An antibacterial silk fibroin gel dressing and a preparation method thereof

Abstract

The application discloses an antibacterial silk fibroin gel dressing and a preparation method thereof, and belongs to the technical field of medical dressings. In order to solve the technical problems that the antibacterial components in the existing antibacterial silk fibroin gel dressing are prone to burst release, leading to initial cytotoxicity and mid-late invalidation, the covalent grafting density is low and the spatial accessibility is poor, and the chemical modification may interfere with the gel-forming performance of the gel, an in-situ functionalization strategy of chemical bonding is adopted, antibacterial active groups are directly introduced into silk fibroin macromolecular side chains through a covalent bond, and then the gel is formed by taking the groups as units, so that each silk fibroin molecular chain constituting the gel network itself becomes a carrier unit of the bactericidal function. The prepared antibacterial silk fibroin gel dressing is not only mild and controllable in the preparation process, and does not need to introduce toxic cross-linking agents or organic solvents, but also is stable and durable in antibacterial activity, high in biological safety, and can provide a novel functional dressing with practicability and advancement for clinical wound infection prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the field of medical dressing technology, specifically relating to an antibacterial silk fibroin gel dressing and its preparation method. Background Technology

[0002] Silk fibroin, a natural structural protein derived from silkworm cocoons, possesses excellent biocompatibility, controllable biodegradation rate, good air and moisture permeability, and biological activity that promotes cell adhesion and proliferation. It has been widely used in wound dressings, tissue engineering scaffolds, and drug delivery systems. Silk fibroin hydrogels provide an ideal moist microenvironment for wound healing, absorbing wound exudate. Its soft and elastic mechanical properties allow for good compatibility with human soft tissues, demonstrating significant advantages in burn, chronic ulcer, and postoperative wound care. However, silk fibroin itself lacks inherent antibacterial activity. Furthermore, the gel dressing is rich in moisture and protein nutrients, making it highly susceptible to becoming a breeding ground for common wound-infecting bacteria such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa during clinical use and storage. Once bacteria proliferate at the dressing-wound interface and form a biofilm, it not only severely delays wound healing, leading to localized purulent inflammation and even systemic sepsis, but also causes additional pain and financial burden for patients due to frequent dressing changes. Therefore, endowing silk fibroin gel dressings with efficient, long-lasting, and safe antibacterial functions has become a core research direction for the field to move from laboratory research to clinical translation.

[0003] Currently, the mainstream technical approach to endowing silk fibroin dressings with antibacterial function is the physical loading method. This involves dispersing active ingredients such as silver ions, nano-zinc oxide, antibiotics, or natural antimicrobial peptides into a silk fibroin gel matrix through impregnation adsorption, in-situ reduction deposition, or simple blending followed by gelation. While this physical embedding method can inhibit bacterial proliferation to some extent in the early stages of dressing application, its inherent technical shortcomings are significant. First, there is the problem of uncontrollable burst release of antibacterial components. Since most antibacterial active substances only have weak hydrogen bonds, hydrophobic stacking, or physical encapsulation with the silk fibroin gel network, upon contact with the protein- and electrolyte-rich exudate of the wound, a large amount of antibacterial agent will burst out in a short period of time, forming a local pulse concentration far exceeding the minimum inhibitory concentration requirement. Literature reports that the cumulative release of silver ions in silver-loaded silk fibroin gel dressings can reach more than 70% of the total amount within 24 hours, and the burst release characteristics of antibiotic-containing silk fibroin gels are even more pronounced. This early burst release not only causes the dressing to lose its antibacterial protective ability during the critical healing period of the following days, but also pushes the local antibacterial agent concentration to cytotoxic levels in the initial stage, potentially inhibiting the activity of keratinocytes and fibroblasts, negatively impacting the growth of new tissue. After the burst release, the antibacterial agent reserves within the dressing itself are almost depleted, and the remaining small amount of active substances is insufficient to continue to cope with the ongoing bacterial challenges. The dressing almost completely loses its antibacterial defense in the mid-to-late stages, requiring frequent dressing changes to compensate, which directly contradicts the clinical principle that ideal dressings should minimize changes that disrupt wound homeostasis. Furthermore, inorganic nanoparticles such as nanosilver also suffer from problems such as aggregation, uneven distribution, and oxidative discoloration in the gel matrix, while antibiotics face an increasingly serious threat of bacterial resistance.

[0004] To slow down the release rate of active substances, researchers have further developed a carrier pre-loading strategy. This involves first loading the antibacterial agent into micro / nanocarriers such as liposomes, polymer microspheres, and mesoporous silica, and then dispersing the drug-loaded carrier within a silk fibroin gel matrix. This method extends the duration of effective antibacterial concentration to some extent; however, the introduction of carriers complicates the preparation process, significantly increases costs, and causes large batch-to-batch fluctuations in drug loading and encapsulation efficiency. More importantly, this method is essentially still a passive release method; the antibacterial activity will eventually be depleted as drug reserves dwindle, failing to achieve long-lasting antibacterial protection that matches the dressing's lifespan.

[0005] The closest existing technology to the concept of this invention is the chemical coupling method that has emerged in recent years, utilizing the residual active side groups on silk fibroin molecules. This type of method typically uses lysine ε-amino groups or tyrosine phenolic hydroxyl groups on the silk fibroin side chains as reaction anchors, and uses carbodiimide chemistry or epoxy crosslinking agents to covalently tether natural antimicrobial peptides or small quaternary ammonium salts such as lysozyme and lactoferrin to the silk fibroin molecules, and then prepares the modified silk fibroin into a gel. Because the antimicrobial groups are covalently linked to the gel network backbone, the loss of active ingredients can be significantly inhibited under conditions of wound exudate immersion and washing, exhibiting a more durable antimicrobial effect than physical blending. However, these existing chemical coupling methods still have substantial technical limitations. First, the number of naturally available reactive groups on silk fibroin molecules is extremely limited. After degumming and regeneration, the lysine content is less than 0.5%, and the reactivity of the tyrosine phenolic hydroxyl groups is moderate, requiring relatively intense activation conditions. The achievable grafting density of antimicrobial groups is far from sufficient to form a dense and effective molecular-level bactericidal interface. Second, directly coupled small-molecule antibacterial agents adhere tightly to the silk fibroin backbone, lacking the buffering effect of flexible spacer arms. Their bactericidal groups are spatially constrained by the folding and stacking of the protein macromolecules, making it difficult for them to extend flexibly and effectively contact the bacterial cell membrane, resulting in insufficient intrinsic antibacterial activity. Third, such coupling reactions often partially consume the already limited active sites on silk fibroin, potentially causing unpredictable disturbances to its subsequent β-sheet assembly and gelation behavior, affecting the mechanical integrity and microstructure of the gel.

