A composite membrane, its preparation method and applications

Abstract

A composite membrane comprises a membrane matrix formed by the stable cross-linking of silk fibroin with β-sheets, and baicalin uniformly dispersed between the segments of the silk fibroin molecular chain, wherein the mass ratio of silk fibroin to baicalin is 100:3-4. The preparation method includes the following steps: 1) taking silkworm cocoons, boiling them several times with sodium carbonate solution, washing them, and drying them to 60°C; 2) dissolving them in a ternary solution, centrifuging and filtering to obtain a clear silk fibroin solution, and drying it to obtain silk fibroin; 3) preparing a silk fibroin solution with a concentration of 1-30 mg / ml, and adjusting the pH to 7.4; 4) adding baicalin to the silk fibroin solution, forming a film, drying it, and annealing it to obtain the composite membrane. The composite membrane of this invention has superior biocompatibility, hemostatic properties, and controllable biodegradability, and can promote M2 polarization of macrophages, shorten the inflammatory period, and effectively promote skin wound healing.

Description

Technical Field

[0001] This invention relates to the field of materials, and in particular to a composite membrane, its preparation method, and its uses. Background Technology

[0002] The skin is the largest organ in the human body and serves as a protective barrier against the external environment. Skin trauma is one of the most common types of injury. Skin trauma can be categorized into acute wounds (such as mechanical, chemical, and surgical wounds) and chronic wounds (such as burns, infections, and diabetes). Chronic wounds, such as those from burns, infections, and diabetes, typically take three months or longer to heal, and are not only slow to heal but also prone to scarring. For severe chronic wounds, healing is slow, and without timely intervention to accelerate healing, the extremities may become necrotic as the disease progresses, sometimes accompanied by severe pain. In such cases, amputation or skin grafting may be necessary. Patients in these situations not only bear enormous medical expenses but also endure prolonged suffering and psychological stress.

[0003] Wound healing comprises four distinct phases: the coagulation phase, the inflammation phase, the proliferative phase, and the tissue remodeling phase. Macrophages and fibroblasts play indispensable roles in skin healing. M1 macrophages trigger inflammatory responses and kill pathogens, while M2 macrophages promote inflammation resolution and skin tissue formation. The balance of macrophage polarization towards M1 and M2 types is crucial for rapid, scarless wound healing. Fibroblasts are among the main cells in the dermis. After skin injury, fibroblasts are recruited in large numbers to the wound site and differentiate into myofibroblasts under the stimulation of relevant growth factors and cytokines. Myofibroblasts secrete large amounts of collagen at the wound site and provide strong tensile strength to promote tissue fibrosis, thereby accelerating wound contraction and healing. However, the continued presence of myofibroblasts can lead to scar formation. Developing wound repair materials that can regulate the inflammatory response and fibroblast differentiation during the wound healing process holds promise for achieving rapid, scarless healing of difficult-to-heal wounds. Currently, there are various treatment methods for skin injuries, such as autologous transplantation, allogeneic transplantation, and wound dressings. However, difficult-to-heal wounds and the formation of scar tissue after wound healing remain clinical challenges.

[0004] Therefore, developing a drug that can promote wound healing, especially shorten the inflammatory period, is one of the directions that those skilled in the art hope to achieve. Summary of the Invention

[0005] One of the objectives of this invention is to address the shortcomings of existing technologies by providing a composite membrane that has superior biocompatibility, hemostatic properties, and controllable biodegradability, and can promote M2 polarization of macrophages, shorten the inflammatory period, and effectively promote skin wound healing.

[0006] The second objective of this invention is to provide a method for preparing the above-mentioned composite membrane, which is simple and convenient and suitable for large-scale industrial production.

[0007] One of the technical solutions to achieve the objective of this invention is: a composite membrane comprising a membrane matrix formed by the stable cross-linking of silk fibroin with each other, and baicalin uniformly dispersed between the silk fibroin molecular segments, wherein the mass ratio of silk fibroin to baicalin is 100:3-4.

