Silk fibroin gel loaded with polyhydroxy phenolic compound as well as preparation method and application of silk fibroin gel

Abstract

The silk fibroin gel loaded with the polyhydroxy phenolic compound is prepared from the polyhydroxy phenolic compound and silk fibroin in a glycerin-containing solvent system, the concentration of the silk fibroin in the gel is 1.5%-13%, the proportion of glycerin is 35%-55%, and the preferable scheme is that the concentration of the polyhydroxy phenolic compound is not lower than 0.1%. The gel provided by the invention can load high-concentration polyhydroxy phenolic compounds with far over-saturated solubility, and especially has a more remarkable effect on insoluble polyhydroxy phenolic compounds; in terms of efficacy, the gel can complete full-thickness defect skin repair, has the effects of resisting bacteria, resisting infection, preventing adhesion and the like, and can be applied to preparation of a preparation with a wound healing effect. According to the preparation method of the silk fibroin gel loaded with the polyhydroxy phenolic compounds, provided by the invention, the silk fibroin can realize a self-gelling process under the condition that auxiliary materials are not required to be added and external force is applied at a relatively low concentration (1.5-2.5%), and the self-gelling speed can be remarkably accelerated.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and particularly relates to a silk fibroin gel loaded with polyhydroxyphenol compounds, and a preparation method and application thereof. Background Art

[0002] Polyphenols are a class of natural compounds with diverse biological activities found widely in plants, including tea polyphenols and catechins. In recent years, the application of polyphenols in antimicrobial and wound healing applications has garnered significant attention. Polyphenols possess broad-spectrum antimicrobial activity, effectively inhibiting the growth of a wide range of bacteria and fungi. For example, tea polyphenols have been shown to exhibit a variety of pharmacological effects, including antioxidant, antimutagenic, antitumor, lipid-lowering, hepatoprotective, anti-aging, and immune-modulating properties. They also possess broad-spectrum and potent antimicrobial activity. Furthermore, the antimicrobial properties of tea polyphenols extend beyond traditional bacteria and include the ability to eliminate drug-resistant bacteria, a crucial aspect given the increasing prevalence of antibiotic resistance. The antimicrobial mechanisms of polyphenols are diverse, including disrupting bacterial cell membrane integrity, inhibiting normal bacterial energy metabolism, and affecting the synthesis of biomacromolecules. For example, tea polyphenols interact with bacterial cell membranes, disrupting their structure and thereby inhibiting bacterial growth and reproduction.

[0003] Polyphenols not only have antibacterial effects, but also show significant effects in wound repair. Due to their rich phenolic hydroxyl groups, polyphenol materials can effectively scavenge free radicals, regulate macrophage polarization, and combine with the photothermal effect of iron ions to perform antibacterial activities, ultimately promoting rapid wound repair. For example, polyphenol-derived biomaterials have been widely used in the field of wound repair due to their unique antioxidant, adhesion and antibacterial functions. Studies have shown that polyphenol-metal functionalized hydrogel dressings can be used to treat chronic wounds, have sustained-release, antibacterial and antioxidant properties, can scavenge excess reactive oxygen species in cells, provide controllable antioxidant bioactivity, and have reliable biocompatibility.

[0004] However, the solubility of polyphenols based on traditional methods is limited, and their use in many aspects is restricted. Therefore, the present invention adopts glycerol as a solvent and combines ultrasonic crushing to improve the solubility of polyphenols, thereby obtaining a uniform and stable solution system, thereby making it possible to form a new preparation with other substances.

[0005] Silk fibroin (SF) is a natural high-molecular polymer extracted from silk. Due to its excellent biocompatibility, controllable biodegradability, and good mechanical properties, it has been widely studied and applied in the fields of wound repair and antibacterial treatment. Biocompatibility and degradability in wound repair: Silk fibroin has excellent biocompatibility and can perfectly integrate with human tissue without producing rejection reactions. It can also be gradually degraded and absorbed and metabolized in the human body. In addition, its degradation and mechanical properties are adjustable, making it an ideal matrix material for wound repair. Promotion of cell proliferation and collagen synthesis: Silk fibroin has the ability to induce collagen synthesis, thus exhibiting the ability to actively induce wound healing. It accelerates wound healing by providing a favorable microenvironment, promoting cell proliferation and migration.

[0006] Silk fibroin itself does not have antibacterial activity, but its antibacterial properties can be significantly enhanced by combining it with other antibacterial materials, such as nanosilver, antimicrobial peptides, and curcumin. Polyphenols themselves are excellent antibacterial substances, so it is possible to combine silk fibroin with polyphenols to prepare anti-infection preparations.

