Injectable pH-responsive silk-based photothermal antibacterial hydrogel and preparation method and application thereof

Abstract

This invention discloses an injectable pH-responsive silk-based photothermal antibacterial hydrogel, its preparation method, and its applications. The preparation method involves dissolving an antimicrobial peptide, levodopa, and a soluble calcium salt in an aqueous solution of silk fibroin to obtain a mixed solution. This solution is placed in a sealed container, and ammonium salt is used as a gas source. Heating decomposes the solution to produce ammonia and carbon dioxide, resulting in a reaction system containing quaternary hybrid microparticles. Ultrasonic treatment then induces a conformational change in the silk fibroin, forming a gel network, yielding the injectable pH-responsive silk-based photothermal antibacterial hydrogel with excellent injectability and biocompatibility. This invention constructs a dual synergistic antibacterial system combining immediate bactericidal action and long-lasting antibacterial activity. Through the synergistic effect of photothermal therapy and chemical antibacterial action, it achieves highly efficient anti-infection, demonstrating significant clinical application potential in addressing complex wound infections and infections associated with drug-resistant bacteria.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to an injectable pH-responsive silk-based photothermal antibacterial hydrogel, its preparation method, and its application. Background Technology

[0002] Hydrogels, with their extracellular matrix-like three-dimensional network structure, high water content, and good biocompatibility, are considered ideal wound dressing matrices. Silk fibroin, as a natural protein-based biomaterial, possesses excellent mechanical properties, biodegradability, and low immunogenicity, making it widely used in soft tissue repair. However, pure silk fibroin hydrogels lack inherent antibacterial activity and require loading with antibacterial agents to achieve anti-infection function. Existing modification methods mainly involve physical blending and chemical grafting: physical blending is prone to drug "burst release" or slow release, failing to provide effective bactericidal concentrations in the early stages of infection or maintain an antibacterial environment in the later stages of healing; chemical grafting may destroy drug activity, and the synthesis process is complex, with residual cross-linking agents exhibiting cytotoxicity. Furthermore, traditional pre-formed hydrogels are difficult to adapt to irregular deep wounds, easily leaving dead spaces that can lead to secondary infections. Injectable hydrogels with shear-thinning properties are more suitable for minimally invasive clinical applications.

[0003] Photothermal therapy (PTT), as a non-antibiotic antibacterial strategy, utilizes photothermal agents to convert light energy into heat energy, disrupting bacterial membrane structure and protein function through localized high temperatures. It offers advantages such as broad-spectrum bactericidal activity and low susceptibility to inducing drug resistance. Inorganic photothermal agents (such as gold nanorods and carbon nanotubes) pose risks of long-term in vivo retention and potential toxicity, while bio-based photothermal agents such as polydopamine (PDA) and melanin-like substances exhibit superior biocompatibility and photothermal stability. However, photothermal therapy alone only provides immediate bactericidal action; once light exposure ceases, sustained bacterial inhibition is lost, leading to frequent recurrences. Combining photothermal therapy with chemotherapy using antibacterial drugs (such as antimicrobial peptides) to construct a synergistic antibacterial system of "immediate killing + long-term inhibition" represents an ideal solution for enhancing anti-infection efficacy.

[0004] In the field of controlled drug release, utilizing the microenvironmental characteristics of infected wounds, such as weak acidity and high enzyme concentration, to construct intelligent responsive carriers is a core direction for improving treatment efficiency. Existing pH-responsive hydrogels mostly rely on polymer chain protonation / deprotonation to induce swelling changes or dynamic chemical bond breaking to achieve drug release. However, the response rate and release kinetics are limited by the diffusion barrier of the polymer network, making it difficult to achieve rapid drug release in the early stages of bacterial infection. Calcium carbonate, as a biomineralizing material, possesses excellent pH sensitivity and can decompose to produce calcium ions and carbon dioxide gas under acidic conditions. Current research mostly uses calcium carbonate as a calcium supplement or a simple drug carrier, with few reports on utilizing its acid degradation-induced "bubble kinetics" effect to physically disrupt the gel network and actively accelerate drug release.

[0005] In terms of preparation technology, constructing composite hydrogels containing multiple components such as photothermal agents, antimicrobial peptides, and inorganic minerals typically requires the synthesis of nanoparticles followed by blending with a gel matrix. This process is cumbersome, costly, and prone to uneven particle dispersion, affecting the mechanical and injection properties of the hydrogel. Therefore, how to simultaneously achieve inorganic core growth, organic shell polymerization, functional molecule loading, and induce in-situ gelation of silk fibroin in a mild and simple one-step system remains a pressing technical challenge in this field. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing antibacterial dressings or hydrogels, which struggle to simultaneously achieve injectability, synergistic photothermal-chemotherapy antibacterial properties, and intelligent rapid-response drug release. It also addresses issues such as uncontrollable drug release (prone to burst release or incomplete release), low efficiency of single antibacterial modes, and cumbersome and easily agglomerated preparation processes for multi-component composite materials. This invention overcomes the compatibility and dispersibility challenges between inorganic photothermal precursors, biological antibacterial peptides, and protein hydrogel matrices, providing an injectable, pH-responsive silk-based photothermal antibacterial hydrogel, its preparation method, and its applications. Employing a mild and simple one-step in-situ assembly method, and utilizing a preparation strategy combining gas diffusion-induced assembly with ultrasonic treatment, a quaternary hybrid hydrogel is constructed with silk fibroin, antibacterial peptides, levodopa, and calcium carbonate as its core components. This endows the material with active drug release driven by bubble dynamics and synergistic photothermal and chemotherapy antibacterial functions, achieving an integrated solution of injectability, intelligent response, high-efficiency antibacterial properties, and tissue repair.

[0007] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0008] The first aspect of this invention provides a method for preparing an injectable pH-responsive silk-based photothermal antibacterial hydrogel, comprising the following steps:

[0009] (1) Dissolve the antimicrobial peptide, levodopa and soluble calcium salt in an aqueous solution of silk fibroin to obtain a mixed solution;

[0010] (2) The mixed solution obtained in step (1) is placed in a sealed container, and ammonium salt is used as a gas source to carry out the reaction at 25-80℃; the ammonium salt is selected from one or more of ammonium bicarbonate (NH4HCO3), ammonium carbamate (NH2COONH4) and ammonium carbonate ((NH4)2CO3); the gas source is decomposed by heat to produce ammonia (NH3) and carbon dioxide (CO2), which diffuse into the mixed solution and generate calcium carbonate in situ, and induce levodopa to polymerize on the surface of calcium carbonate, while silk fibroin and antimicrobial peptide are co-precipitated and coated to obtain a reaction system containing quaternary hybrid particles;

[0011] (3) The reaction system obtained in step (2) is subjected to ultrasonic treatment to induce conformational change of silk fibroin and form a gel network to obtain the injectable pH-responsive silk-based photothermal antibacterial hydrogel.

[0012] This invention utilizes the dual effects of ammonium salt thermal decomposition products: carbon dioxide diffused into the solution acts as a carbon source, reacting with calcium ions to generate calcium carbonate; simultaneously, diffused ammonia provides an alkaline environment, promoting the auto-oxidative polymerization of levodopa on the calcium carbonate surface to form a poly-levodopa shell. The ammonium salt thermal decomposition gas generation method employed in this invention is mild and controllable. Compared to the severe disturbance caused by direct aeration, this invention utilizes the molecular diffusion of gas to form a dynamically balanced micro-reaction zone at the liquid-gas interface, allowing the nucleation reaction of carbon dioxide and calcium ions, the oxidative polymerization of levodopa, and the co-precipitation process of silk fibroin and antimicrobial peptides to proceed synergistically and simultaneously. This gas-liquid-solid multiphase interface assembly method can precisely control the distribution of quaternary hybrid components on the calcium carbonate surface, forming well-structured core-shell hybrid microparticles, providing structural support for the subsequent construction of a stable hydrogel network.

[0013] Further, in step (1), the antimicrobial peptide is selected from one or more of ε-poly-L-lysine, LL-37 acetate, nisin, protamine, poly(L-arginine) hydrochloride, polymyxin B, dermalin 2, bee venom peptide, insect antimicrobial peptide A, and insect antimicrobial peptide B.

[0014] Further, the ε-poly-L-lysine has a weight-average molecular weight of 2000-6000 Da and a molecular weight distribution index (PDI) of 1.1-3.0; the LL-37 acetate is an antimicrobial peptide derivative of human cathepsin inhibitor, with a weight-average molecular weight of 4000-5000 Da; the nisin is derived from lactic acid bacteria, with a weight-average molecular weight of 3000-3500 Da; the protamine is derived from salmon, with a weight-average molecular weight of 4000-5000 Da; the poly(L-arginine) hydrochloride (PArg) has a weight-average molecular weight of 3000-5000 Da or 7000-15000 Da; the polymyxin B has a weight-average molecular weight of 10000-15000 Da; and the Magainin 2 is derived from frog skin, with a weight-average molecular weight of 2000-3000 Da. Da; the melittin has a weight-average molecular weight of 2500-3000 Da; the cecropin A is derived from insects and is also known as cecropin A, with a weight-average molecular weight of 3500-4500 Da; the cecropin B is derived from insects and has a weight-average molecular weight of 3500-4000 Da.

[0015] Further, in step (1), the soluble calcium salt is calcium chloride, preferably calcium chloride dihydrate or anhydrous calcium chloride.

[0016] Further, in step (1), the silk fibroin aqueous solution is prepared by degumming raw silk, dissolving it in lithium bromide solution, dialysis, centrifugation, and then osmotically concentrating it in polyethylene glycol solution.

[0017] Further, in step (1), the concentration of the polyethylene glycol solution is 20-50 wt%, and the weight-average molecular weight of the polyethylene glycol is 20000-40000 Da.

[0018] In a specific embodiment, in step (1), the silk fibroin aqueous solution is prepared by the following method: after degumming raw silk, it is dissolved in lithium bromide solution, and after dialysis and centrifugation to remove impurities, a regenerated silk fibroin aqueous solution is obtained. Finally, the regenerated silk fibroin aqueous solution is placed in a high-concentration polyethylene glycol solution for permeation concentration.

[0019] Further, in step (1), the concentration of the antimicrobial peptide in the mixed solution is 0.2-30 mg / mL.

[0020] Further, in step (1), the concentration of levodopa in the mixed solution is 0.5-10 mg / mL.

[0021] Further, in step (1), the concentration of soluble calcium salt in the mixed solution is 5-30 mg / mL.

[0022] Further, in step (1), the molar concentration of calcium ions in the mixed solution is 34-204 mmol / L.

[0023] Further, in step (1), the concentration of silk fibroin in the mixed solution is 10-50 mg / mL.

[0024] Furthermore, in step (2), the gas source is placed at the bottom of the sealed container or in a separate container, and does not come into direct contact with the mixed solution.

[0025] Further, in step (2), the ratio of the gas source to the mixed solution is 0.27-2.15 g : 1 mL.

[0026] Further, in step (2), the molar ratio of the gas source to the calcium ions in the mixed solution is (150-250):1.

[0027] Furthermore, in step (2), the reaction time is 12-72 h.

[0028] Ammonium salts decompose upon heating, simultaneously producing ammonia and carbon dioxide, which can be used as a stable and mild gas source. This invention employs a gas generation scheme based on the thermal decomposition of ammonium salts. By controlling the ambient temperature to achieve the slow release and diffusion of the gas, the reaction kinetic rate can be significantly reduced, enabling in-situ nucleation and assembly of quaternary hybrid particles at a milder liquid-gas interface. This process not only simplifies the requirements for reaction equipment but also improves the dispersibility and interfacial bonding of hybrid components by optimizing the mass transfer process, achieving efficient control over the morphology and function of the particles.

