pickering emulsion (PE) and positive control Jingwanhong (JWH). The negative control group was cleaned with normal saline every day, and the wounds of the other groups were cleaned with normal saline before each administration to remove the residual drugs, and then the corresponding drugs were applied to the wounds. The dosage of each group was referred to Table 1. The wounds were kept open after medication. The rats in each group were kept in a single cage, fed and drank water normally, and were administered once a day for 19 consecutive days.

[0128] Table 1 Dosage of drugs for each group of rats

[0129] Control BP LO SC PE JWH Normal saline 100 μL 20 μL 0.10 g 0.10 g 0.10 g

[0130] (6) Observation on burn healing in rats

[0131] Within 19 days after injury, wound photos were taken on days 0, 7, 14, and 19. Image J was used to measure wound healing, and the wound healing rate was calculated as wound healing rate (%) = (original wound area - measured wound area) ÷ original wound area × 100%.

[0132] (7) Animal tissue sampling

[0133] Mice were selected and photographed on the 7th, 14th, and 19th days, and the hair around the wound was removed and the broken hair was wiped off with alcohol cotton. The mice were killed by spinal dislocation, and tissues with a diameter of 4 mm and a depth of 10 mm were drilled from the wound surface of the mice for pathological analysis.

[0134] (8) HE staining

[0135] (8.1) Sectioning: Tissue samples were routinely embedded in paraffin and cut into 4 μm thick slices using a paraffin slicer. The slices were immersed in 40°C warm water to allow the tissue to fully expand, and then excess water was wiped off and dried.

[0136] (8.2) Dewaxing: Soak the sample in xylene for 10 min, then replace the xylene and continue soaking for 10 min to remove the paraffin in the tissue.

[0137] (8.3) Hydration: After dewaxing, the sample was immersed in anhydrous ethanol for 5 min, and then immersed in 95%, 85%, and 75% ethanol for 4 min respectively.

[0138] (8.4) Hematoxylin staining: The hydrated sections were soaked in PBS three times for 5 min each time, then stained in hematoxylin for 10 min. After washing, the sections were differentiated with 1% hydrochloric acid, blueing solution was added to restore the blueing, and then rinsed with distilled water.

[0139] (8.5) Eosin staining: Add eosin for 3 min, followed by gradient washing and dehydration.

[0140] (8.6) Sealing: After dehydration, the sample is immersed in xylene twice and then sealed with a neutral resin.

[0141] (8.7) Microscope observation

[0142] (9) Masson staining

[0143] (9.1) Dewax and hydrate the sections according to the method in (8).

[0144] (9.2) Stain with iron hematoxylin for 5 min, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, and rinse with tap water to turn blue.

[0145] (9.3) Ponceau staining: Use Ponceau acid fuchsin solution for 10 min, then rinse with distilled water.

[0146] (9.4) Phosphomolybdic acid: Treat with 1% phosphomolybdic acid aqueous solution for about 3 min.

[0147] (9.5) Aniline blue staining: No need to wash with water, and re-stain with aniline blue for 5 minutes.

[0148] (9.6) Differentiation: Treat with 1% hydrochloric acid for 1 min.

[0149] (9.7) Sealing: Wash with 95% ethanol I, 95% ethanol II, anhydrous ethanol I, anhydrous ethanol II, and then soak in xylene I and xylene II for 2 min each. After air drying, seal with resin.

[0150] (9.8) Microscopic examination, image acquisition and analysis.

[0151] (10) TNF-α and IL-6 immunofluorescence staining

[0152] (10.1) Dewax and hydrate the sections according to the method in (8).

[0153] (10.2) Immunofluorescence staining: Heat the citrate buffer in a microwave for 5 min → Cool in a 4℃ refrigerator for 20 min → Repeat the above steps 3 times → Wash with PBST 3 times, 3 min each time → PBS for 3 min → Circle the tissue with an immunohistochemistry pen → Soak in 3% H2O for 10 min → Wash with PBS 3 times, 2 min each time → Incubate with 5% BSA for 30 min → Incubate with primary antibody in a 4℃ wet box overnight → Incubate with secondary antibody for 60 min → Seal the slides with sealing glue and let dry in a fume hood.

[0154] (10.3) Microscopic examination.

[0155] (11) ROS in wounds

[0156] The wound skin was collected on the 5th day after scald to determine the content of reactive oxygen in the wound of rats. The fresh tissue samples were washed with PBS, and then 50 mg of tissue samples were weighed and placed in 1 mL of homogenization buffer A. The samples were homogenized thoroughly with a homogenizer, centrifuged at 1000xg and 4°C for 10 min, and the supernatant was taken. 20 μL of the supernatant and 2 μL of DMSO solution were added to a 96-well plate, and the mixture was mixed thoroughly with a pipette, and incubated at 37°C in the dark for 30 min. The fluorescence intensity was detected using a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 610 nm.