[0007] Hydrogels can provide a moist environment and are a commonly used dosage form in wound healing, promoting the progress of wound healing. For example, hydrogels can absorb exudate and keep the wound moist, thereby avoiding adhesion when changing dressings. In addition, hydrogels can also be used as drug delivery carriers to deliver antimicrobial agents, cytokines and other drugs to the wound, thereby accelerating wound healing. Some studies have shown that hydrogels can induce angiogenesis, reduce inflammatory responses, and promote the repair of skin tissue. Hydrogel materials generally have good biocompatibility and biodegradability, which allows them to be used safely in the body and eventually degraded. For example, hydrogels based on self-assembly cross-linking not only have high water absorption and high water retention, but also have good biocompatibility and biodegradability. This achieves the purpose of both antibacterial and wound repair, making the gel model an advantageous dosage form for wound repair.

[0008] The present invention discovered that the polyhydroxy structures in polyphenols will first fully combine with glycerol to increase their solubility. This may be due to the primary binding completed by hydrogen bonds or other chemical bonds. Silk fibroin will exhibit a self-stacking and assembly process in the solution, and the free amino acid chains have sites for binding to hydroxyl groups, thereby providing the possibility for silk fibroin to construct a gel with a glycerol-polyphenol solution. Summary of the Invention

[0009] The purpose of the present invention is to construct a gel with a three-dimensional network structure by combining a silk fibroin solution and a glycerol-polyhydroxyphenol compound solution, thereby completing the functions of wound repair, antibacterial and anti-infection, and anti-adhesion.

[0010] The present invention provides a silk fibroin gel loaded with polyhydroxyphenol compounds, which is prepared by using polyhydroxyphenol compounds and silk fibroin in a solvent system containing glycerol. The concentration of the silk fibroin in the gel is 1.5% to 13%, and the proportion of the glycerol is 35% to 55%. Preferably, the concentration of the polyhydroxyphenol compound in the gel is not less than 0.1%.

[0011] As an improvement, the polyhydroxyphenol compound includes one or more of ellagic acid, resveratrol, quercetin, baicalein, kaempferol, lignans, curcumin, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapinic acid, rutin, myricetin, magnolic acid, fisetin, puerarin, rhein, honeysuckle glycoside, proanthocyanidin, p-coumaric acid, vanillic acid, hesperidin, naringenin, luteolin, genistein, emodin, oleanolic acid, salvianol, epigallocatechin gallate, gallic acid, pyrogallol, catechin, tannic acid, tea polyphenols, acacia glycoside, and epigallocatechin.

[0012] As a further improvement, the polyhydroxyphenol compound is a poorly soluble polyhydroxyphenol compound, including one or more of ellagic acid, resveratrol, quercetin, baicalein, kaempferol, lignans, curcumin, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapinic acid, rutin, myricetin, magnolic acid, fisetin, puerarin, rhein, honeysuckle, proanthocyanidins, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, emodin, oleanolic acid, salvianolic acid, and epigallocatechin gallate.

[0013] The present invention also provides a method for preparing the above-mentioned silk fibroin gel loaded with polyhydroxyphenol compounds, which is characterized by comprising three steps: step (1) preparing a silk fibroin solution: the silk fibroin is prepared into a silk fibroin solution with pure water; step (2) constructing a polyhydroxyphenol compound-glycerol system: the polyhydroxyphenol compound is mixed with glycerol and then crushed by ultrasonic wave, so that the polyhydroxyphenol compound is completely dissolved in the glycerol and presents a clear and transparent liquid phase, which is the polyhydroxyphenol compound-glycerol system; step (3): self-gelation: the silk fibroin solution in step (1) and the polyhydroxyphenol compound-glycerol system in step (2) are fully mixed, and a gel is formed after standing.

[0014] As an improvement, in step (3), after the silk fibroin solution and the polyhydroxyphenol compound-glycerol system are fully mixed, a method selected from vortexing, ultrasonic oscillation, and low-frequency shaking is used to promote the formation of the gel; among which ultrasonic oscillation is a more preferred option.

[0015] The polyhydroxyphenol compound-loaded silk fibroin gel provided by the present invention can be applied to preparations with wound healing efficacy, and can also be applied to preparations with antibacterial, anti-infection, and / or anti-adhesion efficacy.

