A SOD-containing lipid colloid and its preparation method, use method and application in chronic wound healing

Abstract

The invention relates to the field of colloid technology, and in particular to a SOD-containing lipid colloid and a preparation method, a use method and an application thereof in chronic wound healing. The SOD-containing lipid colloid of the invention adopts superoxide dismutase SOD as a basis, uses a high molecular polymer material with excellent biocompatibility prepared by lecithin, cholesterol and polyethylene glycol 400 as a carrier, combines chitosan and propolis, and is prepared into nano liposomes loaded with active ingredients, and is prepared into a novel dressing with multiple functions such as being able to fuse and penetrate biological membranes into damaged cells, scavenging oxygen free radicals, reducing oxidative damage to cells and tissues, thereby improving inflammatory reactions, promoting healing, inhibiting the development of lesions, etc., and becomes a continuous or responsive high-efficiency drug delivery system.

Description

Technical Field

[0001] The invention relates to the technical field of colloids, and in particular to a SOD-containing lipid colloid, a preparation method thereof, a use method thereof and application in chronic wound healing. Background Art

[0002] With the aging of Chinese society, chronic and difficult-to-heal wounds have become a social problem that cannot be ignored. Not only is the incidence of classic chronic wounds such as diabetic foot ulcers, lower limb ulcers and pressure sores increasing rapidly, but the number of patients with traumatic wounds, postoperative wounds and cancerous wounds is also large. However, due to the lack of professional wound management personnel and backward models, the huge demand cannot be met and many patients cannot receive timely and correct treatment, which aggravates the problem of chronic wounds.

[0003] In clinical practice, wounds that do not heal and have no tendency to heal after one month of regular treatment are usually called chronic wounds. Chronic wounds have many causes, and their pathogenesis is complex, the course of the disease is long, and many disciplines are involved. Studies have shown that chronic wounds delay healing and are susceptible to infection by pathogens such as Staphylococcus aureus, Escherichia coli, and Enterobacter cloacae. Biofilms can form and adhere to the wound, leading to chronic infection. At the same time, studies have also shown that excessive free radicals produced in the body due to various reasons, especially superoxide anion free radicals, are related to many diseases such as inflammation, radiation sickness, autoimmune diseases, tumors and aging. SOD is an antioxidant enzyme present in organisms and a superoxide anion free radical scavenger. It can decompose highly active peroxides (reactive oxygen species, ROS) into less active oxygen and hydrogen peroxide, maintain the redox potential of physiological conditions, and prevent ROS from oxidizing and damaging membranes and proteins. However, the current medical application of SOD is restricted by poor stability and difficulty in preservation, and cannot be effectively used in chronic wound healing. Summary of the invention

[0004] The purpose of the present invention is to provide a SOD-containing lipid colloid and its preparation method, use method and application in chronic wound healing in view of the deficiencies in the prior art. The colloid has good stability and is effective in chronic wound healing.

[0005] The purpose of the present invention is achieved through the following technical solutions: A SOD-containing lipid colloid, comprising the following raw materials in parts by weight: Superoxide dismutase SOD 0.5-2.5 parts 1,2-Hexanediol 2.5-6.5 parts Lecithin 46.0 parts - 65.0 parts Cholesterol 5.5-7.5 parts Polyethylene glycol 400 9.5 parts - 16.5 parts Chitosan 5.5-9.5 parts Tween-80 3.5 parts - 6.5 parts Propolis 0.9 parts - 4.0 parts.

[0006] Preferably, it comprises the following raw materials in parts by weight: Superoxide dismutase SOD 1.5 parts 4.5 parts of 1,2-hexanediol 60.0 parts of lecithin Cholesterol 6.0 Macrogol 400 11 parts Chitosan 7 parts Tween-80 6 parts Propolis 4.0 parts.

