Silk fibroin-alginate composite hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to silk fibroin-alginate composite hydrogel as well as a preparation method and application thereof. The silk fibroin-alginate composite hydrogel is prepared by adopting a unique process and raw materials, and as a material with good biocompatibility, biodegradability and biological activity, the silk fibroin-alginate composite hydrogel can promote wound healing, support tissue regeneration and realize the function of controllably releasing drugs. A new solution is provided for the fields of tissue engineering, drug delivery, wound repair and the like, and human health and life quality can be improved.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a silk fibroin-alginate composite hydrogel and a preparation method and application thereof. Background Art

[0002] Wound repair and tissue regeneration are important topics in the medical field. Traditional treatment methods have some limitations, such as difficulty in controlling drug release and inability to provide sufficient support and guidance. Therefore, it is necessary to develop new biomedical materials that can promote wound healing, support tissue regeneration, and have the function of controlled drug release.

[0003] Hydrogel is a material with a three-dimensional network structure that can absorb a large amount of water and form a gel state. Hydrogel has soft and elastic properties similar to biological tissues and is widely used in the biomedical field. In the silk fibroin-alginate repair hydrogel, hydrogel technology is used to construct a scaffold structure, which has good biocompatibility and mechanical properties.

[0004] Silk fibroin is a natural protein produced by animals such as insects and spiders. It has good biocompatibility, biodegradability and bioactivity, and is widely used in tissue engineering and regenerative medicine. Silk fibroin can promote cell adhesion, proliferation and differentiation, and also has good mechanical properties and stability.

[0005] Sodium alginate is a polysaccharide compound extracted from seaweed with good biocompatibility and biodegradability. It has a variety of biological activities, such as anti-inflammatory, antibacterial and wound healing. Sodium alginate can form a complex with silk fibroin to improve the stability and function of the composite hydrogel. Summary of the invention

[0006] At present, there are few reports on combining silk fibroin with sodium alginate for the development of hydrogels. The present invention uses a unique process and raw materials to prepare a silk fibroin-alginate composite hydrogel, which, as a material with good biocompatibility, biodegradability and bioactivity, can promote wound healing, support tissue regeneration, and achieve the function of controlled drug release. This provides new solutions for the fields of tissue engineering, drug delivery and wound repair, and helps improve human health and quality of life.

[0007] Specifically, the present invention provides a method for preparing a silk fibroin-alginate composite hydrogel, which comprises the following steps:

[0008] (1) Preparation of silk fibroin composite solution:

[0009] a. The cocoons are heated in an alkaline solution to degumming, and the silk fibroin fibers are obtained after washing and drying;

[0010] b. dissolving the silk fibroin fiber in a solvent, dialyzing, and freeze-drying to obtain silk fibroin;

[0011] c. preparing a silk fibroin solution and a solution of hydrogen peroxide and horseradish peroxidase, and mixing the two to obtain a silk fibroin composite solution;

[0012] (2) Preparation of alginate composite solution:

[0013] i. dissolving polyethyleneimine and 2-formylphenylboronic acid in a buffer to obtain a mixed solution;

[0014] ii. dissolving alginate in water to obtain an alginate solution;

[0015] iii. mixing the mixed solution of i with the alginate solution of ii to obtain an alginate composite solution;

[0016] (3) Preparation of silk fibroin-alginate composite hydrogel: After mixing the silk fibroin composite solution and the alginate composite solution, pour the mixture into a mold and let it stand to obtain the silk fibroin-alginate composite hydrogel.

[0017] Furthermore, the alkaline solution is a Na2HCO3 aqueous solution with a mass fraction of 0.01-1%.

[0018] Furthermore, the heating treatment includes boiling.

[0019] Furthermore, the solvent in step b is a mixed solution of calcium chloride, ethanol and water.

[0020] Furthermore, the molar ratio of calcium chloride, ethanol and water is 0.01-20:0.1-15:0.5-60.

