A wound patch for preventing skin scar formation and a preparation method thereof

Abstract

The application relates to the technical field of biomedical materials, in particular to a wound paste for preventing skin scar formation and a preparation method thereof. The wound paste for preventing skin scar formation comprises an integrated composite film comprising an adhesion layer and a functional layer; the adhesion layer comprises first silk fibroin and metal ions; the metal ions have at least one of the effects of antibiosis, anti-inflammation and pro-angiogenesis; the functional layer comprises composite fibers with a core-shell structure; the core of the composite fiber comprises second silk fibroin and an angiogenesis inhibitor, and the shell comprises third silk fibroin. The wound paste is good in biocompatibility, convenient to apply, gradually releases effective components, adapts to the healing process of a wound, and is more scientific and effective in preventing scar formation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a wound dressing for preventing skin scar formation and a preparation method thereof. BACKGROUND

[0002] Hypertrophic scar (HS) is a pathological scar that may occur during the wound healing process, which poses a major challenge to clinical treatment. This type of scar is usually characterized by hard and abnormally raised tissue, and has significant changes in pigmentation and tissue elasticity compared to the surrounding normal skin. Hypertrophic scars not only affect the appearance of patients, but also can cause symptoms such as itching and chronic pain, and in severe cases, can even affect joint movement and limb function, causing serious impact on the daily life and psychological state of patients.

[0003] The formation mechanism of hypertrophic scars is not yet fully understood, and the formation of hypertrophic scars is related to multiple factors, including inflammatory response, the role of growth factors such as TGF-β1, changes in extracellular matrix, and genetic factors, in addition, the trend of neovascularization and regression during wound healing has a significant impact on wound healing regression, and through segmented regulation of the wound healing process, it may help to prevent scar formation.

[0004] Wound dressings that inhibit scar formation are a special type of dressing that promotes wound healing and reduces scar formation by providing a suitable healing environment. Clinically used wound dressings mainly include silicone-based wound dressings and hydrogel-based dressings, both of which have good adhesion to wounds and can help isolate external contaminants, thereby preventing infection. However, these dressings still need to be improved in terms of breathability, biocompatibility, and cost.

[0005] Therefore, it is necessary to develop a wound dressing that can prevent scar formation, has good biocompatibility and breathability. SUMMARY

[0006] Therefore, one or more embodiments of the present application provide a wound dressing for preventing skin scar formation and a preparation method thereof. The wound dressing can prevent skin scar formation and also has the effect of promoting wound healing. In addition, the material is biodegradable, has good biocompatibility and breathability, and has good adhesion and adhesion to the wound, which can reduce the discomfort of patients.

[0007] The technical solution of the present application includes the following content:

[0008] One embodiment of the present application provides a wound dressing for preventing skin scar formation, which comprises a composite film comprising an adhesive layer and a functional layer.

[0009] The composition of the adhesion layer comprises a first silk fibroin and metal ions; the metal ions have at least one of the effects of antibiosis, anti-inflammation and pro-angiogenesis; the composition of the functional layer comprises composite fibers with a core-shell structure;

[0010] The composition of the core of the composite fiber comprises a second silk fibroin and an angiogenesis inhibitor, and the composition of the shell comprises a third silk fibroin.

[0011] In one embodiment, the weight ratio of the first silk fibroin and the metal ions is (0.25-1.5):1.

[0012] In one embodiment, the weight ratio of the second silk fibroin and the angiogenesis inhibitor is (10-500):1; and / or

[0013] The weight ratio of the core and the shell of the composite fiber is 1:(2-20).

[0014] In one embodiment, the metal ions comprise one or more of Ca 2+ , Zn 2+ , Cu 2+ , Ag + , Mg 2+ , Eu 3+ , Fe 3+ and Mn 2+ ; and / or

[0015] The angiogenesis inhibitor comprises at least one of green tea polyphenols, quercetin, consolide, apigenin, delphinidin, zephyranthine, cyanidin, pelargonidin, ipomoea, peony glycoside, chlorogenic acid, p-coumaric acid, red raspberry, bevacizumab, ramucirumab, olaratumab, anlotinib and sorafenib.

[0016] In one embodiment, the composition of the shell further comprises a plant polyphenol;

[0017] Optionally, the weight ratio of the third silk fibroin and the plant polyphenol is (10-1000):1.

[0018] Another embodiment of the present application provides a preparation method of the wound dressing described above, comprising:

[0019] After mixing the first silk fibroin, the metal salt and the first solvent, the adhesion layer is prepared through a first film-forming treatment;

[0020] The second silk fibroin, the angiogenesis inhibitor and the second solvent are mixed to obtain a core layer spinning solution;

[0021] The third silk fibroin and the third solvent are mixed to obtain a shell layer spinning solution;

[0022] The core layer spinning solution and the shell layer spinning solution are electrospun by a coaxial electrospinning process to obtain the composite fiber, and the composite fiber is formed into a functional layer on the adhesion layer by taking the adhesion layer as a receiving plate, so that the composite film is prepared.

[0023] In one embodiment, the weight ratio of the solutes of the core layer spinning solution and the shell layer spinning solution is 1: (2-20).

[0024] In one embodiment, after the third silk fibroin and the third solvent are mixed, plant polyphenols are further added to obtain the shell layer spinning solution.

