Preparation method and application of environment-friendly silk fibroin-based antibacterial hydrogel

Abstract

The application provides a preparation method and application of an environment-friendly silk fibroin-based antibacterial hydrogel, and belongs to the technical field of medical materials. The hydrogel is obtained by reacting a silk fibroin derivative grafted with polylysine with carboxymethyl chitosan in situ under the condition that the pH is 7.0-7.4 and a natural oxidized polysaccharide is used as a dynamic crosslinking agent. The preparation process does not need to add an exogenous chemical crosslinking agent, significantly increases the crosslinking sites, improves the reaction activity and the crosslinking density, and thus the hydrogel is endowed with good mechanical properties.

Description

Technical Field

[0001] This application relates to a method for preparing and applying an environmentally friendly silk fibroin-based antibacterial hydrogel, belonging to the field of medical materials technology. Background Technology

[0002] Chronic wounds such as diabetic foot ulcers are susceptible to microbial invasion and infection due to prolonged exposure, leading to delayed healing and increased morbidity and mortality. Antibiotics used clinically to treat wound infections generally suffer from poor structural stability, significant toxic side effects, and a tendency to induce drug resistance, failing to meet practical treatment needs. Therefore, there is an urgent need to develop a wound dressing that combines highly effective antibacterial activity with tissue repair functions.

[0003] Hydrogels, with their unique three-dimensional network structure, can effectively absorb wound exudate, maintain a moist healing environment, promote cell migration, and achieve local antibacterial regulation, making them one of the most promising dressing candidates in wound repair. However, existing hydrogel dressings still face the following challenges when applied to infected wounds or chronic, difficult-to-heal wounds: (1) Crosslinking methods are difficult to balance mechanical properties and biosafety: Hydrogels prepared by physical crosslinking have poor stability and mechanical strength, making it difficult to meet the mechanical requirements of dynamic wound dressings; although chemical crosslinking can improve mechanical properties, the residue of crosslinking agents may be toxic or irritating to organisms. Schiff base crosslinking reaction has received widespread attention due to its advantages such as mild conditions and no toxic byproducts, but the crosslinking density and mechanical strength of conventional Schiff base hydrogels are still insufficient.

[0004] (2) Antibacterial strategies are difficult to achieve long-lasting and broad-spectrum antibacterial effects: Strategies that introduce inorganic nano-antibacterial agents or load antibiotics generally suffer from problems such as burst release and rapid depletion of antibacterial agents, potential cytotoxicity, and difficulty in synergistic integration of antibacterial activity and healing-promoting function. Antibacterial hydrogels constructed directly from natural antibacterial materials (such as chitosan derivatives) often have limited antibacterial efficiency, making it difficult to meet the clinical needs of infected wounds for efficient and long-lasting antibacterial action.

[0005] (3) The inherent properties of biomacromolecule substrates have limitations: Although silk fibroin has excellent biocompatibility and degradability, it lacks antibacterial activity and provides limited cross-linking sites in the hydrogel network; although carboxymethyl chitosan has certain moisturizing, antibacterial and healing-promoting abilities, its mechanical strength and tissue adhesion are insufficient to independently support the construction of high-performance functionalized hydrogels. Combining the above materials through simple physical mixing or conventional cross-linking methods makes it difficult to simultaneously achieve the integration of multiple functions such as long-lasting antibacterial, mechanical enhancement and tissue repair. In particular, under the premise of avoiding the use of toxic chemical cross-linking agents, constructing antibacterial hydrogel dressings with excellent mechanical properties still faces great challenges.

[0006] Therefore, how to construct a hydrogel dressing that combines good mechanical strength, tissue adhesion, broad-spectrum and long-lasting antibacterial function, and excellent healing-promoting properties without introducing toxic chemical cross-linking agents is a technical problem that urgently needs to be solved in this field. This invention is proposed precisely to address the aforementioned technical needs. Summary of the Invention

[0007] In view of this, the first objective of this application is to provide a method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel. This method eliminates toxic chemical cross-linking agents and introduces naturally derived oxidized polysaccharides as dynamic cross-linking agents. All components possess excellent biocompatibility and biodegradability. By grafting polylysine onto the silk fibroin molecular chain, functionalized derivatives rich in free amino groups are obtained, significantly increasing the cross-linking sites, enhancing reactivity and cross-linking density, thereby endowing the hydrogel with good mechanical properties.