[0157] (12) EGF content in wound surface

[0158] Recombinant human epidermal growth factor (EGF) has the effect of wound repair. The rat epidermal growth factor ELISA kit was used to determine the EGF content in the wound tissue on the 7th, 14th and 19th days after scald. The specific operation was to rinse the tissue with pre-cooled PBS to remove residual blood, and then cut the tissue into pieces after weighing. The cut tissue and the corresponding volume of PBS were added to a cryogenic grinder, fully ground on ice, and finally the homogenate was centrifuged at 5000 xg for 10 minutes, and the supernatant was taken for detection.

[0159] (13) CD31 immunohistochemical staining

[0160] (13.1) Dewax and hydrate the sections.

[0161] (13.2) Immunohistochemical staining: Heat in citrate buffer in microwave for 5 min → Cool in refrigerator at 4℃ for 20 min → Repeat the above steps 3 times → Wash with PBS 3 times, 3 min each time → Circle tissue with immunohistochemical pen → Soak in 3% distilled water for 10 min → Wash with PBS 3 times, 2 min each time → Block with serum for 1 h → Incubate with primary antibody overnight in a 4℃ wet box → Incubate with secondary antibody for 60 min → Wash with PBS 3 times, 3 min each time → DAB color development → Hematoxylin staining for 6 min → Rinse with running water for 3 min → Alcohol gradient elution → Transparent in xylene II and xylene I for 5 min each → Seal with sealing glue and air dry in a fume hood.

[0162] (13.3) Microscopic examination.

[0163] (14) Data processing

[0164] Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test using SPSS 25.0. All experiments were repeated three times and the results are presented as mean ± SD.

[0165] 2.2 Results Analysis

[0166] (1) Cytotoxicity

[0167] Effects of giant salamander liver oil-giant salamander skin collagen-giant salamander bone polypeptide Pickering emulsion and its components on the survival rate of L929 cells Figure 8 As shown in the figure, the components of giant salamander liver oil (LO), giant salamander skin collagen gel (SC) and giant salamander bone polypeptide (BP) in the emulsion showed no toxicity to cells, and the cell survival rate was higher than that of the control group, and had a certain proliferation effect. The cell survival rate of the emulsion was the highest. This shows that the prepared Pickering emulsion is non-toxic to cells and has good biocompatibility.

[0168] (2) Wound healing rate of rats

[0169] Effects of giant salamander liver oil-giant salamander skin collagen-giant salamander bone polypeptide Pickering emulsion and its components on the healing of deep second-degree burn wounds in rats Figure 8 The rats were scalded on the back with hot water at 97-100℃ for 15 seconds, resulting in a circular wound with a diameter of 1.2 cm and a slightly raised white wound surface.

[0170] From the contraction trajectory of the wound at different time periods ( Figure 9 C), it can be found that after 7 doses of medication, the edges of the scabs in the BP, LO, PE, and JWH groups lifted and fell off. The scabs in the PE group lifted and began to fall off, and the wound contraction rate was greater than that of the other groups, while the degree of scab lifting in the Control and SC treatment groups was less.

[0171] At the same time, as shown in Figure 9 A, on the 7th day, the wound healing rate of the drug group was higher than that of the Control group, and there was a significant difference. Among them, the PE, BP, LO and JWH groups had significant differences, indicating that after seven days of medication, the drug group can promote the repair of burn wounds, and the repair effect is greater than that of commercially available burn ointment.

[0172] As shown in Figure 9 B, after 14 days of medication, the wound surface of each group of rats had contracted and had formed visible differences. The scabs in the Control group rose and fell off, and the wound surface contracted and healed. The healing rate was only 55.95%, while the wound healing rate in the PE group was 96.71%, the wound healing rates of BP, LO, and SC were 86.3%, 93.27%, and 88.94%, respectively, and the JWH group was 80.4%. The wound healing rates of the drug groups were all greater than those of the commercially available scald ointment group, and there were significant differences. This is because BP has an antioxidant effect, which can remove ROS during wound healing and reduce the occurrence of inflammation. LO has antibacterial and anti-inflammatory effects, and more than 70% of fatty acids have the function of promoting wound healing. Collagen is a commonly used skin scaffold in dressings. Type I collagen is an extracellular matrix component in various connective tissues. It is often used as a scaffold for damaged tissue regeneration and has excellent biocompatibility, biodegradability, and low antigenicity. Since PE combines BP, LO, and SC to form a synergistic effect, it improves the drug efficacy. Close contact with the skin can enhance the drug efficacy and slowly release it, promoting wound healing. After 19 days of medication, the wounds in the PE group had healed completely, and the wound healing rates in the BP, LO, and SC groups were 97%, 98.17%, and 97.08%, respectively, while the healing rates in the JWH and control groups were 95.49% and 92.64%, respectively. The healing rates were similar to the results of Shen et al. These results show that the scald repair function of BP, LOD, and SC is significantly greater than that of the Control and JWH groups, but from the 14th to the 19th day, its repair effect is not much different from that of the Control and JWH. PE's ability to promote the healing of scald wounds is greater than that of the commercial scald ointment group and the control group, and can shorten the wound healing time.