[0029] Further, in step (2), the quaternary component hybrid microparticles have a core-shell structure; wherein the core is calcium carbonate, the shell is poly-L-DOPA formed by the oxidative polymerization of L-DOPA, and silk fibroin and antimicrobial peptides are coated on the surface of the microparticles by electrostatic adsorption and co-precipitation.

[0030] The antimicrobial peptides introduced in this invention partially participate in the formation of quaternary hybrid microparticles, while the remainder are encapsulated in a free state by a gel network formed by silk fibroin.

[0031] Further, in step (3), the power of the ultrasonic treatment is 100-200 W, the frequency is 15-25 kHz, and the amplitude is 10-30%; the mode of the ultrasonic treatment is intermittent ultrasound (on for 3-5 s, off for 3-5 s), and the total treatment time is 60-1200 s.

[0032] Furthermore, in step (3), the temperature of the reaction system is controlled at 2-20 ℃ during the ultrasonic treatment process, preferably by using an ice-water bath for temperature control.

[0033] Ultrasonic treatment has a dual function: on the one hand, it can disperse quaternary hybrid particles and prevent aggregation; on the other hand, it can break the hydrogen bonds within silk fibroin molecules, inducing their conformation to change from random coil to β-sheet, thereby forming a physically cross-linked hydrogel network.

[0034] The second aspect of the present invention provides an injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared by the preparation method described in the first aspect.

[0035] The injectable pH-responsive silk-based photothermal antibacterial hydrogel provided by this invention has shear-thinning properties and exhibits a significant photothermal heating effect under near-infrared light irradiation. In a weakly acidic environment with pH < 6.0, the calcium carbonate inside the hydrogel degrades and generates bubbles, disrupting the gel network structure and thus accelerating the release of antimicrobial peptides.

[0036] The third aspect of the present invention provides the application of the injectable pH-responsive silk-based photothermal antibacterial hydrogel described in the second aspect in the preparation of anti-infective wound dressings, soft tissue repair materials or drug delivery carriers.

[0037] The above-described technical solution of the present invention has the following beneficial effects:

[0038] 1. The gas diffusion-in-situ assembly preparation method provided by this invention abandons the cumbersome steps of traditionally synthesizing nanoparticles first and then blending them with gels. It utilizes gas diffusion following the thermal decomposition of ammonium salts to simultaneously complete calcium carbonate nucleation, L-DOPA polymerization and coating, and drug and protein co-precipitation loading within a single reaction system. This method is mild and simple, requires no organic solvents, and the in-situ formed core-shell hybrid microparticles are uniformly dispersed in the gel matrix, solving the technical problem of easy agglomeration in traditional physical blending.

[0039] 2. The bubble dynamics-based drug release mechanism proposed in this invention relies on the pH-responsive characteristics of the calcium carbonate core. In the weakly acidic microenvironment (pH < 6.0) caused by bacterial infection, calcium carbonate degrades to produce calcium ions and release a large number of carbon dioxide bubbles. The generation and expansion of these bubbles physically disrupt the dense structure of the gel network, resulting in a significant cell disruption effect and greatly accelerating the diffusion and release of the internally loaded antimicrobial peptides. Compared to passive diffusion relying solely on concentration gradients, this invention's active physical disruption drug release method offers a faster response and higher release efficiency.

[0040] 3. This invention constructs a dual synergistic antibacterial system combining immediate sterilization and long-term antibacterial action, achieving highly efficient anti-infection through the synergistic effect of photothermal therapy and chemical antibacterial action. Specifically, the poly-L-DOPA shell formed by L-DOPA polymerization endows the hydrogel with excellent photothermal conversion capabilities, rapidly heating up under near-infrared light to achieve immediate sterilization; the antimicrobial peptides released through bubble disruption exert their effect by disrupting bacterial cell membranes, continuously inhibiting bacterial proliferation after light exposure ceases, effectively preventing infection recurrence.

[0041] 4. The injectable pH-responsive silk-based photothermal antibacterial hydrogel provided by this invention possesses excellent injectability and biocompatibility. Based on the physical cross-linking network of silk fibroin, the hydrogel exhibits significant shear-thinning properties, allowing it to be filled into irregular deep wounds via syringe, meeting the needs of minimally invasive drug delivery. Simultaneously, silk fibroin, levodopa, and calcium ions all demonstrate good biocompatibility, resulting in excellent overall biocompatibility of the hydrogel, effectively promoting wound healing and soft tissue regeneration. Attached Figure Description

[0042] Figure 1 This is a schematic diagram illustrating the preparation of an injectable pH-responsive silk-based photothermal antibacterial hydrogel and its intelligent response and synergistic antibacterial mechanism according to the present invention.

[0043] Figure 2The images are FT-IR images of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9 and 17, the DHMs-embedded silk-based hydrogels prepared in Comparative Examples 7-9, and the pure silk-based hydrogels prepared in Comparative Example 15 after freeze-drying; where (A) represents Examples 9, 17, and Comparative Example 15, and (B) represents Comparative Examples 7-9 and Comparative Example 15.

[0044] Figure 3 The images show the XRD patterns of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, the DHMs-embedded silk-based hydrogel prepared in Comparative Examples 7-9, the pure silk-based hydrogel prepared in Comparative Example 15, the CCMs prepared in Comparative Example 20, the Pdopa@CCMs prepared in Comparative Example 21, and the EPL@Pdopa@CCMs prepared in Comparative Example 22 after freeze-drying; where (A) represents Example 9, Comparative Examples 7-9, and Comparative Example 15, and (B) represents Comparative Examples 20-22.

[0045] Figure 4 The thermogravimetric analysis (TGA) curves of mass loss rate and mass change rate are shown for the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, the THMs-embedded silk-based hydrogel prepared in Comparative Example 2, the pure silk-based hydrogel prepared in Comparative Example 13, and the EPL@Pdopa@CCMs prepared in Comparative Example 22 after freeze-drying. Among them, (A) is Comparative Example 13, (B) is Example 9, (C) is Comparative Example 2, and (D) is Comparative Example 22.

[0046] Figure 5 The image shows a cross-sectional SEM image (scale bar is 2 μm) of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9.

[0047] Figure 6 The above is a GPC diagram of the EPL used in this invention; where (A) is the chromatographic elution curve and (B) is the molecular weight distribution curve.

[0048] Figure 7 The graph shows the gelation time test results after ultrasonic treatment during the preparation of injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 7-9, 19-21, and 34-38, as well as DHMs-embedded silk-based hydrogels prepared in Comparative Examples 7-9 and pure silk-based hydrogels prepared in Comparative Examples 13-16. Among them, (A) is Comparative Examples 13-16, (B) is Examples 9 and 34-38, (C) is Examples 9, Comparative Examples 7-9 and Comparative Example 13, and (D) is Examples 7-9 and 19-21.

[0049] Figure 8This is an inverted photograph of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Examples 44-45.

[0050] Figure 9 The dynamic rheological frequency scanning curves are those of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 7-9 and 19-21, and the pure silk-based hydrogels prepared in Comparative Examples 13-14; wherein, (A) are Examples 7-9 and Comparative Example 13, and (B) are Examples 19-21 and Comparative Example 14.

[0051] Figure 10 The graph shows the storage modulus data of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9 and 39-41 at an angular frequency of 10 rad / s.

[0052] Figure 11 Viscosity-shear rate curves of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 7 and 9, and the pure silk-based hydrogel prepared in Comparative Example 13.

[0053] Figure 12 The images show extruded specimens of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9 and 13, and the pure silk-based hydrogel prepared in Comparative Example 13.

[0054] Figure 13 The in vitro degradation curves of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9 and 21 in phosphate buffer solutions of different pH values ​​are shown.

[0055] Figure 14 The image shows the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 5, and its dynamic rheological time-scan curves after being immersed in phosphate buffer solutions of different pH values ​​for 120 h.

[0056] Figure 15 The cumulative release curves of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 42 and the pure silk-based hydrogel loaded with FITC-EPL prepared in Comparative Example 17 in phosphate buffer solutions at different pH values ​​are shown. Among them, (A) is Example 42 and Comparative Example 17 under pH 5.5 conditions, and (B) is Example 42 under pH 5.5 and pH 7.4 conditions.

[0057] Figure 16UV-Vis images of the Pdopa-NPs solution prepared in Comparative Example 19, the EPL@Pdopa@CCMs solution prepared in Comparative Example 22, and the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, collected after 120 h of sustained release in phosphate buffer solution.

[0058] Figure 17 The photothermal heating curves are for the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9, 13, 21, and 25, the THMs-embedded silk-based hydrogels prepared in Comparative Examples 3 and 6, and the pure silk-based hydrogels prepared in Comparative Examples 13 and 14; wherein, (A) is for Examples 9, 13, 3, and 13, and (B) is for Examples 21, 25, 6, and 14.

[0059] Figure 18 The photothermal cycling stability curves of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9 and 21 are shown; where (A) is Example 9 and (B) is Example 21.

[0060] Figure 19 The images show the colony growth of Staphylococcus aureus after plate-coating culture of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, the THMs-embedded silk-based hydrogel prepared in Comparative Example 2, the DHMs-embedded silk-based hydrogel prepared in Comparative Example 8, and the pure silk-based hydrogel prepared in Comparative Example 13.

[0061] Figure 20 The graph shows the concentration of viable bacterial cultures obtained by plate count after Staphylococcus aureus was cultured on plates after being spread on plates by the following data: injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, THMs-embedded silk-based hydrogel prepared in Comparative Example 2, DHMs-embedded silk-based hydrogel prepared in Comparative Example 8, pure silk-based hydrogel prepared in Comparative Example 13, and blank control group (PBS-treated group). Detailed Implementation

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0063] This invention provides a method for preparing an injectable pH-responsive silk-based photothermal antibacterial hydrogel, comprising the following steps:

[0064] (1) Dissolve the antimicrobial peptide, levodopa and soluble calcium salt in an aqueous solution of silk fibroin to obtain a mixed solution;

[0065] (2) Place the mixed solution obtained in step (1) in a sealed container, and use NH4HCO3, NH2COONH4 or (NH4)2CO3 as a gas source to carry out the reaction at 25-80 °C; the gas source decomposes upon heating to produce NH3 and CO2, which diffuse into the mixed solution to generate calcium carbonate in situ and induce levodopa to polymerize on the surface of calcium carbonate. At the same time, silk fibroin and antimicrobial peptides are co-precipitated and coated to obtain a reaction system containing quaternary hybrid microparticles.

[0066] (3) The reaction system obtained in step (2) is subjected to ultrasonic treatment to induce conformational change of silk fibroin and form a gel network to obtain the injectable pH-responsive silk-based photothermal antibacterial hydrogel.

[0067] A schematic diagram of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared according to this invention and its intelligent response and synergistic antibacterial mechanism is shown below. Figure 1 As shown, a reaction mixture solution was prepared by mixing silk fibroin, antimicrobial peptides, L-DOPA, and soluble calcium salts as raw materials. Using ammonium salts as the gas source, ammonia and carbon dioxide were generated through thermal degradation in a sealed system, and in-situ induced assembly was achieved via gas diffusion. Specifically, the carbon dioxide diffused into the mixture solution reacted in-situ with calcium ions to form calcium carbonate as an inorganic core. The alkaline environment provided by ammonia promoted the oxidative polymerization of L-DOPA on the calcium carbonate surface, forming a hydrophilic poly-L-DOPA shell. During this process, silk fibroin and antimicrobial peptides were efficiently loaded via co-precipitation, resulting in well-dispersed quaternary hybrid microparticles. Subsequently, the reaction system was ultrasonically treated to maintain the dispersion stability of the microparticles while inducing conformational changes and physical cross-linking of the silk fibroin, ultimately obtaining a composite hydrogel with silk fibroin and antimicrobial peptides as the gel matrix network and uniformly encapsulated hybrid microparticles.