[0025] Optionally, the weight ratio of the third silk fibroin and the plant polyphenols is (10-1000): 1.

[0026] In one embodiment, after the first fiber, the metal salt and the solvent are mixed, the step of preparing the adhesion layer by a first film-forming treatment includes:

[0027] After the first fiber, the metal salt and the solvent are mixed, a first solution is obtained.

[0028] The first solution is prepared into a film by a first electrospinning or a method of laying flat and evaporating dry to prepare the adhesion layer.

[0029] Optionally, the injection speed of the first solution is 0.05 mL / h-2 mL / h.

[0030] Optionally, the voltage of the first electrospinning is 10 kV-25 kV, and the collection distance is 10 cm-20 cm.

[0031] In one embodiment, the step of electrospinning the core layer spinning solution and the shell layer spinning solution by a coaxial electrospinning process to obtain the composite fiber includes:

[0032] The core layer spinning solution and the shell layer spinning solution are second electrospun by a coaxial electrospinning device, the outer needle inner diameter of the nozzle of the coaxial electrospinning device is 0.86 mm-1.4 mm, and the inner needle outer diameter is 0.31 mm-0.6 mm.

[0033] Optionally, the injection speed of the core layer spinning solution is 0.05 mL / h-2 mL / h, and the injection speed of the shell layer spinning solution is 0.1 mL / h-5 mL / h.

[0034] Optionally, the voltage of the second electrospinning is 10 kV-25 kV, and the collection distance is 10 cm-20 cm.

[0035] The wound patch for preventing skin scar formation of the present application has at least the following beneficial effects:

[0036] (1) The wound patch of the present application can be conveniently and flexibly adhered to the wound surface, without the need for additional fixing devices, and has the advantages of convenient use and a wide range of application scenarios.

[0037] (2) The adhesive layer and the functional layer of the wound patch of the present application are integrated, and due to the shielding of the adhesive layer and the core-shell structure design of the functional layer, the anti-angiogenic active ingredients of the functional layer can achieve the function of delayed release, so that sequential regulation and step-by-step regulation can be achieved throughout the wound healing process: in the early stage of wound healing, the metal ions in the adhesive layer are released first to play a biological activity function, and have the effects of antibacterial, anti-inflammatory and pro-angiogenic, etc., to promote wound healing, and in some embodiments, the active substances of the shell layer of the functional layer are also gradually released at this stage, and together play a role in preventing infection and promoting wound healing; in the later stage of wound healing, the anti-angiogenic agent in the core of the functional layer is released to inhibit neovascularization and excessive collagen proliferation, thereby effectively inhibiting scar formation. Thus, it has the effects of early promotion of healing, avoidance of infection, and prevention of scars in the later stage, thereby adapting to the entire sequential period of normal wound healing.

[0038] (3) The composite film of the wound patch of the present application is integrally formed and has good adhesion and suitable mechanical properties, good adhesion to the wound, and is overall made of natural high molecular fiber material silk fibroin, has good air permeability and low rejection, greatly alleviating the itching, stinging and other problems that may occur during wound healing in patients. BRIEF DESCRIPTION OF DRAWINGS

[0039] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings based on these drawings without creative labor.

[0040] Figure 1 is a laser confocal scanning diagram of the wound patch of Example 1 of the present application;

[0041] Figure 2 is a transmission electron microscope diagram of the wound patch of Example 2 and Comparative Example 1 of the present application;

[0042] Figure 3 is a scanning electron microscope diagram of the wound patch prepared in Example 1 of the present application;

[0043] Figure 4Figure 1 is a graph of the release profile of anti-angiogenic substance EGCG from the wound dressing prepared in Example 1 of the present application;

[0044] Figure 5 Figure 2 is the results of the rabbit ear scar model healing test of the wound dressing prepared in Example 1 and Comparative Example 1 of the present application;

[0045] Figure 6 Figure 3 is the results of the rat dorsal wound model healing test of the wound dressing prepared in Example 2 and Comparative Example 1 of the present application;

[0046] Figure 7 Figure 4 is the results of the rat dorsal wound model healing test of the wound dressing prepared in Example 3 and Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0047] The present application will be further illustrated by the following embodiments and examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. Furthermore, it should be understood that one of ordinary skill in the art can make various changes and modifications to the present application after having read and understood the content of the present application, and these equivalent forms are also within the scope of the appended claims of the present application.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description of the present application herein only for the purpose of describing the specific embodiments of the present application and is not intended to limit the present application.

[0049] Terminology

[0050] Unless otherwise indicated or unless the context clearly indicates otherwise, the terms or phrases used herein have the following meanings:

[0051] The selection range of the term "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or all relevant listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B, and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, and D, i.e., includes the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (i.e., the technical solution connected by "logical and").

[0052] In the present application, "preferably", "more preferably", "even more preferably", and the like are only used to describe the embodiments or examples with better effects, and should be understood as not constituting a limitation on the protection scope of the present application.

[0053] In the present application, "further", "even further", "in particular", and the like are used for the purpose of description, and should not be understood as constituting a limitation on the protection scope of the present application.

[0054] In the present application, the terms "first", "second", "third", "fourth", and the like are only used for the purpose of description, and should not be understood as indicating or implying relative importance or quantity, and also should not be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0055] In the present application, the technical features described in an open manner include the closed technical solution composed of the listed features, and also includes the open technical solution containing the listed features.