[0173] (3) HE and Masson staining

[0174] Burn healing is a complex process, which mainly includes hemostasis, inflammation, cell proliferation / granulation formation and remodeling stages. The results of HE staining and Masson staining of wound tissue at different time periods are shown in Figure 10. After the 7th day of burn, a large number of fibroblasts grew in each group, there were a large number of gaps in the dermis, and there was inflammatory infiltration. This is due to the necrosis of dermal tissue caused by burns. Neutrophils, as the main regulatory factor in the inflammatory period, transform monocytes into macrophages, and macrophages phagocytize and digest tissue fragments and remaining neutrophils. Among them, the Control group was the most serious, followed by JWH, BP, LO and SC groups. The PE treatment group had clear tissue layers and mild inflammatory infiltration. The bubbles formed after the burn were filled with collagen fibers.

[0175] The 14th day after the burn is in the wound proliferation stage, during which the inflammatory cells decrease, the scab gradually falls off, and the epithelium grows slowly. Fig. 10AAs shown in the figure, the crusts of the Control and JWH treatment groups were thicker, with a large number of inflammatory cells, and fewer fibroblasts in the dermis. The inflammatory response of the BP, LO, SC, and PE treatment groups was milder. Except for the SC treatment group, which had a thicker crust and no epidermal growth, epidermal growth was observed in the other groups. A large number of fibroblasts grew in the dermis of the SC and PE treatment groups, which may be because the presence of collagen in the skin of giant salamanders is conducive to cell growth. In addition, the PE treatment group contained a small amount of hair growth.

[0176] After the 19th day of scald, the wounds in each group had healed, and the epidermal tissue had become thinner than that on the 14th day. The epidermal tissue of the Control group and JWH was thicker, while the epidermis after treatment with BP, LO, SC and PE was thinner. Among them, the epidermis of the PE group was the thinnest, and blood vessels and hair follicles were generated. The wound recovered better and the healing process was basically completed, indicating that PE has a good ability to promote the healing of scald wounds.

[0177] Depend on Fig. 10B It can be seen that on the 7th and 14th days after scald, collagen fibers were deposited in all groups, and the PE and SC groups had the most collagen deposition. On the 19th day, although the collagen deposition in the LO, SC and PE treatment groups was less than that in the control group, the collagen in the dermis and epidermis was arranged in an orderly manner, and the growth of blood vessels and hair follicles was observed in the PE treatment group, indicating that the PE burn repair ability was higher than that of the other groups.

[0178] (4) Immunofluorescence staining of TNF-α and IL-6

[0179] The inflammatory microenvironment plays a key role in wound healing and tissue regeneration. The length of the wound inflammatory phase will affect the transition time from the wound to the proliferation phase. Too long a time will slow down the wound healing rate. TNF-α and IL-6 are typical inflammatory cytokines. Therefore, immunofluorescence staining was used to analyze the content of TNF-α and IL-6 in tissue sections, and HE staining was combined to analyze the inflammatory microenvironment of the wound on the fifth day. The results are as follows: FIG. 11A to FIG. 11C shown.

[0180] like Fig.11A After the treatment of Control and JWH, the burn wound still had a large amount of TNF-α and IL-6 inflammatory cytokines, while the other groups could inhibit the production of TNF-α and IL-6 inflammatory cytokines. Among them, the fluorescence intensity of TNF-α and IL-6 in the wound of rats treated with PE was significantly lower than that in the other groups. To further analyze the intensity of each group, Image J was used to quantify the fluorescence intensity of TNF-α and IL-6. The results are shown in the figure. Fig. 11B and Fig. 11CAs shown. The fluorescence intensity of TNF-α in the BP, LO, SC and PE treatment groups was significantly lower than that in the Control and JWH treatment groups, among which the fluorescence intensity in the PE treatment group was the lowest. In the analysis of IL-6 fluorescence intensity, it was found that the fluorescence in the JWH, BP, LO and SC treatment groups was slightly lower than that in the Control group, but there was no significant difference between them, while the fluorescence intensity in the PE treatment group was the lowest, and there was a significant difference with the other groups. Combined with HE staining analysis, the number of inflammatory cells in the Control group and the JWH group was more than that in the other treatment groups, indicating that the BP, LO and SC groups can reduce the occurrence of inflammation, and the PE treatment group has the mildest inflammatory response. These results show that PE can synergistically exert the efficacy of each component, better reduce the inflammatory response, and promote wound healing.