[0068] The hydrogel prepared by this invention has significant shear-thinning properties, enabling minimally invasive injection drug delivery. It also possesses a unique pH-responsive intelligent drug release mechanism and dual synergistic antibacterial function: on the one hand, the poly-L-DOPA structure formed by L-DOPA polymerization endows the material with excellent photothermal conversion capabilities, which can rapidly heat up under near-infrared light irradiation to achieve immediate sterilization; on the other hand, under the weakly acidic conditions of the bacterial infection microenvironment, the calcium carbonate inside the gel degrades and generates a large number of carbon dioxide bubbles, which physically destroys the dense structure of the gel matrix, thereby accelerating the release and diffusion of antimicrobial peptides and achieving long-term antibacterial effect through the cell membrane disruption mechanism.

[0069] The preparation method provided by this invention is mild and simple, and the resulting hydrogel integrates injectability, photothermal therapy and environmentally responsive drug delivery functions, and has broad application prospects in the fields of anti-infective wound dressings, soft tissue repair materials and drug delivery carriers.

[0070] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0072] The ε-poly-L-lysine used in the following examples has a weight-average molecular weight (Mw) of 3969 Da and a polydispersity index (PDI) of 2.37; LL-37 acetate has a weight-average molecular weight of 4500 Da; and melitrix has a weight-average molecular weight of 2800 Da.

[0073] The FITC-labeled ε-poly-L-lysine (EPL) used in the following examples was prepared by the following method: 1 g of EPL was dissolved in deionized water, and the pH was adjusted to 6.5 using 1 mol / L dilute hydrochloric acid and 1 mol / L sodium hydroxide solution. The volume was then brought to 100 mL to prepare an EPL solution with a concentration of 10 mg / mL and a pH of 6.5. FITC fluorescent dye was added to the EPL solution to achieve a concentration of 0.2% of the EPL concentration. This mixture was protected from light and stirred at room temperature for 4 h. After the reaction was completed, the obtained FITC-labeled EPL solution was purified by dialysis, and after freeze-drying, FITC-labeled EPL powder was obtained, designated as FITC-EPL, and stored at -20 °C protected from light.

[0074] The specific method for preparing dry samples by freeze-drying hydrogels in the following test examples includes: freezing the hydrogel at -40°C until complete crystallization, followed by sublimation drying under a low pressure of 0.5 mbar, and finally removing residual adsorbed water at 30°C. The hydrogel samples obtained by this method have intact structures and uniform pores, making them suitable for further testing and analysis.

[0075] Example 1

[0076] A method for preparing an aqueous solution of silk fibroin includes the following steps:

[0077] Shredded raw silk yarn was boiled in a 0.02 mol / L sodium carbonate aqueous solution for 40 min to degummify it, with a raw silk yarn to sodium carbonate aqueous solution ratio of 1 g : 10 mL. This degumming process was repeated 3 times. The silk fibroin fibers were then thoroughly washed with deionized water and dried at 50 °C. The degummed silk fibroin fibers were dissolved in a 9.3 mol / L lithium bromide solution (60 °C, 4 h), with a silk fibroin fiber to lithium bromide aqueous solution ratio of 1 g : 10 mL. After cooling, the solution was dialyzed in pure water for 3 days (dialysis bag molecular weight cutoff 7000 Da) to remove salts and other impurities. The purified silk fibroin aqueous solution was concentrated with a concentrated polyethylene glycol solution (40 wt%, weight-average molecular weight 40,000 Da, linear polyethylene glycol) at 4 °C. The mass ratio of the silk fibroin aqueous solution to the concentrated polyethylene glycol solution was 1:10, and the concentration time was 4 h, yielding a concentrated silk fibroin solution with a concentration of 87.6 mg / mL. The concentration of the silk fibroin (SF) solution was determined by measuring a certain volume of the solution, placing it in a glass dish, drying it to constant weight in a constant-temperature drying oven at 105 °C, measuring its dry weight, and dividing the obtained SF dry weight by its corresponding initial volume. Finally, by adding deionized water to the concentrated silk fibroin solution, silk fibroin aqueous solutions of any concentration range of 10-50 mg / mL could be prepared and stored at 4 °C for later use.

[0078] Example 2-33

[0079] A method for preparing an injectable pH-responsive silk-based photothermal antibacterial hydrogel includes the following steps:

[0080] (1) A certain mass of ε-poly-L-lysine (EPL), L-Dopa and calcium chloride dihydrate (CaCl2·2H2O) were dissolved together in 10 mL of an aqueous solution of silk fibroin (SF) with a specific initial concentration prepared in Example 1 to obtain a homogeneous mixed solution;

[0081] (2) Quaternary hybrid microparticles are induced to assemble in a mixed solution by gas diffusion. The quaternary components include two introduced natural antimicrobial peptides and proteins, EPL and SF, as well as calcium carbonate (CaCO3) and poly-L-DOPA generated during the assembly process. These four components are integrated together to form a quaternary hybrid microparticle. First, the mixed solution obtained in step (1) and a specific mass of ammonium bicarbonate powder are placed in a sealed container, and ammonium bicarbonate (NH4HCO3) is used as a gas source. The ammonium bicarbonate reacts with calcium ions (CaCO3) in the mixed solution. 2+The molar ratio of ammonium bicarbonate to carbon dioxide is 200:1. The ammonia (NH3) and carbon dioxide (CO2) gases produced by the thermal decomposition of ammonium bicarbonate diffuse into the mixed solution. The presence of NH3 creates an alkaline pH environment, which increases the Ca2+ content at the liquid-gas interface. 2+ Ions react mildly with CO2 to generate calcium carbonate nanonuclei, inducing L-Dopa to polymerize on the surface of the calcium carbonate nanonuclei to form a Pdopa shell structure. Simultaneously, free SF and EPL are co-precipitated within the core-shell structure, forming the final quaternary hybrid microparticles, denoted as QHMs. The assembly temperature of QHMs in the mixed solution, i.e., the temperature at which NH4HCO3, as the gas source, decomposes to produce NH3 and CO2, was set to 35 °C, and the reaction time was set to 48 h.

[0082] (3) The reaction system obtained in step (2) was removed from the sealed container and subjected to ultrasonic treatment in an open environment using a probe-type ultrasonic instrument (a Branson Digital Sonifier 250 cell disruptor). This induced a conformational change in the silk fibroin and rapidly formed a gel network, allowing the QHMs generated in step (2) to disperse and embed themselves in the silk matrix gel network under ultrasonication, ultimately yielding an injectable pH-responsive silk-based photothermal antibacterial hydrogel. The ultrasonic treatment output power was 200W, the frequency was 20 kHz, the amplitude was 30%, and the ultrasonic treatment mode was intermittent ultrasonication (on for 3 seconds, off for 3 seconds). The total treatment time was 600 s. During the ultrasonic treatment, the solution was placed in an ice-water bath to control the system temperature at 10 ℃. The prepared hydrogel sample was stored in a 4 ℃ refrigerator.

[0083] The feed table for preparing injectable pH-responsive silk-based photothermal antibacterial hydrogels in Examples 2-33 is shown in Table 1:

[0084] Table 1

[0085]

[0086] Example 34

[0087] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 9, except that the total treatment time of ultrasonic treatment in step (3) is 60 s.

[0088] Example 35

[0089] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 9, except that the total treatment time of ultrasonic treatment in step (3) is 120 s.

[0090] Example 36

[0091] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 9, except that the total treatment time of ultrasonic treatment in step (3) is 300 s.

[0092] Example 37

[0093] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 9, except that the total treatment time of ultrasonic treatment in step (3) is 900 s.

[0094] Example 38

[0095] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 9, except that the total treatment time of ultrasonic treatment in step (3) is 1200 s.

[0096] Example 39

[0097] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 9, except that in step (2), the ambient temperature at which QHMs are assembled in the mixed solution is 25 °C.

[0098] Example 40

[0099] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 9, except that in step (2), the ambient temperature at which QHMs are assembled in the mixed solution is 30 °C.

[0100] Example 41

[0101] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 9, except that in step (2), the ambient temperature at which QHMs are assembled in the mixed solution is 40 °C.

[0102] Example 42

[0103] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 9, except that in step (1), EPL is replaced with FITC-EPL.

[0104] Example 43

[0105] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 21, except that in step (1), EPL is replaced with FITC-EPL.

[0106] Example 44

[0107] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 21, except that in step (1), EPL is replaced with LL-37 acetate.

[0108] Example 45

[0109] The preparation method of an injectable pH-responsive silk-based photothermal antibacterial hydrogel is basically the same as that in Example 21, except that in step (1), EPL is replaced with bee venom peptide.

[0110] Comparative Examples 1-6

[0111] A method for preparing a silk-based hydrogel with ternary component hybrid microparticles (THMs) embedded, comprising the following steps:

[0112] (1) Dissolve any two of the three raw materials, EPL, L-Dopa and CaCl2·2H2O, in 10 mL of SF aqueous solution of a specific initial concentration prepared in Example 1 to obtain a homogeneous mixed solution;

[0113] (2) The ternary hybrid particles are induced to assemble in a mixed solution by gas diffusion. The ternary components are based on SF and combined with any two of EPL, in-situ generated CaCO3 and Pdopa to achieve synergistic assembly of the three components, forming structurally stable ternary hybrid particles, denoted as THMs. First, the mixed solution obtained in step (1) and a specific mass of ammonium bicarbonate powder are placed together in a sealed container, and NH4HCO3 is used as the gas source. The NH3 and CO2 gases generated by the thermal decomposition of ammonium bicarbonate diffuse into the mixed solution. The ambient temperature for the assembly of THMs in the mixed solution, that is, the temperature at which NH4HCO3 is used as the gas source to thermally decompose and generate NH3 and CO2 gases, is set to 35 °C, and the reaction time is set to 48 h.

[0114] (3) The reaction system obtained in step (2) was removed from the sealed container and subjected to ultrasonic treatment using a probe-type ultrasonic instrument (cell disruptor) in an open environment to induce conformational change of silk fibroin and rapidly form a gel network. This allowed the THMs generated in step (2) to disperse and embed into the silk matrix gel network under ultrasonication, ultimately obtaining a silk-based hydrogel with THMs embedded. The output power of the ultrasonic treatment was 200 W, the frequency was 20 kHz, the amplitude was 30%, the ultrasonic treatment mode was intermittent ultrasonication (on for 3 s, off for 3 s), and the total treatment time was 600 s. During the ultrasonic treatment, the solution was placed in an ice-water bath to control the system temperature at 10 ℃. The prepared hydrogel sample was stored in a refrigerator at 4 ℃.

[0115] Table 2 shows the feed table for the preparation of THMs-embedded silk-based hydrogels in Comparative Examples 1-6:

[0116] Table 2

[0117]

[0118] Comparative Examples 7-12

[0119] A method for preparing a silk-based hydrogel with embedded binary component hybrid microparticles (DHMs) includes the following steps:

[0120] (1) Dissolve any one of the three raw materials, EPL, L-Dopa and CaCl2·2H2O, in 10 mL of SF aqueous solution of a specific initial concentration prepared in Example 1 to obtain a homogeneous mixed solution;

[0121] (2) Binary hybrid microparticles are induced to assemble in a mixed solution by gas diffusion. The binary component is based on SF and combines any one of EPL, in-situ generated CaCO3, and Pdopa to achieve synergistic assembly of the two components, forming structurally stable binary hybrid microparticles, denoted as DHMs. First, the mixed solution obtained in step (1) and a specific mass of ammonium bicarbonate powder are placed together in a sealed container, and NH4HCO3 is used as the gas source. The NH3 and CO2 gases generated by the thermal decomposition of ammonium bicarbonate diffuse into the mixed solution. The ambient temperature for DHMs to assemble in the mixed solution, i.e. the temperature at which NH4HCO3 is used as the gas source to thermally decompose and generate NH3 and CO2 gases, is set to 35 °C, and the reaction time is set to 48 h.