[0006] In summary, existing technologies for endowing silk fibroin gel dressings with antibacterial function face the following pressing technical challenges: physical loading methods suffer from initial cytotoxicity risks and mid-to-late-stage antibacterial failure due to uncontrolled burst release of antibacterial components; carrier preloading strategies are complex and still suffer from eventual depletion of active substances; and existing chemical coupling schemes are limited by insufficient available reaction sites in silk fibroin itself, low grafting density, poor spatial accessibility of antibacterial groups, and potential interference with gelation behavior. Therefore, there is an urgent need in this field to develop a novel antibacterial silk fibroin gel dressing and its preparation method that can achieve high-density, highly spatially accessible covalent bonding of antibacterial groups on the three-dimensional gel network without sacrificing the gelling properties and biocompatibility of silk fibroin, thereby obtaining a durable, efficient, and safe antibacterial effect and meeting the urgent clinical need for long-lasting antibacterial protection in dressings for wound care. Summary of the Invention

[0007] To address the technical challenges of existing antibacterial silk fibroin gel dressings, such as the easy burst release of antibacterial components leading to initial cytotoxicity and mid-to-late-stage failure, low covalent grafting density and poor spatial accessibility, and the potential interference of chemical modifications with gelation properties, this invention employs a chemically bonded in-situ functionalization strategy. This strategy directly introduces antibacterial active groups into the side chains of silk fibroin macromolecules via covalent bonds, and then uses these as building blocks for gelation. The aim is to achieve the long-term, stable integration of broad-spectrum, highly efficient, and safe antibacterial activity into the three-dimensional network of the gel in a non-migrating manner.

[0008] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a method for preparing an antibacterial silk fibroin gel dressing, the method comprising the following steps: S1. Preparation of regenerated silk fibroin aqueous solution: Silkworm cocoons are boiled in an alkaline aqueous solution to degummify them. After degumming, they are washed with water until neutral and then dried to obtain degummed silk. The degummed silk is dissolved in a lithium salt aqueous solution to obtain a crude silk fibroin solution. The crude silk fibroin solution is filtered, dialyzed, centrifuged, and then filtered again to obtain a regenerated silk fibroin aqueous solution. S2. Preparation of antibacterial silk fibroin solution: The regenerated silk fibroin aqueous solution obtained in S1 is diluted with a buffer solution to a mass fraction of 0.5-2.0%. Under inert gas protection at 0-10℃, an acrylate-N-succinimide ester solution prepared by dimethyl sulfoxide is added, and the reaction is carried out for 10-14 h to obtain an activated silk fibroin intermediate. An amphoteric monomer and an initiator are added to the silk fibroin intermediate, and the reaction is stirred for 20-30 h under inert gas protection and at room temperature to obtain a reaction product. The reaction product is filtered, dialyzed, and concentrated to obtain an antibacterial silk fibroin solution with a mass fraction of 5-25%. The amphoteric monomer is 2-(methacryloyloxy)ethyl-2-(trimethylamino)ethyl phosphate, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide, or 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate. S3. Physical cross-linking and gelation: Mix ethanol with the antibacterial silk fibroin solution prepared in S2 at a volume ratio of 1:30 to 1:50, let stand at room temperature for 20 to 30 hours to gel, and then immerse the gel in deionized water for 20 to 30 hours, changing the soaking solution 1 to 5 times during the process to obtain antibacterial silk fibroin hydrogel dressing.

[0009] In one embodiment of the present invention, the alkaline aqueous solution in S1 is a sodium carbonate aqueous solution with a mass fraction of 0.1-2%.

[0010] In one embodiment of the present invention, the boiling time in S1 is 20 to 40 minutes, and the degumming step is repeated 1 to 3 times.

[0011] In one embodiment of the present invention, the lithium salt in S1 is selected from at least one of lithium bromide, lithium chloride, and lithium iodide, and the concentration of the lithium salt is 8~10 mol / L.

[0012] In one embodiment of the present invention, the dissolution temperature in S1 is 30~50°C and the dissolution time is 30~60 min.

[0013] Further specifying the steps of S1, the following steps are taken: Cleaned silkworm cocoons are immersed in a 0.5% sodium carbonate aqueous solution, heated to boiling and maintained for 30 minutes to achieve degumming; after removal, they are repeatedly washed three times with warm water at approximately 40°C to remove the peeled sericin and residual alkali; to ensure complete degumming, the fibers are immersed again in a freshly prepared 0.5% sodium carbonate aqueous solution, boiled for 30 minutes, then washed three times with warm water at 40°C, followed by two washes with deionized water, and dried in a 40°C oven for 24 hours to obtain clean, dry degummed silk; the degummed silk is dissolved in a 9.3 mol / L lithium bromide aqueous solution and heated in a 40°C water bath for 40 minutes. The process involves centrifuging the silk fibroin (SF) into individual molecular chains, allowing it to dissociate gradually and dissolve in the aqueous phase. After dissolution, the solution is filtered through eight layers of medical gauze to remove small amounts of insoluble impurities and incompletely dissolved fiber fragments. The solution is then placed in a dialysis bag with a molecular weight cutoff of 12-14 kDa and dialyzed in a large volume of deionized water for three days, with the external solution being replaced periodically to thoroughly remove lithium bromide salts and small molecule degradation products. The dialyzed silk fibroin solution is centrifuged at 8000 r / min for 10 min and filtered again through eight layers of gauze to obtain a clear and transparent regenerated silk fibroin solution with a concentration of approximately 4 wt%.

[0014] In one embodiment of the present invention, the amount of N-succinimide acrylate added in S2 is 3 to 5% of the amount of regenerated silk fibroin protein.

[0015] In one embodiment of the present invention, the amount of zwitterionic monomer added in S2 is 15-25% of the amount of regenerated silk fibroin protein.

[0016] In one embodiment of the present invention, the initiator in S2 is potassium persulfate, and the amount added is 0.3 to 0.4% of the regenerated silk fibroin protein.

[0017] In one embodiment of the present invention, the filtration in step S2 is performed using 6-10 layers of medical gauze; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 12-14 kDa, continuously dialyzing in deionized water for 48-72 hours, with the deionized water being replaced every 8-12 hours; the concentration is performed using a dialysis bag with a molecular weight cutoff of 12-14 kDa, and reverse dialysis concentration is carried out in a 15% polyethylene glycol solution.

[0018] Further specifying the steps of S2, the following steps are taken: The regenerated silk fibroin solution obtained in S1 is diluted to a mass fraction of 1% using a phosphate buffer solution. 50 mL of the diluted silk fibroin solution is taken, and the system temperature is lowered to approximately 4°C using an ice-salt bath. Under an inert atmosphere of 4°C and nitrogen protection, 1.0 mL of a pre-prepared N-succinimide acrylate solution (20 mg / mL) in dimethyl sulfoxide (DMSO) is added dropwise to the silk fibroin solution, and the reaction is continued for 12 h. 0.1 g of 2-(methacryloyloxy)ethyl-2-(trimethylamino)ethyl phosphate and 2 mg of potassium persulfate as the initiator are added, and the reaction is continued with stirring at room temperature for 24 h under a nitrogen atmosphere. After the reaction is complete, the product solution is filtered through 8 layers of medical gauze to remove any small amounts of large particle aggregates that may be generated, and then loaded into a container with a molecular weight cutoff of 12-14. In a kDa dialysis bag, the solution was dialyzed in deionized water for 3 days to thoroughly remove unreacted monomers, ungrafted homopolymers, residual initiator salts, and N-hydroxysuccinimide byproducts released in the first step of the reaction. After dialysis, the antibacterial silk fibroin solution was again dialyzed with 15% polyethylene glycol (PEG). M n The 20000) concentrated solution was concentrated to approximately 20 wt% by reverse dialysis and stored at 4°C.

