A UV-crosslinked silk protein hydrogel fiber, its preparation method and application

Abstract

This invention discloses a UV-crosslinked regenerated silk fibroin hydrogel fiber, its preparation method, and its applications. Using silk fibroin as the main raw material, the silk fibroin is first regenerated to obtain regenerated silk fibroin. Then, the regenerated silk fibroin fibers are collected using electrospinning and a rotating collection roller. These fibers are then soaked in a solution containing a photoinitiator and placed under UV light for crosslinking to form regenerated silk fibroin hydrogel fibers. Finally, the fibers are washed and soaked to obtain the regenerated silk fibroin hydrogel fiber. The advantages are: good mechanical strength and softness, suitable for use as wound dressings or tissue scaffolds; high water content and good swelling properties, capable of absorbing wound exudate; the hydrogel fiber membrane has good drug release performance, can be loaded with drugs for antibacterial or wound repair promotion; it has excellent biocompatibility and mechanical properties, and has broad application prospects as a drug carrier and tissue repair material in tissue engineering and drug release fields.

Description

Technical Field

[0001] This invention relates to an ultraviolet light crosslinked regenerated silk protein hydrogel fiber, its preparation method, and its application, belonging to the field of biomedical material preparation technology. Background Technology

[0002] Trauma leads to a series of skin lesions, all of which are covered with wound dressings after treatment. Hydrogels can serve as wound dressings, tissue engineering scaffolds, and drug or cell delivery carriers, providing spatial and temporal control over the release of loaded components, including chemotherapeutic drugs, proteins, or cells. In drug delivery and tissue engineering scaffolds, hydrogel materials play a crucial role in controlling drug release rates, modulating cell behavior, and providing physical support. Therefore, hydrogel-based skin alternatives have attracted widespread attention due to their unique ability to mimic the natural skin microenvironment. However, traditionally, hydrogels have suffered from poor mechanical properties, poor cell adhesion, and low air permeability.

[0003] Silk fibroin (SF) possesses excellent biocompatibility and degradability, and SF-based nanofibers prepared by electrospinning can provide a favorable environment for cell adhesion, growth, and proliferation. Traditional methods for preparing SF hydrogels (ultrasound, high temperature, alcohol induction) suffer from drawbacks such as long gelation times and poor mechanical strength, making them unsuitable for electrospinning hydrogel fiber preparation. How to combine silk fibroin with nanofibers to prepare specific silk fibroin hydrogel fibers as drug carriers and tissue repair materials is a pressing technical challenge that needs to be addressed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an ultraviolet light crosslinked regenerated silk protein hydrogel fiber with excellent biocompatibility, mechanical properties and drug release performance, and which can be used as a drug carrier and tissue scaffold to promote wound repair, as well as its preparation method and application.

[0005] To solve the above-mentioned technical problems, the present invention provides a method for preparing ultraviolet crosslinked regenerated silk protein hydrogel fibers. Using silk protein as the main raw material, the method first regenerates the silk protein to obtain regenerated silk protein, then electrospinning and collecting the silk protein fibers using a rotating collecting roller, then immersing them in a solution containing a photoinitiator and placing them under ultraviolet light for crosslinking to form regenerated silk protein hydrogel fibers, and finally washing and soaking them to obtain the regenerated silk protein hydrogel fibers.

[0006] The specific preparation steps are as follows:

[0007] Step A: Natural silk protein fiber is chemically modified to obtain regenerated silk protein. Then, the regenerated silk protein is dissolved in an organic solvent as an electrospinning precursor solution. At the same time, a cross-linking agent and a photoinitiator are added and stirred until completely dissolved before use.

[0008] Step B: Transfer the electrospinning precursor solution to a conventional electrospinning injector. The electrospinning injector has a speed of 5-12 μL / min, an applied voltage of 15-21 kV, a distance of 6-13 cm between the needle of the electrospinning injector and the collecting roller, and a rotation speed of 200-500 rpm to obtain regenerated silk protein fibers.

[0009] Step C: Place the regenerated silk protein fiber in an ethanol solution containing a photoinitiator, then place a UV lamp above the solution and carry out a cross-linking reaction at 25℃-37℃ to obtain cross-linked regenerated silk protein hydrogel fiber.

[0010] Step D: Wash the cross-linked regenerated silk protein hydrogel fibers multiple times with the washing solution, and then soak them in deionized water. During this process, the deionized water is changed several times to remove unreacted cross-linking agents, and the regenerated silk protein hydrogel fibers are collected.

[0011] In step A, the natural silk protein fiber is obtained by degumming silkworm cocoons with an alkaline solution, and the concentration of the alkaline solution is 0.02-0.05 mol / L.