[0122] (3) The reaction system obtained in step (2) was removed from the sealed container and subjected to ultrasonic treatment using a probe-type ultrasonic instrument (cell disruptor) in an open environment to induce conformational change of silk fibroin and rapidly form a gel network. This allowed the DHMs generated in step (2) to disperse and embed into the silk matrix gel network under ultrasonication, ultimately obtaining a silk-based hydrogel with DHMs embedded in it. The output power of the ultrasonic treatment was 200 W, the frequency was 20 kHz, the amplitude was 30%, the ultrasonic treatment mode was intermittent ultrasonication (on for 3 s, off for 3 s), and the total treatment time was 600 s. During the ultrasonic treatment, the solution was placed in an ice-water bath to control the system temperature at 10 ℃. The prepared hydrogel sample was stored in a refrigerator at 4 ℃.

[0123] The feed table for the preparation of DHMs-embedded silk-based hydrogels in Comparative Examples 7-12 is shown in Table 3:

[0124] Table 3

[0125]

[0126] Comparative Example 13

[0127] A method for preparing a pure silk-based hydrogel includes the following steps:

[0128] (1) At 35 °C, 10 mL of SF aqueous solution with a concentration of 30 mg / mL prepared in Example 1 and 21.5 g of ammonium bicarbonate powder were placed together in a sealed container, and NH4HCO3 was used as the gas source. The NH3 and CO2 gases generated by the thermal decomposition of ammonium bicarbonate diffused into the mixed solution and reacted for 48 h.

[0129] (3) The reaction system obtained in step (1) was removed from the sealed container and subjected to ultrasonic treatment using a probe-type ultrasonic instrument (cell disruptor) in an open environment to induce conformational change of silk fibroin and rapidly form a gel network, ultimately obtaining pure silk-based hydrogel. The output power of the ultrasonic treatment was 200 W, the frequency was 20 kHz, the amplitude was 30%, the ultrasonic treatment mode was intermittent ultrasonic treatment (on for 3 s, off for 3 s), and the total treatment time was 600 s. During the ultrasonic treatment, the solution was placed in an ice-water bath to control the system temperature at 10 ℃. The prepared hydrogel sample was stored in a refrigerator at 4 ℃.

[0130] Comparative Example 14

[0131] A method for preparing a pure silk-based hydrogel includes the following steps:

[0132] (1) At 35 °C, 10 mL of SF aqueous solution with a concentration of 15 mg / mL prepared in Example 1 and 21.5 g of ammonium bicarbonate powder were placed together in a sealed container, and NH4HCO3 was used as the gas source. The NH3 and CO2 gases generated by the thermal decomposition of ammonium bicarbonate diffused into the mixed solution and reacted for 48 h.

[0133] (3) The reaction system obtained in step (1) was removed from the sealed container and subjected to ultrasonic treatment using a probe-type ultrasonic instrument (cell disruptor) in an open environment to induce conformational change of silk fibroin and rapidly form a gel network, ultimately obtaining pure silk-based hydrogel. The output power of the ultrasonic treatment was 200 W, the frequency was 20 kHz, the amplitude was 30%, the ultrasonic treatment mode was intermittent ultrasonic treatment (on for 3 s, off for 3 s), and the total treatment time was 600 s. During the ultrasonic treatment, the solution was placed in an ice-water bath to control the system temperature at 10 ℃. The prepared hydrogel sample was stored in a refrigerator at 4 ℃.

[0134] Comparative Example 15

[0135] A method for preparing a pure silk-based hydrogel includes the following steps:

[0136] The SF aqueous solution with a concentration of 30 mg / mL prepared in Example 1 was placed in a constant temperature chamber at 35 °C to allow the SF to naturally gel in the aqueous solution without any additional physical or chemical stimulation, thus obtaining a pure silk-based hydrogel.

[0137] Comparative Example 16

[0138] A method for preparing a pure silk-based hydrogel includes the following steps:

[0139] The SF aqueous solution with a concentration of 15 mg / mL prepared in Example 1 was placed in a constant temperature chamber at 35 °C to allow the SF to naturally gel in the aqueous solution without any additional physical or chemical stimulation, thus obtaining a pure silk-based hydrogel.

[0140] Comparative Example 17

[0141] A method for preparing a pure silk-based hydrogel loaded with FITC-EPL includes the following steps:

[0142] (1) At 35 °C, 120 mg of FITC-EPL was dissolved in 10 mL of SF aqueous solution with a concentration of 30 mg / mL prepared in Example 1. The resulting mixed solution was placed together with 21.5 g of NH4HCO3 powder in a sealed container, and NH4HCO3 was used as a gas source. The NH3 and CO2 gases generated by the thermal decomposition of NH4HCO3 diffused into the mixed solution and reacted for 48 h.

[0143] (3) The reaction system obtained in step (1) was removed from the sealed container and subjected to ultrasonic treatment using a probe-type ultrasonic instrument (cell disruptor) in an open environment to induce conformational change of silk fibroin and rapidly form a gel network, ultimately obtaining a pure silk-based hydrogel loaded with FITC-EPL. The output power of the ultrasonic treatment was 200 W, the frequency was 20 kHz, the amplitude was 30%, the ultrasonic treatment mode was intermittent ultrasonic treatment (on for 3 s, off for 3 s), and the total treatment time was 600 s. During the ultrasonic treatment, the solution was placed in an ice-water bath to control the system temperature at 10 ℃. The prepared hydrogel sample was stored in a refrigerator at 4 ℃.

[0144] Comparative Example 18

[0145] A method for preparing a pure silk-based hydrogel loaded with FITC-EPL includes the following steps:

[0146] (1) At 35 °C, 120 mg of FITC-EPL was dissolved in 10 mL of SF aqueous solution with a concentration of 15 mg / mL prepared in Example 1. The resulting mixed solution was placed together with 21.5 g of NH4HCO3 powder in a sealed container, and NH4HCO3 was used as a gas source. The NH3 and CO2 gases generated by the thermal decomposition of NH4HCO3 diffused into the mixed solution and reacted for 48 h.

[0147] (3) The reaction system obtained in step (1) was removed from the sealed container and subjected to ultrasonic treatment using a probe-type ultrasonic instrument (cell disruptor) in an open environment to induce conformational change of silk fibroin and rapidly form a gel network, ultimately obtaining a pure silk-based hydrogel loaded with FITC-EPL. The output power of the ultrasonic treatment was 200 W, the frequency was 20 kHz, the amplitude was 30%, the ultrasonic treatment mode was intermittent ultrasonic treatment (on for 3 s, off for 3 s), and the total treatment time was 600 s. During the ultrasonic treatment, the solution was placed in an ice-water bath to control the system temperature at 10 ℃. The prepared hydrogel sample was stored in a refrigerator at 4 ℃.

[0148] Comparative Example 19

[0149] A method for preparing poly-L-DOPA nanoparticles (Pdopa-NPs) includes the following steps:

[0150] 40 mg of L-Dopa was dissolved in 10 mL of pure water to obtain a homogeneous L-Dopa solution. L-Dopa was then induced to assemble in the solution via gas diffusion to form poly(L-Dopa) nanoparticles. At an ambient temperature of 35 °C, using NH4HCO3 as a gas source, the NH3 and CO2 gases generated by the thermal decomposition of ammonium bicarbonate diffused into the solution. The presence of NH3 created an alkaline pH environment, inducing the direct polymerization of L-Dopa to form Pdopa nanoparticles. The polymerization reaction time was 48 h, and the amount of NH4HCO3 used was 21.5 g. After the reaction was complete, Pdopa nanoparticles were separated by centrifugation (18000 rpm, 15 min), washed with pure water, and then ultrasonically dispersed to ensure uniform dispersion. The ultrasonic output power was 200 W, the frequency was 20 kHz, the amplitude was 30%, and the ultrasonication mode was intermittent (3 s on, 3 s off), with a total treatment time of 600 s. During the ultrasonication, the nanoparticle solution was placed in an ice-water bath to control the system temperature at 10 ℃. The completely dispersed Pdopa-NPs solution was then freeze-dried to obtain Pdopa-NPs powder.

[0151] Comparative Example 20

[0152] A method for preparing calcium carbonate microparticles (CCMs) includes the following steps:

[0153] 200 mg CaCl₂·2H₂O was dissolved in 10 mL of pure water to obtain a homogeneous CaCl₂ solution; CaCl₂ was induced by gas diffusion. 2+ In solution, calcium carbonate particles, denoted as CCMs, are assembled. At an ambient temperature of 35 °C, using NH4HCO3 as a gas source, the NH3 and CO2 gases produced by the thermal decomposition of ammonium bicarbonate diffuse into the solution. The presence of NH3 creates an alkaline pH environment, which facilitates the formation of calcium carbonate particles at the liquid-gas interface. 2+ Ions reacted gently with CO2 to generate CCMs over a period of 48 h, using 21.5 g of NH4HCO3. After the reaction, the CCMs were separated by centrifugation (18000 rpm, 15 min) and washed with pure water. Subsequently, the CCMs were thoroughly dispersed by ultrasonic treatment. The ultrasonic output power was 200 W, the frequency was 20 kHz, the amplitude was 30%, and the ultrasonic treatment mode was intermittent (3 s on, 3 s off), with a total treatment time of 600 s. During ultrasonic treatment, the CCMs solution was placed in an ice-water bath to maintain the system temperature at 10 °C. The completely dispersed CCMs solution was then freeze-dried to obtain CCMs powder.

[0154] Comparative Example 21

[0155] A method for preparing poly(L-DOPA)-coated calcium carbonate microparticles (Pdopa@CCMs) includes the following steps:

[0156] 40 mg L-Dopa and 200 mg CaCl2·2H2O were dissolved together in 10 mL of pure water to obtain a homogeneous mixed solution. Binary hybrid particles, including CaCO3 and Pdopa generated during the assembly process, were induced to assemble in the mixed solution via gas diffusion. These particles formed a stable core-shell structure. At an ambient temperature of 35 °C, using NH4HCO3 as the gas source, the NH3 and CO2 gases produced by the thermal decomposition of ammonium bicarbonate diffused into the solution. The presence of NH3 created an alkaline pH environment, which facilitated the fusion of CaCO3 and Pdopa at the liquid-gas interface. 2+Ions reacted mildly with CO2 to generate calcium carbonate nanonuclei, which induced L-Dopa to polymerize on the surface of the calcium carbonate nanonuclei, forming a Pdopa shell structure, thus creating the final binary hybrid microparticles, denoted as Pdopa@CCMs. The reaction time was 48 h, and the amount of NH4HCO3 used was 21.5 g. After the reaction was complete, the Pdopa@CCMs were separated by centrifugation (18000 rpm, 15 min), washed with pure water, and then ultrasonically dispersed to ensure uniform dispersion. The ultrasonic output power was 200 W, the frequency was 20 kHz, the amplitude was 30%, and the ultrasonic treatment mode was intermittent (3 s on, 3 s off), with a total treatment time of 600 s. During the ultrasonic treatment, the microparticle solution was placed in an ice-water bath to control the system temperature at 10 °C. The completely dispersed microparticle solution was then freeze-dried to obtain Pdopa@CCMs powder.