[0007] The preparation method of the above-mentioned SOD-containing lipid colloid comprises the following preparation steps: Step (1): drying the formulated amount of 1,2-hexanediol at 65°C in a forced air drying oven to remove moisture, adding the formulated amount of SOD, and mixing in a shaker at -20°C until coagulation to obtain a SOD embedding material for later use, so that SOD can be embedded in 1,2-hexanediol to improve the stability of SOD; Step (2): dissolving the formulated amount of lecithin, the formulated amount of cholesterol and 20% of the formulated weight of polyethylene glycol 400 in 50-100 parts of anhydrous ethanol and mixing them evenly; transferring to a rotary vacuum evaporator at -1.5 MPa to evaporate until a uniform lipid film is formed on the bottle wall, adding 10-15 parts of citric acid-citric acid buffer, gently shaking until completely hydrated, sonicating in a cold water bath for 5-10 minutes, and filtering through a 0.45 mm filter membrane to obtain blank liposomes; Step (3): The SOD encapsulated material of step (1) is sealed in a refrigerator and allowed to warm up, and then slowly added dropwise to the blank liposomes of step (2), and sodium bicarbonate solution is added dropwise to make the pH value of the system 7.0, and the mixture is shaken at 45°C for 1-2 hours to obtain SOD liposomes; Step (4): add a formulated amount of propolis to the remaining polyethylene glycol, mix evenly by ultrasonication at 55°C to obtain propolis-polyethylene glycol; swell a formulated amount of chitosan in 55-95 parts of 75°C water for 3-6 hours, freeze-thaw repeatedly at -20°C for 6-8 times, add a formulated amount of Tween-80, stir in a water bath at 75°C until completely dissolved, add propolis-polyethylene glycol, and stir to obtain a uniform colloid; after the uniform colloid is cooled to room temperature, dropwise add the SOD liposomes obtained in step (3) under slow stirring, and stir evenly to obtain a SOD-containing lipid colloid.

[0008] Preferably, the concentration of the citric acid-citric acid buffer is 0.1 M and the pH is 4.4.

[0009] Preferably, the mass percentage concentration of the sodium bicarbonate solution is 5%.

[0010] The method for using the SOD-containing lipid colloid is to use the SOD-containing lipid colloid in combination with ultraviolet irradiation.

[0011] The use of the SOD-containing lipid colloid is to apply the SOD-containing lipid colloid to chronic wound healing.

[0012] The superoxide dismutase (SOD) of the present invention removes oxygen free radicals and reduces oxidative damage to cells and tissues, thereby improving inflammatory reactions, promoting healing, and inhibiting the development of lesions.

[0013] The 1,2-hexanediol of the present invention captures water molecules through the hydrophilic group, reduces the hydration degree around SOD, thereby reducing the hydrolysis of SOD and improving the stability of SOD.

[0014] The lecithin of the present invention increases the phase transition temperature of liposomes, supports the formation of lamellar lipid bilayer structures and stabilizes the structural arrangement thereof.

[0015] The cholesterol of the present invention has strong membrane fusibility and promotes the intracellular uptake and cytoplasmic entry of SOD.

[0016] The polyethylene glycol 400 of the present invention moistens the propolis to increase its viscosity, improves its hydrophilicity, avoids rapid clearance by the immune system, prevents particle aggregation, and increases stability.

[0017] The chitosan of the invention is used as a slow-release and controlled-release carrier to control the release of liposomes.

[0018] The Tween-80 of the invention improves the fluid plasticity of the colloid, increases the gap between propolis in the colloid, and solidifies the surface tension of the colloid, so that the liposome can be evenly dispersed therein to form a kinetically stable system.

[0019] The propolis of the invention has the function of softening and degrading biological membranes, and promotes liposomes to quickly pass through the biological membranes and enter damaged cells.

[0020] Beneficial effects of the present invention: The present invention provides a SOD-containing lipid colloid, which is based on superoxide dismutase SOD, uses a high molecular polymer material with excellent biocompatibility prepared from lecithin, cholesterol and polyethylene glycol 400 as a carrier, combines chitosan and propolis, and is prepared into nanoliposomes loaded with active ingredients. The new dressing has multiple functions such as being able to fuse and penetrate biological membranes into damaged cells, scavenging oxygen free radicals, reducing oxidative damage to cells and tissues, thereby improving inflammatory reactions, promoting healing, inhibiting the development of lesions, etc., and becomes a continuous or responsive high-efficiency drug delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The antibacterial performance diagram of the colloid is shown; Figure 2 This is a diagram showing the effect of colloid in promoting chronic wound healing. DETAILED DESCRIPTION

[0022] The present invention is further described in conjunction with the following examples.

