Preparation method and application of polydatin-silk fibroin hydrogel

Abstract

The present application relates to the technical field of high polymer materials, and aims to solve the problem that the existing dressing mainly provides physical covering and isolation protection, and is difficult to actively regulate the wound microenvironment, so the curative effect is poor, the present application provides a preparation method and application of a scirpusin-silk fibroin hydrogel, silk fibroin aqueous solution is prepared first, then scirpusin is added to the prepared silk fibroin aqueous solution, and the mixture is uniformly placed until the hydrogel is formed; a hydrogel with physical barrier and anti-inflammatory, antioxidant and anti-aging functions is obtained, which can be used as a wound dressing to promote chronic wound healing.

Description

Technical Field

[0001] This invention relates to the field of polymer materials technology, specifically to a method for preparing a polydipsia glycoside-silk fibroin hydrogel and its application. Background Technology

[0002] Wound healing is an important and complex process. The persistent inflammation, high oxidative stress, and accumulation of senescent cells in the wound environment exacerbate this, creating a microenvironment that promotes high oxidative stress, inflammation, and aging. This causes healing to stall at the inflammatory stage, resulting in a chronic wound. Severe chronic wounds, if left untreated, can lead to amputation, sepsis, or even death.

[0003] Most existing dressings primarily provide physical coverage and isolation protection, making it difficult to actively regulate the wound microenvironment, thus resulting in poor therapeutic effects. Summary of the Invention

[0004] To address the issue that existing dressings primarily provide physical coverage and isolation protection, but lack the ability to actively regulate the wound microenvironment, resulting in poor therapeutic effects, this invention provides a method for preparing a polysaccharide-silk fibroin hydrogel and its application. This method produces a hydrogel that combines physical barrier properties with anti-inflammatory, antioxidant, and anti-aging functions, which can be used as a wound dressing to promote the healing of chronic wounds.

[0005] The technical solution of the present invention is as follows: a method for preparing a polygalactosidase-silk fibroin hydrogel, the preparation method comprising the following steps: (1) Prepare an aqueous solution of silk fibroin, wherein the mass-volume percentage of the aqueous solution of silk fibroin is 8%-20% w / v; Currently, silk fibroin is prepared into aqueous solutions through solution concentration and freeze-drying. However, it is difficult to obtain higher concentrations using either method. As a preferred method, the preparation of the aqueous solution of silk fibroin involves the following steps: First, silkworm cocoons are cut into small pieces and boiled in a sodium carbonate solution repeatedly to obtain degummed silk fibroin. This is then rinsed several times with pure water and dried for later use. Second, the dried silk fibroin is dissolved in a lithium bromide solution to adjust the concentration to 8-20% W / V. Finally, the resulting solution is placed in a dialysis bag, dialyzed in pure water, and then placed back into the dialysis bag for osmotic concentration in a PEG2000 aqueous solution to obtain an 8%-20% W / V aqueous solution of silk fibroin.

[0006] Sodium carbonate solution acts as a degumming agent, preferably with a molar concentration of 0.01-0.05M; lithium bromide acts as a dissolving agent, completely destroying the β-sheet structure of silk fibroin to obtain regenerated SF with a random coil / α-helix conformation, with a molar concentration of 8-10M; PEG2000 aqueous solution has a concentration of 5%~30% w / v, and acts as a permeation concentration agent.

[0007] As a preferred option, the dialysis bag has a molecular weight cutoff of 3000-5000 Da. Dialysis is performed in ultrapure water for 3-5 days, with the dialysis solution being changed several times a day to remove LiBr salt ions and obtain a pure SF aqueous solution.

[0008] (2) Preparation of polydipsia-silk fibroin hydrogel: First, add polydipsia to the silk fibroin aqueous solution prepared in step (1), mix evenly and let stand until hydrogel is formed to obtain polydipsia-silk fibroin hydrogel.

[0009] Preferably, the addition amount of polygalactoside-silk fibroin is 1 ml of 8%-20% w / v silk fibroin aqueous solution for every 5-20 mg of polygalactoside. Polygalactoside and silk fibroin first self-assemble to form fibers, and then further self-assemble to form a hydrogel. The two must be within a specific ratio range; too high a ratio will lead to precipitation, and too low a ratio will prevent gel formation. Polygalactoside molecules + silk fibroin chains (when the concentration reaches a critical value) undergo molecular recognition and orientation to form one-dimensional linear aggregation (β-sheet conformation induction), leading to the formation of nanofibers. The fibers entangle / crosslink to form a three-dimensional network, and finally, a hydrogel is formed.