[0181] (5) Analysis of ROS in wound surface

[0182] When normal skin tissue is damaged, due to the body's defense mechanism, phagocytosis of microorganisms or foreign debris by inflammatory cells may lead to the production of high concentrations of ROS, inducing oxidative stress, leading to a series of adverse effects such as delayed cell behavior, damage to proteins and DNA, prolonged inflammation, reduced re-epithelialization and reduced angiogenesis. In order to reduce the effect of high concentrations of ROS on wound healing in rats, Pickering emulsion was prepared using BP, LO, and SC, and the ROS content in the wound tissue of each group was detected on the 5th day after injury. The results are shown in Figure 2. Fig.12 shown.

[0183] After the burn wound was treated with BP, LO, SC and PE, the ROS content in the wound was significantly reduced. Among them, the ROS content in the PE treatment group was the lowest, but there was no significant difference between the two groups, which showed that the addition of BP was very helpful in removing ROS. The LO and SC treatment groups were also able to reduce the ROS content, probably because oil and collagen had certain antioxidant activity. In addition, combined with the content of inflammatory cytokines, the ROS content in the wound was consistent with the fluorescence intensity trend of TNF-α and IL-6, indicating that PE can remove ROS content, thereby slowing down inflammation and promoting wound healing.

[0184] (6) Determination of EGF content in rat wounds

[0185] EGF is one of the main factors that promote epithelial growth. It is a heat-resistant single-chain polypeptide that can bind to cell membrane receptors and play a variety of biological roles, promoting epithelial cell proliferation and division, improving collagen construction, regulating protein synthesis, and thus accelerating wound healing. The ELISA method was used to detect the EGF content in the wound tissue. The results are as follows Fig.13 shown.

[0186] On the 7th day of healing, the EGF content in the wound tissue of each group was higher than that of the Control group and the JWH group, among which the PE treatment group had the highest content, which was significantly different from the other groups, indicating that PE can heal the wound faster. On the 14th day of healing, there were significant differences between the SC and PE groups and the BP, LO, Control and JWH groups. This may be due to the presence of collagen in the skin of giant salamanders, which formed a strong cross-link with EGF, making the collagen fibers resistant to damage and promoting wound healing. On the 19th day after injury, the EGF content in the wound of the PE treatment group was higher than that of the other groups, and there was a significant difference. In summary, PE can regulate the secretion of EGF, promote epidermal regeneration, and accelerate the healing process of burn wounds.

[0187] (7) CD31 immunohistochemical staining

[0188] Angiogenesis is the key to wound healing. It is the result of multiple signals acting on endothelial cells. In the process of angiogenesis, the formation of new blood vessels occurs through the budding or sprouting mechanism of intact blood vessels at the edge of the wound, and requires endothelial cell proliferation. CD31 is a marker protein of endothelial cells and is mainly expressed in endothelial cells. Therefore, immunohistochemical staining was used to observe the effects of different drugs on the expression of CD31 protein in the wound surface and to study the angiogenesis of the wound surface. The results are as follows: Fig.14 shown.

[0189] On the 7th day, the expression of CD31 in the wound after PE treatment was lower than that in the other groups. This is because after the skin undergoes deep second-degree burns, the blood vessels and hair follicles in the dermis are damaged. During the healing process, the necrotic part needs to be digested and absorbed before new tissues and organs can grow. This shows that the necrotic blood vessels in the wound tissue of the PE treatment group have been absorbed, and wound healing has entered the next stage.

[0190] On the 14th day after injury, after PE treatment, a large amount of CD31 was expressed on the wound surface, while only a small amount was expressed in the other groups. On the 19th day after injury, a large amount of CD31 was expressed in the BP, LO, SC and PE groups. Among them, the CD31 content in the wound surface after PE treatment was higher than that in the other groups, while the CD31 expression in the wound surface of the Control and JWH groups was less, which shows that BP, LO and SC can promote blood vessel growth, and PE combines the three well, plays a synergistic role, and promotes blood vessel growth to the greatest extent, thereby promoting wound repair.

