Preparation method of composite film-forming liquid for promoting healing of diabetic skin wound

Abstract

The invention discloses a preparation method of a composite film-forming liquid for promoting healing of diabetic skin wounds, and belongs to the technical field of regenerative medicine. According to the specific method, chitosan, polyvinylpyrrolidone, hydroxy propyl cellulose, gelatin, calcium peroxide, active protein, growth factors and genipin are utilized to construct a novel composite film forming solution which has excellent film forming ability and slowly releases the active protein, the growth factors and oxygen. The composite film-forming liquid is easy to smear on skin wounds, can quickly form films, recover skin physical barriers, preserve moisture and inhibit bacteria, promotes angiogenesis by slowly releasing active proteins, growth factors and oxygen, improves the repairing capacity and migration of skin cells, further promotes healing of diabetic skin wounds, and has a good application prospect. The method has important clinical practical application significance.

Description

Technical Field

[0001] The invention belongs to the technical field of regenerative medicine, and in particular relates to a preparation method and application of a composite film-forming liquid for promoting healing of diabetic skin wounds. Background Art

[0002] Diabetes is a chronic metabolic disease with a high incidence worldwide. One of its complications, diabetic skin wounds, brings great pain to patients. The harm of diabetic skin wounds is mainly reflected in the following aspects: First, the wounds heal slowly, which is easy to cause infection and further aggravate the condition. Secondly, long-term non-healing wounds will seriously affect the patient's quality of life, increase the patient's pain and economic burden. In addition, severe diabetic skin wounds may even lead to amputation, endangering the patient's life.

[0003] Diabetic skin wounds have the following characteristics: the local high-sugar environment of the wound produces advanced glycation end products (AGEs) through a complex glycosylation process. AGEs bind to their main cell surface receptor, the receptor for advanced glycation end products (RAGE), to induce oxidative stress, aggravate inflammation, and affect cell function and metabolism. In addition, diabetic wounds have reduced angiogenesis, which is characterized by reduced blood vessel and capillary density. During wound repair, neutrophils release an appropriate amount of oxygen free radicals (ROS) in the early stage of inflammation to remove bacteria and foreign matter, but the accumulation of AGEs inhibits the migration of neutrophils and promotes their apoptosis. At the same time, the phenotypic transformation of macrophages in diabetic wounds is blocked, the number of M1 macrophages that mainly secrete proinflammatory factors increases, and the number of M2 macrophages that secrete anti-inflammatory factors and pro-repair cytokines decreases, resulting in reduced proliferation and migration of repair cells and blocked wound healing.

[0004] At present, the treatment methods for diabetic skin wounds mainly include debridement, dressing application, drug therapy, etc. However, these methods have defects. Although debridement can remove necrotic tissue, excessive debridement may cause the wound to expand further and increase the difficulty of healing. Although existing dressings can provide a moist environment, they are not adequate for the special pathological mechanisms of diabetic wounds, such as high-sugar environment, inflammatory response, insufficient angiogenesis, etc., and the effect is limited. In terms of drug treatment, although some drugs can regulate macrophage polarization and promote ulcer healing, the efficacy and safety of these drugs still need to be further verified.

[0005] In view of the above problems, it is particularly important to develop new dressings that promote the healing of diabetic skin wounds.