[0008] Specifically, this application is implemented through the following scheme: A method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel involves using natural oxidized polysaccharide as a dynamic crosslinking agent. Under pH conditions of 7.0–7.4, polylysine-grafted silk fibroin derivatives are reacted with carboxymethyl chitosan and in situ molded to obtain the hydrogel.

[0009] Furthermore, as a preferred option: The degree of oxidation of the natural oxidized polysaccharide is 10-70%; more preferably, the degree of oxidation is 20-60%.

[0010] The natural oxidized polysaccharide molecular chain contains an aldehyde group, which reacts with the amino groups on polylysine-grafted silk fibroin derivatives and carboxymethyl chitosan to form Schiff base bonds.

[0011] The natural oxidized polysaccharide is at least one of sodium alginate, oxidized dextran, or oxidized hyaluronic acid.

[0012] The reaction and molding process is as follows: First, polylysine-grafted silk fibroin derivatives are mixed with carboxymethyl chitosan to form a precursor solution; then, natural oxidized polysaccharides are added to the precursor solution, and a three-dimensional interpenetrating network structure of antibacterial hydrogel is formed in situ using the Schiff base reaction between aldehyde and amino groups. The Schiff base reaction is carried out in the aqueous phase under mild reaction conditions, without the need for any toxic chemical crosslinking agents.

[0013] More preferably: Before adding the natural oxidized polysaccharide, the concentration of the precursor solution was 2–10% w / v.

[0014] In the precursor solution, the mass ratio of polylysine-grafted silk fibroin derivative to carboxymethyl chitosan is 1:9 to 6:4.

[0015] The volume ratio of the precursor solution to the natural oxidized polysaccharide is 1:4 to 4:1. More preferably, the volume ratio of the precursor solution to the natural oxidized polysaccharide is 1:3 to 3:1, and the natural oxidized polysaccharide is added in the form of an oxidized polysaccharide solution with a concentration of 1 to 15% w / v.

[0016] The polylysine-grafted silk fibroin derivative and carboxymethyl chitosan were dissolved separately in PBS solution and then mixed to obtain a precursor solution.

[0017] The synthesis process of the polylysine-grafted silk fibroin derivative is as follows: ε-polylysine is added to a silk fibroin solution in the presence of a condensing agent, and grafting modification is carried out through an amidation reaction. After purification, the polylysine-grafted silk fibroin derivative is obtained. By grafting polylysine onto the silk fibroin molecular chain through the above amidation reaction, a functionalized derivative rich in free amino groups is obtained. More preferably: The mass ratio of ε-polylysine to silk fibroin is 1:4 to 40.

[0018] The molecular weight of the ε-polylysine is 1–10 kDa.

[0019] The molecular weight of the silk fibroin is 50-200 kDa, more preferably 100-200 kDa.

[0020] The condensing agent is a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0021] The second objective of this application is to provide an antibacterial hydrogel prepared by the above method, wherein the antibacterial hydrogel has a three-dimensional interpenetrating network structure, which endows the hydrogel with excellent mechanical strength and tissue adhesion, and has a broad-spectrum and long-lasting contact bactericidal function.

[0022] A third objective of this application is to provide an application of the aforementioned antibacterial hydrogel in medical dressings. Specifically, the aforementioned medical dressing can effectively inhibit wound infection, promote angiogenesis and collagen deposition, and therefore can be used for angiogenesis and wound healing, including but not limited to infected wounds and chronic, difficult-to-heal wounds (such as foot ulcers caused by diabetes).

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention abandons the traditional toxic chemical crosslinking agent and uses naturally sourced oxidized polysaccharide as a dynamic crosslinking agent. All components have excellent biocompatibility and degradability, avoiding the cytotoxicity problem caused by crosslinking agent residue.

[0024] (2) In this invention, amino-rich ε-polylysine is grafted onto the silk fibroin molecular chain through an amidation reaction, which significantly increases the cross-linking sites, thereby improving the cross-linking density, mechanical strength and tissue adhesion of the hydrogel.

[0025] (3) The present invention avoids the problems of easy burst release and easy depletion of antibacterial agents in physical loading methods, has a broad-spectrum and long-lasting contact sterilization function, and is not easy to induce bacterial resistance.