[0021] Furthermore, the mass concentration of silk fibroin in the silk fibroin solution is 1-20%.

[0022] Furthermore, the silk fibroin solution is an aqueous solution of silk fibroin.

[0023] Furthermore, in the solution of hydrogen peroxide and horseradish peroxidase, the mass concentration of hydrogen peroxide is 0.01-5%, and the concentration of horseradish peroxidase is 0.01-20 mg / mL.

[0024] Furthermore, the solution of hydrogen peroxide and horseradish peroxidase is a PBS buffer solution in which hydrogen peroxide and horseradish peroxidase are dissolved.

[0025] Furthermore, the molar ratio of polyethyleneimine to 2-formylphenylboronic acid is 0-5:1.

[0026] Furthermore, the buffer solution is PBS with a pH of 6-8.

[0027] Furthermore, the alginate includes sodium alginate.

[0028] Furthermore, the mass concentration of the alginate solution is 1-5wt%.

[0029] Furthermore, in step iii, the mixing ratio (volume ratio) of the mixed solution of polyethyleneimine and 2-formylphenylboronic acid to the alginate solution is 0.5-5:1.

[0030] Furthermore, in step (3), the ratio of silk fibroin to alginate in the silk fibroin composite solution and the alginate composite solution is 0.1-5:0.1-20 (w / w).

[0031] Furthermore, in addition to silk fibroin and alginate, other biocompatible and biodegradable materials are added, such as collagen, gelatin, etc. The purpose of repairing hydrogel is achieved by mixing different materials or preparing composite materials.

[0032] Furthermore, other functional ingredients, such as growth factors, antibacterial agents, anti-inflammatory agents, etc., are added to the silk fibroin-sodium alginate hydrogel to enhance the repair effect or provide additional therapeutic functions.

[0033] Compared with the prior art, the method for preparing silk fibroin-alginate composite hydrogel of the present invention optimizes the preparation process of hydrogel, such as changing the concentration, temperature, pH value and other conditions of the crosslinking agent, controlling the rate and structure of gel formation, so as to obtain better performance and applicability.

[0034] Furthermore, the method for preparing the silk fibroin-alginate composite hydrogel of the present invention can also be combined with other technical means, such as nanotechnology, 3D printing, etc., to further improve the microstructure and function of the hydrogel, and enhance the repair effect and treatment accuracy.

[0035] In other aspects, the present invention also provides a silk fibroin-alginate composite hydrogel prepared by the method described herein.

[0036] Furthermore, the hydrogel also contains other biocompatible and biodegradable materials, such as collagen, gelatin, etc.

[0037] Furthermore, the hydrogel also contains other functional components, such as growth factors, antibacterial agents, anti-inflammatory agents, etc., to enhance the repair effect or provide additional therapeutic functions.

[0038] In other aspects, the present invention also provides the use of the silk fibroin-alginate composite hydrogel as described herein in the preparation of a drug carrier.

[0039] In other aspects, the present invention also provides a pharmaceutical composition, which includes the silk fibroin-alginate composite hydrogel as described herein and a drug encapsulated in the hydrogel.

[0040] Beneficial Effects of the Invention

[0041] The silk fibroin-sodium alginate composite hydrogel prepared by the present invention is a new type of biomedical material, which is mainly used in the fields of tissue engineering, drug delivery and wound healing. The composite hydrogel solves the following technical problems:

[0042] 1. Controlled release of drugs: Composite hydrogels can be used as drug carriers to release drugs in wounds or tissues. By regulating the structure and properties of composite hydrogels, sustained and controlled release of drugs can be achieved, thereby improving the efficacy of drugs.

[0043] 2. Tissue repair and regeneration: Silk fibroin-sodium alginate composite hydrogel has good biocompatibility and biodegradability, and can provide a scaffold structure to promote the adhesion, proliferation and differentiation of tissue cells, and promote tissue repair and regeneration.