[0157] Comparative Example 22

[0158] A method for preparing calcium carbonate ternary component hybrid microparticles (EPL@Pdopa@CCMs) co-coated with poly-L-dopa and ε-poly-L-lysine includes the following steps:

[0159] 120 mg EPL, 40 mg L-Dopa, and 200 mg CaCl2·2H2O were dissolved together in 10 mL of pure water to obtain a homogeneous mixed solution. The assembly of ternary hybrid microparticles in this mixed solution was induced by gas diffusion. The ternary components included the introduced natural antimicrobial peptide EPL, and CaCO3 and Pdopa generated during the assembly process, forming a stable core-shell structure. At an ambient temperature of 35 °C, NH4HCO3 was used as the gas source. The NH3 and CO2 gases produced by the thermal decomposition of NH4HCO3 diffused into the solution. The presence of NH3 created an alkaline pH environment, which facilitated the assembly of CaCl2·2H2O at the liquid-gas interface. 2+Ions reacted mildly with CO2 to generate calcium carbonate nanonuclei, inducing L-Dopa to polymerize on the surface of the calcium carbonate nanonuclei to form a Pdopa shell structure. Simultaneously, free EPL was co-precipitated within the core-shell structure, forming the final ternary hybrid microparticles, denoted as EPL@Pdopa@CCMs. The reaction time was 48 h, and the amount of NH4HCO3 used was 21.5 g. After the reaction, the EPL@Pdopa@CCMs were separated by centrifugation (18000 rpm, 15 min), washed with pure water, and then ultrasonically dispersed to ensure uniform dispersion. The ultrasonic output power was 200 W, the frequency was 20 kHz, the amplitude was 30%, and the ultrasonic treatment mode was intermittent (3 s on, 3 s off), with a total treatment time of 600 s. During ultrasonic treatment, the microparticle solution was placed in an ice-water bath, and the system temperature was controlled at 10 ℃. The completely dispersed microparticle solution was freeze-dried to obtain EPL@Pdopa@CCMs microparticle powder.

[0160] Test Example 1

[0161] Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and gel permeation chromatography (GPC) were used to systematically characterize the chemical structure, crystal composition, thermal stability, microstructure, and EPL molecular weight distribution of the hydrogel and microparticles. FT-IR measurements were performed at room temperature, with a wavenumber range of 400–4000 cm⁻¹. -1The KBr pellet method was used for sample analysis. XRD analysis was performed using Cu Kα rays as the radiation source (λ = 0.15406 nm), scanning within the diffraction angle range of 2θ = 5°–80°. The crystal structure characteristics of the samples were evaluated by the changes in diffraction peak positions and intensities. TGA analysis was conducted under a nitrogen atmosphere at a temperature range of room temperature to 800 °C at a heating rate of 10 °C / min. The thermal decomposition process and thermal stability of the samples were assessed using mass loss rate curves (TG, %) and mass change rate curves (DTG, %). The method for observing the cross-section of hydrogels using SEM is as follows: The hydrogel is cut into cylinders of appropriate size (approximately 10-20 mm in diameter), and excess surface moisture is absorbed with filter paper. The sample is then immersed in liquid nitrogen and frozen until completely brittle. Rapid fracture is then performed in a predetermined direction under liquid nitrogen conditions to obtain a fresh, flat cross-section. The fractured sample is immediately transferred to a freeze dryer for freeze-drying to remove internal moisture and maintain the original microstructure. After freeze-drying, the sample is fixed with the cross-section facing upwards on the scanning electron microscope stage and gold sputtering is performed to improve the conductivity of the sample. Finally, the cross-sectional morphology of the hydrogel is observed and photographed under a scanning electron microscope, with the accelerating voltage typically set to 3-10 kV. GPC testing was performed using a gel permeation chromatography column (TSKgel G2500PWxl) suitable for water-soluble polymers, with a 0.1 mol / L NaNO3 aqueous solution as the mobile phase and a constant flow rate. The sample solution was filtered through a 0.22 μm filter before injection. The chromatographic system was calibrated using polyethylene glycol (PEG) standard samples. The relative number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (PDI = Mw / Mn) of EPL were calculated based on the correspondence between retention time and response value. The molecular weight distribution curve was automatically calculated by the chromatographic analysis software.

[0162] The FT-IR images of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9 and 17, the DHMs-embedded silk-based hydrogels prepared in Comparative Examples 7-9, and the pure silk-based hydrogel prepared in Comparative Example 15 after freeze-drying are shown below. Figure 2 As shown, all hydrogel samples were at 1600-1700 cm⁻¹ -1 (Amide I band), 1500-1600 cm -1 (Amide II band) and 1200-1300 cm -1 The (amide III band) region showed characteristic absorption peaks of silk fibroin, indicating that silk fibroin constitutes the main framework of the hydrogel. Compared with pure silk-based hydrogel (Comparative Example 15), the spectrum of Example 9 showed characteristic absorption peaks belonging to calcite-type calcium carbonate: among which, located at 1402 cm⁻¹... -1 The strong and broad absorption band in the vicinity is attributed to carbonate (CO3). 2-Asymmetric stretching vibration (v3 mode); located at 877 cm. -1 The sharp characteristic peak at 877 cm⁻¹ is attributed to the out-of-plane bending vibration of the carbonate ion (v₂ mode). -1 The presence of this characteristic location clearly confirms that the inorganic mineral phase formed in the system is a thermodynamically stable calcite crystal form. At 1446 cm⁻¹ -1 The observed distinct absorption peak further confirms the presence of the Pdopa component in the system. This peak is mainly attributed to the C=C and C=N skeletal stretching vibrations of the aromatic structures (aromatic rings and indole rings) in the Pdopa molecule. Simultaneously, this position corresponds to the CO32- concentration in calcite-type calcium carbonate. 2- The overlapping of asymmetric stretching vibration regions and the strong signal superposition phenomenon reflect the tight interfacial bonding between the Pdopa shell and the CaCO3 inorganic core. The synergistic presentation of the above series of characteristic peaks provides a complete spectroscopic criterion for the successful in-situ polymerization and coating of Pdopa on the mineral surface. This indicates that during the gas diffusion-induced assembly process, the mineralization deposition of calcium carbonate and the oxidative polymerization of L-DOPA exhibit a high degree of spatiotemporal synchronicity, which promotes the stable integration of the formed quaternary hybrid microparticles into the three-dimensional physical network of silk fibroin, forming a structurally complete composite system.

[0163] The XRD patterns of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, the DHMs-embedded silk-based hydrogels prepared in Comparative Examples 7-9, the pure silk-based hydrogel prepared in Comparative Example 15, the CCMs prepared in Comparative Example 20, the Pdopa@CCMs prepared in Comparative Example 21, and the EPL@Pdopa@CCMs prepared in Comparative Example 22 after freeze-drying are shown below. Figure 3 As shown, Figure 3Analysis of the hydrogel matrix and composite system in (A) shows that the pure silk-based hydrogel (Comparative Example 15) exhibits typical broad and diffuse peaks in the diffraction angle range of 2θ = 20°-25°, which are attributed to the crystalline or amorphous structure of Silk I fibroin, indicating that the matrix has good flexible network properties. The spectra of Comparative Example 7 (containing only Pdopa) and Comparative Example 8 (containing only EPL) are similar to those of the pure silk-based hydrogel, both dominated by gentle amorphous broad peaks, with no obvious inorganic crystal diffraction signals observed. In contrast, Comparative Example 9 (containing only CaCO3) and Example 9 (containing QHMs), which introduce inorganic components, show a series of clear and sharp characteristic diffraction peaks while retaining the amorphous background of silk fibroin. Among them, the diffraction peaks located at diffraction angles 2θ ≈ 29.4° (104), 39.4° (113), 47.5° (018), and 48.5° (116) are in high agreement with the standard card for calcite-type calcium carbonate (JCPDS No. 05-0586). This result confirms that, under the induction of gas diffusion and EPL / L-Dopa, the system generates a thermodynamically stable calcite phase in situ, rather than a metastable aragonite. At the same time, these characteristic peaks do not show abnormal enhancement or high texture, and are superimposed on a distinct amorphous background, indicating that the generated calcium carbonate crystals are not large-sized independent precipitations, but rather grow under restricted conditions and are uniformly dispersed in the organic gel network. Furthermore, the extremely strong peak at 32.6° and the associated peak at 23.0° in the spectrum correspond to the (110) and (100) crystal planes of the byproduct ammonium chloride (NH4Cl), respectively. This strong signal directly confirms that CaCl2, NH3, CO2 and H2O react in situ to generate CaCO3 and NH4Cl (i.e. CaCl2 + NH3 + CO2 + H2O → CaCO3 + NH4Cl). Figure 3(B) further reveals the evolution of hybrid particles from inorganic minerals to organic-inorganic composite structures. The spectrum of Comparative Example 20 shows an extremely strong 32.6° diffraction peak and significant characteristic peaks at 23.0°, 29.4°, and 39.4°, confirming the formation of highly crystalline calcite-type calcium carbonate and ammonium chloride products. With the introduction of organic components, Comparative Example 21 (containing Pdopa) and Comparative Example 22 (containing Pdopa and EPL) exhibit obvious signal modulation characteristics while maintaining the characteristic peak positions: on the one hand, the relative intensity of all characteristic peaks decreases with increasing organic matter content, and the baseline in the low-angle region slightly rises, strongly proving that the amorphous or long-range ordered Pdopa shell effectively physically encapsulates the internal inorganic core, producing significant physical shielding and signal attenuation effects; on the other hand, the precise overlap of characteristic peak positions confirms that the in-situ polymerization process did not destroy the lattice structure of calcium carbonate, but rather achieved deep anchoring of organic matter on the inorganic crystal plane through intermolecular interactions. The spectral characteristics of this strong diffraction inorganic core coexisting with the regulated organic shell fully outline the successful construction of quaternary hybrid microparticles (QHMs), providing solid microstructural evidence for their chemical stability in complex biological environments.

[0164] Thermogravimetric analysis (TGA) curves of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, the THMs-embedded silk-based hydrogel prepared in Comparative Example 2, the pure silk-based hydrogel prepared in Comparative Example 13, and the EPL@Pdopa@CCMs prepared in Comparative Example 22 after freeze-drying are shown below. Figure 4 As shown, the thermal degradation behavior, thermal stability, and multiphase component characteristics of different systems were analyzed. Figure 4 As shown in Figure (A), the pure silk-based hydrogel (Comparative Example 13) exhibits a typical three-stage weight loss: in addition to the water loss peak near 78 °C, its main chain pyrolysis peak is located at 298 °C, accompanied by a significant shoulder peak at 429 °C. This shoulder peak is attributed to the carbonized residue or deep degradation of the stable crystalline region after the thermal pyrolysis of the β-sheet structure of silk fibroin, indicating that the pure protein matrix has a relatively uniform thermal degradation pathway. Figure 4 The spectrum of (D) (Comparative Example 22) reveals its unique "core-shell" thermal characteristics: First, a distinctive decomposition peak appears at 138 °C, which... Figure 4In the gel system of (A)-(C), no early thermal desorption of the side chain amino groups of EPL molecules attributable to the high-density coating on the surface of the microparticles or the release of interlayer bound water was observed. Secondly, the main organic phase decomposition peak split into 277°C and 308°C, and the high-temperature carbonization peak shifted to 435°C. These temperatures are significantly higher than those of the gel system, proving that the hard inorganic calcium carbonate core has a significant "thermal shielding effect" and interface anchoring effect on the external Pdopa and EPL organic coating shell, which enhances the thermal stability of the organic components. Finally, the strong decomposition peak at 714°C is strong evidence that calcium carbonate decomposes into calcium oxide and carbon dioxide, confirming the existence of highly crystalline calcite-type calcium carbonate. Figure 4 (B) (Example 9, containing Pdopa, EPL and CaCO3 components) and Figure 4 Example C (Comparative Example 2, containing Pdopa and CaCO3 components, but without EPL) demonstrates the complex thermal behavior of the hybrid microparticles combined with the silk fibroin matrix. Both exhibited significant splitting of their organic phase decomposition peaks and a shift to lower temperatures (Example 9 splits to 256 °C and 281 °C; Comparative Example 2 splits to 247 °C and 290 °C), and both retained carbonized shoulder peaks at approximately 409-422 °C. This change in degradation temperature, peak broadening, and splitting strongly suggests a strong intermolecular interaction (such as hydrogen bond competition and electrostatic attraction) between the silk fibroin and the introduced EPL, Pdopa, and other components, disrupting the crystalline integrity of some SF components and resulting in a matrix thermal stability slightly lower than that of pure SF at 298 °C. Furthermore, the inorganic decomposition temperature of the pure microparticles (714 °C) is higher than that of the gel system (696 °C in Example 9 and 689 °C in Comparative Example 2), indicating that the microparticles dispersed in the gel matrix are microscopically regulated by the surrounding organic network. The inorganic decomposition peak and residual shoulder peak temperatures of Example 9 are slightly higher than those of Comparative Example 2 without EPL, further confirming that the introduction of EPL enhances the bonding strength of the organic-inorganic interface. In summary, the TGA results not only quantitatively confirm the effective loading and in-situ integration of hybrid microparticles in the hydrogel, but also thermodynamically demonstrate the tight interfacial fusion and structural integration between the organic matrix and the inorganic filler, endowing the composite material with superior overall thermal stability.