[0010] Preferably, the mixed solution is first vortexed to homogenize it, then placed in a constant temperature shaker and heated to 80-120°C until it becomes clear, and then left to stand at room temperature until a hydrogel forms.

[0011] The hydrogel contains silk fibroin (SF) and polydextrin (PD). Due to the protein structure of silk fibroin, it has natural compatibility with human tissues. The small polypeptide chains and peptides produced by the degradation of SF do not cause significant hypersensitivity reactions in organisms, making it a biocompatible material. Studies have shown that SF fibers exhibit minimal inflammatory tissue responses in the absence of sericin.

[0012] In recent years, there has been an increasing amount of research on the pharmacological effects and mechanisms of polygala glycoside. Polygala glycoside is the main active ingredient of Polygonum cuspidatum and has a variety of pharmacological activities, including anti-inflammatory, bone marrow protection, wound healing promotion, and anti-radiation effects.

[0013] The above-mentioned hydrogel is used as a wound dressing in the treatment of chronic wounds.

[0014] Compared with the prior art, the beneficial effects of the present invention are: (1) The hydrogel prepared by the present invention has injectable properties, can dynamically conform to irregular wounds, and can deliver hydrophobic drugs PD through its gel network while acting as a physical barrier. (2) The SF-PD hydrogel of the present invention can exert a synergistic effect of improving local oxidative stress in wounds, regulating the inflammatory microenvironment and anti-aging. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the molecular docking between silk fibroin and polygalactoside. Figure 2 (A) is a schematic diagram of the injectability of the silk fibroin and polydipsia hydrogel; (B) is a scanning electron microscope image of the silk fibroin and polydipsia hydrogel; (C) is a release curve of polydipsia in the silk fibroin and polydipsia hydrogel. Figure 3 Figure showing the detection results of the effects of silk fibroin and polydipsia glycoside hydrogels on oxidative stress and inflammatory factors in wound tissue: Among them: (A) lipid peroxide MDA; (B) superoxide dismutase SOD activity; (C) TNF-α content; (D) IL-1β content; Figure 4 Image showing the results of immunofluorescence staining of P53, CD68, CD86 and CD206 in wound tissue; Figure 5 Figure showing the results of promoting wound healing in diabetic rats using silk fibroin and polydipsia glycoside hydrogel. Figure 6 Figure showing how silk fibroin and polygalactosin hydrogels promote wound healing in aging rats. Detailed Implementation

[0016] The technical problems to be solved, the technical solutions, and the beneficial effects of the present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

[0017] All raw materials used in the examples are commercially available or prepared using conventional methods.

[0018] Example 1

[0019] (1) Cut the silkworm cocoons into pieces, boil them in 0.02M sodium carbonate solution for 1 hour, repeat the degumming process twice, then soak and wash them in ultrapure water 5 times, and dry them for later use; add the dried and degummed SF to 9.3M lithium bromide solution and react at 65℃ for 2 hours to dissolve it; adjust the SF concentration to 10% (W / V); put the obtained solution into a 3500Da dialysis bag and dialyze it in ultrapure water for 3 days, changing the dialysate 5 times a day during the period, then put it back into the dialysis bag and place it in PEG 2000 aqueous solution for osmotic concentration to obtain 10% SF (W / V) aqueous solution; (2) Preparation of SF-PD hydrogel: Add 1 ml of 10% SF (W / V) solution for every 10 mg PD, vortex until homogeneous, place in a constant temperature shaker and heat at 100°C until clear, let stand at room temperature until hydrogel is formed, and obtain SF-PD hydrogel 1.

[0020] Example 2

[0021] (1) Cut the silkworm cocoons into pieces, boil them in 0.04M sodium carbonate solution for 2 hours, repeat the degumming process twice, then soak and wash them in ultrapure water 5 times, and dry them for later use; add the dried and degummed SF to 9M lithium bromide solution and react at 65℃ for 2 hours to dissolve it; adjust the SF concentration to 15% (W / V); put the obtained solution into a 4000Da dialysis bag and dialyze it in ultrapure water for 3 days, changing the dialysate 5 times a day during the period, then put it back into the dialysis bag and place it in PEG2000 aqueous solution for osmotic concentration to obtain 15% (W / V) SF aqueous solution; (2) Preparation of SF-PD hydrogel: Add 1 ml of 15% (W / V) SF solution for every 15 mg PD, vortex until homogeneous, place in a constant temperature shaker and heat at 100°C until clear, let stand at room temperature until hydrogel is formed, and obtain SF-PD hydrogel 2.