[0008] The membrane matrix has a thickness of 0.05-0.2 mm and a unit area mass of 2.5-4.0, preferably 3.73 mg / cm³. 2 The mass ratio of silk fibroin to baicalin is 100:3-4.

[0009] The second technical solution to achieve the objective of this invention is: a method for preparing any of the above-mentioned composite membranes, comprising the following steps:

[0010] 1) Take silkworm cocoons, boil them several times with sodium carbonate solution, wash them, and dry them at 60℃;

[0011] 2) Dissolve the silk fibroin in a ternary solution, centrifuge and filter to obtain a clear solution of silk fibroin, and dry to obtain silk fibroin;

[0012] 3) Prepare a silk fibroin solution with a concentration of 1-30 mg / ml and adjust the pH to 7.4;

[0013] 4) Add baicalin to the silk fibroin solution, and after film formation, dry and anneal to obtain a composite film.

[0014] Step 1) The concentration of sodium carbonate solution is 0.5 wt%, and the dissolution is carried out three times, with each dissolution time being 25-30 min. Wash with clean water.

[0015] Furthermore, the ternary solution in step 2) is a mixture of calcium chloride, ethanol, and water, with a molar ratio of calcium chloride, ethanol, and water of 1:2:8.

[0016] Preferably, in step 2), the solution is dissolved in a water bath at 60°C for 1-2 hours. After complete dissolution, the solution is naturally cooled to room temperature and centrifuged at 8000 rpm for 10 minutes. The solution is filtered using 400-500 mesh nylon cloth and then freeze-dried at -20°C for 48 hours to obtain silk fibroin.

[0017] Preferably, in step 3), the freeze-dried silk fibroin is dissolved in ultrapure water to prepare a silk fibroin solution with a concentration of 14 mg / ml, and the pH is adjusted using 1M sodium hydroxide-1M acetic acid.

[0018] Further, in step 4), baicalin is prepared as a baicalin solution, the solvent is a silk fibroin solution with a pH of 7.4 and a concentration of 3 wt%, and the mass ratio of baicalin to silkworm cocoon is 3:125-140.

[0019] Further, in step 4), the film is formed by casting, dried at 42°C for 40-44 hours, and then annealed at 70-80% humidity for 4-5 hours.

[0020] The present invention also provides the use of any of the above-described composite films in the preparation of medicaments for the repair of chronic skin wounds.

[0021] Furthermore, the medication is a dressing.

[0022] The above technical solution has the following beneficial effects:

[0023] 1. The composite membrane of this invention comprises a membrane matrix of silk fibroin interlinked by stable β-sheets, and baicalin uniformly dispersed between the silk fibroin molecular segments on the membrane matrix. The β-sheets are water-insoluble structures, preventing wound adhesion. Furthermore, once formed, the β-sheets are irreversible, meaning this membrane is highly stable and easy to transport and store. The molecular segments of the β-sheets form several spaces within which baicalin is released easily and exerts its therapeutic effect rapidly. By controlling the mass ratio of silk fibroin to baicalin to 100:3-4, biocompatibility is maximized while ensuring efficacy. If the mass ratio is too high, the efficacy is insufficient; if the mass ratio is too low, cytotoxicity is high and safety is low. The silk fibroin in the composite membrane possesses superior biocompatibility, good tensile strength, excellent hemostatic properties, controllable biodegradability, and non-cytotoxicity. Its derived implants also exhibit good immunogenicity. Furthermore, the degradation products of silk fibroin materials consist of soluble peptides and free amino acids, which are easily metabolized and absorbed by the human body, making them excellent repair materials. Baicalin is a flavonoid active substance extracted from the root of the traditional Chinese medicine Scutellaria baicalensis. It is inexpensive, easy to extract, and possesses anti-inflammatory and anti-infective activities. In addition, Scutellaria baicalensis promotes M2 polarization of giant cells, which can shorten the inflammatory phase. However, silk fibroin alone has low anti-inflammatory activity and slows wound healing, while baicalin alone has low bioavailability and is not easily effective. Combining the two achieves highly efficient utilization and potent healing.