[0165] Cross-sectional SEM image of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9 is shown below. Figure 5As shown, a large number of spherical or near-spherical microparticles are uniformly dispersed and embedded in the pore walls or encapsulated within the network within the layered matrix composed of silk fibroin. These microparticles are QHMs formed through gas diffusion-induced assembly. The microparticles are tightly bound to the gel matrix, with no obvious interfacial delamination. The uniform distribution of QHMs in the gel network and their tight bonding with the matrix fully demonstrate the highly uniform microstructure advantage of this composite material, providing a solid structural guarantee for its photothermal stability and intelligent response release.

[0166] The GPC diagram of the EPL used in this invention is as follows: Figure 6 As shown, the calculated Mw of this EPL raw material is 3969 Da, Mn is 1674 Da, and PDI is 2.37. This molecular weight range (2000-6000 Da) is within the range where EPL exhibits optimal antibacterial activity. At the same time, the relatively wide molecular weight distribution facilitates the rapid diffusion of small molecular weight components in the early stage of gel degradation and the long-term retention of large molecular weight components in the later stage, thereby achieving a stepwise antibacterial effect.

[0167] Test Example 2

[0168] The gelation time after ultrasonic treatment was tested for the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 7-9, 19-21, 34-38, and 44-45, as well as the DHMs-embedded silk-based hydrogels prepared in Comparative Examples 7-9 and the pure silk-based hydrogels prepared in Comparative Examples 13-16. The gelation time was recorded using the inverted observation method: the precursor solution after ultrasonic treatment was allowed to stand at room temperature; when the sample tube was inverted, the solution stopped flowing, indicating that the gelation transition was complete, and this moment was recorded as the gelation time.

[0169] The test results systematically evaluated the effects of ultrasonication time, component composition, and raw material concentration on hydrogel gelation kinetics, such as... Figure 7 As shown. Figure 7 (A) first demonstrated that ultrasound treatment, as a physical triggering method, can significantly shorten the natural gelation process of silk fibroin from several days to minutes. Figure 7 (B) reveals the nonlinear relationship between ultrasonic time and gelation rate: although extending ultrasonic time generally accelerates the process, its marginal effect diminishes; when ultrasonic time is extended from 600 s to 900 s or even 1200 s, the reduction in gelation time is no longer significant, indicating that 600 s can provide sufficient energy to induce conformational change and is determined to be the preferred process parameter. Figure 7In section (C), the interference effect of hybrid components on the gelation process was investigated. The results showed that the pure silk-based hydrogel (Comparative Example 13) had the fastest gelation rate, while the introduction of quaternary hybrid microparticles (QHMs) or binary hybrid microparticles (DHMs) delayed gelation to some extent. Further comparison revealed that the groups containing EPL (Example 9 and Comparative Example 8) had the slowest gelation rate, while the groups without EPL (Comparative Example 7 and Comparative Example 9) had a slower gelation rate than pure SF but faster than the EPL-containing group. This phenomenon suggests that the introduction of EPL may have a certain hindering effect on the orderly assembly of silk fibroin molecular chains into the β-sheet structure through steric hindrance or charge repulsion. Figure 7 In addition to confirming that high-concentration (30 mg / mL) silk fibroin solutions exhibit faster gelation rates due to increased molecular chain collision probability, the study (D) also revealed, through intra-group comparisons of Examples 7-9 (30 mg / mL group) and Examples 19-21 (15 mg / mL group), that gelation time increases with increasing EPL dosage, further confirming the negative impact of EPL on SF gelation. Nevertheless, this hindering effect did not diminish the clinical applicability of the material. Under the preferred 600 s ultrasound conditions, the gelation time for all 30 mg / mL concentration systems was controlled within 10-15 min, while the 15 mg / mL system could also complete curing within 30 min, fully meeting the time window requirements for injectable hydrogels in practical applications.

[0170] An inverted photograph of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Examples 44-45. Figure 8 As can be seen, after replacing LL-37 acetate or melitin with the antimicrobial peptide ε-poly-L-lysine, the system can still successfully form an injectable hydrogel with a uniform appearance and stable structure through gas diffusion-in situ assembly and ultrasonic treatment strategy. The hydrogel does not flow or collapse when inverted, which proves the universality and compatibility of the preparation method of the present invention for different antimicrobial peptides.

[0171] Test Example 3

[0172] Rheological tests were performed on the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 5, 7-9, 13, 19-21, and 39-41, as well as the pure silk-based hydrogels prepared in Comparative Examples 13-14.

[0173] First, the dynamic rheological frequency scanning curves of the fully gelled hydrogel were tested using a rotational rheometer, and the storage modulus (G') and loss modulus (G") under different angular frequency conditions were compared to evaluate its mechanical properties and gelation stability. The test machine used was a Thermo Fisher HAAKE MARS-40, and the test parameters were: flat plate fixture (circular, 20 mm in diameter), modulus-angular frequency scanning in oscillation mode, fixture spacing of 500 μm, test temperature of 37 ℃, and strain of 1%. After gelation in a special mold, the sample was demolded to obtain a cylindrical gel with a diameter of 20 mm and a thickness of 500 μm.

[0174] Figure 9 The influence of different components on the viscoelastic properties of the hydrogel was analyzed by dynamic frequency sweep testing using a rotational rheometer. The dynamic rheological frequency sweep curves showed that, within the test frequency range (0.1-100 rad / s), the G' values ​​of the hydrogel samples from Examples 7-9, Examples 19-21, and the relevant comparative examples were consistently significantly higher than G', and were insensitive to changes in stress frequency, exhibiting typical and stable elastic solid behavior, demonstrating the formation of a complete hydrogel network. Furthermore, Figure 9 (A) and Figure 9 The hydrogels in (B) exhibit a consistent trend: as the mass of EPL increases (corresponding to Examples 7-9 and 19-21), the G' of the hydrogel gradually decreases. This rheological phenomenon reveals the intrinsic mechanism by which EPL influences the construction of the SF gel network from a mechanical perspective. Its essence is not merely due to steric hindrance or charge repulsion between macromolecular chains, but more fundamentally, the hydrogen bond competition between EPL and SF molecular chains. Because the EPL molecular side chains are rich in high-density amino groups, they readily form strong intermolecular heterogeneous hydrogen bonds with the amide groups (carbonyl or imino) on the SF peptide chains; this process directly competes with the homogeneous hydrogen bonds required for the SF molecular chains to construct their β-sheet structure, thereby interfering with the regular arrangement and orientation of the SF molecular chains and inhibiting the formation and development of the β-sheet crystalline regions, which serve as physical cross-linking points. Figure 7Analysis reveals that this competitive mechanism not only slows down the gelation process but also significantly reduces the effective cross-linking density of the gel network, ultimately manifesting as a continuous decrease in storage modulus G' with increasing EPL content in the macroscopic mechanical response. This result elucidates the crucial regulatory role of inter-component interactions on the evolution of hydrogel network structure and mechanical properties at the molecular scale. Although the introduction of EPL weakens the mechanical strength of the hydrogel to some extent, G' of all components remains significantly higher than G' throughout the entire testing frequency range, and the modulus exhibits good stability with frequency changes, indicating that the material as a whole still maintains stable solid-like gel characteristics. More importantly, this result strongly confirms the excellent mechanical tunability of the material system of this invention: by simply adjusting the loading of the functional component EPL, precise control of the hydrogel's hardness and elasticity can be achieved within a certain range, enabling the material to flexibly adapt to the differentiated mechanical performance requirements of different soft tissues, such as potential clinical applications ranging from low-modulus tissues like skin to high-modulus environments like deep tissues.

[0175] Figure 10 The influence of temperature (25 ℃ to 40 ℃) during the gas diffusion assembly stage on the final hydrogel G' was demonstrated. With increasing assembly temperature, the G' of the hydrogel showed a significant upward trend. From a reaction kinetics perspective, moderately increasing the assembly temperature significantly accelerated the thermal degradation rate of the precursor ammonium bicarbonate and improved the gas diffusion efficiency of NH3 and CO2, thereby shortening the nucleation and growth cycle of the quaternary hybrid microparticles. Under high-temperature conditions, the crystallinity of the calcium carbonate mineralization nuclei was higher, and the oxidative polymerization of L-DOPA was more complete, significantly improving the "maturity" of the microparticles. These fully developed hybrid microparticles, acting as physical cross-linking cores, effectively promoted the tight entanglement and aggregation of surrounding silk fibroin and EPL molecular chains, ultimately forming a denser and more stable three-dimensional composite network structure. Furthermore, a gradient comparison of the examples revealed that the storage modulus of the hydrogel showed a significant increase from Example 40 (25 °C) to Example 9 (35 °C). However, when the assembly temperature was further increased from 35 °C (Example 9) to 40 °C (Example 41), the increase in modulus narrowed significantly, exhibiting a clear diminishing marginal effect. This indicates that at 35 °C, the in-situ generation of microparticles and the pre-assembly of the gel matrix within the system have reached a relatively ideal equilibrium, and further heating has limited marginal contribution to network densification. This conclusion not only confirms the strengthening effect of temperature on the mechanical properties of composite materials but also provides a scientific basis for the precise optimization of process parameters during large-scale preparation.

[0176] Secondly, the shear thinning behavior of the fully formed hydrogel was detected by measuring its viscosity-shear rate curve, which reflects its injectability. The test mode was the viscosity-shear rate curve mode in rotation mode, the diameter of the flat fixture was 20 mm, the fixture spacing was 500 μm, and the test temperature was 37 ℃. After the sample was gelled in a special mold, it was demolded to obtain a cylindrical gel with a diameter of 20 mm and a thickness of 500 μm.

[0177] Figure 11 The viscosity-shear rate curves illustrate the relationship between the apparent viscosity of different component hydrogel systems and the shear rate, which can be used to evaluate the minimally invasive injection performance of the materials. Comparative Example 13, as well as Examples 7 and 9, all exhibit significant shear-thinning characteristics. With increasing shear rate, the system viscosity rapidly decreases, indicating that the hydrogel has good flowability under high shear conditions, which is beneficial for injection through fine needles. After the shear effect disappears, the system viscosity recovers rapidly, contributing to the stable molding of the material at the target site. Further comparison shows that the viscosity levels of each system show a trend of Example 9 being higher than Example 7, and both being higher than Comparative Example 13, indicating that the introduction of the functional component EPL and its increased loading can effectively improve the viscosity of the hydrogel system. This result shows that by adjusting the amount of EPL added, the structural stability of the material under low shear conditions can be enhanced while maintaining good injectability, thereby achieving effective control of the rheological properties of the hydrogel. Example 9 combines high static viscosity and significant shear-thinning behavior, demonstrating excellent applicability for minimally invasive injection and molding stability, meeting the comprehensive performance requirements of injectable functional materials in clinical applications.