[0056] In the present application, when a numerical interval (i.e. a numerical range) is involved, the optional numerical values are considered to be continuous within the numerical interval and include both numerical end points (i.e. the minimum value and the maximum value) of the numerical range and each numerical value between the two numerical end points, unless otherwise specified. When a numerical interval refers only to integers within the numerical interval, the two end point integers and each integer between the two end points are included, unless otherwise specified. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be interpreted as including any and all sub-ranges therein.

[0057] In the present application, the weight can be μg, mg, g, kg, and other mass units known in the chemical industry.

[0058] In the present application, unless otherwise specified, the size, particle size, diameter generally refers to the average value.

[0059] In the present application, unless otherwise specified, the molecular weight is the average molecular weight, and further, unless otherwise specified, the index is the average molecular weight.

[0060] An embodiment of the present application provides a wound paste for preventing scar formation on skin, which comprises a composite film comprising an adhesion layer and a functional layer;

[0061] The composition of the adhesion layer comprises a first silk fibroin and metal ions having antibacterial and anti-inflammatory effects;

[0062] The composition of the functional layer comprises composite fibers having a core-shell structure;

[0063] The composition of the core of the composite fiber comprises a second silk fibroin and an angiogenesis inhibitor, and the composition of the shell comprises a third silk fibroin.

[0064] In the technical solution of the embodiment of the present application, the composite film takes silk fibroin as a substrate, so that it has flexibility, suitable mechanical properties, and is conducive to the close adhesion of each layer to each other, is integrally formed, and also has good biocompatibility, and has high adhesion to the wound. In use, the adhesion layer in the composite film is directly adhered to the wound, has good adhesion, and the released metal ions can have the effects of anti-inflammatory, antibacterial, and promoting angiogenesis, thereby promoting wound healing. The functional layer disposed above the adhesion layer comprises composite fibers having a core-shell structure, and after a period of wound healing, the active ingredient angiogenesis inhibitor in the core is gradually released, thereby avoiding scar formation.

[0065] In the technical solution of the embodiment of the present application, the adhesion layer is a fibrous layer taking silk fibroin as a substrate, contains a suitable amount of metal ions having antibacterial and anti-inflammatory effects, and the metal ions having antibacterial and anti-inflammatory effects include Ca 2+ , Zn2+ Cu 2+ Ag + Mg 2+ Eu 3+ Fe 3+ and Mn 2+ .

[0066] Optionally, the weight ratio of the first silk fibroin and the metal ion is (0.25-0.5):1, so that the metal ion and the silk fibroin base have more suitable electrostatic interaction and chelation binding force, and better antibacterial and anti-inflammatory effects can be achieved in use.

[0067] Optionally, the weight ratio of the first silk fibroin and the metal ion can be (0.25-1.5):1, such as 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1, etc.

[0068] Optionally, the metal ion with antibacterial and anti-inflammatory effects includes at least one of Ca 2+ and Zn 2+ and Mg 2+ , further, the molar proportion of Ca 2+ in the metal ion with antibacterial and anti-inflammatory effects is 10%-20%, and can further be 10%-15%, so that, in combination with the structural characteristics of the silk fibroin base of the adhesive layer, better infection prevention effects can be achieved in the early stage of wound healing, and the metal ion can also improve the adhesion of the silk fibroin base adhesive layer and improve the biocompatibility of the silk fibroin.

[0069] In the technical scheme of the embodiments of the present application, the functional layer includes a composite fiber with a core-shell structure, the composition of the core includes a second silk fibroin and an angiogenesis inhibitor, and the composition of the shell includes a third silk fibroin.

[0070] In one embodiment, the composition of the shell further includes a plant polyphenol, which can work together with the metal ion with antibacterial and anti-inflammatory effects as described above to prevent infection and promote wound healing.

[0071] Optionally, the plant polyphenol and the angiogenesis inhibitor are different in composition.

[0072] Optionally, the weight ratio of the third silk fibroin and the plant polyphenol can be selected from (100-1000):1, such as 10:1, 20:1, 50:1, 100:1, 200:1, 500:1, 1000:1, etc.

[0073] In one embodiment, the composition of the core includes a second silk fibroin and an angiogenesis inhibitor, and the weight ratio of the second silk fibroin and the angiogenesis inhibitor is (10-500):1.

[0074] Optionally, the weight ratio of the second silk fibroin and the angiogenesis inhibitor can be (10-500): 1, such as 10:1, 50:1, 100:1, 200:1, 300:1, 500:1, etc. In the technical solution of the embodiments of the present application, the weight ratio of the core and the shell of the composite fiber can be 1:(2-20), such as 1:2, 1:5, 1:8, 1:10, 1:12, 1:15, 1:18, 1:20, etc. In this way, the anti-angiogenic active ingredient in the core can achieve a delayed release, and the release amount will not be reduced due to the hindering of the shell layer, thereby affecting the effect of preventing scar formation.

[0075] The angiogenesis inhibitor includes at least one of green tea polyphenol, quercetin, consolide, apigenin, delphinidin, zephyranthine, cyanidin, pelargonidin, petunidin, peonidin, chlorogenic acid, p-coumaric acid, red raspberry, bevacizumab, ramucirumab, olaratumab, anlotinib, and sorafenib.