[0026] (4) The present invention constructs a three-dimensional interpenetrating network structure by dynamically crosslinking polylysine grafted silk fibroin derivatives, carboxymethyl chitosan and oxidized polysaccharides, which not only significantly enhances the moisturizing and tissue repair capabilities of hydrogels, but also effectively induces angiogenesis and collagen deposition, thereby accelerating the repair of chronic wounds such as diabetic foot ulcers. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application.

[0028] Figure 1 The figure shows the microstructure of the hydrogel dressing prepared in Example 1. Part (A) is a SEM image and part (B) is a pore size distribution map.

[0029] Figure 2 The polylysine-grafted silk fibroin derivative in Example 1 1 H NMR spectrum.

[0030] Figure 3 The effect of the mass ratio of ESF to CMCS in the precursor solution of Example 3 on the antibacterial properties of the hydrogel.

[0031] Figure 4 To illustrate the repair effect of the hydrogel of this application as a dressing on the wounds of diabetic rats, part (A) of the figure shows the repair photograph, part (B) shows the wound healing rate 12 days after surgery, part (C) shows HE staining, Masson staining and CD31 staining images, part (D) shows inflammatory infiltration, and part (E) shows the CD31 positivity rate.

[0032] Figure 5 This is a diagram illustrating the adhesion behavior of the hydrogel of this application on pig skin. Detailed Implementation

[0033] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only for explaining this application and are not intended to limit the technical solutions of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.

[0034] The raw materials used in this embodiment, such as silk fibroin, ε-polylysine, carboxymethyl chitosan, sodium alginate, and dextran, can all be obtained through commercial channels or prepared according to conventional methods in the field.

[0035] Silk fibroin can be prepared using conventional methods in the art, such as dissolving degummed silkworm cocoons in a lithium bromide solution, followed by dialysis, centrifugation, and filtration to obtain a regenerated silk fibroin solution. The molecular weight of ε-polylysine is preferably 1–10 kDa, and the molecular weight of silk fibroin is preferably 100–200 kDa. The degree of substitution of carboxymethyl chitosan is preferably 0.6–1.2.

[0036] The preparation method of oxidized polysaccharide is as follows: Sodium alginate, dextran, hyaluronic acid, and other polysaccharides are dissolved in deionized water. A certain amount of sodium periodate is added, and the reaction is carried out in the dark for a certain time. Ethylene glycol is added to terminate the reaction. The oxidized polysaccharide is obtained by dialysis and lyophilization. By adjusting the amount of sodium periodate and the reaction time, the oxidation degree of the oxidized polysaccharide can be controlled to be 10-70%, and more preferably 20-60%.

[0037] Example 1

[0038] This embodiment provides a method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel, the process of which is as follows: Step 1: Preparation of the oxidized polysaccharide solution: Sodium alginate was dissolved in deionized water to prepare a 2% w / v solution. An equal volume of sodium periodate (NaIO4) solution was added to bring the concentration in the reaction system to 0.65% w / v, and the mixture was stirred for 24 hours in the dark. Ethylene glycol was then added at a volume ratio of 1:50, and stirring was continued for 30 minutes to terminate the oxidation reaction. The reaction solution was transferred to a dialysis bag (molecular weight cutoff 3.5 kDa), dialyzed thoroughly with deionized water, and then freeze-dried to obtain oxidized sodium alginate. The oxidation degree was determined to be approximately 60% by hydroxylamine hydrochloride-potentiometric titration. It was stored in a dark, sealed container at 4°C for later use.

[0039] Step 2, preparation of hydrogel: S1, Preparation of precursor solution: Polylysine-grafted silk fibroin derivative and carboxymethyl chitosan were mixed at a mass ratio of 4:6 and dissolved in PBS solution (pH=7.4) to obtain a precursor solution with a concentration of 6% w / v.

[0040] S2, In-situ Crosslinking: The oxidized sodium alginate prepared in step one was dissolved in PBS solution to prepare an 8% w / v oxidized polysaccharide solution. The precursor solution and the oxidized polysaccharide solution were mixed evenly at a volume ratio of 2:1 and allowed to stand at room temperature for about 3 minutes to form a hydrogel.