[0019] Further specifying, the specific steps of S3 are as follows: Take 0.5 mL of anhydrous ethanol and slowly and evenly mix it into 20 mL of the antibacterial silk protein solution prepared in S2 at room temperature (it can be gently stirred to disperse it evenly), and then let it stand for 24 hours to gel; after the gel is formed, immerse it in a large amount of deionized water for 24 hours, changing the deionized water several times during the process, to obtain the antibacterial silk protein hydrogel dressing.

[0020] A second objective of this invention is to provide an antibacterial silk fibroin gel dressing obtained by the above preparation method.

[0021] The beneficial effects of this invention are: This invention abandons the conventional approach of physical blending and passive soaking loading, instead employing an in-situ functionalization strategy based on chemical bonding. Antibacterial active groups are directly introduced into the side chains of silk fibroin macromolecules via covalent bonds, and then gelled using these as building blocks. Each silk fibroin molecular chain constituting the gel network becomes a carrier unit for bactericidal function. The antibacterial functional groups are no longer free guest molecules suspended in the gel pores awaiting dissolution, but are integrated with the gel matrix through stable covalent connections such as carbon-carbon single bonds or amide bonds. In the hydrated and swollen state, the antibacterial side groups bonded to the silk fibroin backbone are fully exposed at the gel-wound interface and the surface of the internal pores as the molecular chains expand, killing invading bacteria through a contact mechanism without diffusion or loss due to exudate soaking or repeated rinsing. Therefore, the antibacterial efficacy of the dressing decays synchronously with its own degradation cycle, rather than being rapidly lost within hours, fundamentally solving the dilemma of "initial burst release, later failure" in existing physically loaded silk fibroin gel dressings.

[0022] This invention completely eliminates the problem of burst release and loss of antibacterial active ingredients. By pre-grafting high-density, long-segment zwitterionic polymers or quaternary ammonium salt polymers onto the silk fibroin molecular chain via covalent bonds, the antibacterial polymer chains are anchored to the silk fibroin backbone by carbon-carbon single bonds, and their antibacterial lifespan is completely synchronized with the degradation cycle of the gel bulk. Simultaneously, this invention breaks the dilemma in traditional antibacterial strategies where "high-concentration bactericidal effect" and "low cytotoxicity" are mutually exclusive. The selected zwitterionic polymer, because its repeating unit contains both a positively charged group and a negatively charged group, possesses a unique electrostatically induced highly hydrated layer structure. Its repulsive force against protein, microorganism, and bacterial surfaces is far stronger than that of polyethylene glycol-dependent hydrogen bond-induced hydrated layers, exhibiting excellent anti-protein non-specific adsorption and antibacterial adhesion properties even under complex physiological conditions such as serum or blood. When zwitterionic polymers are grafted onto a silk fibroin gel network in the form of a high-density polymer brush, a stable hydration barrier formed at the gel-wound interface can effectively repel bacteria in the first step of their attempt to contact and colonize, blocking the formation of biofilms at the source without relying on the release of any bactericidal substances, thus exhibiting zero toxicity to normal tissue cells.

[0023] Furthermore, all functional modifications in this invention are performed without substantially impairing the inherent superior properties of silk fibroin. Through a carefully designed reaction route, the grafting process of the antibacterial polymer is completed in a mild aqueous or mixed solvent system. The selection of grafting sites and control of reaction conditions prioritize preserving the integrity of the silk fibroin backbone and its β-sheet gelation ability. The resulting antibacterial silk fibroin gel dressing not only fully retains its natural biological activities of promoting cell proliferation, mimicking the extracellular environment, and inhibiting scar formation, but also possesses the intrinsic dual-function synergistic protection against biocontamination and antibacterial activity, independent of drug release and without inducing bacterial resistance. The preparation process is mild and controllable, requiring no introduction of toxic cross-linking agents or organic solvents. It exhibits stable and long-lasting antibacterial activity and high biosafety, providing an ideal dressing solution for the prevention and treatment of clinical wound infections, combining safety, durability, broad-spectrum antibacterial properties with active healing promotion. Attached Figure Description

[0024] Figure 1 The chemical structural formulas of NAS, MPC, SBMA, and CBMA are shown. Figure 2 The chemical reaction equation for silk fibroin RSF and N-succinimide acrylate NAS; Figure 3 The chemical reaction equation for the graft copolymerization reaction of MPC on silk fibroin; Figure 4 Infrared spectrum of MPC antibacterial functionalized silk fibroin; Figure 5 The chemical reaction equation for the graft copolymerization of SBMA onto silk fibroin; Figure 6 Infrared spectrum of SBMA-functionalized silk fibroin; Figure 7 The chemical reaction equation for the graft copolymerization of CBMA onto silk fibroin; Figure 8 Infrared spectrum of CBMA antibacterial functionalized silk fibroin; Figure 9 The infrared spectrum of pure silk fibroin; Figure 10 The image shows the cytotoxicity evaluation results of the silk fibroin gel dressing. Figure 11 This is a drug release curve for silk fibroin gel dressing. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. In the art, any embodiments obtained by those skilled in the art without creative effort are protected by this invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0027] The materials and pharmaceutical information used in this invention are as follows: Silkworm cocoons: purchased from Nantong City, Jiangsu Province; Sodium carbonate, specification: GC, ≥99.0%, purchased from Sinopharm Chemical Reagent Co., Ltd. Lithium bromide, specification: ACS, ≥99.0%, purchased from Aladdin Reagent Co., Ltd. Polyethylene glycol, specification: GC, ≥99.0%, purchased from Sinopharm Chemical Reagent Co., Ltd.; Dimethyl sulfoxide (DMSO), specification: GC, ≥99.0%, purchased from Sinopharm Chemical Reagent Co., Ltd. N-Succinimidyl acrylate (NAS), specification: GC, ≥98.0%, purchased from Aladdin Reagent Co., Ltd. 2-(methacryloyloxy)ethyl-2-(trimethylamino)ethyl phosphate (MPC), specification: ACS, ≥98.0%, purchased from Aladdin Reagent Co., Ltd. [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide (SBMA), specification: ACS, ≥98.0%, purchased from Maclean's Reagent Co., Ltd. 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), specification: HPLC, ≥98.0%, purchased from Aladdin Reagent Co., Ltd.; Potassium persulfate (KPS), specification: ACS, ≥99.0%, purchased from Aladdin Reagent Co., Ltd. Anhydrous ethanol, specification: AR, ≥99.7%, purchased from Sinopharm Chemical Reagent Co., Ltd. The chemical structural formulas of NAS, MPC, SBMA, and CBMA are as follows: Figure 1 As shown.