[0012] In step A, the regenerated silk protein is a carbon-carbon double bond modified regenerated silk protein with a modification degree of 10%-40%.

[0013] In step A, the concentration of regenerated silk protein in the electrospinning precursor solution is 6%-12% w / v, the concentration of crosslinking agent is 3%-5% wt, and the concentration of photoinitiator is 1%-2% w / v. The crosslinking agent includes N,N-methylenebisacrylamide, and the photoinitiator includes 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.

[0014] In step C, the concentration of the photoinitiator in the ethanol solution containing the photoinitiator is 5%-10% w / v, the volume fraction of ethanol is 99% w / v, the ultraviolet lamp is a cold light source with a power of 50W-75W, the distance between the ultraviolet lamp and the surface of the ethanol solution is 3cm-5cm, and the ultraviolet light irradiation time is 5-10min.

[0015] In step D, the washing solution includes anhydrous ethanol and deionized water solution. The solution is used for washing for 5-10 minutes, and the soaking time in deionized water is 12-36 hours.

[0016] In step D, the soaking time in deionized water is 18-24 hours.

[0017] The ultraviolet-crosslinked regenerated silk protein hydrogel fiber obtained by the preparation method described above.

[0018] The ultraviolet-crosslinked regenerated silk protein hydrogel fibers obtained by the above preparation method can be used as drug carriers and tissue repair materials in the fields of drug release and tissue engineering.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] SF hydrogel fibers, possessing the dual properties of hydrogel softness and electrospun fiber structure, were prepared through electrospinning and ultraviolet crosslinking. These fibers, with micron-sized diameters, consist of individual fibers with diameters of 1-2 μm, forming micron-sized hydrogel fibers. Unreacted crosslinking agents and initiators were removed by washing with ethanol and deionized water, resulting in excellent biocompatibility and suitability for cell growth. Furthermore, the photocrosslinking reaction conditions were mild and the reaction rate was fast. In addition, the photocrosslinked SF hydrogel overcame the brittleness commonly found in physical crosslinking, thus exhibiting good mechanical strength and softness, making it suitable as a wound dressing or tissue scaffold. Its high water content and good swelling properties allow it to absorb wound exudate. The hydrogel fiber membrane also exhibits good drug release properties, enabling it to load drugs for antibacterial or wound-healing purposes. The prepared regenerated silk protein hydrogel fibers combine the advantages of hydrogels and nanofibers, possessing excellent biocompatibility and mechanical properties, and show broad application prospects as drug carriers and tissue repair materials in tissue engineering and drug release fields. Attached Figure Description

[0021] Figure 1 The image shows the microstructure of the regenerated silk protein hydrogel fiber prepared according to the present invention under a scanning electron microscope.

[0022] Figure 2 The mechanical properties of the regenerated silk protein hydrogel fiber membrane prepared according to the present invention are shown in the figure.

[0023] Figure 3 The swelling properties of the regenerated silk protein hydrogel fiber membrane prepared in this invention are shown in the figure.

[0024] Figure 4 This is a diagram showing the drug release performance of the regenerated silk protein hydrogel fiber membrane prepared in this invention. Detailed Implementation

[0025] The preparation method of regenerated silk protein hydrogel fiber of the present invention uses silk protein as the main raw material. First, the silk protein is regenerated to obtain regenerated silk protein. Then, the silk protein fiber is collected by single-channel electrospinning and rotating collection roller. Then, it is soaked in a solution containing a photoinitiator and crosslinked under ultraviolet light to form regenerated silk protein hydrogel fiber. Finally, it is washed and soaked to obtain regenerated silk protein hydrogel fiber. The following describes the ultraviolet crosslinked regenerated silk protein hydrogel fiber, preparation method and application of the present invention in further detail with reference to the accompanying drawings and specific embodiments.

[0026] Example 1:

[0027] 10g of silkworm cocoons were placed in 1L (0.2mol / L) Na₂CO₃ aqueous solution and treated at 100℃ for 30min. After treatment, the cocoons were washed three times with deionized water and then dried at 37℃ to constant weight. 5g of degummed silk fibroin fibers were dissolved in 20mL (9.3mol / L) LiBr solution and dissolved at 60℃ for 4h. After complete dissolution, 1.5mL (GMA) of glycidyl methacrylate was added to the solution, and the reaction continued for 3h. After the reaction, the solution was transferred to a dialysis bag (3500MWCO) and dialyzed for 72h, with the deionized water changed multiple times during dialysis. After dialysis, the solution was centrifuged to remove impurities, and the resulting solution was the regenerated silk fibroin aqueous solution. The solution was freeze-dried to obtain GMA-modified regenerated silk fibroin powder.