[0044] 3. Functional regulation: By regulating the physical and chemical properties of silk fibroin-sodium alginate composite hydrogel, its functions can be regulated. For example, its mechanical properties, biodegradation rate, cell affinity and other properties can be regulated to meet the needs of different application fields.

[0045] 4. Improve biocompatibility: Both silk fibroin and sodium alginate have good biocompatibility and biodegradability, which can reduce the occurrence of immune response and toxic side effects.

[0046] 5. Improve mechanical properties: Silk fibroin has good mechanical properties and stability, and can provide better support and guidance after forming a composite hydrogel with sodium alginate.

[0047] 6. Hydrogel stability: The composite hydrogel should have good stability and be able to maintain its shape and performance for a certain period of time in the body to achieve long-term drug release and tissue repair effects.

[0048] 7. It can integrate more diversified medication schemes, including single-component compounds, combined use of multiple drugs, encapsulation of Chinese herbal extracts, and encapsulation and delivery of exosomes, extracellular vesicles, liposomes and other nanoparticles.

[0049] By solving the above technical problems, silk fibroin-sodium alginate composite hydrogel can play an important role in the medical field and provide new solutions for tissue engineering and wound healing.

[0050] The key points of the silk fibroin-sodium alginate repair hydrogel prepared by the present invention include the following aspects:

[0051] Ratio of silk fibroin and sodium alginate: The ratio of silk fibroin and sodium alginate needs to be precisely adjusted to obtain a composite hydrogel with good biocompatibility, mechanical properties, and bioactivity.

[0052] Selection of therapeutic drugs and carrier forms: The selection of therapeutic drugs and carrier forms need to be personalized according to different diseases and wound types to improve the therapeutic effect.

[0053] Stability and controlled release of hydrogel: The stability and controlled release of hydrogel is one of the key technical points, which needs to be adjusted through material structure and physical and chemical methods to improve the release rate and accuracy of drugs.

[0054] Production process and quality control: Production process and quality control need to be strictly controlled to ensure product stability and consistency while meeting the requirements of relevant regulations and standards.

[0055] Diversified drug delivery modes: single component compounds, multi-drug combination, Chinese herbal extracts, delivery of exosomes, extracellular vesicles, liposomes and other nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 SEM images of the silk fibroin-alginate composite hydrogels prepared in the examples and comparative examples are shown (Figures ad are the silk fibroin-alginate composite hydrogels prepared in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively).

[0057] Figure 2 The results of the pH stability tests of the silk fibroin-alginate composite hydrogels prepared in the examples and comparative examples are shown.

[0058] Figure 3 The degradation performance test results of the silk fibroin-alginate composite hydrogels prepared in the examples and comparative examples are shown. DETAILED DESCRIPTION

[0059] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0060] Embodiment 1:

[0061] A method for preparing a silk fibroin-alginate composite hydrogel comprises the following steps:

[0062] 1. Preparation of silk protein solution: weigh 7.5g of anhydrous Na2HCO3 and put it into 1.5L of boiling deionized water. After it is fully dissolved, add 20g of pre-cut (size 5mm) 2 ) domestic silkworm cocoons, boil and soak for 30 minutes for degumming, wash with deionized water after 30 minutes, repeat the above operation 3 times; after thorough washing, loosen the silk fibroin fibers, put them in a 60°C oven for drying to obtain crude silk fibroin; prepare a mixed solution of calcium chloride, ethanol and water in a molar ratio of 8:2:1, add 1g of crude silk fibroin to 40mL of the mixed solution, heat to 65°C and keep stirring for 30-40min, and obtain a mixed solution of silk fibroin after dissolution; after cooling to room temperature, dialyze the mixed solution of silk fibroin in deionized water for 3-4 days; after the dialysis is completed, high-speed centrifuge at a low temperature at a speed of 2000r for 10min, and freeze-dry the obtained silk fibroin solution to obtain silk fibroin.

[0063] 2. Add 6 g of silk fibroin, 1 g of hydrogen peroxide and 1 g of horseradish peroxidase into 100 mL of deionized water, fully dissolve and shake to obtain a silk fibroin composite solution.