[0006] The new dressing should have the following functions: first, strengthen wound closure, protection and antibacterial, prevent the invasion and proliferation of external bacteria and pollutants, and reduce infection; second, enhance angiogenesis, improve the blood supply to the wound by promoting angiogenesis, and provide necessary nutrition and oxygen for wound healing; third, regulate macrophages and improve the repair ability and migration of fibroblasts to accelerate wound healing; fourth, enhance the continuous supply of oxygen, improve the local hypoxia of the wound, and promote the repair of skin wound tissue. At present, there is an urgent need to develop new diabetic skin wound treatment dressings with the above functions to improve the wound treatment effect and quality of life of diabetic patients. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides a preparation method and application of a composite film-forming liquid for promoting the healing of diabetic skin wounds, aiming to enhance the wound film-forming protection and antibacterial ability, while promoting angiogenesis by sustained-release of active proteins, growth factors and oxygen, improving the repair ability and migration of skin cells, thereby further promoting the healing of diabetic skin wounds.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0009] The present invention discloses a method for preparing a composite film-forming liquid for promoting healing of diabetic skin wounds, which is characterized by comprising the following steps: S1. A polyvinyl pyrrolidone solution having a molecular weight of 320,000 to 500,000 Da was added to the chitosan solution, and the mixture was stirred and mixed to obtain a mixed solution A; S2. Hydroxypropyl cellulose having a molecular weight of 100,000 to 500,000 Da is added to the mixed solution A in S1, and the mixture is stirred and mixed to obtain a mixed solution B; S3. The gelatin was added to the mixed solution B in S2, and the mixture was stirred to obtain a mixed solution C; S4. The calcium peroxide was added to the mixed solution C in S3, and the mixture was stirred to obtain a mixed solution D; S5. The active protein was added to the mixed solution D in S4, and the mixture was stirred to obtain a mixed solution F; S6. The growth factor was added to the mixed solution F in S5, and the mixture was stirred to obtain a mixed solution G; S7. Add genipin to the mixture G in S6, stir and mix evenly to obtain a composite film-forming liquid for promoting the healing of diabetic skin wounds.

[0010] Furthermore, the pH of the chitosan solution in step S1 is 4.0-6.2, and the molecular weight of chitosan is 100000-400000 Da.

[0011] Furthermore, the gelatin in step S3 is a mixture of alkaline gelatin and acidic gelatin, the molecular weight of gelatin is 100,000 to 500,000 Da, and the mass ratio of the alkaline gelatin to the acidic gelatin in the mixture is 1:4 to 4:1.

[0012] Furthermore, the particle size of the calcium peroxide in step S4 is 20 nm to 200 μm.

[0013] Furthermore, the active protein in step S5 is one or more of Thbs1, fibronectin, CCN1, Andersonin-W1, and silk fibroin.

[0014] Furthermore, in step S6, the growth factor is one or more of VEGF, PDGF, EGF, bFGF, TGF-β1, KGF, IGF, and CGRP.

[0015] Furthermore, the specific stirring conditions in steps S1-S7 are: the stirring and mixing time is 10 to 19 minutes; the stirring speed is 2000 to 10000 rpm.

[0016] Furthermore, in the step S7, the concentration of chitosan in the composite film-forming solution is 0.4-1.2% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 11-20% (w / v, g / mL), the concentration of hydroxypropyl cellulose is 3-10% (w / v, g / mL), the concentration of gelatin is 4-12% (w / v, g / mL), the concentration of calcium peroxide is 3-10% (w / v, g / mL), the concentration of each active protein is 0.01-0.5% (w / v, g / mL), the concentration of each growth factor is 20-200 ng / mL, and the concentration of genipin is 0.02-0.08% (w / v, g / mL).

[0017] The present invention also discloses the use of a composite film-forming liquid prepared by any one of the preparation methods described above in the preparation of a film-forming agent for promoting the healing of diabetic skin wounds.

[0018] The present invention also discloses a composite film-forming liquid for promoting the healing of diabetic skin wounds, characterized in that it is prepared by any of the preparation methods described above; the composite film-forming liquid is applied to diabetic skin wounds to promote the healing of the wounds.

[0019] Compared with the prior art, the present invention has the following beneficial effects.