[0023] Example 1

[0024] A SOD-containing lipid colloid, comprising the following raw materials in parts by weight: Superoxide dismutase SOD 0.5 parts 2.5 parts of 1,2-hexanediol 46.0 parts of lecithin Cholesterol 5.5 parts 9.5 parts of polyethylene glycol 400 Chitosan 5.5 parts Tween-80 3.5 parts 0.9 parts of propolis.

[0025] The preparation method of the above-mentioned SOD-containing lipid colloid comprises the following preparation steps: Step (1): drying the formulated amount of 1,2-hexanediol in a forced air drying oven at 65°C to remove moisture, adding the formulated amount of SOD, and mixing in a shaker at -20°C until coagulation to obtain a SOD embedding material for later use; Step (2): add the formulated amount of lecithin, the formulated amount of cholesterol and 20% of the formulated weight of polyethylene glycol 400 into 50 parts of anhydrous ethanol and dissolve them, and mix them evenly; transfer them to a rotary vacuum evaporator at -1.5 MPa to evaporate until a uniform lipid film is formed on the bottle wall, add 10 parts of citric acid-citric acid buffer, gently shake until completely hydrated, perform sonication in a cold water bath for 5 minutes, and filter through a 0.45 mm filter membrane to obtain blank liposomes; Step (3): The SOD encapsulated material of step (1) is sealed in a refrigerator and allowed to warm up, and then slowly added dropwise to the blank liposomes of step (2), and sodium bicarbonate solution is added dropwise to make the pH value of the system 7.0, and the mixture is shaken at 45°C for 1 hour to obtain SOD liposomes; Step (4): add the formulated amount of propolis to the remaining polyethylene glycol, mix evenly by ultrasonication at 55°C to obtain propolis-polyethylene glycol; swell the formulated amount of chitosan in 55 parts of 75°C water for 3 hours, freeze-thaw repeatedly at -20°C for 6 times, add the formulated amount of Tween-80, stir in a water bath at 75°C until completely dissolved, add propolis-polyethylene glycol, and stir to obtain a uniform colloid; after the uniform colloid is cooled to room temperature, dropwise add the SOD liposomes obtained in step (3) under slow stirring, and stir evenly to obtain a SOD-containing lipid colloid.

[0026] The concentration of the citric acid-citric acid buffer is 0.1 M, and the pH is 4.4.

[0027] The use of the SOD-containing lipid colloid is to apply the SOD-containing lipid colloid to chronic wound healing.

[0028] Example 2

[0029] A SOD-containing lipid colloid, comprising the following raw materials in parts by weight: Superoxide dismutase SOD 1.5 parts 4.5 parts of 1,2-hexanediol 60.0 parts of lecithin Cholesterol 6.0 Macrogol 400 11 parts Chitosan 7 parts Tween-80 6 parts Propolis 4.0 parts.

[0030] The preparation method of the above-mentioned SOD-containing lipid colloid comprises the following preparation steps: Step (1): drying the formulated amount of 1,2-hexanediol in a forced air drying oven at 65°C to remove moisture, adding the formulated amount of SOD, and mixing in a shaker at -20°C until coagulation to obtain a SOD embedding material for later use; Step (2): add the formulated amount of lecithin, the formulated amount of cholesterol and 20% of the formulated weight of polyethylene glycol 400 into 80 parts of anhydrous ethanol and dissolve them, and mix them evenly; transfer them to a rotary vacuum evaporator at -1.5 MPa to evaporate until a uniform lipid film is formed on the bottle wall, add 12 parts of citric acid-citric acid buffer, gently shake until completely hydrated, perform ultrasound in a cold water bath for 8 minutes, and filter through a 0.45 mm filter membrane to obtain blank liposomes; Step (3): The SOD encapsulated material of step (1) is sealed in a refrigerator and allowed to warm up, and then slowly added dropwise to the blank liposomes of step (2), and sodium bicarbonate solution is added dropwise to make the pH value of the system 7.0, and the mixture is shaken at 45°C for 2 hours to obtain SOD liposomes; Step (4): add the formulated amount of propolis to the remaining polyethylene glycol, mix evenly by ultrasonication at 55°C to obtain propolis-polyethylene glycol; swell the formulated amount of chitosan in 70 parts of 75°C water for 5 hours, freeze-thaw repeatedly at -20°C for 7 times, add the formulated amount of Tween-80, stir in a water bath at 75°C until completely dissolved, add propolis-polyethylene glycol, and stir to obtain a uniform colloid; after the uniform colloid is cooled to room temperature, dropwise add the SOD liposomes obtained in step (3) under slow stirring, and stir evenly to obtain a SOD-containing lipid colloid.