[0016] Beneficial effects

[0017] The polyhydroxyphenol compound-loaded silk fibroin gel provided by the present invention can load high concentrations of polyhydroxyphenol compounds that far exceed the compound's saturated solubility, and this effect is particularly significant for poorly soluble polyhydroxyphenol compounds. The gel can repair full-thickness skin defects while also possessing antibacterial, anti-infection, and anti-adhesion properties, making it suitable for use in the preparation of wound healing preparations. The polyhydroxyphenol compound-loaded silk fibroin gel preparation method provided by the present invention enables the silk fibroin to self-gel at relatively low concentrations (1.5% to 2.5%) without the addition of auxiliary materials or the application of external forces, and significantly accelerates the rate of self-gel formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Silk fibroin gel loaded with polyhydroxyphenol compounds

[0019] Figure 2 Effect of ellagic acid concentration on gel formation. The percentage in the figure represents the concentration of ellagic acid glycerol solution during the preparation process. For example, 0.1% means the formula of the gel is "5% silk fibroin solution + 0.1% silk fibroin glycerol solution"

[0020] Figure 3 Wound contraction rate, representing the wound healing effect (n=3, compared with the blank group, *P<0.05, **P<0.01) Figure 4 、 Figure 5 In vitro antibacterial effect (compared with the blank group, *P<0.05, **P<0.01, ***P<0.001) DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further illustrated below with reference to specific embodiments, which are not intended to limit the technical solution.

[0022] Example 1 Glycerol Rapidly Dissolves Polyhydroxyphenol Compounds

[0023] Quercetin, ellagic acid, syringic acid, chlorogenic acid, tea polyphenols, sinapinic acid, and other polyhydroxyphenol compounds were solubilized in glycerol using ultrasonication to prepare supersaturated solutions. Their saturated solubilities were measured by UV-spectrophotometry, as shown in Table 1. It can be seen that the solubility of polyhydroxyphenol compounds in glycerol was significantly improved, with ellagic acid and chlorogenic acid showing the best results.

[0024] Table 1 Saturated solubility of polyhydroxyphenol compounds in glycerol

[0025]

[0026] Example 2 Preparation of Silk Fibroin Gel Loaded with Polyhydroxyphenol Compounds

[0027] The silk fibroin obtained by extracting from natural silk cocoons is used to prepare a silk fibroin solution with a mass concentration of 3% to 23% with pure water. If the concentration is too high, it is diluted with pure water in proportion. The method in Example 1 is used to prepare a glycerol solution of a high concentration of polyhydroxyphenol compounds. At this time, the solution should be clear and transparent, without suspended particles and impurity precipitation. After the above-mentioned silk fibroin solution and the glycerol solution of polyhydroxyphenol compounds are fully mixed, they are allowed to stand at room temperature until the gelation is completely completed. During this period, one of the methods of vortexing, ultrasonic oscillation, and low-frequency shaking can be used to accelerate the formation of the gel; the proportion of glycerol in the gel is 35% to 55%, the concentration of silk fibroin is 1.5% to 13%, and the concentration of polyhydroxyphenol compounds is not less than 0.1%. The prepared gel is a transparent and clear colloidal substance. Figure 1 Shown are gels prepared using the above method with multiple polyhydroxyphenol compounds. The silk fibroin gel serves as a blank control, meaning the polyhydroxyphenol compounds were removed from the gels. This indicates that under the same experimental conditions, silk fibroin gels can form without the addition of polyhydroxyphenol compounds, but they still exhibit a certain degree of fluidity, indicating weak gelation properties. However, the addition of multiple polyhydroxyphenol compounds significantly improves gelation properties.

[0028] Example 3 Effect of polyhydroxyphenol compounds on gel formation speed

[0029] A 5% silk fibroin solution was prepared for later use, and an appropriate amount of glycerol was taken for later use. A 0.1% to 0.7% ellagic acid glycerol solution was prepared using the method in Example 1. Ellagic acid-silk fibroin gels loaded with different concentrations were prepared using the method in Example 2. During the gel formation process, the gel was allowed to stand at room temperature. The overall color change of the system was observed every 0.5 h, and the centrifuge tube was inverted to check the gel formation. The time required for gelation was recorded. The results are shown in Table 2. Compared with the 5% silk fibroin solution + glycerol group, the gelation time required for the group with ellagic acid added was reduced by at least 2 h, and as the ellagic acid concentration increased, the gelation time was shorter. It can be seen that the addition of polyhydroxyphenol compounds can significantly accelerate the self-gelation speed of silk fibroin, and the concentration of polyhydroxyphenol compounds is one of the important factors controlling the gel formation speed. Figure 2 The state of the gel prepared under different ellagic acid concentration conditions is shown, which also reflects the important influence of the concentration of polyhydroxyphenol compounds on gel formation.