[0028] Example 1: This embodiment provides an antibacterial silk fibroin gel dressing, the preparation of which revolves around a mild and precise two-step chemical bonding strategy. The overall technical route is as follows: A regenerated silk fibroin aqueous solution is extracted and purified from natural silkworm cocoons; then, N-succinimide acrylate (NAS) is used to site-activated the active amino groups on the side chains of the silk fibroin, covalently introducing acryloyl double bonds that can further participate in free radical polymerization onto the protein macromolecule; based on this, 2-(methacryloyloxy)ethyl-2-(trimethylamino)ethyl phosphate (MPC), a methacrylate monomer containing zwitterionic phosphocholine groups, is added to the system, and under the action of an initiator, it undergoes a free radical graft copolymerization reaction with the double bonds already anchored on the protein molecules, forming a polyzwitterionic polymer brush firmly bound by covalent bonds on each silk fibroin molecular chain; finally, the functionalized silk fibroin solution undergoes a sol-gel transition through ethanol-induced physical cross-linking, and residual ethanol is replaced by thorough soaking in deionized water, obtaining an intrinsic silk fibroin gel dressing with both durable anti-biofouling and contact antibacterial functions.

[0029] The specific preparation method of the antibacterial silk fibroin gel dressing provided in this embodiment is as follows: 1. Preparation of regenerated silk fibroin aqueous solution First, the silkworm cocoons undergo thorough degumming to remove sericin and waxy impurities. Specifically, 30 g of cleaned silkworm cocoons are weighed and immersed in a 0.5% sodium carbonate aqueous solution, heated to boiling, and maintained for 30 minutes. The alkaline environment of sodium carbonate allows the sericin surrounding the silk fibers to fully swell and hydrolyze, thus achieving degumming. After removal, the cocoons are repeatedly washed three times with warm water at approximately 40°C to remove the detached sericin and residual alkali. To ensure complete degumming, the fibers are immersed again in a freshly prepared 0.5% sodium carbonate aqueous solution, boiled for 30 minutes, and then washed three times with warm water at 40°C, followed by two washes with deionized water. Finally, the fibers are dried in a 40°C oven for 24 hours to obtain clean, dry, degummed silk.

[0030] Subsequently, degummed silk was dissolved in a 9.3 mol / L lithium bromide aqueous solution and heated in a 40°C water bath for 40 min. This process disrupted the hydrogen bonds between silk fibroin molecules through the strong hydration of the high concentration of lithium and bromide ions, causing the silk fibroin (SF) to gradually dissociate into individual molecular chains and dissolve in the aqueous phase. After dissolution, the solution was filtered through eight layers of medical gauze to remove small amounts of insoluble impurities and incompletely dissolved fiber fragments. It was then placed in a dialysis bag with a molecular weight cutoff of 12–14 kDa and dialyzed in a large volume of deionized water for 3 days, with the external solution replaced periodically to thoroughly remove lithium bromide salts and small molecule degradation products. The dialyzed silk fibroin solution was centrifuged at 8000 r / min for 10 min and filtered again through eight layers of gauze to obtain a clear, transparent, approximately 4 wt% regenerated silk fibroin dilute solution.

[0031] 2. Synthesis of antibacterial functionalized silk fibroin The core of this stage of chemistry lies in the stepwise implementation of two continuous molecular bonding processes: active ester-amino condensation activation and free radical graft copolymerization.

[0032] The first step is the activation of silk fibroin, which involves site-directed acylation modification of the active primary amino groups on the side chains of silk fibroin using NAS, introducing polymerizable acrylate double bonds into the silk fibroin molecule. Specifically, the RSF solution obtained in step 1 was diluted to a mass fraction of 1 wt% with phosphate-buffered saline (PBS). 50 mL of the diluted silk fibroin solution was placed in a reaction vessel, and the system temperature was lowered to approximately 4°C using an ice-salt bath. NAS is the key reagent in this reaction step. Its molecular structure consists of an acryloyl group and an N-succinimide ester leaving group linked by a carbonyl group. The acryloyl group has a polymerizable carbon-carbon double bond CH2=CH- at its end, while the N-succinimide ester moiety is a highly activated ester group capable of selective condensation with primary amines under mild conditions. Under an inert atmosphere protected by nitrogen at 4°C, 1.0 mL of a pre-prepared NAS solution with a concentration of 20 mg / mL in dimethyl sulfoxide (DMSO) was added dropwise to the silk fibroin solution, and the reaction was continued for 12 h. In this low-temperature microenvironment, the ε-primary amino group derived from the lysine residue side chain in the silk fibroin molecule acts as a nucleophile, selectively attacking the activated carbonyl carbon of the NAS ester, undergoing a nucleophilic substitution reaction. The N-succinimide group, as an excellent leaving group, is removed, generating an acrylamide group covalently linked by a stable amide bond. The chemical equation for this activation reaction is as follows: Figure 2 As shown. N-hydroxysuccinimide is a byproduct of the reaction and can be completely removed in the subsequent dialysis step. Through this reaction, an acrylamide group with an active carbon-carbon double bond at the end was successfully covalently introduced into the side chain of the silk fibroin macromolecule, yielding the activated silk fibroin intermediate (RSF-NAS).

[0033] The second step is the free radical graft copolymerization of zwitterionic monomers, aiming to grow polyphosphocholine brushes with excellent anti-biofouling properties onto silk fibroin molecular chains via chemical bonds. The selected functional monomer is MPC, whose molecular structure contains three functional regions: one end is a methacrylate group, providing a CH2=C(CH3)-COO- double bond that can participate in free radical polymerization; the middle is an ethyl linker arm -O-CH2CH2- providing appropriate flexibility and spatial spacing; and the other end is a zwitterionic head group of phosphocholine -O-PO2. - -O-CH2CH2-N + The (CH3)3 head group contains both a phosphate anion and a quaternary ammonium cation, forming a typical zwitterionic structure. The charge balance within the repeating MPC unit gives it a strong electrostatically induced hydration capacity, enabling the formation of a dense and stable hydration barrier on the material surface. During the operation, 0.1 g of MPC and 2 mg of potassium persulfate (KPS) initiator were directly added to the same reaction system after the acylation reaction. The reaction was carried out under nitrogen atmosphere with continuous stirring at room temperature for 24 h to ensure sufficient monomer conversion and a considerable grafting density. Potassium persulfate undergoes gradual homolytic cleavage in the aqueous system, generating sulfate anion radicals SO42-. - These primary free radicals first attack the CH2=CH- double bond of the acrylamide group on the side chain of the activated silk fibroin molecule, opening the π electron cloud of the double bond and generating a large silk fibroin free radical with a carbon atom as the active center. Simultaneously, the CH2=C(CH3)- methacrylate double bond of the free phosphocholine monomer in solution is also attacked by primary free radicals or the already generated large protein free radicals, triggering chain initiation and chain growth. The growing polyphosphocholine chain terminates through chain transfer reactions to the double bonds on the silk fibroin surface or through direct coupling with the large protein free radical, ultimately covalently tethered to the lysine residue side chain end of the silk fibroin backbone in the form of carbon-carbon single bonds. The overall chemical equation for the MPC graft copolymerization reaction is as follows: Figure 3 As shown in the diagram, in the structural formula of the reaction product, the repeating unit represents a poly(2-(methacryloyloxy)ethyl-2-(trimethylamino)ethyl phosphate) graft chain, whose main chain is a typical vinyl polymer carbon-carbon backbone. Each repeating unit has a complete phosphocholine zwitterion head group suspended at the end of its side chain. The grafting point is located on the ε-amino nitrogen atom of the lysine residue side chain of silk fibroin, and is connected to the first carbon atom of the polymer main chain via an acrylamide bridge bond -NH-CO-CH2-CH-.