[0076] An embodiment of the present application provides a preparation method of the wound dressing above, comprising:

[0077] After the first silk fibroin, the metal salt, and the first solvent are mixed, a first film-forming treatment is performed to obtain the adhesion layer;

[0078] The second silk fibroin, the angiogenesis inhibitor, and the second solvent are mixed to obtain the core layer spinning solution;

[0079] The third silk fibroin and the third solvent are mixed to obtain the shell layer spinning solution;

[0080] The core layer spinning solution and the shell layer spinning solution are electrospun by the coaxial electrospinning process to obtain the composite fiber, and the adhesion layer is used as a receiving plate to form the functional layer on the adhesion layer.

[0081] It can be understood that the material of the core layer spinning solution forms the core of the composite fiber, the material of the shell layer spinning solution forms the shell of the composite fiber, and the coaxial electrospinning process is used to prepare the composite fiber with the corresponding core-shell structure by electrospinning the core layer spinning solution and the shell layer spinning solution from the appropriate nozzle under suitable electrospinning conditions.

[0082] In one embodiment, the weight ratio of the solutes of the core layer spinning solution and the shell layer spinning solution is 1:(2-20).

[0083] In one embodiment, the third silk fibroin, the third solvent, and the plant polyphenol are mixed to obtain the shell layer spinning solution;

[0084] The plant polyphenol and the angiogenesis inhibitor have different components;

[0085] Optionally, the weight ratio of the third silk fibroin and the plant polyphenol is (20-1000):1.

[0086] In one embodiment, after mixing the first fiber, the metal salt and the solvent, the step of preparing the first film by the first film-forming treatment comprises:

[0087] After mixing the first fiber, the metal salt and the solvent, a first solution is obtained;

[0088] The first solution is prepared into a film by electrospinning or flat laying and evaporating dry to obtain the first film.

[0089] In one embodiment, the step of electrospinning the core layer spinning solution and the shell layer spinning solution by the coaxial electrospinning process to obtain the composite fiber comprises:

[0090] The core layer spinning solution and the shell layer spinning solution are electrospun by the coaxial electrospinning equipment, the outer needle inner diameter of the nozzle of the coaxial electrospinning equipment is 0.86mm-1.4mm, and the outer needle outer diameter is 0.31mm-0.6mm;

[0091] The push injection speed of the core layer spinning solution is set to 0.05mL / h-2mL / h, and the push injection speed of the shell layer spinning solution is set to 0.1mL / h-5mL / h;

[0092] Optionally, the voltage of the electrospinning is 10kV-25kV, and the collection distance is 10cm-20cm.

[0093] The wound plaster adopting the technical solution of the present application can delay the initial release of the angiogenesis inhibitor (EGCG) relative to the traditional multi-layer wound plaster, and there is basically no release in 0-3 days, the release is accelerated in about 3-7 days and continues to about 14 days for stable release. This is because in the initial stage, the adhesion layer (the layer in contact with the wound) hinders the penetration of water molecules, thereby delaying the release of EGCG. As the wound heals, the adhesion layer gradually dissolves, and a small amount of EGCG physically adsorbed or weakly combined on the double-layer film nanofiber of the core-shell structure is continuously released. After 3-7 days, the release speed of EGCG is accelerated, but due to the diffusion mechanism of the silk fibroin nanofiber and the slow degradation speed thereof, the release of EGCG in the double-layer core-shell wound plaster can continue for nearly 14 days.

[0094] The wound plaster adopting the technical solution of the present application reduces the initial burst release, prolongs the drug release curve, allows the release of EGCG to be controlled for more than 14 days, realizes the initial slow release and on-demand release, and provides support for the anti-inflammatory and antioxidant effects of a small amount of EGCG in wound healing in the early stage and the sustained release of EGCG for inhibiting angiogenesis in the later stage.

[0095] The following are some specific embodiments.

[0096] The experimental parameters not written in the following specific examples are preferably referred to the guidance given in this application document, and can also be referred to the experimental manual in the art or other experimental methods known in the art, or the experimental conditions recommended by the manufacturer.

[0097] The raw materials and reagents involved in the following specific examples can be obtained commercially or prepared by those skilled in the art according to known means.

[0098] Example 1

[0099] This example provides a preparation method of the wound dressing of the present application and its application in wound healing, which is as follows:

[0100] 1.1 Preparation of the wound dressing

[0101] (1) Dissolve NaHCO3 in 2 L distilled water and boil, add 10 g of cut silkworm cocoons, and boil for 30 min under boiling condition. Wash the silk with 2 L distilled water (60-70 °C) for 4-5 times, and dry in an oven at (40-60 °C). Soak the degummed silk in a 9.3 mol / L lithium bromide solution, dissolve for 4 hours in a dry box at 60 °C, and dialyze in deionized water for three days using a 1.2 KD dialysis bag. Freeze-dry to white fibers to prepare purified silk fibroin.

[0102] (2) Dissolve 0.2 g of calcium chloride, 0.02 g of zinc chloride, and 0.8 g of silk fibroin in 8 mL of formic acid to prepare a first film by first electrospinning.