[0024] 2. The method for preparing the composite membrane of the present invention involves repeatedly boiling silkworm cocoons in a 0.5wt% Na2CO3 solution to remove the sericin from the cocoons, which facilitates subsequent dissolution. After washing with water, the cocoons are dried to 60°C to facilitate subsequent dissolution. Drying prevents the addition of water from disrupting the subsequent ternary solution system.

[0025] 3. The ternary solution prepared in this invention, by disrupting the β-sheet of the silkworm cocoon, facilitates the subsequent reshaping and formation of the membrane shape. After film formation, annealing at a humidity of 70-80% for 4-5 hours is conducive to the formation of silk fibroin β-sheets, ultimately yielding the composite membrane of the structure of this invention.

[0026] The following is a further explanation of specific implementation methods. Attached Figure Description

[0027] Figure 1 The infrared spectrum and peak fitting diagram of the composite film prepared in Example 1;

[0028] Figure 2 The X-ray diffraction pattern of the composite film prepared in Example 2;

[0029] Figure 3 Transmittance test graphs of the composite membrane prepared in Example 2 and the blank membrane of the control example;

[0030] Figure 4 Photograph of the composite membrane in Example 2. Detailed Implementation

[0031] In this invention, the silkworm cocoons used were obtained from the National Key Laboratory for Efficient Breeding and Utilization of Resource Insects; the baicalin purity was 90% and it was obtained from Aladdin Reagent (Shanghai) Co., Ltd.; the anhydrous calcium chloride was obtained from JESK with a purity of 99.99%; the anhydrous ethanol was obtained from Chengdu Jinshan Chemical Reagent Co., Ltd. with batch number 20240806; and the ultrapure water was prepared by an instrument (Sartorius).

[0032] Prepare a ternary solution: CaCl2 (111g): CH3CH2OH (92.14mL): H2O (144mL) = 1:2:8 (molar ratio).

[0033] Example 1

[0034] 1) Take 10g of silkworm cocoons and boil them three times with 0.5wt% Na2CO3 solution. Each boiling time is 25min to remove the sericin from the silkworm cocoons. After washing with water, dry them at 60℃ to facilitate subsequent dissolution.

[0035] 2) Dissolve in 300ml of ternary solution;

[0036] 3) Centrifuge at 8000 rpm for 10 min, use a 1000 μL pipette to aspirate the supernatant, then filter the supernatant through a 400-500 mesh nylon cloth, dialyze the obtained supernatant through a dialysis bag for 3 days at 8000-14000 rpm, and then freeze-dry it in a freeze dryer for 48 h (sample temperature -20℃) to obtain freeze-dried silk fibroin.

[0037] 4) Take 70 mg of lyophilized silk fibroin, add 5 ml of ultrapure water, and prepare 5 ml of silk fibroin solution with a concentration of 14 mg / ml;

[0038] 5) Depending on the pH of the pure water used, adjust the pH of the silk fibroin solution to 7.4 using 1M NaOH-1M CH3COOH;

[0039] 6) Take 152.1 mg of baicalin solution and add it to silk fibroin solution. Mix well using a vortex mixer to obtain a mixed solution. The concentration of baicalin solution is 3 wt%.

[0040] 7) Take 10 ml of the mixture and cast it into a 9 cm polyethylene petri dish to form a film, ensuring there are no air bubbles;

[0041] 8) Dry in an oven at 42℃ for 39-42 hours;

[0042] 9) Close the oven and place a beaker containing 100-120ml of distilled water into the oven. Anneal for 4-5 hours.