[0020] 1. The present invention uses chitosan, polyvinyl pyrrolidone, hydroxypropyl cellulose, gelatin and genipin to construct a composite film-forming liquid for sustained-release active proteins, growth factors and oxygen. Chitosan has antibacterial properties, can effectively inhibit the growth of bacteria on the wound surface, and stabilize the healing environment. In addition, chitosan has certain film-forming properties, but the stability of the film is insufficient and the strength is low, which makes it difficult to meet the requirements of rapid, stable and uniform film formation after application on diabetic skin wounds. In order to enhance the film-forming ability, polyvinyl pyrrolidone, hydroxypropyl cellulose and gelatin are added to the present invention. Polyvinyl pyrrolidone, hydroxypropyl cellulose and gelatin all have strong hydrogen bonds and electrostatic interactions with chitosan, and they can form a more stable composite network structure with chitosan, thereby significantly improving the film-forming property of the film-forming liquid, shortening the film-forming time, and greatly enhancing the stability, mechanical properties and barrier properties of the film. Polyvinyl pyrrolidone is uncharged, and it is first compounded with chitosan, which can effectively disperse the entanglement between chitosan molecules, and is more conducive to the compounding of chitosan molecular chains with hydroxypropyl cellulose and gelatin molecular chains, so that the three-dimensional network is more uniform and the structure of the film is more homogeneous. In addition, hydroxypropyl cellulose can also increase the adhesion of the film to the wound tissue, so that the formed film adheres to the wound more stably. In addition, gelatin is a product of collagen hydrolysis, which retains the activity of collagen to a certain extent. The composite film containing chitosan and gelatin is similar to the extracellular matrix in composition and structure, and the formed film is conducive to the migration and function of skin repair cells. The use of genipin can moderately cross-link the three-dimensional network composed of chitosan, polyvinyl pyrrolidone, hydroxypropyl cellulose and gelatin, thereby further improving the stability of the film in the harsh environment of the skin wound. Thus, the composite film-forming liquid prepared by the present invention can form a film quickly, stably and evenly on the diabetic skin wound, and play a better barrier role.

[0021] 2. The present invention adds calcium peroxide to the composite film-forming liquid constructed by chitosan, polyvinyl pyrrolidone, hydroxypropyl cellulose, gelatin and genipin, so that the formed film can better release oxygen on the skin wound surface, effectively improve the hypoxic state of the wound surface, so as to improve antibacterial properties, promote angiogenesis, accelerate collagen formation, reduce inflammatory reactions, and facilitate wound healing. In the composite film-forming liquid of the present invention, polyvinyl pyrrolidone also serves as a stabilizer for calcium peroxide, which can regulate the dissolution rate of calcium peroxide. At the same time, hydroxypropyl cellulose and gelatin, as hydrophilic polymers, can also control the slow release of oxygen by regulating the dissolution rate of calcium peroxide. This multi-component synergistic slow release of oxygen mechanism can effectively prolong the release time of oxygen, help to improve the hypoxic state of the skin wound surface for a long time, and promote wound healing.

[0022] 3. The present invention adds active proteins (one or more of Thbs1, fibronectin, CCN1, Andersonin-W1, and silk fibroin) and growth factors (one or more of VEGF, PDGF, EGF, bFGF, TGF-β1, KGF, IGF, and CGRP) to the composite film-forming solution constructed by chitosan, polyvinyl pyrrolidone, hydroxypropyl cellulose, gelatin, and genipin, so that the formed film can better release active proteins and growth factors on the skin wound surface, thereby better regulating the phenotype and function of immune cells, scavenging free radicals, reducing oxidative stress, promoting angiogenesis, promoting cell proliferation and migration, and accelerating wound healing. Under physiological conditions, chitosan and acidic gelatin are positively charged, and alkaline gelatin is negatively charged, so the active proteins and growth factors with positive or negative charges can be bound to the composite film by electrostatic action, thereby better realizing the sustained release of active proteins and growth factors on the wound surface, prolonging the action time, and accelerating wound healing.

[0023] 4. The composite film-forming liquid proposed in the present invention is easy to use and does not require the use of special devices. It can form a film and take effect by evenly applying it on the skin wound surface. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail in conjunction with the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] The present invention uses chitosan, polyvinyl pyrrolidone, hydroxypropyl cellulose, gelatin, calcium peroxide, active protein, growth factor and genipin to construct a novel composite film-forming liquid with excellent film-forming ability and slow release of active protein, growth factor and oxygen. The composite film-forming liquid is easy to be applied to the skin wound surface, can quickly form a film, restore the skin physical barrier, moisturize, inhibit bacteria, and promote angiogenesis by slow release of active protein, growth factor and oxygen, improve the repair ability and migration of skin cells, thereby further promoting the healing of diabetic skin wounds.

[0026] Embodiment 1.