[0041] The resulting hydrogel is translucent, with a high water content (>90%) and a low swelling ratio (<400%). After freeze-drying, it combines... Figure 1 The scanning electron microscope results show that the hydrogel has a three-dimensional porous network structure, confirming that the prepared hydrogel has a three-dimensional interpenetrating network structure with a uniform pore size distribution (1-10 μm).

[0042] In the above process, polylysine-grafted silk fibroin derivatives can be prepared by the following method: Silk fibroin (molecular weight approximately 150 kDa) was dissolved in deionized water to prepare a 2% w / v silk fibroin solution, which was then cooled and stabilized in an ice bath to approximately 4 °C. Morpholine ethanesulfonic acid buffer was added to the solution to adjust the pH to 6.5. Subsequently, condensing agents EDC and NHS were added, with a total mass of 7.5% of the silk fibroin weight and a mass ratio of 2:1. After stirring and activating for 30 minutes, ε-polylysine (molecular weight approximately 5 kDa) was added, at a concentration of 10% of the silk fibroin weight. The reaction was continued with stirring in an ice bath for 6 hours. After the reaction was complete, the reaction solution was transferred to a dialysis bag (molecular weight cutoff 8-14 kDa) and dialyzed in deionized water for 48 hours, changing the dialysate every 6 hours to remove unreacted ε-polylysine, EDC, NHS, and byproducts. After dialysis, the product was freeze-dried to obtain a polylysine-grafted silk fibroin derivative.

[0043] The prepared polylysine-grafted silk fibroin derivative was analyzed by 1H NMR spectroscopy (NMR spectroscopy). 1 Characterized by 1H NMR. The results are as follows: Figure 2 As shown: Based on the relationship between polylysine-grafted silk fibroin derivatives and ε-polylysine... 1 The percentage of peak areas at δ=1.43, 1.61 and 1.79 ppm in the 1H NMR spectrum that belong to methylene (-CH2-CH2-CH2-) was used to calculate the grafting rate of polylysine to be approximately 8.9%.

[0044] Example 2

[0045] This embodiment provides a method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel, the process of which is as follows: Step 1: Preparation of the oxidized polysaccharide solution: Dissolve the natural polysaccharide in deionized water to prepare a 2% w / v solution. Add an equal volume of sodium periodate (NaIO4) solution to achieve a concentration of 0.65% w / v in the reaction system, and stir the mixture under light-protected conditions for 24 hours. Then, add ethylene glycol at a volume ratio of 1:50 and continue stirring for 30 minutes to terminate the oxidation reaction. Transfer the reaction solution to a dialysis bag (molecular weight cutoff 3.5 kDa), dialyze thoroughly with deionized water to obtain the oxidized polysaccharide. Freeze-dry the polysaccharide and store it sealed at 4°C under light-protected conditions for later use.

[0046] Step 2, preparation of hydrogel: S1, Preparation of precursor solution: Polylysine grafted silk fibroin derivative (ESF) and carboxymethyl chitosan (CMCS) were mixed at a mass ratio of 4:6 and dissolved in PBS solution (pH=7.4) to obtain a precursor solution with a concentration of 6% w / v.

[0047] S2, In-situ crosslinking: The oxidized polysaccharide prepared in step one was dissolved in PBS solution to prepare an 8% w / v oxidized polysaccharide solution. The precursor solution and the oxidized polysaccharide solution were mixed evenly at a volume ratio of 2:1 and allowed to stand at room temperature for about 3 minutes to form a hydrogel.

[0048] In the above process, polylysine-grafted silk fibroin derivatives can be prepared by the following method: Silk fibroin (molecular weight approximately 150 kDa) was dissolved in deionized water to prepare a 2% w / v silk fibroin solution, which was then cooled and stabilized in an ice bath to approximately 4 °C. Morpholine ethanesulfonic acid buffer was added to the solution to adjust the pH to 6.5. Subsequently, condensing agents EDC and NHS were added, with a total mass of 7.5% of the silk fibroin weight and a mass ratio of 2:1. After stirring and activating for 30 minutes, ε-polylysine (molecular weight approximately 5 kDa) was added, at a concentration of 10% of the silk fibroin weight. The reaction was continued with stirring in an ice bath for 6 hours. After the reaction was complete, the reaction solution was transferred to a dialysis bag (molecular weight cutoff 8-14 kDa) and dialyzed in deionized water for 48 hours, changing the dialysate every 6 hours to remove unreacted ε-polylysine, EDC, NHS, and byproducts. After dialysis, the product was freeze-dried to obtain a polylysine-grafted silk fibroin derivative.