[0043] 10) Remove the culture dish, use tweezers to demold, and obtain the composite membrane. Its infrared spectrum and peak fitting are as follows: Figure 1 As shown. From Figure 1 It is known that the addition of baicalin does not affect the stability of the material. Its beta-fold content is as high as 49.3%, and it has excellent mechanical properties. It is a very stable material that is easy to transport.

[0044] Example 2

[0045] 1) Take 15g of silkworm cocoons and boil them three times with 0.5wt% Na2CO3 solution to remove the sericin from the cocoons. After washing with ultrapure water, boil them once more with ultrapure water and dry them to 60℃ to facilitate subsequent dissolution.

[0046] 2) Dissolve in 300ml of ternary solution;

[0047] 3) Centrifuge at 8000 rpm for 10 min, use a 1000 μL pipette to aspirate the supernatant, then filter the supernatant through a 400-500 mesh nylon cloth, dialyze the obtained supernatant through a dialysis bag for 3 days at 8000-14000 rpm, and then freeze-dry it in a freeze dryer for 48 h (sample temperature -20℃) to obtain freeze-dried silk fibroin.

[0048] 4) Take 140 mg of lyophilized silk fibroin, add 10 ml of ultrapure water, and prepare 10 ml of silk fibroin solution with a concentration of 14 mg / ml;

[0049] 5) Depending on the pH of the pure water used, adjust the pH of the silk fibroin solution to 7.4 using 1M NaOH-1M CH3COOH;

[0050] 6) Take 304.2 mg of baicalin solution and add it to silk fibroin solution. Mix well using a vortex mixer to obtain a mixed solution. The concentration of baicalin solution is 3 wt%.

[0051] 7) Take 10 ml of the mixture and cast it into a 9 cm polyethylene petri dish to form a film, ensuring there are no air bubbles;

[0052] 8) Dry in an oven at 42℃ for 44-48 hours;

[0053] 9) Close the oven and place a beaker containing 150-200ml of distilled water into the oven. Anneal for 4-5 hours.

[0054] 10) Remove the culture dish, use tweezers to demold, and obtain the composite membrane. Its X-ray diffraction pattern is shown below. Figure 2 As shown. From Figure 2 It can be seen that baicalin may maintain a crystalline state after being loaded into a pure silk fibroin membrane, indicating that baicalin can exist stably in the silk fibroin membrane and exert its medicinal effect.

[0055] Compare with Example 1

[0056] Same as Example 2, except that step 6) is omitted to prepare a pure silk fibroin membrane.

[0057] Compare with Example 2

[0058] Similar to Example 2, except that step 9) is omitted, and the prepared membrane material dissolves directly in water, making it impossible to verify in animal experiments.

[0059] Compare with Example 3

[0060] Similar to Example 1, except that a beaker containing distilled water is not placed in step 9). The resulting membrane material dissolves in water, but at a slower rate than in Control Example 2.

[0061] Example 4

[0062] The transmittance of the composite membrane prepared in Example 2 and the blank membrane in Control Example 1 were tested, and the results are as follows: Figure 3 As shown, from Figure 3 It can be seen that the baicalin-loaded silk fibroin membrane has good light transmittance, which can reach up to about 85%. A photograph of the composite membrane in Example 2 is shown below. Figure 4 As shown, the school emblem can be clearly seen through the baicalin-loaded silk fibroin membrane, and the good light transmittance is beneficial for detecting the wound healing status.

[0063] Example 5

[0064] SD rats were divided into three groups: the first group was the blank control group (repeated three times); the second group was the experimental group, using the composite film prepared in Example 2 as the dressing (repeated three times); and the third group used the control group (Example 1). The specific procedures are as follows:

[0065] ① Male Kunming rats (14 weeks old) from Enswell Biotech were anesthetized for 10 minutes using isoflurane (2.5%) (there may be slight errors depending on the weight of the rats);

[0066] ②The operating table and surgical instruments are disinfected by spraying with 75% alcohol;

[0067] ③ Lay a disposable sterile surgical drape over the operating table.