[0031] The concentration of the citric acid-citric acid buffer is 0.1 M, and the pH is 4.4.

[0032] The use of the SOD-containing lipid colloid is to apply the SOD-containing lipid colloid to chronic wound healing.

[0033] Example 3

[0034] A SOD-containing lipid colloid, comprising the following raw materials in parts by weight: Superoxide dismutase SOD 2.5 servings 6.5 parts of 1,2-hexanediol 65.0 parts of lecithin Cholesterol 7.5 parts 16.5 parts of polyethylene glycol 400 Chitosan 9.5 parts Tween-80 6.5 parts Propolis 4.0 parts.

[0035] The preparation method of the above-mentioned SOD-containing lipid colloid comprises the following preparation steps: Step (1): drying the formulated amount of 1,2-hexanediol in a forced air drying oven at 65°C to remove moisture, adding the formulated amount of SOD, and mixing in a shaker at -20°C until coagulation to obtain a SOD embedding material for later use; Step (2): adding a formula amount of lecithin, a formula amount of cholesterol and 20% of the formula weight of polyethylene glycol 400 to 100 parts of anhydrous ethanol to dissolve and mix evenly; transferring to a rotary vacuum evaporator at -1.5MPa to evaporate until a uniform lipid film is formed on the bottle wall, adding 15 parts of a citric acid-citric acid buffer system, wherein 1-2 parts by weight of silk fibroin are dissolved in the system, gently shaking until completely hydrated, ultrasonicating in a cold water bath for 10 minutes, and filtering through a 0.45mm filter membrane to obtain a composite blank liposome, which can further reduce the hydrolysis of SOD and improve the stability of SOD; Step (3): The SOD encapsulated material of step (1) is sealed in a refrigerator and allowed to warm up, and then slowly added dropwise to the composite blank liposomes of step (2), and sodium bicarbonate solution is added dropwise to make the pH value of the system 7.0, and the mixture is shaken at 45°C for 2 hours to obtain SOD liposomes; Step (4): add the formulated amount of propolis to the remaining polyethylene glycol, mix evenly by ultrasonication at 55°C to obtain propolis-polyethylene glycol; swell the formulated amount of chitosan in 95 parts of 75°C water for 6 hours, freeze-thaw repeatedly at -20°C for 8 times, add the formulated amount of Tween-80, stir in a water bath at 75°C until completely dissolved, add propolis-polyethylene glycol, and stir to obtain a uniform colloid; after the uniform colloid is cooled to room temperature, dropwise add the SOD liposomes obtained in step (3) under slow stirring, and stir evenly to obtain a SOD-containing lipid colloid.

[0036] The concentration of the citric acid-citric acid buffer is 0.1 M, and the pH is 4.4.

[0037] The use of the SOD-containing lipid colloid is to apply the SOD-containing lipid colloid to chronic wound healing.

[0038] Comparative Example 1

[0039] A SOD-containing lipid colloid, comprising the following raw materials in parts by weight: Superoxide dismutase SOD 1.5 parts 4.5 parts of 1,2-hexanediol 8.8 parts of polyethylene glycol Chitosan 7 parts Tween-80 6 parts Propolis 4.0 parts.