[0049] Among them, the natural oxidized polysaccharides were sodium alginate oxidization, dextran oxidization, and hyaluronic acid oxidization, respectively. Three oxidation degrees (20%, 40%, and 60%) were set for each natural oxidized polysaccharide to investigate the effects of different oxidized polysaccharides and oxidation degrees on the gelation time and rheological behavior of hydrogels. The results are shown in Table 1.

[0050] Table 1: Effects of different oxidized polysaccharides .

[0051] As can be seen from Table 1: The gelation time was determined using the inverted test tube method. The results showed that the gelation time significantly decreased with increasing oxidation degree of the oxidized polysaccharide. For oxidized sodium alginate with an oxidation degree of 20%, the gelation time was approximately 3.5 minutes; at an oxidation degree of 40%, the gelation time decreased to about 2 minutes; and at an oxidation degree of 60%, the gelation time further decreased to within 1 minute.

[0052] The rheological behavior of the obtained hydrogels at 25 °C was characterized and analyzed using a rheometer. The results showed that, within the frequency range of 0.1–100 rad / s, the storage modulus (G') of all hydrogels was consistently greater than the loss modulus (G''), confirming that they were in a stable gel state. Furthermore, the G' / G'' ratio increased with increasing oxidation degree, indicating the formation of a stable elastic gel, and the network structure was more compact under high oxidation degree.

[0053] Example 3

[0054] This embodiment has the same setup as Embodiment 1, except that the mass ratio of ESF to CMCS is replaced by 0:10 (i.e., containing only CMCS), 2:8, and 6:4, respectively, instead of 4:6. The effect of the mass ratio of ESF to CMCS in the precursor solution on the cell compatibility and antibacterial properties of the hydrogel was investigated.

[0055] In vitro cytotoxicity tests were conducted using the CCK-8 assay with L929 fibroblasts as a model cell. Results showed that cell viability for all formulations was above 90%, confirming the good biocompatibility of the hydrogels.

[0056] Colony counting was used, with Escherichia coli (E. coli) as the primary bacterial species. E. coli ATCC 6538), Staphylococcus aureus ( S. aureus ATCC 8739) and methicillin-resistant Staphylococcus aureus (MRSA) MRSA Antimicrobial tests were conducted using ATCC 43300 as indicator bacteria. Results are as follows: Figure 3 As shown, the antibacterial activity of the hydrogel increases with the increasing ratio of ESF to CMCS. When the mass ratio of ESF to CMCS is 6:4, the resulting hydrogel exhibits [high antibacterial activity].E. coli , S. aureus and MRSA The antibacterial rate reached over 99%, confirming that the hydrogel of this invention has excellent broad-spectrum antibacterial activity and wound infection inhibition function.

[0057] Application Examples

[0058] In this embodiment, a diabetic rat model was established by streptozotocin induction to evaluate the repair effect of the hydrogel of the present invention on full-thickness skin defects in diabetic rats.

[0059] Nine SD rats with blood glucose levels of 18–20 mmol / L were selected for the experiment. Three full-thickness skin defects, each 10 mm in diameter, were created on the back of each rat, with an interval of at least 1 cm between the defects. The rats were randomly divided into three groups: Blank control group: No treatment was performed.

[0060] Negative control group 1: Hydrogel covered with silk fibroin without polylysine grafting.

[0061] Experimental group: Covered with the hydrogel prepared in Example 1.