[0034] After the reaction was complete, the product solution was filtered through eight layers of medical gauze to remove any small amounts of large aggregates that might have formed. It was then placed in a dialysis bag with a molecular weight cutoff of 12-14 kDa and dialyzed in deionized water for 3 days to thoroughly remove unreacted monomers, ungrafted homopolymers, residual initiator salts, and the N-hydroxysuccinimide byproduct released in the first step of the reaction. After dialysis, the antibacterial silk fibroin solution was again filtered with 15% polyethylene glycol (PEG). M n A 20000 g / L solution was concentrated by reverse dialysis to approximately 20 wt%. The concentration was accurately determined by weighing to obtain a concentrated antibacterial silk fibroin solution suitable for direct gel preparation. This solution was then stored at 4°C. The infrared spectrum of MPC antibacterial functionalized silk fibroin is shown below. Figure 4 As shown, the infrared spectrum of MPC antibacterial functionalized silk fibroin shows a characteristic absorption peak that can be attributed to zwitterionic phosphate, indicating that the functional monomer MPC was successfully grafted onto the silk fibroin molecular chain.

[0035] 3. Physical cross-linking and gelation Take 0.5 mL of anhydrous ethanol and slowly and evenly mix it into 20 mL of the antibacterial silk fibroin solution prepared in step 2 at room temperature (slight stirring can be used to ensure uniform dispersion). Then allow it to stand for 24 hours to gel. During this process, ethanol, as a mild physical cross-linking promoter and conformation inducing agent, weakens the hydrogen bonding between water molecules and silk fibroin chains, reduces the hydration degree of the protein chains, and partially disrupts the hydration layer of silk fibroin molecules, thereby reducing its solubility and conformational freedom in the aqueous phase. This promotes the slow transformation of silk fibroin macromolecules from a random coil conformation to an antiparallel β-sheet conformation. The intermolecular hydrogen bond network formed between chain segments acts as physical cross-linking points, and the β-sheet layers gradually increase and interconnect, ultimately forming a three-dimensional gel network throughout the entire system. The entire gelation process relies entirely on the physical conformational transformation and intermolecular physical cross-linking of silk fibroin, without introducing any chemical cross-linking agents, fundamentally ensuring the dressing's excellent biocompatibility and tissue safety. After gel formation, the gel is immersed in a large amount of deionized water for 24 hours, with the deionized water being changed multiple times during this period. The concentration gradient is used to fully displace residual ethanol from the gel network, allowing the gel to reach hydration equilibrium. This results in a swellable, pure antibacterial silk fibroin hydrogel dressing. In this dressing, each silk fibroin molecular chain forming the three-dimensional network framework is covalently grafted with a high-density polyphosphocholine zwitterionic polymer brush (phosphobetaine type). When the gel, in its hydrated state, comes into contact with the wound, these flexible zwitterionic side chains fully extend to the gel-wound interface and the inner surface of the pores, inducing a highly stable, dense hydrated layer through electrostatic attraction. This prevents non-specific protein adsorption and bacterial / microbial adhesion and colonization from the source, thus achieving intrinsic anti-biocontamination and antibacterial dual-function synergistic protection that is independent of drug release and does not induce bacterial resistance.

[0036] Example 2: This embodiment provides an antibacterial silk fibroin gel dressing. The difference between the preparation method of this antibacterial silk fibroin gel dressing and that of Example 1 is that the zwitterionic monomer used in the graft copolymerization stage is replaced with [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide (SBMA). This monomer belongs to the sulfonate betaine type zwitterionic compound. One end of its molecule is a methacrylate double bond that can participate in free radical polymerization. The middle is connected to a tertiary amine cation via an ethyl linker arm. Two methyl groups and one 3-carbon sulfonyl propyl chain are attached to the quaternary ammonium nitrogen atom. The end is a sulfonate group with a permanent negative charge. From the perspective of charge distribution and hydration chemistry, although both the sulfonate betaine-type zwitterion and the phosphate choline-type zwitterion in the main scheme are isoelectrically neutral inner salt structures, the negative charge density and geometry of the sulfonate anion differ from those of the phosphate ester anion. The sulfonate anion is trigonal pyramidal, and the negative charge distribution of its three sulfur-oxygen bonds is highly symmetrical, enabling it to bind a large number of water molecules through strong electrostatic interactions, constructing an extremely dense and stable hydration barrier layer on the polymer brush surface. Furthermore, sulfonate betaine exhibits excellent chemical stability over a wide pH range and at high temperatures, without undergoing the acid-catalyzed hydrolysis side reactions that may occur with phosphate ester groups. Therefore, this material demonstrates more robust performance retention in dressing applications requiring long-term storage or facing complex acid-base environments on wound surfaces.

[0037] The specific preparation method of the antibacterial silk fibroin gel dressing provided in this embodiment is as follows: 1. Preparation of regenerated silk fibroin aqueous solution Same as Example 1.

[0038] 2. Synthesis of antibacterial functionalized silk fibroin The RSF solution obtained in step 1 was diluted to 1 wt% with phosphate-buffered saline (PBS). 50 mL of the diluted silk fibroin solution was placed in a reaction vessel, and the system temperature was lowered to approximately 4°C using an ice-salt bath. Under an inert atmosphere protected by nitrogen at 4°C, 1.0 mL of NAS solution pre-prepared with DMSO at a concentration of 20 mg / mL was added dropwise to the silk fibroin solution, and the reaction was continued for 12 h.

[0039] In the subsequent graft copolymerization functionalization stage, 0.1 g of SBMA was directly added to the reaction solution after the activation reaction, followed by 2 mg of potassium persulfate as a free radical initiator. The reaction was carried out under nitrogen atmosphere and stirred continuously at room temperature for 24 h. Potassium persulfate gradually homolytically cleaved in the aqueous phase to generate sulfate anion radicals SO4. -The primary free radical simultaneously attacks the acrylamide double bond (CH2=CH-) on the activated silk fibroin side chain and the methacrylate double bond (CH2=C(CH3)-) at the end of the free monomer, activating both the protein macromolecule and the monomer to form corresponding free radical active centers. The silk fibroin macromolecule free radical then initiates chain growth reactions on the surrounding sulfonate betaine monomers. Simultaneously, the homopolymer growth chains in solution can also achieve chemical grafting through chain transfer to the double bonds on the protein surface or coupling termination. The chemical equation for the entire graft copolymerization reaction is as follows: Figure 5 As shown in the structural formula, in this product, the ε-amino group of the lysine residue side chain of the silk fibroin backbone is connected to the first carbon atom of the polymer backbone via an acrylamide bridge bond -NH-CO-CH2-CH-, and the end of the repeating unit side chain is suspended with a zwitterionic head group -N-sulfobetaine. + (CH3)2-CH2CH2CH2-SO3 - The head base forms a dense hydration protective layer in the physiological environment through strong electrostatic hydration.