[0028] Example 2:

[0029] A 12% w / v RSF-GMA / HFIP solution was prepared, and then N,N-methylenebisacrylamide (5% wt of RSF-GMA) and photoinitiator w / v 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959, 1% w / v) were added. After stirring evenly, the solution was transferred into a syringe (spinning needle inner diameter of 0.6 mm) for spinning. The spinning parameters were set as follows: voltage of 21 kV, push speed of 12 μL / min, distance between roller and needle of 13 cm, and roller speed of 400 rpm. After spinning, the fiber membrane is placed in anhydrous ethanol containing 10% w / v Irgacure 2959 and irradiated under a 365nm ultraviolet cold light source (75W) for 5 minutes to induce cross-linking of the fibers. After irradiation, the fiber membrane is washed with ethanol and deionized water and then soaked in an aqueous solution for 24 hours to finally obtain regenerated silk protein hydrogel fibers. See the attached instruction manual. Figure 1 .

[0030] Example 3:

[0031] A 12% w / v RSF-GMA / HFIP solution was prepared, and then N,N-methylenebisacrylamide (5% wt of RSF-GMA) and photoinitiator w / v 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959, 1% w / v) were added. After stirring evenly, the solution was transferred to a syringe (spinning needle inner diameter of 0.6 mm) for spinning. The spinning parameters were set as follows: voltage of 15 kV, push speed of 5 μL / min, distance between roller and needle of 13 cm, and roller speed of 400 rpm. After spinning, the fiber membrane was placed in anhydrous ethanol containing 10% w / v Irgacure 2959 and irradiated under a 365 nm UV cold light source (75 W) for 5 min to induce cross-linking of the fibers. After irradiation, the fiber membrane was washed with ethanol and deionized water and soaked in aqueous solution for 24 h to finally obtain regenerated silk protein hydrogel fibers.

[0032] Example 4:

[0033] A 12% w / v RSF-GMA / HFIP solution was prepared, and then N,N-methylenebisacrylamide (5% wt of RSF-GMA) and photoinitiator w / v 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959, 1% w / v) were added. After stirring evenly, the solution was transferred to a syringe (spinning needle inner diameter of 0.6 mm) for spinning. The spinning parameters were set as follows: voltage of 18 kV, push speed of 8 μL / min, distance between roller and needle of 13 cm, and roller rotation speed of 400 rpm. After spinning, the fiber membrane was placed in anhydrous ethanol containing 10% w / v Irgacure 2959 and irradiated under a 365 nm ultraviolet cold light source (75 W) for 5 min to induce cross-linking of the fibers. After irradiation, the fiber membrane was washed with ethanol and deionized water and soaked in aqueous solution for 24 h to finally obtain regenerated silk protein hydrogel fibers.

[0034] Example 5:

[0035] The regenerated silk protein hydrogel membrane from Example 2 was fabricated into rectangular strips of 50mm x 10mm. The mechanical properties of the samples were tested using a universal tensile testing machine at a tensile speed of 0.2mm / min and a gauge length of 20mm. The experiment was divided into two groups: one group consisted of dried regenerated silk protein hydrogel fiber membranes, and the other group consisted of regenerated silk protein hydrogel fiber membranes under swelling equilibrium conditions (see attached instruction manual). Figure 2 .

[0036] Example 6:

[0037] The regenerated silk protein hydrogel fiber membrane from Example 2 was dried and weighed, and the weight was recorded as W0. The sample was then immersed in PBS solution (10 mL, 0.01 mol / L, pH = 7.4). At different time intervals, the surface moisture was absorbed using filter paper, and the weight of the wet fiber hydrogel was accurately recorded as W. t Calculate the swelling ratio according to the formula provided in the instruction manual. Figure 3 .

[0038] Swelling ratio (%) = (W t -W0) / W0×100%

[0039] Example 7:

[0040] Prepare a 12% w / v RSF-GMA / HFIP solution, then add N,N-methylenebisacrylamide (5% wt of RSF-GMA) and photoinitiator w / v 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (Irgacure 2959, 1% w / v), then add 2 mg tetracycline hydrochloride. After stirring evenly, transfer the solution into a syringe (spinning needle inner diameter of 0.6 mm) for spinning. Set the spinning parameters as follows: voltage of 21 kV, push speed of 12 μL / min, distance between roller and needle of 13 cm, and roller speed of 400 rpm. After spinning, the fiber membrane was placed in anhydrous ethanol containing 10% w / v Irgacure 2959 and irradiated for 5 min under a 365 nm UV cold light source (75 W) to induce cross-linking of the fibers. After irradiation, the fiber membrane was washed with ethanol and deionized water and soaked in an aqueous solution for 24 h to finally obtain regenerated silk protein hydrogel fibers. Example 7 differs from Example 2 in that tetracycline hydrochloride was added to the spinning solution to verify the sustained-release performance of the material. The RSF hydrogel fiber membrane loaded with tetracycline hydrochloride obtained in the examples was placed in PBS solution (5 mL, 0.01 mol / L, pH = 7.4) and placed in a constant-temperature shaker. At fixed time intervals (1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h), the solution in the test tube was replaced with fresh PBS. The release amount of tetracycline was calculated by measuring the change in absorbance of the simulated drug tetracycline hydrochloride in the solution. The results are shown in the appendix of the instruction manual. Figure 4 .