[0030] Table 2 Effect of different ellagic acid concentrations on gel formation speed

[0031]

[0032] Example 4 Investigation of Skin Wound Healing Effect

[0033] The silk fibroin gel of polyhydroxyphenol compound was prepared by the method in Example 2, and its skin wound healing effect was assessed using a full-thickness skin defect wound model, with the glycerol-silk fibroin gel without polyhydroxyphenol compound as a control. C57B mice were divided into 9 groups, namely, blank group, glycerol-silk fibroin (Gly-SF) group, poly-polymerized complex sugar medical glue material (MCPGL) group, glycerol-ellagic acid-silk fibroin (Gly-EA-SF) group, glycerol-syringic acid-silk fibroin (Gly-Syr-SF), glycerol-tea polyphenols-silk fibroin (Gly-TP-SF), glycerol-quercetin-silk fibroin (Gly-Que-SF), glycerol-chlorogenic acid-silk fibroin (Gly-CGA-SF), glycerol-sinapic acid-silk fibroin (Gly-SA-SF). Mice were anesthetized with an air anesthesia machine, their back hair was removed, and the back area was disinfected with iodine tin and alcohol. Subsequently, the healing pad was sutured to the back of the mouse, and a circular skin defect (d = 8 mm) was created in the back pad of each mouse using a skin punch. 0.5 mL of gel was administered to each group on day 0, and the blank group was not treated. All wounds were fixed with breathable medical dressings to prevent the drug from moving. On days 0, 3, 7, and 14, the wounds were photographed with a digital camera and the dressings were changed. The wound size was measured using Image J software. In order to evaluate the healing process, the wound contraction rate was evaluated, as shown in Figure 2. Figure 3 As shown in the figure, the repair effect of the gel formed by silk fibroin and glycerol is more significant than that of the blank group, and is almost equivalent to that of the positive control group. The addition of polyhydroxyphenols also has a significant improvement compared with the blank group, and the repair effect of the Gly-SF group and the positive control group has also been well improved. It can be seen that the silk fibroin gel loaded with polyhydroxyphenol compounds has a significant improvement in wound repair.

[0034] Example 5 In vitro antibacterial performance investigation

[0035] The antibacterial ability of the hydrogel was tested by contact antibacterial test, according to the grouping in Example 4. 100 μL of bacterial suspension and 900 μL of sterile PBS were mixed with the hydrogel in a 24-well plate, and then 100 μL of bacterial suspension and 900 μL of sterile PBS without material were used as a control. After incubating the plate at 37°C for 18-24 hours, the colony forming units (CFU) were counted on the culture dish after dilution with PBS. CFU was calculated as follows: CFU = C / (V×N)×100%, where C represents the colony count on the culture dish after dilution, V represents the volume of the bacterial suspension on the culture dish, and N represents the multiple of PBS dilution. The results are expressed as antibacterial rate (%): Antibacterial rate (%) = (1 survivor bacterial count in the experimental group / bacterial count in the control)×100%. All these tests were repeated 3 times.

[0036] Figure 4 and Figure 5 This chart compares the antibacterial rates of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) using the direct contact method. The blank group showed no antibacterial effect, while the positive control group exhibited only a weak inhibitory effect. Gly-SF exhibited significant antibacterial activity, and the addition of polyhydroxyphenols increased the antibacterial effect by at least one-third. This demonstrates that the gel constructed by dissolving polyhydroxyphenols in glycerol and combining them with silk fibroin exhibits excellent antibacterial properties.

[0037] Example 6 Anti-blocking performance investigation

[0038] The in vivo anti-adhesion effect of a silk fibroin gel loaded with the polyhydroxyphenol compound ellagic acid (Gly-EA-SF) was evaluated in a rat lateral wall defect-cecal abrasion model. SD rats were anesthetized with sodium pentobarbital (50 mg / kg) and their abdominal hair was shaved. A 5-cm incision was then made along the midline of the abdominal wall using surgical scissors. The cecum was isolated, and its serosal surface was gently rubbed with sterile surgical gauze until punctate bleeding occurred. A 1 cm × 2 cm peritoneal defect was created on the corresponding lateral side of the abdominal wall using a scalpel. In the Gly-EA-SF group, 1 mL of Gly-EA-SF sample was placed on the injured abdominal wall and cecum. In the negative control group, 1 mL of sterile saline was sprayed on the wound surface. In the positive control group, the abdominal wall defect was covered with a commercially available polylactic acid anti-adhesion membrane. Four rats in each group were euthanized after 7 and 14 days, and the peritoneum was opened and examined for adhesions. Adhesions formed between the cecum and the abdominal wall were scored using a standard scoring system (Table 3).