[0040] The preparation method of the above-mentioned SOD-containing lipid colloid comprises the following preparation steps: Step (1): drying the formulated amount of 1,2-hexanediol in a forced air drying oven at 65°C to remove moisture, adding the formulated amount of SOD, and mixing in a shaker at -20°C until coagulation to obtain a SOD embedding material for later use; Step (2): add a formulated amount of propolis to 8.8 parts of polyethylene glycol, mix evenly under ultrasonication at 55°C to obtain propolis-polyethylene glycol; swell a formulated amount of chitosan in 70 parts of 75°C water for 5 hours, freeze-thaw repeatedly at -20°C for 7 times, add a formulated amount of Tween-80, stir in a water bath at 75°C until completely dissolved, add propolis-polyethylene glycol, and stir to obtain a uniform colloid; after the uniform colloid is cooled to room temperature, drop the SOD embedding material obtained in step (1) into it while slowly stirring, and stir evenly to obtain a SOD colloid.

[0041] The use of the SOD-containing colloid is to apply the SOD-containing colloid to the healing of chronic wounds.

[0042] Comparative Example 2

[0043] The difference between the present invention and Example 2 is that when used, the SOD-containing lipid colloid is used in combination with ultraviolet irradiation, rather than being used alone as in Example 2.

[0044] Experimental part:

[0045] In this experiment, the colloid of comparative example 1 is referred to as sample 0, the colloid of embodiment 2 is referred to as sample 1, and comparative example 2 is referred to as sample 1+Light.

[0046] 1. Evaluation of the antibacterial properties of colloids

[0047] An ideal chronic wound dressing should have excellent broad-spectrum antibacterial ability. In this experiment, sample 0, sample 1, and sample 1+Light were incubated with E. coli, S. aureus, and P. aeruginosa suspensions for 24 h, and 70 μL of bacterial suspension was evenly applied to the solid culture medium. After another 24 h of incubation, the growth of the colonies on the agar plate was observed and recorded. In addition, 100 μL of E. coli, S. aureus, and P. aeruginosa bacterial suspensions were taken, and the OD600 value was tested with a UV spectrophotometer, and the antibacterial rate of different hydrogels against the three bacteria was calculated.

[0048] Depend on Figure 1 (a) It can be seen that no colonies appeared on the agar plates of the three bacteria treated with sample 1+Light group; a small number of colonies appeared on the agar plates of the three bacteria treated with sample 1 group; a large number of bacterial colonies appeared in both sample 0 group and Control group, but the number of colonies in sample 0 was significantly less than that in the Control group. This is because sample 1+Light group achieved a very good antibacterial effect through the combined treatment of photothermal and antibacterial agents; the antibacterial ability of sample 1 group was better than that of sample 0 group because the blank lipids formed by lecithin, cholesterol and polyethylene glycol were added, which reduced the hydrolysis of SOD and improved the stability of SOD.

[0049] Depend on Figure 1 (bd) It can be seen that the survival rates of E. coli, S. aureus and P. aeruginosa treated by sample 1+Light group were 3.4%, 2.9% and 2.0% respectively; the survival rates of the three bacteria treated by sample 1 group were 10.2%, 11.8% and 19% respectively; the survival rates of the three bacteria treated by sample 0 group were 66.5%, 42.0% and 80.8% respectively. This colloid achieved a comprehensive antibacterial effect through the synergistic treatment of photothermal and antibacterial agents.

[0050] 2. Colloid promotes the healing effect of chronic wounds

[0051] Depend on Figure 2(a) It can be seen that, except for the negative control group (clean wounds cleaned with 0.9% saline), the other four groups were all seriously infected wounds on day 0; after 3 days, the infection degree of the wounds in the positive control group was still very serious, while the wounds in the colloid group and the negative control group had scabs, but the scab thickness of the sample 0 group was the thinnest, and the wound size did not change. The wounds of sample 1, sample 1+Light and the negative control group not only scab but also contracted to a certain extent; after 7 days, the infection in the positive control group was relieved due to autoimmunity, and the wounds began to scab. The wound healing area of ​​sample 1, sample 1+Light and the negative control group increased significantly, and the healing speed of sample 1+Light and the negative control group was similar, proving that the good antibacterial effect of the material in the early stage promoted the rapid healing of the wound and achieved the effect of synchronous healing with the uninfected wound; after 14 days, the new full-thickness skin tissue of sample 1+Light and the negative control group almost filled the entire wound area, while the positive control group and sample 0 group did not heal completely. The results show that colloid dressings with good antibacterial properties can effectively promote the healing of chronic wounds in diabetic mice.