[0191] In summary, the present application example constructs a rat deep second-degree burn model and treats it with Pickering emulsion, which is prepared from giant salamander bone antioxidant polypeptides that can remove ROS, giant salamander liver oil with burn repair potential, and giant salamander skin collagen, and can adhere closely to the skin to create a moist environment that is conducive to wound recovery. The results are as follows:

[0192] (1) In vitro cytotoxicity tests showed that BP, LO, SC and emulsion were non-toxic to L929 cells and could promote the proliferation of L929 cells. The effect of PE on the proliferation of L929 cells was significantly greater than that of the other groups.

[0193] (2) Images of the wounds of rats were taken at different time periods and their healing rates were analyzed. The results showed that the wounds of rats treated with Pickering emulsion had a higher healing rate than those of the other groups at all stages. The wounds of rats treated with BP, LO and SC had a higher healing rate than those of the Control group and the JWH group.

[0194] (3) H&E staining and Masson staining showed that Pickering emulsion treatment can reduce inflammatory response, promote angiogenesis and epidermal regeneration, and increase collagen deposition.

[0195] (4) Immunofluorescence staining analysis showed that Pickering emulsion could inhibit the expression of TNF-α and IL-6 and reduce inflammatory response. The determination of ROS content in the wound surface showed that Pickering emulsion could remove reactive oxygen species, reduce inflammation and promote wound healing.

[0196] (5) Pickering lotion can regulate the secretion of EGF in wound tissue and promote epidermal growth. Immunohistochemical staining analysis showed that Pickering lotion can promote the expression of CD31 and promote blood vessel growth, thereby promoting wound repair.

[0197] In the description of this specification, specific features, structures, materials or characteristics may be combined in an appropriate manner in any one or more embodiments or examples.

[0198] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a Pickering emulsion, characterized in that: The following steps are involved: A. Dissolving giant salamander skin collagen in acetic acid to obtain a giant salamander skin collagen solution, and dialyzing out the acetic acid to obtain a giant salamander skin collagen gel for later use; The concentration of the giant salamander skin collagen solution is 70-100 mg / mL; The method for preparing giant salamander skin collagen comprises the following steps: removing fat and pigment from giant salamander skin, then adding 0.5 mol / L acetic acid to the giant salamander skin according to a solid-liquid ratio of 1:10-30, then adding pepsin with a mass fraction of 0.2-2%, extracting, filtering and centrifuging, taking the supernatant, adding 5 mol / L NaCl solution to the supernatant to make the concentration of NaCl 0.9 mol / L, standing for 24 hours to separate layers, then centrifuging at 8000 r / min for 15 minutes, recovering flocs, adding 0.5 mol / L acid to the flocs to dissolve them, taking the supernatant, adding 5 mol / L NaCl to the supernatant to make the concentration of NaCl 0.9 mol / L, standing for 24 hours to separate layers, then centrifuging at 10000 r / min for 20 minutes to recover the flocs, and dialyzing and freeze-drying them to obtain giant salamander skin collagen; B. Add the giant salamander bone polypeptide and giant salamander liver oil to the giant salamander skin collagen gel obtained in step A and homogenize to obtain a dispersed Pickering emulsion; The preparation method of giant salamander liver oil comprises the following steps: Add 11000 U / g of neutral protease to the liver of giant salamander, and control the enzymatic hydrolysis temperature to 50℃, the enzymatic hydrolysis time to 3 h and the solid-liquid ratio to 1:1.5, then adjust the pH value, inactivate the enzyme and centrifuge to recover the upper oil to obtain crude oil, which needs to be degummed, deacidified and decolorized to obtain giant salamander liver oil; The method for preparing the giant salamander bone polypeptide comprises the following steps: The giant salamander bones were cleaned and pre-treated with decalcification, and the pre-treated giant salamander bones were enzymatically hydrolyzed with alkaline protease at a solid-liquid ratio of 5 mg / mL at 55°C, an enzyme addition amount of 9000 U / g, and a pH value of 12 for 3 h, the pH value was adjusted, the enzyme was inactivated, the supernatant was centrifuged, and the filtrate was collected by ultrafiltration membrane interception, and the giant salamander bone polypeptide was obtained after freeze-drying; The mass ratio of the giant salamander skin collagen, giant salamander bone polypeptide and giant salamander liver oil is (0.4-2):0.4:(10-50); The concentration of the giant salamander skin collagen is 15-25 mg / mL, and the oil-to-water ratio is between 2:8 and 4:

6.

2. A Pickering emulsion prepared by the preparation method of claim 1, characterized in that: The Pickering lotion is used for burns and scalds.

Images