[0039] The negative control group exhibited severe, unremovable adhesions on days 7 and 14, with weakened adhesion observed between the abdominal wall and cecum. This may be due to the commercially available polylactic acid anti-adhesion membrane being applied as a solid sheet, which failed to fully cover the injured surface, a drawback of this type of conventional anti-adhesion barrier material. The Gly-EA-SF group showed no signs of adhesion on days 7 and 14. Furthermore, on day 14, the injured abdominal wall and cecum in the Gly-EA-SF group had returned to normal. In Table 7, the Gly-EA-SF group achieved the lowest score of the three groups, demonstrating its superior anti-adhesion efficacy.

[0040] Table 3 Adhesion scores of ellagic acid-loaded silk fibroin gels on the 7th and 14th days after treatment.

[0041]

Claims

1. A silk fibroin gel loaded with a polyhydroxyphenol compound, characterized in that: The gel is prepared by using polyhydroxyphenol compounds and silk fibroin in a solvent system containing glycerol. The concentration of the silk fibroin in the gel is 1.5% to 13%, and the proportion of the glycerol is 35% to 55%.

2. The silk fibroin gel according to claim 1, wherein: The concentration of the polyhydroxyphenol compound in the gel is not less than 0.1%.

3. The silk fibroin gel according to claim 1 or 2, characterized in that: The polyhydroxyphenol compounds include one or more of ellagic acid, resveratrol, quercetin, baicalein, kaempferol, lignans, curcumin, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapinic acid, rutin, myricetin, magnolic acid, fisetin, puerarin, rhein, honeysuckle glycoside, proanthocyanidin, p-coumaric acid, vanillic acid, hesperidin, naringenin, luteolin, genistein, emodin, oleanolic acid, salvianol, epigallocatechin gallate, gallic acid, pyrogallol, catechin, tannic acid, tea polyphenols, acacia glycoside, and epigallocatechin.

4. The silk fibroin gel according to claim 1 or 2, characterized in that: The polyhydroxyphenol compounds in the gel are poorly soluble polyhydroxyphenol compounds, including one or more of ellagic acid, resveratrol, quercetin, baicalein, kaempferol, lignans, curcumin, chlorogenic acid, ferulic acid, caffeic acid, syringic acid, sinapinic acid, rutin, myricetin, magnolic acid, fisetin, puerarin, rhein, honeysuckle, proanthocyanidins, p-coumaric acid, vanillic acid, hesperetin, naringenin, luteolin, genistein, emodin, oleanolic acid, salvianolic acid, and epigallocatechin gallate.

5. The method for preparing the silk fibroin gel according to any one of claims 1 to 4, characterized in that The method comprises three steps: step (1) preparing a silk fibroin solution: the silk fibroin is prepared into a silk fibroin solution with pure water; step (2) constructing a polyhydroxyphenol compound-glycerol system: the polyhydroxyphenol compound is mixed with glycerol and then crushed with ultrasound, so that the polyhydroxyphenol compound is completely dissolved in the glycerol and presents a clear and transparent liquid phase, which is the polyhydroxyphenol compound-glycerol system; step (3): self-gelling: the silk fibroin solution in step (1) and the polyhydroxyphenol compound-glycerol system in step (2) are fully mixed and allowed to stand to form a gel.

6. The method for preparing the silk fibroin gel according to claim 5, wherein: In step (3), the silk fibroin solution and the polyhydroxyphenol compound-glycerol system are fully mixed and then a method selected from vortexing, ultrasonic oscillation and low-frequency shaking is used to promote the formation of gel.

7. The method for preparing the silk fibroin gel according to claim 6, wherein: In step (3), the silk fibroin solution and the polyhydroxyphenol compound-glycerol system are fully mixed and then ultrasonic vibration is used to promote the formation of gel.

8. A silk fibroin gel loaded with polyhydroxyphenol compounds prepared by the preparation method according to any one of claims 5 to 6.

9. Use of the silk fibroin gel according to any one of claims 1 to 4 and 8 in preparing a preparation having wound healing efficacy, characterized in that: The invention comprises the silk fibroin complex according to any one of claims 1 to 4 and 8 and a pharmaceutically acceptable excipient.

10. Use of the silk fibroin gel according to any one of claims 1 to 4 and 8 in the preparation of a preparation having antibacterial, anti-infective, and / or anti-adhesion effects, characterized in that: The invention comprises the silk fibroin complex according to any one of claims 1 to 4 and 8 and a pharmaceutically acceptable excipient.

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