[0052] The healing of chronic wounds treated with different materials was then evaluated by hematoxylin and eosin staining (H&E staining) at 3, 7, and 14 days. Figure 2 (b) As can be seen, as time goes by, the inflammation of each wound is improved due to the body's own immune function and the antibacterial effect of the material. And the number of inflammatory cells in the sample 1+Light group is significantly reduced compared with the sample 1 group and the sample 0 group, proving that the colloid has greatly reduced the infection of the wound site through the combined treatment of photothermal and chemical therapy, and has made a huge contribution to the wound healing process. And at 3 days and 7 days, the inflammation in the sample 1+Light group is lighter than that in the negative group because the colloid dressing can fit the wound tightly and protect the wound from bacterial invasion, thereby reducing the occurrence of inflammation and promoting wound healing.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A SOD-containing lipid colloid, characterized in that: It includes the following raw materials in parts by weight: Superoxide dismutase SOD 0.5-2.5 parts 1,2-Hexanediol 2.5-6.5 parts Lecithin 46.0 parts - 65.0 parts Cholesterol 5.5-7.5 parts Polyethylene glycol 400 9.5 parts - 16.5 parts Chitosan 5.5-9.5 parts Tween-80 3.5 parts - 6.5 parts Propolis 0.9 parts - 4.0 parts.

2. A SOD-containing lipid colloid according to claim 1, characterized in that: It includes the following raw materials in parts by weight: Superoxide dismutase SOD 1.5 parts 4.5 parts of 1,2-hexanediol 60.0 parts of lecithin Cholesterol 6.0 parts Macrogol 400 11 parts Chitosan 7 parts Tween-80 6 parts Propolis 4.0 parts.

3. A method for preparing a SOD-containing lipid colloid as claimed in claim 1, characterized in that: It includes the following preparation steps: Step (1): drying the formulated amount of 1,2-hexanediol in a forced air drying oven at 65°C to remove moisture, adding the formulated amount of SOD, and mixing in a shaker at -20°C until coagulation to obtain a SOD embedding material for later use; Step (2): dissolving the formulated amount of lecithin, the formulated amount of cholesterol and 20% of the formulated weight of polyethylene glycol 400 in 50-100 parts of anhydrous ethanol and mixing them evenly; transferring to a rotary vacuum evaporator at -1.5 MPa to evaporate until a uniform lipid film is formed on the bottle wall, adding 10-15 parts of citric acid-citric acid buffer, gently shaking until completely hydrated, sonicating in a cold water bath for 5-10 minutes, and filtering through a 0.45 mm filter membrane to obtain blank liposomes; Step (3): The SOD encapsulated material of step (1) is sealed in a refrigerator and allowed to warm up, and then slowly added dropwise to the blank liposomes of step (2), and sodium bicarbonate solution is added dropwise to make the pH value of the system 7.0, and the mixture is shaken at 45°C for 1-2 hours to obtain SOD liposomes; Step (4): add a formulated amount of propolis to the remaining polyethylene glycol, mix evenly by ultrasonication at 55°C to obtain propolis-polyethylene glycol; swell a formulated amount of chitosan in 55-95 parts of 75°C water for 3-6 hours, freeze-thaw repeatedly at -20°C for 6-8 times, add a formulated amount of Tween-80, stir in a water bath at 75°C until completely dissolved, add propolis-polyethylene glycol, and stir to obtain a uniform colloid; after the uniform colloid is cooled to room temperature, dropwise add the SOD liposomes obtained in step (3) under slow stirring, and stir evenly to obtain a SOD-containing lipid colloid.

4. The method for preparing a SOD-containing lipid colloid according to claim 1, wherein: The concentration of the citric acid-citric acid buffer is 0.1 M, and the pH is 4.

4.

5. A method for using the SOD-containing lipid colloid as claimed in claim 1, characterized in that: The SOD-containing lipid colloid was used in combination with ultraviolet irradiation.

6. An application of the SOD-containing lipid colloid as claimed in claim 1, characterized in that: The SOD-containing lipid colloid is applied to chronic wound healing.

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