[0103] (3) Add 0.01 g of EGCG to 5 mL of silk fibroin formic acid solution with a concentration of 10% to prepare a core layer spinning solution. Use 10 mL of silk fibroin with a concentration of 10% as a shell layer spinning solution, and prepare a composite fiber using coaxial electrospinning process conditions of the electrospinning instrument, with the first film as the receiving layer and a power supply voltage of 20 kV. The injection speed of the core layer spinning solution is 0.5 mL / h, and the injection speed of the shell layer spinning solution is 1.5 mL / h. The inner diameter of the outer needle is 1 mm, the inner diameter of the inner needle is 0.4 mm, and the collection distance is 15 cm. The electrospun collection film is fully dried to remove the solvent, and the wound dressing S1 is obtained.

[0104] Example 2

[0105] This example provides a preparation method of the wound dressing of the present application and its application in wound healing, which is as follows:

[0106] 1.1 Preparation of the wound dressing

[0107] (1) Take NaHCO3 dissolved in 2L distilled water, add 10g cut silkworm chrysalis, cook for 30min under boiling state, wash the silk with 2L distilled water (60-70℃) for 4-5 times, dry in (40-60℃) oven, immerse the degummed silk into 9.3mol / L lithium bromide solution, dissolve for 4 hours in 60℃ drying box, use 1.2KD dialysis bag to dialyze in deionized water for three days, freeze-dry into white fiber to prepare purified silk fibroin.

[0108] (2) Take 0.2 calcium chloride, 0.02 magnesium chloride, 0.8 silk fibroin, dissolve in 8 mL formic acid to prepare the first film by first electrospinning; the conditions of first electrospinning include: electrospinning machine power voltage 20 kV, solution injection speed 0.5 mL / h, collection distance 15 cm.

[0109] (3) Add 0.05g quercetin to 5mL silk fibroin formic acid solution with a concentration of 10% to prepare the core layer spinning solution; dissolve 0.05g tannic acid in 10mL silk fibroin with a concentration of 10% to prepare the shell layer spinning solution, use coaxial electrospinning process to prepare composite fibers, use the first film as the receiving layer, power voltage 20 kV; the injection speed of core layer spinning solution is 0.5 mL / h, the injection speed of shell layer spinning solution is 2 mL / h; the inner diameter of outer needle is 1 mm, the inner diameter of inner needle is 0.5 mm; the collection distance is 15 cm; after electrospinning collection film and fully drying the volatile solvent, the wound paste S2 is obtained.

[0110] Example 3

[0111] This example provides a preparation method of the wound paste of the present application and its application in wound healing, as follows:

[0112] 1.1 Preparation of wound paste

[0113] (1) Take NaHCO3 dissolved in 2L distilled water, add 10g cut silkworm chrysalis, cook for 30min under boiling state, wash the silk with 2L distilled water (60-70℃) for 4-5 times, dry in (40-60℃) oven, immerse the degummed silk into 9.3mol / L lithium bromide solution, dissolve for 4 hours in 60℃ drying box, use 1.2KD dialysis bag to dialyze in deionized water for three days, freeze-dry into white fiber to prepare purified silk fibroin.

[0114] (2) Take 0.2 grams of calcium chloride, 0.02 grams of zinc chloride, and 2 grams of silk fibroin and dissolve them in 20 mL of formic acid to form a first film by first electrospinning. The conditions for the first electrospinning include: a power supply voltage of 20 kV for the electrospinning machine, a push injection speed of 0.5 mL / h for the solution, and a collection distance of 15 cm.

[0115] (3) Add 0.02 grams of EGCG to 5 mL of a 10% silk fibroin formic acid solution to prepare a core layer spinning solution. Dissolve 0.1 grams of anthocyanin in 10 mL of a 10% silk fibroin solution to prepare a shell layer spinning solution. Use the coaxial electrospinning process of the electrospinning machine to prepare the composite fiber, with the first film as the receiving layer and a power supply voltage of 25 kV. The push injection speed of the core layer spinning solution is 0.5 mL / h, and the push injection speed of the shell layer spinning solution is 2 mL / h. The inner diameter of the outer needle is 1 mm, the inner diameter of the inner needle is 0.4 mm, the collection distance is 15 cm, and the electrospun film is fully dried to remove the solvent. The resulting product is a wound paste S3.

[0116] Comparative Example 1

[0117] This comparative example provides a preparation method of a wound paste according to the present application and its application in wound healing, as follows:

[0118] 1.1 Preparation of the wound paste

[0119] (1) Dissolve NaHCO3 in 2L distilled water and boil, add 10g of cut silkworm cocoons, and cook for 30min under boiling conditions. Wash the silk with 2L of distilled water (60-70℃) for 4-5 times, and dry it in an oven at (40-60℃). Soak the degummed silk in a 9.3mol / L lithium bromide solution, dissolve it in a 60℃ drying box for 4 hours, and then dialyze it in a 1.2KD dialysis bag for three days in deionized water. Freeze-dry the white fibers to prepare purified silk fibroin.

[0120] (2) Take 0.8 grams of silk fibroin and dissolve it in 8 mL of formic acid to form a first film by first electrospinning. The conditions for the first electrospinning include: a power supply voltage of 20 kV for the electrospinning machine, a push injection speed of 0.5 mL / h for the solution, and a collection distance of 15 cm.