[0178] Figure 12 The extrusion photographs visually demonstrate the injectability of the hydrogel of this invention: after the formed gel is loaded into a syringe, it can be smoothly extruded by applying force. The extruded gel quickly recovers its shape and remains intact, proving that the hydrogel can fill irregular wounds in a minimally invasive manner. Furthermore, observing the appearance of the hydrogel, the hydrogel shows a significant color deepening trend with the increase of L-Dopa dosage. The L-Dopa dosage in Example 9 was twice that in Example 13, and the hydrogel product prepared from it was a deep brownish-black, while that in Example 13 was light brown. This difference in macroscopic color depth is highly consistent with the initial concentration of L-Dopa, visually confirming the increase in Pdopa generation in the system and demonstrating the adjustability of the material composition.

[0179] Test Example 4

[0180] The in vitro degradation performance and mechanical stability of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 5, 9, and 21 were tested. The in vitro degradation performance was tested as follows: 2 g of hydrogel was weighed and immersed in 4 mL of 0.5 mol / L PB solution (corresponding to pH 5.5 and pH 7.4, respectively), and incubated in a 37 ℃ constant temperature shaking incubator (100 rpm). During degradation, to maintain the activity of the degradation medium and the stability of the pH value, the immersion solution was completely removed and replaced with an equal volume of fresh PB solution every 12 h. Hydrogel samples were removed at predetermined time intervals, the surface moisture was dried, and the weight was measured. The percentage of remaining gel mass (n = 3) was calculated by comparing the remaining mass at a specific time point with the initial mass, and a degradation curve was plotted as a function of time. The mechanical stability test method is as follows: The injectable pH-responsive silk-based photothermal antibacterial hydrogel (a cylindrical hydrogel sample with a diameter of 20 mm and a thickness of 500 μm) prepared in Example 5 of the present invention was immersed in 5 mL of phosphate buffer solution (PB, 0.5 mol / L) with different pH values ​​(pH 5.5 and pH 7.4) for 120 hours. The dynamic rheological time-scan curve was tested by rotational rheometer. The hydrogel in the initial state (unimmersed) was used as a control sample to reflect its mechanical stability in solution environments with different pH values.

[0181] The in vitro degradation curves of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9 and 21 in phosphate buffer solutions of different pH values ​​are shown below. Figure 13 As shown, the degradation behavior of the hydrogel exhibits a significant pH dependence. Under conditions simulating a normal physiological environment (pH 7.4), the hydrogel experiences slow mass loss and demonstrates good stability. However, under weakly acidic conditions simulating the microenvironment of an infected wound (pH 5.5), the degradation rate of the hydrogel is significantly accelerated. This is because the calcium carbonate core within the hydrogel is acid-sensitive and undergoes a decomposition reaction (CaCO3 + 2H+) at pH 5.5. + → Ca 2+ The formation and escape of carbon dioxide bubbles (H₂O + CO₂↑) generate a large number of carbon dioxide bubbles. The formation and escape of these bubbles physically disrupt the gel network (i.e., the "bubble kinetics" effect), thereby accelerating the disintegration and mass loss of the gel matrix.

[0182] Example 5 shows the injectable pH-responsive silk-based photothermal antibacterial hydrogel and its dynamic rheological time-scan curves after immersion in phosphate buffer solutions of different pH values ​​for 120 h. Figure 14As shown, after soaking in a phosphate buffer solution at pH 7.4 for 120 h, the G' value of the hydrogel in Example 5 showed little change; however, after soaking in a phosphate buffer solution at pH 5.5 for the same time, its G' value decreased significantly. The decrease in mechanical strength is related to... Figure 13 The results of mass loss were consistent with those of the previous studies, confirming that the bubble effect generated by the decomposition of calcium carbonate under acidic conditions disrupted the crosslinking density and integrity of the gel network.

[0183] Test Example 5

[0184] The in vitro drug release performance of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 42 and the pure silk-based hydrogel loaded with FITC-EPL prepared in Comparative Example 17 was tested. The test method was as follows: 2 g of drug-loaded hydrogel sample was weighed and placed in a container containing 4 mL of 0.5 mol / L phosphate buffer solution (pH 5.5 and pH 7.4), and placed in a constant temperature environment of 37 ℃ for release experiments. The rotation speed of the constant temperature shaking chamber was 100 rpm. At the predetermined time point, 500 μL of release liquid was taken from the release medium as the test sample, and an equal volume of fresh PB solution was immediately added to keep the medium volume constant. The 500 μL of release liquid was transferred to a volumetric flask and diluted to 50 mL with PB (100-fold dilution). The fluorescence intensity of the diluted solution was measured using a fluorescence spectrophotometer, with the excitation wavelength set at 495 nm and the emission wavelength at 519 nm. The fluorescence intensity was converted into drug concentration according to the pre-established FITC-EPL standard curve, and then the cumulative percentage of drug release at different time points (n = 3) was calculated and the release curve was plotted.

[0185] The cumulative release curves of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 42 and the pure silk-based hydrogel loaded with FITC-EPL prepared in Comparative Example 17 in phosphate buffer solutions at different pH values ​​are shown below. Figure 15 As shown, the pH sensitivity of the hydrogel and the gas-driven release mechanism are revealed. Figure 15 (A) compared the release kinetics of different formulations in a simulated infection microenvironment (pH 5.5). The drug release rate of Example 42 was significantly higher than that of Comparative Example 17. At pH 5.5, the release curve of Example 42 exhibited a clear explosive growth characteristic, while Comparative Example 17 showed a relatively slow passive diffusion behavior. This significant difference confirms the unique proton-triggered-gas-driven mechanism of this invention: the calcium carbonate hybrid particles integrated in Example 42 are highly sensitive to acidic environments, especially in H... +Under induced degradation, CO2 bubbles are generated in situ. The rapid nucleation and expansion of these microbubbles within the gel create an active micro-driving force for cell disruption, physically widening the pore size of the silk fibroin network and even opening up closed pore structures, thereby significantly reducing the mass transfer resistance of the drug diffusion process (EPL). In contrast, Comparative Example 17, lacking this gas generation kinetic mechanism, still confines the drug molecules within a dense gel network, relying solely on slow concentration gradient permeation for release, resulting in a much lower release amount under the same acidic stimulation compared to Example 42. Figure 15 (B) further demonstrates the differences in release behavior of Example 42 under different pH conditions. The cumulative drug release rate of Example 42 at pH 5.5 is significantly higher than that at pH 7.4. At pH 5.5, the cumulative release rate exceeds 40% within 120 h, while under the neutral physiological condition of pH 7.4, the release curve tends to be stable, with a cumulative release rate of only about 30%. This indicates that under normal physiological pH, the hybrid microparticles maintain structural integrity and can effectively lock the drug within the gel network, avoiding the side effects of burst release; while in the locally acidified environment caused by bacterial infection, the hydrogel can respond rapidly and open the release channels. In summary, Figure 15 The results fully demonstrate that the hydrogel possesses the intelligent characteristic of on-demand drug delivery: through the comparison between Example 42 and Comparative Example 17, the core role of the acid-sensitive gas generation mechanism in accelerating drug release was confirmed, enabling it to accurately adapt to the acidic microenvironment in the early stage of bacterial infection and achieve rapid and efficient delivery of antibacterial drugs.

[0186] Test Example 6

[0187] The optical absorption characteristics of nanoparticle solutions (Pdopa-NPs prepared in Comparative Example 19 and EPL@Pdopa@CCMs prepared in Comparative Example 22) and the release solution collected after 120 h of sustained release in phosphate buffer solution (PB, pH 5.5, 0.5 mol / L) prepared in Example 9 were detected by ultraviolet absorption spectroscopy (UV-Vis). The test temperature was controlled at 25 °C. The nanoparticle solution of Comparative Example 19 was prepared by dispersing Pdopa-NPs in pure water (0.2 mg / mL); the nanoparticle solution of Comparative Example 22 was prepared by dispersing EPL@Pdopa@CCMs in pure water (0.2 mg / mL); Example 9 (release solution) was the release solution collected after 1 g of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9 was released for 120 h in 2 mL of phosphate buffer solution (PB, pH 5.5, 0.5 mol / L), the release temperature was 37 °C, and the rotation speed of the constant temperature shaking incubator was 100 rpm.

[0188] Test results are as follows Figure 16 As shown, both Comparative Example 19 (Pdopa-NPs) and Example 9 (release liquid) exhibit a characteristic absorption peak at 276 nm, which is completely consistent with the typical π-π* electronic transition behavior of Pdopa. The absorption spectrum of Comparative Example 22 (EPL@Pdopa@CCMs) shows a significant redshift of its characteristic peak to 335 nm. This phenomenon can be attributed to coordination and electrostatically induced redshift effects: within the hybrid particles, the catechol groups of Pdopa interact with Ca... 2+ Strong chelating coordination occurred, and a tight hydrogen bond and electrostatic network formed with the high-density EPL side-chain amino groups. This strong intermolecular interaction perturbed the conjugated electron system of Pdopa, lowering its excitation energy level, thus causing the absorption peak to shift towards longer wavelengths (335 nm). However, during the release process in Example 9, due to the decrease in ambient pH (pH 5.5), CaCO3 decomposed in an acidic environment, and the EPL component was released slowly. This process effectively dismantled the original hybrid network, allowing the Pdopa component to dissociate from the complex coordination environment. Spectroscopic results showed that the absorption peak of the release solution in Example 9 returned to 276 nm. This peak recovery phenomenon strongly demonstrates that despite the hybridization and release process, the core chemical structure of Pdopa was not destroyed, maintaining its excellent photothermal activity. It also confirms that the expected structural disintegration and component release of the hybrid particles occurred under acidic conditions. In summary, UV-Vis not only quantitatively tracked the release behavior of photothermal components, but also revealed the dynamic processes of hybrid particle assembly, response, and dissociation from the perspective of molecular energy levels, providing theoretical support for the material to achieve intelligent drug release and stable photothermal therapy in complex pathological environments.

[0189] Test Example 7

[0190] The injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9, 13, 21, and 25, as well as the THMs-embedded silk-based hydrogels prepared in Comparative Examples 3 and 6, and the pure silk-based hydrogels prepared in Comparative Examples 13 and 14, were subjected to photothermal performance tests. The photothermal heating curves and photothermal cycling stability curves of the hydrogels under near-infrared laser irradiation were recorded to verify their photothermal conversion ability and stability. The test conditions for the photothermal heating curves were as follows: a cylindrical hydrogel sample with a diameter of 4 cm and a thickness of 1 cm was placed on the test platform, and a near-infrared laser with a wavelength of 808 nm was used to vertically irradiate the sample surface, with the laser power density set to 1.5 W / cm². 2 The sample temperature was recorded in real time with irradiation time using an infrared thermal imager, with a continuous irradiation time of approximately 330 s. The test conditions for the photothermal cycling stability curve were as follows: under the same laser parameters (808 nm, 1.5 W / cm²),...2 The cylindrical hydrogel samples were subjected to a laser "on-off" cyclic irradiation test, that is, the laser was turned on to irradiate the sample until the temperature reached equilibrium, and then the laser was turned off to allow it to cool naturally to room temperature. This process was repeated for 4 cycles, and the temperature change data over time was recorded throughout the process.