[0027] Add a polyvinyl pyrrolidone solution with a molecular weight of 320,000Da to a chitosan solution (pH 4.0) with a molecular weight of 100,000Da and a deacetylation degree of 90%, and stir and mix evenly. Then, add hydroxypropyl cellulose with a molecular weight of 100,000Da and stir and mix evenly. Then, add alkaline gelatin and acidic gelatin with a molecular weight of 100,000Da, the mass ratio of the two is 1:4, and stir and mix evenly. Then, add calcium peroxide with a particle size of 20nm and stir and mix evenly. Then, add active proteins (Thbs1, fibronectin, CCN1) and stir and mix evenly. Then, add growth factors (VEGF, PDGF, EGF, bFGF) and stir and mix evenly. Finally, add genipin and stir and mix evenly. The stirring time is 10 minutes and the stirring speed is 2000rpm. Thus, a composite film-forming liquid 1# is obtained.

[0028] The concentration of chitosan in this composite film-forming liquid 1# is 1.2% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 20% (w / v, g / mL), the concentration of hydroxypropyl cellulose is 10% (w / v, g / mL), the concentration of gelatin is 12% (w / v, g / mL), the concentration of calcium peroxide is 10% (w / v, g / mL), the concentration of each active protein is 0.5% (w / v, g / mL), the concentration of each growth factor is 200 ng / mL, and the concentration of genipin is 0.08% (w / v, g / mL).

[0029] At the same time, four control composite film-forming solutions were prepared without polyvinyl pyrrolidone, hydroxypropyl cellulose, gelatin, and genipin, while other conditions remained unchanged. They were named control composite film-forming solutions 2#, 3#, 4#, and 5#, respectively.

[0030] 300 μl of each of the composite film-forming liquid 1# and four control composite film-forming liquids 2#, 3#, 4#, and 5# were applied to the wound surface of the diabetic infectious skin wound mouse model (the initial diameter of the back skin wound was 10 mm), and the membrane area retention rate of each group on the 14th day was calculated to evaluate the differences in membrane stability among the groups.

[0031] The experiment found that on the 14th day, the membrane area retention rate of composite membrane-forming liquid 1# was 100%, while the membrane area retention rates of control composite membrane-forming liquids 2#, 3#, 4# and 5# were 47%, 35%, 41% and 24% respectively. This experiment shows that the addition of polyvinyl pyrrolidone, hydroxypropyl cellulose, gelatin or genipin has a significant effect on improving the film stability of the composite membrane, and the stability is best when the four components are added at the same time.

[0032] Embodiment 2.

[0033] Add a polyvinyl pyrrolidone solution with a molecular weight of 500,000Da to a chitosan solution (pH 6.2) with a molecular weight of 400,000Da and a deacetylation degree of 95%, and stir and mix evenly. Then, add hydroxypropyl cellulose with a molecular weight of 500,000Da and stir and mix evenly. Then, add alkaline gelatin and acidic gelatin with a molecular weight of 500,000Da, with a mass ratio of 4:1, and stir and mix evenly. Then, add calcium peroxide with a particle size of 200μm and stir and mix evenly. Then, add active proteins (Thbs1, Andersonin-W1, silk protein) and stir and mix evenly. Then, add growth factors (TGF-β1, KGF, IGF, CGRP) and stir and mix evenly. Finally, add genipin and stir and mix evenly. The stirring and mixing time is 19 minutes, and the stirring speed is 10,000rpm. Thus, a composite film-forming liquid 1# is obtained.

[0034] In this composite film-forming liquid 1#, the concentration of chitosan is 0.4% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 11% (w / v, g / mL), the concentration of hydroxypropyl cellulose is 3% (w / v, g / mL), the concentration of gelatin is 4% (w / v, g / mL), the concentration of calcium peroxide is 3% (w / v, g / mL), the concentration of each active protein is 0.01% (w / v, g / mL), the concentration of each growth factor is 20 ng / mL, and the concentration of genipin is 0.02% (w / v, g / mL).

[0035] At the same time, 8 control composite film-forming solutions containing polyvinyl pyrrolidone concentration of 10% (w / v, g / mL) or 21% (w / v, g / mL), hydroxypropyl cellulose concentration of 2% (w / v, g / mL) or 11% (w / v, g / mL), gelatin concentration of 3% (w / v, g / mL) or 13% (w / v, g / mL), and genipin concentration of 0.01% (w / v, g / mL) or 0.09% (w / v, g / mL) were prepared, while other conditions remained unchanged. They were named control composite film-forming solutions 2-9#.