[0040] After the reaction was complete, the product solution was filtered through eight layers of medical gauze to remove any small amounts of large aggregates that might have formed. It was then placed in a dialysis bag with a molecular weight cutoff of 12-14 kDa and dialyzed in deionized water for 3 days to thoroughly remove unreacted monomers, ungrafted homopolymers, residual initiator salts, and the N-hydroxysuccinimide byproduct released in the first step of the reaction. After dialysis, the antibacterial silk fibroin solution was again filtered with 15% polyethylene glycol (PEG). M n =20000) concentrated solution was concentrated by reverse dialysis to a concentration of approximately 20 wt%. The concentration was accurately determined by weighing to obtain a concentrated antibacterial silk fibroin solution that can be directly used for gel preparation. It was then stored at 4°C. The infrared spectrum of SBMA antibacterial functionalized silk fibroin is as follows: Figure 6 As shown, the infrared spectrum of SBMA-functionalized silk fibroin exhibits a distinct characteristic absorption peak attributable to zwitterionic sulfonate betaine, indicating that the functional monomer SBMA was successfully grafted onto the silk fibroin molecular chain.

[0041] 3. Physical cross-linking and gelation Take 0.5 mL of anhydrous ethanol and slowly and evenly mix it into 20 mL of the antibacterial silk fibroin solution prepared in step 2 at room temperature (you can stir it slightly to disperse it evenly). Then let it stand for 24 hours to gel. After the gel forms, immerse it in a large amount of deionized water for 24 hours, changing the deionized water several times during this period. Use the concentration gradient to fully displace the residual ethanol in the gel network, and at the same time, let the gel reach a hydration equilibrium state, finally obtaining a sulfonate betaine-type antibacterial silk fibroin gel dressing.

[0042] Example 3: This embodiment provides an antibacterial silk fibroin gel dressing. The difference between the preparation method of this antibacterial silk fibroin gel dressing and that of Example 1 is that methacrylic acid betaine is used as the functional monomer in the graft copolymerization stage. Its full name is 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA). Compared with the two zwitterionic monomers mentioned above, the molecular structure of CBMA is characterized by the transformation of its negative charge center from sulfonate or phosphate ester to carboxylate. The pKa value of the carboxylate group is usually in the range of 4 to 5. Under neutral physiological pH conditions, the carboxyl group is deprotonated -COO. - It exists in the form of the quaternary ammonium cation N within the same molecule. + (CH3)2 forms an internal salt-type zwitterionic structure, exhibiting overall electroneutrality and extremely strong hydration capacity. The carboxybetaine group in CBMA is found in natural osmoregulators such as glycine betaine, demonstrating excellent biocompatibility and extremely low immunogenicity, proven through long-term evolution. Compared to sulfonate betaine and phosphocholine, carboxybetaine has a more compact molecular size, less steric hindrance from its negatively charged groups, and can achieve higher packing density in polymer brushes, accommodating more zwitterionic pairs per unit area, thus forming a denser hydration layer at comparable grafting rates. Furthermore, the pH-responsive characteristics of carboxybetaine polymers under specific conditions provide an additional dimension for functional expansion. Although its zwitterionic properties are stable in a neutral physiological environment, this ionizable side group may offer intelligent response potential in material processing or research on the unique microenvironment of wounds.

[0043] The specific preparation method of the antibacterial silk fibroin gel dressing provided in this embodiment is as follows: 1. Preparation of regenerated silk fibroin aqueous solution Same as Example 1.

[0044] 2. Synthesis of antibacterial functionalized silk fibroin The RSF solution obtained in step 1 was diluted to 1 wt% with phosphate-buffered saline (PBS). 50 mL of the diluted silk fibroin solution was placed in a reaction vessel, and the system temperature was lowered to approximately 4°C using an ice-salt bath. Under an inert atmosphere protected by nitrogen at 4°C, 1.0 mL of NAS solution pre-prepared with DMSO at a concentration of 20 mg / mL was added dropwise to the silk fibroin solution, and the reaction was continued for 12 h.

[0045] In the subsequent graft copolymerization functionalization stage, 0.1 g of CBMA was directly added to the reaction solution after the activation reaction, followed by 2 mg of potassium persulfate as a free radical initiator. The reaction was carried out under nitrogen atmosphere with continuous stirring at room temperature for 24 h. During the graft copolymerization stage, the acrylamide double bonds on the activated silk fibroin side chains and the methacrylate double bonds on the CBMA monomers underwent free radical copolymerization initiated by potassium persulfate, generating a covalently anchored polycarboxylate betaine functional layer. The chemical equation for this graft copolymerization reaction is as follows: Figure 7 As shown. The polymer repeating unit side chain of this product has a carboxybetaine zwitterionic head group -N at its end. + (CH3)2-CH2CH2-COO - Its quaternary ammonium cations and carboxylate anions attract and strongly bind surrounding water molecules through electrostatic attraction, forming a dense and ordered interfacial water molecule layer. This hydration layer can effectively resist the non-specific adsorption of proteins, bacteria, and other biomolecules.

[0046] After the reaction was complete, the product solution was filtered through eight layers of medical gauze to remove any small amounts of large aggregates that might have formed. It was then placed in a dialysis bag with a molecular weight cutoff of 12-14 kDa and dialyzed in deionized water for 3 days to thoroughly remove unreacted monomers, ungrafted homopolymers, residual initiator salts, and the N-hydroxysuccinimide byproduct released in the first step of the reaction. After dialysis, the antibacterial silk fibroin solution was again filtered with 15% polyethylene glycol (PEG). M n A 20000 g / L solution was concentrated by reverse dialysis to approximately 20 wt%. The concentration was accurately determined by weighing to obtain a concentrated antibacterial silk fibroin solution suitable for direct gel preparation. This solution was then stored at 4°C. The infrared spectrum of CBMA-functionalized antibacterial silk fibroin is shown below. Figure 8 As shown, the infrared spectrum of CBMA-functionalized silk fibroin exhibits characteristic absorption peaks attributable to zwitterionic carboxylate betaine, indicating that the functional monomer CBMA was successfully grafted onto the silk fibroin molecular chain.

[0047] 3. Physical cross-linking and gelation Take 0.5 mL of anhydrous ethanol and slowly and evenly mix it into 20 mL of the antibacterial silk fibroin solution prepared in step 2 at room temperature (you can stir it slightly to disperse it evenly). Then let it stand for 24 hours to gel. After the gel forms, immerse it in a large amount of deionized water for 24 hours, changing the deionized water several times during this period. Use the concentration gradient to fully displace the residual ethanol in the gel network, and at the same time, let the gel reach a hydration equilibrium state, finally obtaining a carboxybenzene-type antibacterial silk fibroin gel dressing.

[0048] Comparative Example 1: 1. Preparation of regenerated silk fibroin aqueous solution Same as Example 1. The infrared spectrum of pure silk fibroin is as follows: Figure 9 As shown.

[0049] 2. Physical cross-linking gelation Take 0.5 mL of anhydrous ethanol and slowly and evenly mix it into 20 mL of the regenerated silk fibroin aqueous solution prepared in step 1 at room temperature (you can stir it slightly to disperse it evenly). Then let it stand for 24 hours to gel. After the gel is formed, immerse it in a large amount of deionized water for 24 hours, changing the deionized water several times during this period to fully displace the residual ethanol in the gel network.