[0121] (3) 5 mL of 10% concentration of silk fibroin formic acid solution was prepared as the core layer spinning solution; 10 mL of 10% concentration of silk fibroin was used as the shell layer spinning solution, and a coaxial electrospinning process was used to prepare the composite fiber, with the first film as the receiving layer, a power supply voltage of 20 kV; the injection speed of the core layer spinning solution was 0.5 mL / h, and the injection speed of the shell layer spinning solution was 2 mL / h; the inner diameter of the outer needle was 1 mm, the inner diameter of the inner needle was 0.31 mm; the collection distance was 15 cm; after the electrospun film was fully dried and the volatile solvent was removed, the wound paste S4 was obtained.

[0122] Comparative Example 2

[0123] This comparative example provides a preparation method of the wound paste of the present application and its application in wound healing, as follows:

[0124] 1.1 Preparation of the wound paste

[0125] (1) NaHCO3 was dissolved in 2 L of distilled water and boiled, 10 g of cut silkworm cocoons were added, and boiled for 30 min under boiling condition, the silk was washed with 2 L of distilled water (60-70℃) for 4-5 times, and dried in an oven at (40-60℃), the degummed silk was immersed in a 9.3 mol / L lithium bromide solution, dissolved in a 60℃ drying box for 4 hours, and then dialyzed in a 1.2 KD dialysis bag for three days, and then freeze-dried into white fibers to prepare purified silk fibroin.

[0126] (2) 0.5 g of calcium chloride, 0.1 g of zinc chloride, and 0.8 g of silk fibroin were dissolved in 8 mL of formic acid to prepare a first film by electrospinning; the first electrospinning conditions included a power supply voltage of 20 kV, a solution injection speed of 0.5 mL / h, and a collection distance of 15 cm.

[0127] (3) 0.02 g of EGCG and 0.1 g of anthocyanin were added to 15 mL of 10% concentration of silk fibroin formic acid solution to prepare a spinning solution; the first film was used as the receiving layer, and the power supply voltage was 20 kV; the injection speed of the spinning solution was 0.5 mL / h, and the collection distance was 15 cm; after the electrospun film was fully dried and the volatile solvent was removed, the wound paste S5 was obtained.

[0128] II. Performance characterization

[0129] (1) Characterization of the internal structure of the functional layer of the wound paste

[0130] The composite fibers were prepared according to the step (3) of Reference Example 1, except that rhodamine B and calcein were used to replace the inner layer EGCG and silk fibroin, respectively, and the composite fibers D1 were prepared using the coaxial electrospinning process. A part of the fibers was placed on a glass slide and observed under a laser scanning confocal microscope. The structure is shown in Figure 1 According to the structure of the composite fibers, Figure 1 the core layer emits red fluorescence under 540 nm excitation, and the shell layer emits green fluorescence under 495 nm excitation, which confirms that the core-shell structure is successfully prepared by coaxial electrospinning. The core-shell structure formed by coaxial electrospinning allows the slow release of the carried therapeutic drugs or substances to meet the drug concentration requirements during the wound repair process of the tissue.

[0131] The composite fibers of Example 2 and Comparative Example 1 were observed under a transmission electron microscope. The results are shown in Figure 2 According to the structure of the composite fibers, Figure 2 the core layer and the shell layer of the composite fibers prepared in Example 2 have different densities, resulting in different brightness under the transmission electron microscope, which confirms that the core-shell structure is successfully prepared by coaxial electrospinning. The core-shell structure formed by coaxial electrospinning allows the slow release of the carried therapeutic drugs or substances to meet the drug concentration requirements during the wound repair process of the tissue.

[0132] (2) Overall appearance characterization of wound dressing

[0133] The wound dressing S1 of Example 1 was taken, fractured by liquid nitrogen, and the cross-sectional front surface was adhered to a conductive glue. After gold spraying, the sample was placed on a scanning electron microscope instrument for observation. The results are shown in Figure 3

[0134] According to the structure of the composite fibers, Figure 3 it can be found that the lower adhesive layer of the wound dressing S1 is a thin film formed by a large number of silk fibroin filaments, and calcium and zinc ions are uniformly distributed therein. The functional layer of the upper layer is composed of composite fibers with a core-shell structure. The average size of the composite fibers is about 900 nm, the morphology is uniform, and the surface is smooth.

[0135] (3) Active substance release test

[0136] The wound dressing S1 of Example 1 was taken, cut to an appropriate size, and attached to a transwell chamber. The transwell chamber was immersed in a PBS solution in a 24-well plate at 37°C. At the predetermined time point, the amount of EGCG released was measured by a Shimadzu UV-2600 ultraviolet-visible spectrophotometer. The results are shown in Figure 4

[0137] According to the structure of the composite fibers, Figure 4 ​​It was found that the monolayer non-core-shell layer, monolayer core-shell layer, Example 1 double-layer core-shell adhesive patch placed on the Transwell chamber filter membrane, the monolayer non-core-shell layer fiber showed a typical one-step release, a higher proportion of EGCG was completely released in a short time (12 h). In contrast, the monolayer core-shell layer fiber showed a two-stage release behavior, 58% of EGCG was released in the first day, followed by a sustained slow release, reaching a cumulative release rate of 100% by the 7th day. In the case of the double-layer adhesive patch of Example 1, the presence of the lower adhesion layer hindered the penetration of water molecules, thereby delaying the release of EGCG. During the dissolution of the lower layer, a small amount of EGCG physically adsorbed or weakly bound on the double-layer membrane nanofiber of the core-shell structure was continuously released. However, after 3-5 days, the release rate of EGCG accelerated, but due to the diffusion mechanism of SF nanofiber and its slow degradation rate, the release of EGCG in the double-layer core-shell adhesive patch lasted for nearly 14 days. These results show that by using the double-layer core-shell adhesive patch fiber structure, the initial burst release is reduced, the drug release curve is prolonged, allowing the release of EGCG to be controlled for more than 14 days. This allows the initial slow release and on-demand release to provide support for the anti-inflammatory and antioxidant effects of the early small amount of EGCG in wound healing and the sustained release of EGCG to inhibit angiogenesis later.