[0036] 300 μl of each of the composite film-forming liquid 1# and 8 control composite film-forming liquids 2-9# were applied to the wound surface of the diabetic infected skin wound mouse model (the initial diameter of the back skin wound was 10 mm), and the membrane area retention rate of each group on the 14th day was calculated to evaluate the differences in membrane stability among the groups.

[0037] The experiment found that on the 14th day, the membrane area retention rate of composite membrane-forming liquid 1# was 100%, and the membrane area retention rates of control composite membrane-forming liquids 2#, 4#, 6# and 8# were 87%, 80%, 81% and 74% respectively, while the control composite membrane-forming liquids 3#, 5#, 7# and 9# could not be evenly applied on the wound surface due to the very high viscosity of the prepared liquids, so they could not be used. This experiment shows that the concentrations of polyvinyl pyrrolidone, hydroxypropyl cellulose, gelatin and genipin used in the present invention are in the optimal range for skin wound application and membrane stability.

[0038] Embodiment three.

[0039] Add a polyvinyl pyrrolidone solution with a molecular weight of 400,000Da to a chitosan solution (pH 5.1) with a molecular weight of 250,000Da and a deacetylation degree of 95%, and stir and mix evenly. Then, add hydroxypropyl cellulose with a molecular weight of 300,000Da and stir and mix evenly. Then, add alkaline gelatin and acidic gelatin with a molecular weight of 300,000Da, with a mass ratio of 1:1, and stir and mix evenly. Then, add calcium peroxide with a particle size of 100μm and stir and mix evenly. Then, add active proteins (fibronectin, silk protein) and stir and mix evenly. Then, add growth factors (PDGF, bFGF, TGF-β1, IGF) and stir and mix evenly. Finally, add genipin and stir and mix evenly. The stirring time is 15 minutes, and the stirring speed is 6000rpm. Thus, a composite film-forming liquid 1# is obtained.

[0040] The concentration of chitosan in this composite film-forming liquid 1# is 0.8% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 15% (w / v, g / mL), the concentration of hydroxypropyl cellulose is 6.5% (w / v, g / mL), the concentration of gelatin is 8% (w / v, g / mL), the concentration of calcium peroxide is 6.5% (w / v, g / mL), the concentration of each active protein is 0.25% (w / v, g / mL), the concentration of each growth factor is 110 ng / mL, and the concentration of genipin is 0.05% (w / v, g / mL).

[0041] At the same time, 6 control composite film-forming solutions containing polyvinyl pyrrolidone with a molecular weight of 310,000Da or 510,000Da, hydroxypropyl cellulose with a molecular weight of 90,000Da or 510,000Da, and gelatin with a molecular weight of 90,000Da or 510,000Da were prepared respectively, while other conditions remained unchanged, and they were named control composite film-forming solutions 2-7# respectively.

[0042] 300 μl of each of the composite film-forming liquid 1# and six control composite film-forming liquids 2-7# were applied to the wound surface of the diabetic infectious skin wound mouse model (the initial diameter of the back skin wound was 10 mm), and the membrane area retention rate of each group on the 14th day was calculated to evaluate the differences in membrane stability among the groups.

[0043] The experiment found that on the 14th day, the membrane area retention rate of composite membrane-forming liquid 1# was 100%, and the membrane area retention rates of control composite membrane-forming liquids 2#, 4#, and 6# were 85%, 82%, and 80%, respectively. The control composite membrane-forming liquids 3#, 5#, and 7# could not be used because the viscosity of the prepared membrane-forming liquid was too high and could not be evenly applied on the wound surface. This experiment shows that the molecular weights of polyvinyl pyrrolidone, hydroxypropyl cellulose, and gelatin used in the present invention are in the optimal range for application on skin wounds and membrane stability.

[0044] Embodiment 4.