[0022] Example 3

[0023] (1) Cut the silkworm cocoons into pieces, boil them in 0.01M sodium carbonate solution for 2 hours, repeat the degumming process twice, then soak and wash them in ultrapure water 5 times, and dry them for later use; add the dried and degummed SF to 10M lithium bromide solution and react at 65℃ for 2 hours to dissolve it; adjust the SF concentration to 20% (W / V); put the obtained solution into a 5000Da dialysis bag and dialyze it in ultrapure water for 3 days, changing the dialysate 5 times a day during the period, then put it back into the dialysis bag and place it in PEG 2000 aqueous solution for osmotic concentration to obtain 20% (W / V) SF aqueous solution; (2) Preparation of SF-PD hydrogel: Add 1 ml of 20% (W / V) SF solution for every 10 mg PD, vortex until homogeneous, place in a constant temperature shaker and heat at 100°C until clear, let stand at room temperature until hydrogel is formed, and obtain SF-PD hydrogel 3.

[0024] Molecular docking diagrams of the SF-PD hydrogels prepared in Examples 1-3 are shown below. Figure 1As shown, the amide groups (-CO-NH-) of silk fibroin form intermolecular hydrogen bonds with multiple phenolic hydroxyl groups (-OH) of polydextrin. The hydrophobic domains of silk fibroin (such as the alanine-rich region) aggregate with the hydrophobic aglycone of polydextrin. The trans-stilbene backbone of polydextrin interacts with the π-electron clouds of the aromatic amino acid residues (Tyr, Phe, Trp) of silk fibroin. The polar amino acids of silk fibroin exhibit weak electrostatic attraction with the glycosyl moiety of polydextrin. This invention utilizes the amphiphilic nature of silk fibroin, directly dissolving polydextrin in a silk fibroin solution, and relying solely on physical processes to co-assemble polydextrin and silk fibroin into a hydrogel. Neither silk fibroin nor polydextrin itself forms a gel; they must be combined to form a gel. Furthermore, no organic solvents are required, and the process occurs only in a pure aqueous environment.

[0025] Comparative Example 1 Commercially available medical 3M dressings.

[0026] A schematic diagram illustrating the injectability properties of SF-PD hydrogel is shown below. Figure 2 As shown in (A), it can dynamically conform to irregular wounds, acting as a physical barrier while simultaneously delivering hydrophobic drugs (PD) via its gel network; the scanning electron microscope image of the SF-PD hydrogel is shown below. Figure 2 As shown in (B), a dense network structure can be observed (scale bar: 50 μm); the PD release curve in the SF-PD hydrogel is shown in Figure 1. Figure 2 As shown in (C), the polygalactosin in this invention is not adsorbed in the hydrogel, but rather co-assembled with silk fibroin as a hydrogel framework, which is durable and stable, and the release rate is slower than that of adsorption.

[0027] like Figure 3 As shown, compared with existing dressings, the SF-PD hydrogel group showed a decrease in the level of lipid peroxide MDA in the wound tissue and an increase in the activity of superoxide dismutase SOD, indicating that the present invention can effectively reduce oxidative damage and enhance the tissue's antioxidant capacity; the levels of pro-inflammatory factors TNF-α and IL-1β were both reduced, indicating that it can effectively inhibit the chronic inflammatory response of the wound.

[0028] Figure 4 The results showed that, compared with existing dressings, the expression of the pro-inflammatory marker CD86 was weakened in the SF-PD hydrogel group, while the expression of the repair-related marker CD206 was enhanced, indicating that the hydrogel can regulate the transformation of macrophages from a pro-inflammatory phenotype to a repair phenotype and improve the immune microenvironment of the wound. The expression of the aging-related protein P53 was reduced in the SF-PD hydrogel group, indicating that it has the effect of alleviating the aging state.

[0029] In summary, the SF-PD hydrogel of the present invention can exert a synergistic effect of improving local oxidative stress in wounds, regulating the inflammatory microenvironment, and anti-aging.

[0030] Compared with Comparative Example 1, the wound repair model in diabetic rats, such as Figure 5 The wound repair model shown is similar to that of aging rats. Figure 6 As shown, this demonstrates that SF-PD hydrogel has the function of promoting chronic wound healing.