[0138] (4) Rabbit ear scar model healing test

[0139] New Zealand white rabbits (3-4 months old) were anesthetized by intravenous injection of sodium pentobarbital (30 mg / kg), and each ear received three wounds using an 8 mm biopsy punch and completely excised the cartilaginous membrane with a scalpel. Subsequently, they were randomly divided into 3 groups: (1) a blank control group, (2) a Comparative Example 1 group, and (3) an Example 1 group. The New Zealand white rabbits of the blank control group were treated with phosphate buffered saline (10 μL) on the wound surface, and the New Zealand white rabbits of the Comparative Example 1 group and the Example 1 group were completely covered with the film of the Comparative Example 1 group and the wound film of Example 1, respectively, on the rabbit ear wound surface. The wound surface was photographed on days 0, 3, 7, 14, 21, and 28, and the rabbit ear tissue was taken for histological analysis on days 14, 21, and 28, and the results are shown in FIGS. 10A, 10B, and 10C. Figure 5

[0140] According to Figure 5 It was found that the wound healing of the Comparative Example 1 group and the Example 1 group was faster within 1 week, and the wound surface of the Example 1 group was almost completely healed, while the wound healing of the control group was poor, accompanied by exudation and bacterial infection. By the 14th day, the control group was not completely healed, the wound surface was raised, and was deep red in color, and the Example 1 group had no obvious raised area. On the 21st day and the 28th day, the control group was significantly scabbed, and the Comparative Example 1 group had a severe protrusion in a local area. In contrast, the Example 1 group was often flat, with obvious hair growth on the surface.

[0141] ​(5) Rat dorsal scar model healing test

[0142] SD rat full-thickness wound model was established, and experimental animals were randomly divided into blank group, Comparative Example 1 group, and Example 2 group. All animals were anesthetized by intraperitoneal injection of chloral hydrate (0.3 mg / kg), then shaved back with an electric animal shaver, and finally completely exposed the surgical site with depilatory cream. Subsequently, 6 full-thickness wounds (diameter ~ 15 mm) were created on the back of each rat with surgical scissors. After removing the wound skin, Comparative Example 1 group and Example 2 group were covered with SF film prepared by Comparative Example 1 and wound film prepared by Example 2, respectively, and the blank group was treated with 50 μL of pH 7.4 phosphate buffer. The wound healing process was recorded and analyzed by Image J on postoperative days 0, 7, 14, and 21, and the results are shown in FIGS. 8A-8D. Figure 6 and Figure 7

[0143] According to Figure 6 , it was confirmed by rat full-thickness skin defect repair experiment that the adhesive dressing prepared by Example 2 had the effects of promoting wound healing and inhibiting scar formation. According to the image of the rat dorsal wound and the quantitative analysis of the wound diameter by ImageJ, the wound healing trend was monitored on day 0, day 3, day 7, day 14, and day 21. Overall, the wound contraction rate of the adhesive dressing prepared by Example 2 was significantly faster than that of the control group and other groups. By day 7, the difference in healing rate between the adhesive dressing prepared by Example 2 and other groups could already be seen. By day 14, the wound in the adhesive dressing prepared by Example 2 group was basically healed, leaving only a small scar. In contrast, the wound sizes of the other two groups were different, and the unhealed wound area of the control group was the largest. By day 21, all groups except the control group had completely healed. These results showed that the application of integrated silk fibroin-based Janus double-layer core-shell sustained-release adhesive wound plaster can promote wound healing.

[0144] According to Figure 7 ​The effect of the dressing of Example 3 in promoting wound healing and inhibiting scar formation was confirmed by a rat full-thickness skin defect repair experiment, and the dressing prepared in the comparative example had no obvious effect on promoting wound healing. The experimental groups were the dressing of Example 3, the dressing of Comparative Example 2, and the completely untreated group (control group). According to the image of the wound on the back of the rat and the quantitative analysis of the wound diameter by ImageJ, the wound healing trend was monitored on day 0, day 7, and day 14. In the overall trend, the wound contraction rate of the dressing of Example 3 was significantly faster than that of the control group and the Comparative Example 2 group. By day 7, the difference in healing rate between the dressing of Example 3 and the control group and the Comparative Example 1 group could already be seen. By day 14, the wound in the dressing of Example 3 group was basically healed, leaving only a small scar. In contrast, the dressing of Comparative Example 2 group was not much different from the control group, both leaving a larger wound. Comparative Example 2 did not have a core-shell structure to release the anti-angiogenic substance, which prematurely inhibited the formation of blood vessels, resulting in delayed wound healing.