[0045] Add a polyvinyl pyrrolidone solution with a molecular weight of 350,000Da to a chitosan solution (pH 4.5) with a molecular weight of 350,000Da and a deacetylation degree of 97%, and stir and mix evenly. Then, add hydroxypropyl cellulose with a molecular weight of 150,000Da and stir and mix evenly. Then, add alkaline gelatin and acidic gelatin with molecular weights of 160,000Da and 360,000Da, respectively, in a mass ratio of 1:3, and stir and mix evenly. Then, add calcium peroxide with a particle size of 20μm and stir and mix evenly. Then, add active proteins (Thbs1, fibronectin, Andersonin-W1) and stir and mix evenly. Then, add growth factors (VEGF, PDGF, KGF, IGF) and stir and mix evenly. Finally, add genipin and stir and mix evenly. The stirring time is 12 minutes and the stirring speed is 3000rpm. Thus, a composite film-forming liquid 1# is obtained.

[0046] The concentration of chitosan in this composite membrane-forming solution 1# is 0.6% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 17% (w / v, g / mL), the concentration of hydroxypropyl cellulose is 5% (w / v, g / mL), the concentration of alkaline gelatin is 7% (w / v, g / mL), the concentration of acidic gelatin is 10% (w / v, g / mL), the concentration of calcium peroxide is 5% (w / v, g / mL), the concentration of Thbs1 is 0.15% (w / v, g / mL), the concentration of fibronectin is 0.1% (w / v, g / mL), the concentration of Andersonin-W1 is 0.2% (w / v, g / mL), the concentration of VEGF is 50ng / mL, the concentration of PDGF is 70ng / mL, the concentration of KGF is 90ng / mL, the concentration of IGF is 120ng / mL, and the concentration of genipin is 0.06% (w / v, g / mL).

[0047] At the same time, three control composite film-forming solutions containing no calcium peroxide, 2.5% (w / v, g / mL) calcium peroxide, and 11% (w / v, g / mL) calcium peroxide were prepared respectively, while other conditions remained unchanged, and they were named control composite film-forming solutions 2-4#, respectively.

[0048] 300 microliters of composite film-forming liquid 1# and three control composite film-forming liquids 2-4# were applied to the wounds of diabetic infected skin wound mouse models (the initial diameter of the back skin wound was 10 mm), and the wound healing rate of each group on the 14th day was calculated to evaluate the differences in promoting wound healing. In addition, a conventional control group 5# without composite film-forming liquid was set up at the same time.

[0049] The experiment found that on the 14th day, the wound healing rate of composite film-forming liquid 1# was 94%, while the wound healing rates of control composite film-forming liquids 2# and 3# were 51% and 60%, respectively. However, 4# could not be used because of the high concentration of calcium peroxide contained in it, resulting in poor film-forming effect. The wound healing rate of the conventional control group 5# that did not use the composite film-forming liquid was 33%. This experiment shows that the composite film-forming liquid of the present invention has the best effect in promoting wound healing, and the most suitable concentration of calcium peroxide is 3-10% (w / v, g / mL). The film-forming liquid without calcium peroxide has a certain promoting effect compared with the film-forming liquid without using it, but it is significantly worse than the film-forming liquid containing 3-10% (w / v, g / mL) calcium oxide.

[0050] Embodiment five.

[0051] Add a polyvinyl pyrrolidone solution with a molecular weight of 450,000Da to a chitosan solution (pH 5.8) with a molecular weight of 200,000Da and a deacetylation degree of 98%, and stir and mix evenly. Then, add hydroxypropyl cellulose with a molecular weight of 400,000Da and stir and mix evenly. Then, add alkaline gelatin and acidic gelatin with molecular weights of 340,000Da and 220,000Da, respectively, in a mass ratio of 3:2, and stir and mix evenly. Then, add calcium peroxide with a particle size of 80nm and stir and mix evenly. Then, add active proteins (CCN1, silk fibroin) and stir and mix evenly. Then, add growth factors (EGF, bFGF, TGF-β1, CGRP) and stir and mix evenly. Finally, add genipin and stir and mix evenly. The stirring and mixing time is 18 minutes, and the stirring speed is 7500rpm. Thus, a composite film-forming liquid 1# is obtained.