Claims

1. A method for preparing ultraviolet light crosslinked regenerated silk protein hydrogel fibers, characterized in that: Using silk fibroin as the main raw material, the silk fibroin is first regenerated to obtain regenerated silk fibroin. Then, the silk fibroin fibers are collected by electrospinning and rotating collection roller. The fibers are then soaked in a solution containing a photoinitiator and placed under ultraviolet light to crosslink and form regenerated silk fibroin hydrogel fibers. Finally, the fibers are washed and soaked to obtain regenerated silk fibroin hydrogel fibers. The specific preparation steps are as follows: Step A: Natural silk protein fiber is chemically modified to obtain regenerated silk protein. Then, the regenerated silk protein is dissolved in an organic solvent as an electrospinning precursor solution. At the same time, a cross-linking agent and a photoinitiator are added and stirred until completely dissolved before use. Step B: Transfer the electrospinning precursor solution to a conventional electrospinning injector. The electrospinning injector has a speed of 5-12 μL / min, an applied voltage of 15-21 kV, a distance of 6-13 cm between the needle of the electrospinning injector and the collecting roller, and a rotation speed of 200-500 rpm to obtain regenerated silk protein fibers. Step C: Place the regenerated silk protein fiber in an ethanol solution containing a photoinitiator, then place a UV lamp above the solution and carry out a cross-linking reaction at 25 ℃-37 ℃ to obtain cross-linked regenerated silk protein hydrogel fiber; Step D: Wash the cross-linked regenerated silk protein hydrogel fibers with washing solution multiple times, and then soak them in deionized water. During this period, the deionized water is changed multiple times to remove unreacted cross-linking agents. Collect the regenerated silk protein hydrogel fibers. In step A, the crosslinking agent includes N,N-methylenebisacrylamide, the photoinitiator includes 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone; the regenerated silk fibroin is carbon-carbon double bond modified regenerated silk fibroin with a modification degree of 10%-40%; and the ultraviolet light irradiation time is 5-10 min.

2. The method for preparing ultraviolet-crosslinked regenerated silk protein hydrogel fibers according to claim 1, characterized in that: In step A, the natural silk protein fiber is obtained by degumming silkworm cocoons with an alkaline solution, and the concentration of the alkaline solution is 0.02 - 0.05 mol / L.

3. The method for preparing ultraviolet-crosslinked regenerated silk protein hydrogel fibers according to claim 1, characterized in that: In step A, the concentration of regenerated silk protein in the electrospinning precursor solution is 6%-12% w / v, the concentration of crosslinking agent is 3%-5% wt, and the concentration of photoinitiator is 1%-2% w / v.

4. The method for preparing ultraviolet-crosslinked regenerated silk protein hydrogel fibers according to claim 1, characterized in that: In step C, the concentration of the photoinitiator in the ethanol solution containing the photoinitiator is 5%-10% w / v, the volume fraction of ethanol is 99% v / v, the ultraviolet lamp is a cold light source with a power of 50 W-75 W, and the distance between the ultraviolet lamp and the surface of the ethanol solution is 3cm-5cm.

5. The method for preparing ultraviolet-crosslinked regenerated silk protein hydrogel fibers according to claim 1, characterized in that: In step D, the washing solution includes anhydrous ethanol and deionized water solution. The solution is used for washing for 5-10 minutes, and the soaking time in deionized water is 12-36 hours.

6. The method for preparing ultraviolet-crosslinked regenerated silk protein hydrogel fibers according to claim 5, characterized in that: In step D, the soaking time in deionized water is 18-24 hours.

7. Ultraviolet-crosslinked regenerated silk protein hydrogel fiber obtained by the preparation method according to any one of claims 1 to 6.

8. The application of ultraviolet-crosslinked regenerated silk protein hydrogel fibers obtained by the preparation method according to any one of claims 1 to 6 as drug carriers and tissue repair materials in the fields of drug release and tissue engineering.

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