[0145] All the documents mentioned in the present application are incorporated by reference in the present application as if each document is incorporated by reference individually. Unless and to the extent that the documents mentioned in the present application conflict with the purpose and / or technical scheme of the present application, the documents mentioned in the present application are incorporated by reference in their entirety and in their entirety. When the present application refers to the documents mentioned in the present application, the definition of the relevant technical features, terms, names, phrases, etc. in the documents mentioned in the present application are also incorporated by reference. When the present application refers to the documents mentioned in the present application, the examples and preferred modes of the relevant technical features mentioned in the documents mentioned in the present application are also incorporated by reference in the present application, but to the extent that the present application can be implemented. It should be understood that when the content of the reference conflicts with the description in the present application, the present application is used as the reference or is modified according to the description in the present application.

[0146] The technical features of the above-mentioned embodiments and examples can be combined in any suitable manner. In order to make the description simple, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described, however, as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present description.

[0147] The above embodiments only express several implementation ways of the present application, but cannot be understood as limitation to the patent scope. It should be pointed out that, for ordinary skilled in the art, several variations and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. In addition, it should be understood that, after reading the above teaching of the present application, the skilled in the art can make various changes or modifications to the present application, and the equivalent forms also fall within the protection scope of the present application. It should also be understood that, the skilled in the art can obtain the technical solutions on the basis of the technical solutions provided by the present application through logical analysis, reasoning or limited test, which all fall within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A wound dressing for preventing skin scarring, characterized in that, It includes a composite film comprising an adhesive layer and a functional layer; The adhesive layer comprises a first silk fibroin and metal ions; the metal ions have at least one of the following effects: antibacterial, anti-inflammatory, and angiogenesis-promoting. The functional layer is composed of composite fibers with a core-shell structure; The core of the composite fiber comprises a second silk fibroin and an angiogenesis inhibitor, while the shell comprises a third silk fibroin.

2. The wound dressing according to claim 1, characterized in that, The weight ratio of the first silk fibroin to the metal ions is (0.25~1.5):

1.

3. The wound dressing according to claim 1, characterized in that, The weight ratio of the second silk fibroin to the angiogenesis inhibitor is (10~500):1; and / or The weight ratio of the core to the shell of the composite fiber is 1:(2~20).

4. The wound dressing according to any one of claims 1 to 3, characterized in that, The metal ions include Ca. 2+ Zn 2+ Cu 2+ Ag + Mg 2+ Eu 3+ Fe 3+ and Mn 2+ One or more of the following; and / or The angiogenesis inhibitors include at least one of the following: green tea polyphenols, quercetin, delphinidin, apigenin, luteolin, stigmosiderin, anthocyanins, pelargonidin, picaridin, paeonol, chlorogenic acid, p-coumaric acid, red raspberry, bevacizumab, ramucirumab, olalimumab, anlotinib, and sorafenib.

5. The wound dressing according to any one of claims 1 to 3, characterized in that, The shell also includes plant polyphenols; Optionally, the weight ratio of the third silk fibroin to the plant polyphenol is (10~1000):

1.

6. A method for preparing a wound dressing according to any one of claims 1 to 5, characterized in that, include: The first silk fibroin, metal salt and first solvent are mixed and then subjected to a first film-forming treatment to obtain the adhesion layer; The second silk fibroin, the angiogenesis inhibitor, and the second solvent are mixed to obtain the core spinning solution; The third silk fibroin and the third solvent are mixed to obtain the shell spinning solution; The composite fiber is obtained by electrospinning the core spinning solution and the shell spinning solution using a coaxial electrospinning process, and the composite fiber is formed on the adhesive layer as a receiving plate to form a functional layer on the adhesive layer, thus obtaining the composite film.

7. The preparation method according to claim 6, characterized in that, The weight ratio of the solute in the core spinning solution to the shell spinning solution is 1:(2~20).

8. The preparation method according to claim 6 or 7, characterized in that, After mixing the third silk fibroin and the third solvent, plant polyphenols are added and mixed to obtain the shell spinning solution. Optionally, the weight ratio of the third silk fibroin to the plant polyphenol is (10~1000):

1.

9. The preparation method according to claim 6 or 7, characterized in that, The steps of preparing the adhesion layer by mixing the first fiber, metal salt, and solvent, followed by a first film-forming treatment, include: The first fiber, metal salt, and solvent are mixed to obtain the first solution; The first solution is prepared into a thin film by electrospinning or by spreading and then evaporating to obtain the adhesion layer. Optionally, the injection rate of the first solution is 0.05 mL / h to 2 mL / h; Optionally, the voltage of the first electrospinning is 10kV~25kV, and the collection distance is 10cm~20cm.

10. The preparation method according to claim 6 or 7, characterized in that, The step of electrospinning the core spinning solution and the shell spinning solution using a coaxial electrospinning process to obtain the composite fiber includes: The core spinning solution and the shell spinning solution are subjected to a second electrospinning process using a coaxial electrospinning device. The inner diameter of the outer nozzle of the coaxial electrospinning device is 0.86 mm to 1.4 mm, and the outer diameter of the inner nozzle is 0.31 mm to 0.6 mm. Optionally, the injection rate of the core spinning solution is 0.05 mL / h to 2 mL / h, and the injection rate of the shell spinning solution is 0.1 mL / h to 5 mL / h. Optionally, the voltage of the second electrospinning is 10kV~25kV, and the collection distance is 10cm~20cm.

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