[0052] In this composite film-forming solution 1#, the concentration of chitosan is 1% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 14% (w / v, g / mL), the concentration of hydroxypropyl cellulose is 8% (w / v, g / mL), the concentration of alkaline gelatin is 9% (w / v, g / mL), the concentration of acidic gelatin is 6% (w / v, g / mL), the concentration of calcium peroxide is 7% (w / v, g / mL), the concentration of CCN1 is 0.08% (w / v, g / mL), the concentration of silk fibroin is 0.4% (w / v, g / mL), the concentration of EGF is 78ng / mL, the concentration of bFGF is 88ng / mL, the concentration of TGF-β1 is 100ng / mL, the concentration of CGRP is 95ng / mL, and the concentration of genipin is 0.04% (w / v, g / mL).

[0053] At the same time, three control composite film-forming solutions were prepared without active protein, with CCN1 and silk protein concentrations of 0.009% (w / v, g / mL) and 0.6% (w / v, g / mL), while other conditions remained unchanged. They were named control composite film-forming solutions 2-4#.

[0054] 300 μl of each of the composite film-forming liquid 1# and three control composite film-forming liquids 2-4# were applied to the wounds of the diabetic infected skin wound mouse model (the initial diameter of the back skin wound was 10 mm), and the wound healing rate of each group on the 14th day was calculated to evaluate the differences in promoting wound healing among the groups.

[0055] The experiment found that on the 14th day, the wound healing rate of composite film-forming liquid 1# was 93%, while the wound healing rates of control composite film-forming liquids 2# and 3# were 50% and 60%, respectively. However, 4# could not be used because of its poor film-forming effect due to the high concentration of active protein contained in it. This experiment shows that the composite film-forming liquid of the present invention has the best effect in promoting wound healing, and the most suitable concentration of each active protein is 0.01-0.5% (w / v, g / mL).

[0056] Embodiment six.

[0057] Add a polyvinyl pyrrolidone solution with a molecular weight of 410000Da to a chitosan solution (pH 6.1) with a molecular weight of 240000Da and a deacetylation degree of 91%, and stir and mix evenly. Then, add hydroxypropyl cellulose with a molecular weight of 300000Da and stir and mix evenly. Then, add alkaline gelatin and acidic gelatin with molecular weights of 300000Da and 300000Da, respectively, with a mass ratio of 4:1, and stir and mix evenly. Then, add calcium peroxide with a particle size of 180μm and stir and mix evenly. Then, add active proteins (Thbs1, CCN1) and stir and mix evenly. Then, add growth factors (EGF, KGF, IGF) and stir and mix evenly. Finally, add genipin and stir and mix evenly. The stirring and mixing time is 11 minutes, and the stirring speed is 2500rpm. Thus, a composite film-forming liquid 1# is obtained.

[0058] The concentration of chitosan in this composite film-forming liquid 1# is 0.55% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 16.5% (w / v, g / mL), the concentration of hydroxypropyl cellulose is 5.9% (w / v, g / mL), the concentration of alkaline gelatin is 7.9% (w / v, g / mL), the concentration of acidic gelatin is 10.5% (w / v, g / mL), the concentration of calcium peroxide is 8% (w / v, g / mL), the concentration of Thbs1 is 0.35% (w / v, g / mL), the concentration of CCN1 is 0.22% (w / v, g / mL), the concentration of EGF is 35ng / mL, the concentration of KGF is 45ng / mL, the concentration of IGF is 65ng / mL, and the concentration of genipin is 0.05% (w / v, g / mL).

[0059] At the same time, three control composite membrane-forming solutions were prepared without growth factors, with EGF, KGF and IGF concentrations of 19 ng / mL and 210 ng / mL respectively, while other conditions remained unchanged. They were named control composite membrane-forming solutions 2-4# respectively.

[0060] 300 μl of each of the composite film-forming liquid 1# and three control composite film-forming liquids 2-4# were applied to the wounds of the diabetic infected skin wound mouse model (the initial diameter of the back skin wound was 10 mm), and the wound healing rate of each group on the 14th day was calculated to evaluate the differences in promoting wound healing among the groups.