[0064] 3. Dissolve polyethyleneimine (PEI) and 2-formylphenylboronic acid (2-FPBA) in PBS (pH = 7.4) at a molar ratio of 3:1, stir to fully dissolve, and leave at room temperature overnight; prepare a 2wt% sodium alginate (SA) solution: weigh 2g SA and dissolve it in ultrapure water until it is fully dissolved; mix the mixed solution of PEI and 2-FPBA with the prepared sodium alginate solution in a ratio of 1:1 to obtain a sodium alginate composite solution.

[0065] 4. After evenly mixing the silk fibroin composite solution in step 2 and the sodium alginate composite solution in step 3 at a ratio of 1:1, pour the mixture into a container and let stand for about 30 minutes to obtain a silk fibroin-sodium alginate composite hydrogel.

[0066] Comparative Example 1

[0067] A method for preparing a silk fibroin-alginate composite hydrogel comprises the following steps:

[0068] 1. Same as Example 1.

[0069] 2. Same as Example 1.

[0070] 3. Dissolve polyethyleneimine (PEI) and 2-formylphenylboronic acid (2-FPBA) in PBS (pH = 7.4) at a molar ratio of 3:1, stir to fully dissolve, and leave at room temperature overnight; prepare a 6wt% sodium alginate (SA) solution: weigh 6g SA and dissolve it in ultrapure water until it is fully dissolved; mix the mixed solution of PEI and 2-FPBA with the prepared sodium alginate solution at a ratio of 1:1 (v / v) to obtain a sodium alginate composite solution.

[0071] 4. Same as Example 1.

[0072] Comparative Example 2:

[0073] A method for preparing a silk fibroin-alginate composite hydrogel comprises the following steps:

[0074] 1. Preparation of silk protein solution: Weigh 7.5g of anhydrous Na2HCO3 and put it into 1.5L of boiling deionized water. After fully dissolving, add 20g of natural mulberry silk, boil and soak for degumming treatment, wash with deionized water after 30 minutes, and repeat the above operation 3 times; after fully washing, loosen the silk protein fiber and put it into a 60℃ oven for drying to obtain crude silk protein; prepare a mixed solution of calcium chloride, ethanol and water in a molar ratio of 8:2:1, add 1g of crude silk protein to 40mL of the mixed solution, heat to 65℃ and keep stirring for 30-40min, and obtain a mixed solution of silk protein after dissolution; after cooling to room temperature, dialyze the mixed solution of silk protein in deionized water for 3-4 days; after dialysis, centrifuge at a high speed of 2000r for 10min at low temperature, and freeze-dry the obtained silk protein solution to obtain silk protein.

[0075] 2-4. Same as the embodiment.

[0076] Comparative Example 3:

[0077] A method for preparing a silk fibroin-alginate composite hydrogel comprises the following steps:

[0078] 1. Preparation of silk protein solution: Weigh 7.5g of anhydrous Na2HCO3 and put it into 1.5L of boiling deionized water. After fully dissolving, add 20g of natural castor silk, boil and soak for degumming treatment, wash with deionized water after 30 minutes, and repeat the above operation 3 times; after fully washing, loosen the silk protein fiber and put it into a 60°C oven for drying to obtain crude silk protein; prepare a mixed solution of calcium chloride, ethanol and water in a molar ratio of 8:2:1, add 1g of crude silk protein to 40mL of the mixed solution, heat to 65°C and keep stirring for 30-40min, and obtain a mixed solution of silk protein after dissolution; after cooling to room temperature, dialyze the mixed solution of silk protein in deionized water for 3-4 days; after dialysis, centrifuge at a high speed of 2000r for 10min at low temperature, and freeze-dry the obtained silk protein solution to obtain silk protein.

[0079] 2-4. Same as the embodiment.