[0050] Comparative Example 2: 1. Preparation of regenerated silk fibroin aqueous solution Same as Example 1.

[0051] 2. Preparation of antibacterial agent / silk fibroin mixed aqueous solution The regenerated silk fibroin aqueous solution was mixed with 15% polyethylene glycol (PEG) by mass. M n The 20000-fold concentrated solution was concentrated to approximately 20 wt% by reverse dialysis, and the concentration was accurately determined by gravimetric analysis. Then, 0.1 g of MPC was directly added to the above-mentioned regenerated silk fibroin aqueous solution, and the reaction was carried out with continuous stirring at room temperature for 24 hours under nitrogen atmosphere. This yielded an antibacterial agent / silk fibroin mixed aqueous solution that can be directly used for gel preparation and stored at 4°C.

[0052] 3. Physical cross-linking and gelation Take 0.5 mL of anhydrous ethanol and slowly and evenly mix it into 20 mL of the antibacterial agent / silk fibroin mixed aqueous solution prepared in step 2 at room temperature. Then let it stand for 24 hours to gel. After the gel forms, immerse it in a large amount of deionized water for 24 hours, changing the deionized water several times during this period to fully displace the residual ethanol in the gel network.

[0053] The protein adsorption and antibacterial properties of the silk fibroin gel dressings prepared in Examples 1-3 and Comparative Examples 1-2 were tested, and the test results are shown in Table 1.

[0054] The procedure for detecting the protein adsorption kinetics of bovine serum albumin (BSA) on the surface of silk fibroin gel dressing is as follows: First, prepare a 0.1 mol / L PBS buffer solution with pH 7.4. Then, prepare a freshly prepared BSA solution by accurately weighing 1.0 g of bovine serum albumin, dissolving it thoroughly in the PBS buffer solution, and bringing the volume to 1 L to obtain a 1.0 g / L BSA / PBS solution. Simultaneously, cut the silk fibroin gel dressing into 1 cm × 1 cm squares, without drying, and directly immerse them in 0.1 mol / L PBS with pH 7.4. Incubate at 25°C with shaking for 12 h to thoroughly remove contaminants adhering to the membrane surface. After pretreatment, remove the membrane and transfer it to 10 mL of freshly prepared 1.0 g / L BSA / PBS solution. Incubate at 25°C with shaking for another 12 h for static adsorption. Separately, prepare an equal volume of the original BSA solution without the silk fibroin gel dressing and place it under the same conditions as a blank control. After adsorption, remove the membrane and measure the absorbance of the blank control solution at 280 nm using a UV spectrophotometer. C B,0 Absorbance of the solution after the silk fibroin gel dressing is absorbed C B,1 Each sample was measured three times, and the average value was taken. The final adsorption percentage of BSA was calculated by the change in absorbance before and after adsorption, i.e., adsorption percentage = ( C B,0 C B,1 ) / C B,0 ×100%.

[0055] Antibacterial test of silk fibroin gel dressing: Take 200 μL of Staphylococcus aureus bacterial suspension from a -80°C freezer and add it to 5 mL of SOB liquid medium. Immerse different materials in the bacterial suspension and incubate at 37°C for 24 h on a shaker. Remove the materials from the incubator, rinse with PBS to remove bacteria that are not adhering to the surface, and then immerse the membrane in 3% paraformaldehyde solution for 2 h to fix the adhering bacteria. Then, stain with ammonium oxalate crystal violet solution for 30 min to remove excess liquid from the surface of the materials, add a small amount of cedarwood oil, and observe the colonies under a 100x oil immersion microscope.

[0056] Take 200 μL of Escherichia coli bacterial suspension from a -80°C freezer and add it to 5 mL of LB liquid medium. Immerse different materials in the bacterial suspension and incubate at 37°C for 24 h on a shaker. Remove the materials from the incubator, rinse with PBS to remove bacteria that are not adhering to the surface, and then immerse the membrane in 3% paraformaldehyde solution for 2 h to fix the adhering bacteria. Then, stain with eosin for 30 min, remove excess liquid from the surface of the materials, add a small amount of cedarwood oil, and observe the colonies under a 100x oil immersion microscope.

[0057] Table 1. Protein adsorption and antibacterial properties of silk fibroin gel dressings

[0058] As shown in Table 1, the antibacterial silk fibroin gel dressings prepared by the three embodiments all exhibited excellent protective effects in the anti-biofouling performance test. Since the betaine zwitterionic polymer brushes are covalently anchored to each silk fibroin molecular chain constituting the three-dimensional network framework of the gel, there is no diffusion or loss pathway for the active ingredients under conditions of wound exudate immersion and repeated rinsing. In the serum protein adsorption experiment, the protein adsorption on the surface of the gels of both derivative schemes was reduced by more than 90% compared to the unmodified silk fibroin gel, indicating that the hydration layer formed on its surface can effectively repel non-specific protein deposition. In the bacterial adhesion experiment, after incubation with Staphylococcus aureus and Escherichia coli suspensions for 24 hours, the number of bacteria adhering to the surface of the gels of both derivative schemes decreased by more than three orders of magnitude compared to the blank control group. Furthermore, the very few bacteria adhering to the surface were confirmed by live-dead staining to have their cell membrane integrity destroyed through contact, thus losing their proliferative capacity. Two sulfobetaine and carboxybetaine derivative schemes, together with the main scheme's phosphocholine scheme, form a complementary family of antibacterial silk fibroin gel dressings. The most suitable zwitterionic head base structure can be selected according to different wound types, exudate composition, and clinical needs, providing a wide range of material options for personalized wound infection prevention and treatment.

[0059] The cytotoxicity of the silk fibroin gel dressings prepared in Examples 1-3 and Comparative Examples 1-2 was tested, and the results are as follows: Figure 10 As shown.

[0060] Cytotoxicity assay of silk fibroin gel dressing: Mouse fibroblasts (L929) were cultured in TC-treated 25 cm⁻¹ cells. 2 In culture flasks. The culture medium used was DMEM supplemented with 10% fetal bovine serum, containing 100 μg / mL penicillin and 100 μL / mL streptomycin. The incubator temperature was 37°C, and the CO2 concentration was 5%. When the cells spread to approximately 95% of the bottom area of ​​the flask, they were digested with 0.25% trypsin solution and then passaged every 3-4 days.

[0061] First, the silk fibroin gel dressing was sterilized by soaking in 75% ethanol for 24 hours, followed by soaking in PBS buffer for 3 days to remove the ethanol. Then, the sterilized silk fibroin gel dressing was placed in 5 volumes of DMEM medium and soaked at 37°C for 24 hours. The cytotoxicity of the silk fibroin gel dressing extract was quantitatively tested using the CCK-8 assay. Cells were obtained at a density of approximately 1 × 10⁻⁶ cells / day. 5L929 cell suspension at 1 × 10⁶ / mL per well 4 Cells were seeded in 96-well plates, with at least 5 wells per group, and incubated at 37°C with 5% CO2 for 24 h. Then, the complete culture medium in the 9-well plates was removed. 100 μL of DMEM was added to the control group, and 100 μL of 100% extract was added to the experimental groups. Cells were incubated for 24 h, 48 h, and 72 h. Finally, the liquid in the plates was removed, and 100 μL of 10% CCK-8 reagent was added to each well under dark conditions. After 3 h, the absorbance at 450 nm was measured using a microplate reader.