[0068] ④ Fix the rat on the operating table, shave the hair off its back with a blade, wipe the skin on its back with warm water, and finally disinfect it with 75% alcohol;

[0069] ⑤ Using a 10mm diameter sterile punch, mark the location on the back, lift the skin with sterile forceps, and cut the skin along the mark with sterile ophthalmic scissors to create a circular full-thickness skin defect;

[0070] ⑥ For the blank group, apply 3M patch; for the experimental group, apply baicalin-loaded silk fibroin membrane and fix with 3M patch; for the control group, use pure silk fibroin membrane and fix with 3M patch. Change 3M patch and corresponding medicated film daily for 0-3 days on the wound.

[0071] ⑦ Record the wound repair status on the rat's back at days 0, 1, 2, 3, 5, 7, and 11;

[0072] During period ⑧, the rats were fed according to the usual feeding method.

[0073] The wound healing status is shown in the table below:

[0074]

[0075] As can be seen from the table, the repair rate of the baicalin-loaded silk fibroin membrane increased by 4.71% compared with the control group. This shows that the baicalin-loaded silk fibroin membrane plays a role in ordinary wounds and enhances the repair ability, while the pure silk fibroin membrane causes slow wound healing due to air circulation obstruction.

Claims

1. A method for preparing a composite membrane, characterized in that, The composite membrane comprises a membrane matrix formed by the stable cross-linking of silk fibroin with β-sheets, and baicalin uniformly dispersed between the silk fibroin molecular segments. The mass ratio of silk fibroin to baicalin is 100:3-4. The thickness of the membrane matrix is ​​0.05-0.2 mm, and the mass per unit area is 2.5-4.0 mg / cm³. 2 The β-sheet content was 49.3%. The preparation process includes the following steps: 1) Take silkworm cocoons, boil them several times with sodium carbonate solution, wash them, and dry them at 60℃; 2) Dissolve the silk fibroin in a ternary solution, centrifuge and filter to obtain a clear solution of silk fibroin, and dry to obtain silk fibroin; 3) Prepare a silk fibroin solution with a concentration of 1-30 mg / ml and adjust the pH to 7.4; 4) Add baicalin to the silk fibroin solution, cast the film, dry and anneal to obtain a composite film. After film formation, dry at 42℃ for 40-44h and anneal at 70-80% humidity for 4-5h.

2. The preparation method according to claim 1, characterized in that, The mass per unit area is 3.73 mg / cm². 2 .

3. The preparation method according to claim 1, characterized in that, Step 1) The concentration of sodium carbonate solution is 0.5 wt%, and the dissolution is carried out three times, with each dissolution time being 25-30 min. Wash with clean water. Step 2) The ternary solution is a mixture of calcium chloride, ethanol, and water, with a molar ratio of calcium chloride, ethanol, and water of 1:2:

8.

4. The preparation method according to claim 1, characterized in that, Step 2) Dissolve in a 60℃ water bath for 1-2 hours. After complete dissolution, cool naturally to room temperature and centrifuge at 8000 rpm for 10 minutes. Filter using 400-500 mesh nylon cloth and freeze-dry at -20℃ for 48 hours to obtain silk fibroin.

5. The preparation method according to claim 1, characterized in that, Step 3) Dissolve the freeze-dried silk fibroin in ultrapure water to prepare a silk fibroin solution with a concentration of 14 mg / ml. Adjust the pH using 1M sodium hydroxide-1M acetic acid.

6. The preparation method according to claim 1, characterized in that, Step 4) Baicalin is prepared as a baicalin solution, with the solvent being a silk fibroin solution at pH 7.4 and a concentration of 3 wt%. The mass ratio of baicalin to silkworm cocoons is 3:125-140.

7. Use of the composite membrane prepared by any of the preparation methods of claims 1 to 6 in the preparation of a drug for repairing chronic skin wounds.

8. The use according to claim 7, characterized in that, The medication for repairing chronic skin wounds is a dressing.

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