[0191] The photothermal heating curves of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9, 13, 21, and 25, the THMs-embedded silk-based hydrogels prepared in Comparative Examples 3 and 6, and the pure silk-based hydrogels prepared in Comparative Examples 13 and 14 are shown below. Figure 17 As shown, Figure 17 (A) Systematically demonstrates the decisive influence of the photothermal component Pdopa on the temperature rise of the system. The silk-based hydrogels (Comparative Examples 13 and 3) without Pdopa showed almost no temperature fluctuation under continuous irradiation for up to 330 s, proving that the silk fibroin matrix and other inorganic components have extremely high transmittance to 808 nm near-infrared light and extremely low background absorption. In contrast, the hydrogel embedded with QHMs in Example 9 exhibited a rapid and significant temperature rise effect, with the system temperature increasing by more than 50 °C after only 330 s of irradiation, and the final temperature far exceeding the protein denaturation lethal temperature of common pathogenic bacteria (such as Staphylococcus aureus and Escherichia coli). Furthermore, a lateral comparison revealed that the temperature rise of Example 13 (with 50% of the L-Dopa dosage of Example 9) was significantly lower than that of Example 9, exhibiting a clear dose-dependent effect. This demonstrates that the in-situ generated Pdopa shell has excellent photothermal conversion efficiency, and its temperature rise efficiency can be precisely controlled by adjusting the L-Dopa loading. Figure 17 (B) further verified the universality of this pattern in matrices with different silk fibroin concentrations. In systems with lower silk fibroin concentrations (15 mg / mL), both Example 21 (high Pdopa content) and Example 25 (low Pdopa content) exhibited excellent photothermal response characteristics, and... Figure 17 Consistent with the trend in (A), the heating slope and the highest plateau temperature of Example 25 were both lower than those of Example 21. This series of results confirms that regardless of changes in the silk fibroin matrix concentration, the hybrid network constructed in this invention can maintain efficient photothermal conversion potential, thereby endowing the hydrogel with a powerful photothermal bactericidal ability to physically ablate bacterial structures and destroy biofilms through localized high temperatures.

[0192] The photothermal cycling stability curves of the injectable pH-responsive silk-based photothermal antibacterial hydrogels prepared in Examples 9 and 21 are shown below. Figure 18As shown, the photothermal stability of the hydrogel during repeated heating and cooling cycles was further evaluated. The experiment employed a four-cycle laser-on (heating) and off (cooling) mode, testing two silk fibroin concentration systems (Examples 9 and 21) with high and low concentrations. The results showed that after multiple repeated high-temperature shocks, the maximum temperature difference between the two examples did not show significant attenuation, and the heating and cooling curves of each cycle exhibited high consistency and overlap. This phenomenon reveals the superiority of the preparation process of this invention from a mechanistic perspective: the poly-L-DOPA component embedded in QHMs possesses excellent photostability and will not undergo photobleaching or oxidative degradation under continuous excitation by a strong near-infrared laser. Simultaneously, the multi-hybrid structure of QHMs and the physical encapsulation of the silk fibroin network effectively restrict the thermal movement and loss of the photothermal component, preventing it from detaching from the gel matrix during frequent thermal expansion and contraction. This ensures that the hydrogel maintains stable and reliable photothermal bactericidal properties during multiple, intermittent photothermal treatments, laying a solid foundation of safety and reliability for its clinical application as a functional wound dressing.

[0193] Test Example 8

[0194] The antibacterial properties of the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, the THMs-embedded silk-based hydrogel prepared in Comparative Example 2, the DHMs-embedded silk-based hydrogel prepared in Comparative Example 8, and the pure silk-based hydrogel prepared in Comparative Example 13 were tested. Their antibacterial activity against Staphylococcus aureus was quantitatively evaluated using the plate coating method. The test method was as follows: First, a single colony was picked and inoculated into LB liquid medium and cultured with shaking at 37 °C for 16 h; then, the initial concentration was increased to 1.0 × 10⁻⁶. 6 Staphylococcus aureus bacterial suspension at CFU / mL was co-cultured with hydrogel samples from each group in sterile 24-well plates. The blank control group was the PBS-treated group. The sample loading ratio was set at 100 μL of bacterial suspension for every 100 mg of hydrogel. Subsequently, a near-infrared laser with a wavelength of 808 nm (power density 1.5 W / cm²) was used for culturing. 2 The sample surface was vertically irradiated for 10 min. After the light treatment, the sample was transferred to a 37 ℃ incubator for 24 h to examine the long-lasting antibacterial effect of the hydrogel after photothermal intervention. After incubation, sterile PBS buffer was added to each well and the sample was repeatedly agitated to fully wash away bacteria attached to the gel and well walls. The eluent was serially diluted 10-fold, and 100 μL of the diluted solution was evenly spread on LB solid agar plates and incubated upside down at 37 ℃ for 24 h. Finally, images were acquired and counted using an automated colony counter. The bacterial concentration (CFU / mL) before spreading was calculated using the formula: "Bacterial concentration = Plate colony count × 10 × 10⁻⁶". n (10 of them)n The value is calculated by reverse calculation (representing the gradient dilution factor).

[0195] Images of Staphylococcus aureus colony growth after plating with the injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, the THMs-embedded silk-based hydrogel prepared in Comparative Example 2, the DHMs-embedded silk-based hydrogel prepared in Comparative Example 8, and the pure silk-based hydrogel prepared in Comparative Example 13 are shown below. Figure 19 As shown, the culture dish of Comparative Example 13 (without antibacterial agent EPL, no photothermal ability) was filled with extremely dense colonies, indicating that the silk fibroin matrix itself does not possess intrinsic antibacterial activity. In Comparative Example 8 (containing only EPL, no photothermal ability) and Comparative Example 2 (without antibacterial agent EPL, with photothermal ability), although the number of colonies was reduced compared to the control group, a clear colony distribution was still observed, indicating that single antimicrobial peptide therapy or physical thermotherapy is insufficient to completely eliminate pathogenic bacteria. In contrast, Example 9 (synergistic treatment group, EPL combined with photothermal therapy) exhibited a remarkably significant bactericidal effect, with very few colonies on the plate, almost reaching the visual level of sterile growth. This intuitive comparison demonstrates the significant synergistic effect produced by combining physical photothermal ablation with antimicrobial peptide drug killing. This synergistic mode overcomes the limitations of single treatment strategies, physically destroying bacterial structures through the instantaneous high temperature generated by photothermal action, while simultaneously achieving a long-term attack through the sustained release of EPL from the gel network, thus realizing rapid, deep, and thorough elimination of bacteria.

[0196] The injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared in Example 9, the THMs-embedded silk-based hydrogel prepared in Comparative Example 2, the DHMs-embedded silk-based hydrogel prepared in Comparative Example 8, the pure silk-based hydrogel prepared in Comparative Example 13, and the data on the concentration of viable bacteria obtained by plate count method after plating culture of Staphylococcus aureus in the blank control group (PBS treatment group) are shown in the figure. Figure 20 As shown, the bacterial concentration in the blank control group was maintained at 10. 7At a high loading level of approximately CFU / mL, Comparative Examples 8 and 2 reduced bacterial concentration by about two orders of magnitude, exhibiting moderate antibacterial activity. However, the bacterial concentration after treatment in Example 9 showed a precipitous decrease, with a reduction of nearly four orders of magnitude, achieving a bactericidal rate exceeding 99.9%. This superior bactericidal effect stems from the multiple synergistic mechanisms constructed in this invention. The localized heating caused by the photothermal effect not only directly induces pathogen protein denaturation but also significantly enhances the fluidity and permeability of the bacterial cell membrane, thereby providing a more favorable channel for the transmembrane entry of the cationic antimicrobial peptide EPL. Simultaneously, the near-infrared induced thermal effect reduces the mass transfer resistance of the hydrogel network, accelerates the diffusion of EPL from hybrid particles into the bacterial environment, and increases the local effective drug concentration. This forms a synergistic bactericidal strategy on both temporal and spatial scales, first achieving rapid targeted clearance through short-term photothermal treatment and then providing long-term antibacterial protection through the continuous release of EPL.

[0197] In summary, this invention effectively solves the technical problems of multi-component aggregation, uncontrollable drug release, and low single antibacterial efficiency through a gas diffusion-in-situ assembly process. The resulting injectable pH-responsive silk-based photothermal antibacterial hydrogel possesses excellent shear-thinning and injectability, achieves intelligent active drug release based on bubble dynamics, and achieves a bactericidal rate of over 99.9% through the synergistic effect of photothermal and chemical antibacterial action, combining immediate sterilization with long-term bacteriostasis. It has significant clinical application potential in addressing complex wound infections and drug-resistant bacterial infections.

[0198] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing an injectable pH-responsive silk-based photothermal antibacterial hydrogel, characterized in that, Includes the following steps: (1) Dissolve the antimicrobial peptide, levodopa and soluble calcium salt in an aqueous solution of silk fibroin to obtain a mixed solution; (2) The mixed solution obtained in step (1) is placed in a sealed container, and ammonium salt is used as a gas source to carry out the reaction at 25-80 °C; the ammonium salt is selected from one or more of ammonium bicarbonate, ammonium carbamate and ammonium carbonate; the gas source is decomposed by heat to produce ammonia and carbon dioxide, which diffuse into the mixed solution and generate calcium carbonate in situ, and induces levodopa to polymerize on the surface of calcium carbonate, while silk fibroin and antimicrobial peptide are co-precipitated and coated to obtain a reaction system containing quaternary hybrid particles; (3) The reaction system obtained in step (2) is subjected to ultrasonic treatment to induce conformational change of silk fibroin and form a gel network to obtain the injectable pH-responsive silk-based photothermal antibacterial hydrogel.

2. The production method according to claim 1, characterized by, In step (1), the antimicrobial peptide is selected from one or more of ε-poly-L-lysine, LL-37 acetate, nisin, protamine, poly(L-arginine) hydrochloride, polymyxin B, dermalin 2, bee venom peptide, insect antimicrobial peptide A, and insect antimicrobial peptide B; the soluble calcium salt is calcium chloride.

3. The production method according to claim 2, characterized by, In step (1), the weight-average molecular weight of the ε-poly-L-lysine is 2000-6000 Da, and the molecular weight distribution index is 1.1-3.

0.

4. The method of claim 1, wherein, In step (1), the silk fibroin aqueous solution is prepared by degumming raw silk, dissolving it in lithium bromide solution, dialysis, centrifugation, and then osmotically concentrating it in polyethylene glycol solution.

5. The preparation method according to claim 4, characterized in that, In step (1), the concentration of antimicrobial peptide in the mixed solution is 0.2-30 mg / mL; the concentration of levodopa in the mixed solution is 0.5-10 mg / mL; the molar concentration of calcium ions in the mixed solution is 34-204 mmol / L; and the concentration of silk fibroin in the mixed solution is 10-50 mg / mL.

6. The preparation method according to claim 1, characterized in that, In step (2), the gas source is placed at the bottom of the sealed container or in a separate container, and does not come into direct contact with the mixed solution.

7. The preparation method according to claim 1, characterized in that, In step (2), the ratio of the amount of gas source to the mixed solution is 0.27-2.15 g : 1 mL; in step (2), the molar ratio of the gas source to the calcium ions in the mixed solution is (150-250):

1.

8. The preparation method according to claim 1, characterized in that, In step (3), the ultrasonic treatment power is 100-200 W, the frequency is 15-25 kHz, and the amplitude is 10-30%; the ultrasonic treatment mode is intermittent ultrasound, which is on for 3-5 seconds and off for 3-5 seconds, with a total processing time of 60-1200 seconds.

9. An injectable pH-responsive silk-based photothermal antibacterial hydrogel prepared by the preparation method according to any one of claims 1-8.

10. The use of the injectable pH-responsive silk-based photothermal antibacterial hydrogel of claim 9 in the preparation of anti-infective wound dressings, soft tissue repair materials, or drug delivery carriers.

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