[0031] Comparative Example 2: (1) Prepare a 10% (w / v) SF aqueous solution using the same method as in Example 1, step (1); Silkworm cocoons were cut into pieces and boiled in 0.02M sodium carbonate solution for 1 hour. This process was repeated twice to degumm the silk. The silk was then soaked and washed 5 times with ultrapure water and dried for later use. The dried and degummed SF was added to 9.3M lithium bromide solution and reacted at 65°C for 2 hours to dissolve it. The SF concentration was adjusted to 10% (W / V). The resulting solution was placed in a 3500 Da dialysis bag and dialyzed in ultrapure water for 3 days, with the dialysate changed 5 times a day. The solution was then placed back into the dialysis bag and concentrated by osmosis in PEG 2000 aqueous solution to obtain a 10% (W / V) SF aqueous solution. (2) Add 1 ml of 10% (W / V) SF solution for every 3 mg PD, vortex until homogeneous, place in a constant temperature shaker and heat at 100°C until clear, then let stand at room temperature.

[0032] Ultimately, they cannot self-assemble into a hydrogel. Whether it is silk fibroin or polygalactoside, if the amount used is below the protective range, although they can self-assemble into fibers, they cannot further self-assemble into a hydrogel.

[0033] Comparative Example 3 (1) Prepare a 25% (w / v) SF aqueous solution using the same method as in Example 1, step (1); (2) Add 1 ml of 25% (W / V) SF solution for every 10 mg PD, vortex until homogeneous, place in a constant temperature shaker and heat at 100°C until clear, then let stand at room temperature.

[0034] Precipitation occurred, preventing self-assembly into a hydrogel. Whether it's silk fibroin or polygalactoside, if the dosage is too high, they may become insoluble, preventing further self-assembly into a hydrogel.

[0035] This invention involves the self-assembly of silk fibroin and polydipsia glycoside through intermolecular interactions to form fibers, which further self-assemble into hydrogels. These fibers can dynamically adhere to irregular wounds, acting as a physical barrier while delivering hydrophobic drugs (PD) via their gel network. This process can also exert synergistic effects in improving local oxidative stress in wounds, regulating the inflammatory microenvironment, and anti-aging.

Claims

1. A method for preparing a polygalactosidase-silk fibroin hydrogel, characterized in that, The preparation method comprises the following steps: (1) Prepare an aqueous solution of silk fibroin, wherein the mass-volume percentage of the aqueous solution of silk fibroin is 5%-20% w / v; (2) Preparation of multifunctional hydrogel: First, add polysaccharide to the silk fibroin aqueous solution prepared in step (1), mix evenly and let stand until hydrogel is formed.

2. The method for preparing the polygalactosidase-silk fibroin hydrogel according to claim 1, characterized in that, The preparation method of the silk fibroin aqueous solution in step (1) is as follows: First, the silkworm cocoons are cut into pieces, boiled in sodium carbonate solution, and repeated several times to obtain degummed silk fibroin. Then, the silk fibroin is soaked and washed several times with ultrapure water and dried for later use. Second, the degummed silk fibroin is added to lithium bromide solution for dissolution, and the concentration of silk fibroin is adjusted to 5-20% W / V. Finally, the obtained solution is put into a dialysis bag, dialyzed in ultrapure water, and then put back into the dialysis bag and placed in PEG2000 aqueous solution for osmotic concentration to obtain an 8%-20% W / V silk fibroin aqueous solution.

3. The method for preparing the polygalactosidin-silk fibroin hydrogel according to claim 2, characterized in that, The molar concentration of sodium carbonate solution is 0.01-0.05M, the molar concentration of lithium bromide solution is 8-10M, and the concentration of PEG2000 aqueous solution is 5%~30%w / v.

4. The method for preparing the polygalactosidin-silk fibroin hydrogel according to claim 2, characterized in that, The dialysis bag has a molecular weight cutoff of 3000-5000 Da. Dialysis is performed in ultrapure water for 3-5 days, with the dialysis solution changed several times a day during this period.

5. The method for preparing the polygalactosidin-silk fibroin hydrogel according to claim 2, characterized in that, Step (2) The amount of polygalactoside-silk fibroin added is 1 ml of 8%-20% W / V silk fibroin aqueous solution for every 5-20 mg of polygalactoside.

6. The method for preparing the polygalactosidin-silk fibroin hydrogel according to claim 2, characterized in that, Step (2) After the mixed solution is vortexed to homogenize, it is placed in a constant temperature shaker and heated to 80-120℃ until it becomes clear. Then it is left to stand at room temperature until a hydrogel forms.

7. The application of a method for preparing the polygalactosidase-silk fibroin hydrogel as described in any one of claims 1-6 as a wound dressing in the treatment of chronic wounds.

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