[0080] Test Example 1: Structural Characterization of Silk Fibroin-Alginate Composite Hydrogel

[0081] The silk fibroin-alginate composite hydrogels prepared in Example 1 and Comparative Examples 1-3 were observed by scanning electron microscopy. Figure 1 As shown, it can be seen that the silk fibroin-alginate composite hydrogel prepared in Example 1 has a better pore structure and thus has better structural stability.

[0082] Test Example 2: pH stability and degradation performance test of silk fibroin-alginate composite hydrogel

[0083] According to GB / T 16886.12-2017 "Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Materials", the extract of silk fibroin-alginate composite hydrogel was prepared, and the pH meter was used to measure the extract of the hydrogel. The results are as follows Figure 2 As shown, it can be seen that the silk fibroin-alginate composite hydrogel prepared in Example 1 has better pH stability.

[0084] The hydrogel degradation test results are as follows Figure 3 As shown, it can be seen that the silk fibroin-alginate composite hydrogel prepared in Example 1 has a relatively suitable degradation rate and is suitable for tissue repair and wound healing.

[0085] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A method for preparing a silk fibroin-alginate composite hydrogel, characterized in that: The following steps are involved: (1) Preparation of silk fibroin composite solution: a. The cocoons are heated in an alkaline solution to degumming, and the silk fibroin fibers are obtained after washing and drying; b. dissolving the silk fibroin fiber in a solvent, dialyzing, and freeze-drying to obtain silk fibroin; c. preparing a silk fibroin solution and a solution of hydrogen peroxide and horseradish peroxidase, and mixing the two to obtain a silk fibroin composite solution; (2) Preparation of alginate composite solution: i. dissolving polyethyleneimine and 2-formylphenylboronic acid in a buffer to obtain a mixed solution; ii. dissolving alginate in water to obtain an alginate solution; iii. mixing the mixed solution of i with the alginate solution of ii to obtain an alginate composite solution; (3) Preparation of silk fibroin-alginate composite hydrogel: After mixing the silk fibroin composite solution and the alginate composite solution, pour the mixture into a mold and let it stand to obtain the silk fibroin-alginate composite hydrogel.

2. The preparation method according to claim 1, characterized in that: The alkaline solution is a Na2HCO3 aqueous solution with a mass fraction of 0.01-1%; Furthermore, the heating treatment includes boiling.

3. The preparation method according to claim 1, characterized in that: The solvent in step b is a mixed solution of calcium chloride, ethanol and water in a molar ratio of 0.01-20:0.1-15:0.5-60.

4. The preparation method according to claim 1, characterized in that: The silk fibroin solution is an aqueous solution of silk fibroin; Furthermore, in the solution of hydrogen peroxide and horseradish peroxidase, the mass concentration of hydrogen peroxide is 0.01-5%, and the concentration of horseradish peroxidase is 0.01-20 mg / mL; Furthermore, the solution of hydrogen peroxide and horseradish peroxidase is a PBS buffer solution in which hydrogen peroxide and horseradish peroxidase are dissolved.

5. The preparation method according to claim 1, characterized in that: The molar ratio of polyethyleneimine to 2-formylphenylboronic acid is 0-5:1; The buffer solution is PBS with a pH of 6-8.

6. The preparation method according to claim 1, characterized in that: The alginate comprises sodium alginate; The mass concentration of the alginate solution is 1-5wt%; Further, in step iii, the mixing ratio of the mixed solution of polyethyleneimine and 2-formylphenylboronic acid to the alginate solution is 0.5-5:1; Furthermore, in step (3), the ratio of silk fibroin to alginate in the silk fibroin composite solution and the alginate composite solution is 0.1-5:0.1-20.

7. A silk fibroin-alginate composite hydrogel, characterized in that: Obtained by the preparation method described in any one of claims 1 to 6.

8. Use of the silk fibroin-alginate composite hydrogel according to claim 7 in preparing a drug carrier.

9. A pharmaceutical composition, characterized in that The invention comprises the silk fibroin-alginate composite hydrogel according to claim 7, and a drug encapsulated in the hydrogel.

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