[0062] Wound healing was observed on postoperative days 3, 7, and 12. Wound area was measured and healing rate was calculated. Simultaneously, wound tissue was taken for histological analysis (HE staining and Masson staining) and immunohistochemical analysis (CD31 vascular endothelial marker). Results are as follows: Figure 4 The results showed that the wound healing rate in the experimental group was significantly faster than that in the control group. On the 3rd day after surgery, there was no significant difference in wound healing between the blank control and the negative control (healing rate <5%), and their healing rate was much lower than that of the experimental group (healing rate approximately 15%). On the 12th day after surgery, the wound healing effect in the experimental group was significant (healing rate >90%), while the blank control group (healing rate <70%) and the negative control group (healing rate <80%) still had obvious wound residue. Obvious hair growth was observed around the wounds treated in the experimental group. HE staining and inflammatory infiltration results showed that the wound tissue in the blank control group contained a large number of inflammatory cells, the number of inflammatory cells in the negative control group was reduced compared to the blank group, while the inflammatory cell infiltration in the wound tissue of the experimental group was significantly reduced. Masson staining results showed that the collagen fibers in the wound tissue of the experimental group were more orderly and dense, while the collagen fibers in the control group were disordered and sparse, presenting obvious scar-like structures. CD31 staining results showed that the number of new blood vessels in the wound tissue of the experimental group was significantly higher than that in the control group (CD31 microvessel density was approximately twice that of the blank control group).

[0063] On the 7th postoperative day, wound tissue homogenates from each group were collected for bacterial culture and counting. The results showed that the bacterial load in the wound tissue of the blank control group was (1.5±0.3)×10⁻⁶. 7CFU / g, the negative control group was (8.0±1.2)×10 6 CFU / g (no significant difference compared with the blank group), the experimental group was (2.0±0.5)×10 4 The CFU / g level was reduced by approximately three orders of magnitude compared to the blank control group (p<0.001), confirming that the hydrogel of this invention has significant long-lasting anti-infective ability in vivo.

[0064] The above results indicate that the environmentally friendly silk fibroin-based antibacterial hydrogel provided in this application can effectively inhibit wound infection, promote angiogenesis and collagen deposition, and significantly accelerate the healing process of diabetic wounds.

[0065] Comparative Example 1

[0066] CN 119055824 A is used as Comparative Example 1.

[0067] The cross-linking strategy employed in this application differs fundamentally from that of Comparative Example 1. Comparative Example 1 used traditional chemical cross-linking agents such as glutaraldehyde and genipin for cross-linking, and the residue of these agents may cause cytotoxicity. This application completely abandons exogenous chemical cross-linking agents, relying solely on the Schiff base reaction between the aldehyde and amino groups in natural oxidized polysaccharides for in-situ cross-linking. The reaction conditions are mild, with no toxic byproducts, and all components exhibit excellent biocompatibility and degradability.

[0068] The utilization method of silk fibroin in this application is fundamentally different from that in Comparative Example 1. In Comparative Example 1, polylysine is grafted onto the surface of silk nanofibers, and the grafting reaction occurs on the fiber surface, with grafting sites limited by the fiber's specific surface area. In this application, polylysine is directly grafted onto the silk fibroin macromolecular chain, achieving uniform modification at the molecular level. Each silk fibroin molecule can participate in subsequent cross-linking reactions, resulting in a significantly higher density of cross-linking sites.

[0069] The hydrogel of this application has advantages in terms of three-dimensional network structure and multifunctional synergy. Comparative Example 1 is a two-phase composite structure of fiber skeleton and hydrogel matrix, with the fibers and matrix physically embedded. This application forms a three-dimensional interpenetrating network at the molecular level through Schiff base reaction (ESF, CMCS, and oxidized polysaccharide all participate in network construction through covalent bonds), resulting in a more uniform and dense network structure and superior mechanical properties. The main function of Comparative Example 1 is antibacterial. This application achieves multiple functions simultaneously, including antibacterial, angiogenesis promotion, and collagen deposition promotion, through the synergy of three components: ESF (grafted with polylysine to provide antibacterial activity and increase cross-linking sites), CMCS (providing moisturizing and healing-promoting capabilities), and oxidized polysaccharide (dynamically cross-linked to construct the network). Moreover, the functions enhance each other rather than simply adding together.

[0070] Comparative Example 2

[0071] This comparative example provides a silk fibroin / carboxymethyl chitosan hydrogel without polylysine grafting modification. The setup is the same as in Example 1, except that unmodified silk fibroin is used instead of the polylysine-grafted silk fibroin derivative in the precursor solution; that is, the precursor solution is prepared by mixing unmodified silk fibroin and carboxymethyl chitosan at a mass ratio of 4:6.