[0062] Depend on Figure 10 It can be seen that the antibacterial silk fibroin gel dressings prepared by the three embodiment schemes all exhibited excellent biocompatibility in the cytotoxicity test. Furthermore, Comparative Example 1 and Comparative Example 2 also showed excellent biocompatibility in the cytotoxicity test. Specifically, the cytotoxicity test results of Comparative Example 1 showed that silk fibroin itself has good biocompatibility; the cytotoxicity test results of Comparative Example 2 also showed that it was non-toxic. Although the three antibacterial monomers MPC, SBMA, and CBMA were incorporated into the silk fibroin aqueous solution through physical blending, these three antibacterial monomers are non-cytotoxic and also exhibit good biocompatibility.

[0063] The drug sustained-release properties of the silk fibroin gel dressings prepared in Examples 1-3 and Comparative Example 2 were tested, and the test results are as follows: Figure 11 As shown.

[0064] Drug sustained-release performance test of silk fibroin gel dressing: First, equal masses of silk fibroin gel dressing samples were weighed from each group, including Comparative Example 1 (blank silk fibroin aqueous solution), Comparative Example 2 (unmodified silk fibroin gel with physically loaded antibacterial agent), Example 1 (MPC grafting), Example 2 (SBMA grafting), and Example 3 (CBMA grafting). All samples were immersed in pre-prepared phosphate-buffered saline (PBS) solution at pH 7.4, with each sample placed in an independent centrifuge tube, ensuring complete immersion. The centrifuge tubes were then placed in a constant-temperature shaking incubator at 37°C with a constant shaking speed to simulate dynamic body fluid contact under physiological conditions. At preset time points (e.g., 0 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, etc.), 5 mL of solution was taken from each centrifuge tube, and 5 mL of fresh PBS solution was added to maintain a constant total volume. The extracted solutions were analyzed using UV-Vis spectrophotometry or high-performance liquid chromatography to determine the characteristic absorption peak intensity of the released antibacterial components (for physically loaded antibacterial agents in the comparative examples, or the trace amounts of ungrafted monomers that may have detached in the examples). The cumulative release concentration at each time point was calculated based on the standard curve, and the cumulative release rate was then calculated. At least three parallel samples were set for each group, and the results were averaged and the standard deviation was noted. By plotting the cumulative release rate over time, the differences in drug release kinetics between different dressing systems can be visually compared. The entire release experiment lasted for 7 days to fully observe the release behavior of each dressing group in the short and long term. This testing method effectively reflects the essential difference between the chemical bonding strategy and the physical loading strategy in this invention in inhibiting the burst release and loss of antibacterial components.

[0065] Depend on Figure 11 It can be seen that the antibacterial silk fibroin gel dressings prepared in the three embodiments showed a very low cumulative release rate after 7 days of release testing, indicating that the antibacterial monomers were covalently grafted onto the silk fibroin molecular chains, and there was no burst release or loss of antibacterial components. However, the antibacterial silk fibroin gel dressing prepared in Comparative Example 2, because the antibacterial monomers were physically loaded into the silk fibroin, showed a very high cumulative release rate throughout the release test, exhibiting significant burst release and loss of antibacterial components. In summary, in the three embodiments, because the betaine zwitterionic polymer brushes were covalently anchored to each silk fibroin molecular chain constituting the three-dimensional network framework of the gel, there was no diffusion or loss pathway of the active ingredients under conditions of wound exudate immersion and repeated flushing.

[0066] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for preparing an antibacterial silk fibroin gel dressing, characterized in that, Includes the following steps: S1. Preparation of regenerated silk fibroin aqueous solution: Silkworm cocoons are boiled in an alkaline aqueous solution to degummify them. After degumming, they are washed with water until neutral and then dried to obtain degummed silk. The degummed silk is dissolved in a lithium salt aqueous solution to obtain a crude silk fibroin solution. The crude silk fibroin solution was filtered, dialyzed, centrifuged, and then filtered again to obtain a regenerated silk fibroin aqueous solution. S2. Preparation of antibacterial silk fibroin solution: The regenerated silk fibroin aqueous solution obtained in S1 is diluted with a buffer solution to a mass fraction of 0.5-2.0%. Under inert gas protection at 0-10℃, an acrylate-N-succinimide ester solution prepared by dimethyl sulfoxide is added, and the reaction is carried out for 10-14 h to obtain an activated silk fibroin intermediate. An amphoteric monomer and an initiator are added to the silk fibroin intermediate, and the reaction is stirred for 20-30 h under inert gas protection and at room temperature to obtain a reaction product. The reaction product is filtered, dialyzed, and concentrated to obtain an antibacterial silk fibroin solution with a mass fraction of 5-25%. The amphoteric monomer is 2-(methacryloyloxy)ethyl-2-(trimethylamino)ethyl phosphate, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide, or 3-[[2-(methacryloyloxy)ethyl]dimethylammonium]propionate. S3. Physical cross-linking and gelation: Mix ethanol with the antibacterial silk fibroin solution prepared in S2 at a volume ratio of 1:30 to 1:50, let stand at room temperature for 20 to 30 hours to gel, and then immerse the gel in deionized water for 20 to 30 hours, changing the soaking solution 1 to 5 times during the process to obtain antibacterial silk fibroin hydrogel dressing.

2. The preparation method according to claim 1, characterized in that, The alkaline aqueous solution described in S1 is a sodium carbonate aqueous solution with a mass fraction of 0.1-2%.

3. The preparation method according to claim 1, characterized in that, The boiling time in S1 is 20-40 minutes, and the degumming step is repeated 1-3 times.

4. The preparation method according to claim 1, characterized in that, The lithium salt described in S1 is selected from at least one of lithium bromide, lithium chloride, and lithium iodide, and the concentration of the lithium salt is 8~10 mol / L.

5. The preparation method according to claim 1, characterized in that, The dissolution temperature described in S1 is 30~50℃, and the dissolution time is 30~60 min.

6. The preparation method according to claim 1, characterized in that, The amount of N-succinimide acrylate added in S2 is 3-5% of the regenerated silk fibroin protein.

7. The preparation method according to claim 1, characterized in that, The amount of zwitterionic monomer added in S2 is 15-25% of the regenerated silk fibroin protein.

8. The preparation method according to claim 1, characterized in that, The initiator described in S2 is potassium persulfate, and its addition amount is 0.3~0.4% of the regenerated silk fibroin protein.

9. The preparation method according to claim 1, characterized in that, The filtration described in S2 uses 6-10 layers of medical gauze for filtration; the dialysis uses a dialysis bag with a molecular weight cutoff of 12-14 kDa, and is performed continuously in deionized water for 48-72 hours, with the deionized water being replaced every 8-12 hours; the concentration uses a dialysis bag with a molecular weight cutoff of 12-14 kDa, and is performed by reverse dialysis concentration in a 15% polyethylene glycol solution.

10. The antibacterial silk fibroin gel dressing obtained by any of the preparation methods described in claims 1-9.

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