[0061] The experiment found that on the 14th day, the wound healing rate of composite film-forming liquid 1# was 96%, while the wound healing rates of control composite film-forming liquids 2# and 3# were 49% and 58%, respectively. However, 4# cannot be used because it contains too high a concentration of growth factors, resulting in high cytotoxicity and inflammatory reactions. This experiment shows that the composite film-forming liquid of the present invention has the best effect in promoting wound healing, and the most suitable concentration of each growth factor is 20 to 200 ng / mL.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a composite film-forming liquid for promoting healing of diabetic skin wounds, characterized in that: The method comprises the following steps: S1. adding a polyvinyl pyrrolidone solution having a molecular weight of 320,000 to 500,000 Da to a chitosan solution, and stirring and mixing the mixture to obtain a mixed solution A; S2. Hydroxypropyl cellulose having a molecular weight of 100,000 to 500,000 Da is added to the mixed solution A in S1, and the mixture is stirred and mixed to obtain a mixed solution B; S3. The gelatin was added to the mixed solution B in S2, and the mixture was stirred to obtain a mixed solution C; S4. The calcium peroxide was added to the mixed solution C in S3, and the mixture was stirred to obtain a mixed solution D; S5. The active protein was added to the mixed solution D in S4, and the mixture was stirred to obtain a mixed solution F; S6. The growth factor was added to the mixed solution F in S5, and the mixture was stirred to obtain a mixed solution G; S7. Add genipin to the mixture G in S6, stir and mix evenly to obtain a composite film-forming liquid for promoting the healing of diabetic skin wounds.

2. The method for preparing the composite film-forming solution for promoting healing of diabetic skin wounds according to claim 1, characterized in that: The pH of the chitosan solution in step S1 is 4.0-6.2, and the molecular weight of chitosan is 100000-400000 Da.

3. The method for preparing the composite film-forming solution for promoting healing of diabetic skin wounds according to claim 1, characterized in that: The gelatin in step S3 is a mixture of alkaline gelatin and acidic gelatin, the molecular weight of gelatin is 100,000 to 500,000 Da, and the mass ratio of the alkaline gelatin to the acidic gelatin in the mixture is 1:4 to 4:

1.

4. The method for preparing the composite film-forming solution for promoting healing of diabetic skin wounds according to claim 1, characterized in that: The particle size of the calcium peroxide in step S4 is 20 nm to 200 μm.

5. The method for preparing the composite film-forming solution for promoting healing of diabetic skin wounds according to claim 1, characterized in that: In the step S5, the active protein is one or more of Thbs1, fibronectin, CCN1, Andersonin-W1, and silk fibroin.

6. The method for preparing the composite film-forming solution for promoting healing of diabetic skin wounds according to claim 1, characterized in that: In step S6, the growth factor is one or more of VEGF, PDGF, EGF, bFGF, TGF-β1, KGF, IGF, and CGRP.

7. The method for preparing the composite film-forming solution for promoting healing of diabetic skin wounds according to claim 1, characterized in that: The specific stirring conditions in steps S1-S7 are: the stirring and mixing time is 10 to 19 minutes; the stirring speed is 2000 to 10000 rpm.

8. The method for preparing the composite film-forming solution for promoting healing of diabetic skin wounds according to claim 1, characterized in that: In the step S7, the concentration of chitosan in the composite film-forming solution is 0.4-1.2% (w / v, g / mL), the concentration of polyvinyl pyrrolidone is 11-20% (w / v, g / mL), the concentration of hydroxypropyl cellulose is 3-10% (w / v, g / mL), the concentration of gelatin is 4-12% (w / v, g / mL), the concentration of calcium peroxide is 3-10% (w / v, g / mL), the concentration of each active protein is 0.01-0.5% (w / v, g / mL), the concentration of each growth factor is 20-200 ng / mL, and the concentration of genipin is 0.02-0.08% (w / v, g / mL).

9. Use of the composite film-forming liquid prepared by the preparation method according to any one of claims 1 to 8 in preparing a film-forming agent for promoting the healing of diabetic skin wounds.

10. A composite film-forming liquid for promoting healing of diabetic skin wounds, characterized in that: The composite film-forming liquid is prepared by the preparation method according to any one of claims 1 to 8; the composite film-forming liquid is applied to the diabetic skin wound to promote wound healing.