[0072] The compressive strength of the hydrogel in this comparative example was 30 ± 5 kPa, significantly lower than the 125 ± 10 kPa of Example 1. Under flat pigskin conditions, the hydrogel adhered to the pigskin surface, but when the pigskin was bent or curled, the hydrogel fell directly off. Antibacterial performance tests showed that the hydrogel... E. coli , S. aureus The antibacterial rates of both polylysine and MRSA were less than 50%, confirming that grafting polylysine is the key to endowing the hydrogel with antibacterial activity.

[0073] Comparative Example 3

[0074] This comparative example provides a silk fibroin-based hydrogel prepared using the traditional crosslinking agent glutaraldehyde.

[0075] Polylysine-grafted silk fibroin derivatives and carboxymethyl chitosan were mixed at a mass ratio of 4:6 and dissolved in PBS to prepare a 6% w / v solution. Glutaraldehyde (final concentration 0.25% v / v) was added to the above solution, mixed thoroughly, and then allowed to stand at room temperature for crosslinking to obtain a hydrogel.

[0076] The compressive strength of this comparative hydrogel was 100 ± 10 kPa, lower than that of Example 1 (125 ± 10 kPa). Cytotoxicity testing showed that the cell viability of this hydrogel was less than 75%, significantly lower than the 93% of Example 1, confirming that residual glutaraldehyde has a certain degree of cytotoxicity. Furthermore, this comparative hydrogel lacks self-healing properties, and the gelation time is difficult to precisely control.

[0077] The environmentally friendly silk fibroin-based antibacterial hydrogel and its preparation method provided in this application can be used to prepare medical dressings for treating chronic, difficult-to-heal wounds such as infected skin trauma, diabetic foot ulcers, and pressure ulcers. This hydrogel is biocompatible throughout, has a simple preparation process, and controllable performance, demonstrating significant clinical application value and promising prospects for industrial production.

[0078] The above-described embodiments merely illustrate several feasible implementations of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention, nor are the embodiments intended to limit the scope of protection in the claims of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention. All equivalent implementations or changes that do not depart from the present invention should be included in the technology of the present invention.

Claims

1. A method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel, characterized in that: Using natural oxidized polysaccharide as a dynamic crosslinking agent, a hydrogel was obtained by reacting polylysine-grafted silk fibroin derivative with carboxymethyl chitosan and forming it in situ under pH conditions of 7.0–7.

4.

2. The method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel according to claim 1, characterized in that: The oxidation degree of the natural oxidized polysaccharide is 10-70%.

3. The preparation method of an environmentally friendly silk fibroin-based antibacterial hydrogel according to claim 1, characterized in that: The natural oxidized polysaccharide is at least one of sodium alginate, oxidized dextran, or oxidized hyaluronic acid.

4. The method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel according to claim 1, characterized in that: A precursor solution is formed by mixing polylysine-grafted fibroin derivatives with carboxymethyl chitosan, wherein the mass ratio of polylysine-grafted fibroin derivatives to carboxymethyl chitosan in the precursor solution is 1:9 to 6:

4. The volume ratio of the precursor solution to the natural oxidized polysaccharide solution is 1:4 to 4:

1.

5. The method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel according to claim 4, characterized in that: The concentration of the natural oxidized polysaccharide solution is 1% to 15% w / v.

6. The method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel according to claim 1, characterized in that: In the presence of a condensing agent, ε-polylysine is added to a silk fibroin solution, and grafting modification is carried out through an amidation reaction to obtain a polylysine-grafted silk fibroin derivative.

7. The method for preparing an environmentally friendly silk fibroin-based antibacterial hydrogel according to claim 6, characterized in that: The mass ratio of ε-polylysine to silk fibroin is 1:4 to 40, the molecular weight of ε-polylysine is 1 to 10 kDa, and the molecular weight of silk fibroin is 50 to 200 kDa.

8. An environmentally friendly silk fibroin-based antibacterial hydrogel prepared by the method according to any one of claims 1 to 7, characterized in that: The hydrogel has a three-dimensional interpenetrating network structure with Schiff base crosslinking.

9. The application of the environmentally friendly silk fibroin-based antibacterial hydrogel of claim 8 in medical dressings.

10. The application according to claim 9, characterized in that: The medical dressing is used for angiogenesis and wound healing.

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