Spatial separation double-layer hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical antibacterial materials, and particularly relates to spatial separation double-layer hydrogel as well as a preparation method and application thereof. The spatial separation double-layer hydrogel is composed of an inner layer and an outer layer, wherein the inner layer is sepharose gel microspheres loaded with engineering lactic acid bacteria, and the outer layer is methacrylated gelatin and is loaded with a photosensitizer. By designing a loading structure of the lactic acid bacteria and the AIE photosensitizer in the gel, the problem of mutual interference of active components in the multifunctional hydrogel is solved, and the activity of the lactic acid bacteria and the photodynamic effect of the photosensitizer are ensured. The double-layer hydrogel not only can stably release bioactive substances at a wound part, but also can effectively reduce the risk of wound infection so as to promote the healing of a diabetic wound surface. And the hydrogel also has good biocompatibility, antibacterial activity and potential of promoting healing of diabetic refractory wounds, and provides a new thought for application of the multifunctional hydrogel in diabetic wound treatment.

Description

Technical Field

[0001] This application belongs to the technical field of biomedical antibacterial materials, and specifically relates to a spatially separated double-layer hydrogel and its preparation method and application. Background Art

[0002] Diabetic foot ulcers (DFUs) are one of the main complications of diabetic patients, referring to symptoms such as foot infections, suppuration, and ulceration in diabetic patients. DFUs heal slowly, are susceptible to infection, and prone to recurrence. In severe cases, amputation may be caused. Currently, for the treatment of diabetic foot ulcers, clinically, mainly methods such as anti-infection, negative pressure suction, improving local blood flow supply, and local thorough debridement are adopted. Although the current treatment methods are effective to a certain extent, due to reasons such as the difficulty of controlling infection and the poor healing ability of the body, the gangrene of the vast majority of patients still cannot be effectively controlled, and the condition continues to deteriorate. Therefore, it is very necessary to actively explore more reasonable treatment plans and research and develop new treatment methods to improve the treatment effect of DFUs.

[0003] Photodynamic therapy is a treatment method that induces local microbial inactivation through light radiation of a specific wavelength. Different from traditional antibacterial drugs, its mechanism is to transfer the excited energy to molecular oxygen through a photosensitizer, thereby generating reactive oxygen species with strong redox activity. Reactive oxygen species can act on the cell wall, cell membrane, and nucleic acid of bacteria, effectively killing antibiotic-sensitive bacteria and drug-resistant strains, while reducing the formation of drug resistance. Since reactive oxygen species exist for a very short time and the action range is limited to local tissues, this treatment method can effectively target local infections and avoid damage to surrounding healthy tissues. However, traditional organic photosensitizers (such as rose bengal and methylene blue) are limited in their photodynamic antibacterial effects due to defects such as easy aggregation quenching and low ROS generation efficiency. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a spatially separated double-layer hydrogel and its preparation method and application. The specific technical solutions are as follows: In the first aspect, the present invention provides a spatially separated double-layer hydrogel, and the spatially separated double-layer hydrogel includes: An inner layer gel, where the inner layer gel is an agarose gel microsphere loaded with engineered lactic acid bacteria; An outer layer gel, where the outer layer gel includes methacrylated gelatin and a photosensitizer loaded on the methacrylated gelatin; The photosensitizer is TPA-Py + -C4; the TPA-Py + -C4 has the following structural formula: In the present invention, live bacteria (engineered lactic acid bacteria) encapsulated in hydrogel can effectively protect the activity of probiotics and provide a stable microenvironment. The probiotics in the hydrogel can be protected from the adverse effects of the wound environment, thereby prolonging their survival time at the wound site. In addition, the application of photocurable hydrogels in wound treatment further expands the advantages of hydrogels. Such hydrogels can rapidly solidify under light irradiation, providing better adaptability and precisely conforming to wounds of various shapes and sizes. Among them, methacrylated gelatin (GELMA) is an important biomedical material, and its properties are similar to those of natural extracellular matrix. Under light irradiation with a wavelength of 405 nm, GELMA can crosslink to form a stable gel structure, which can serve the purpose of drug release in the present invention.

[0005] Among them, (1) the inner-layer gel is in the form of microspheres, loaded with engineered lactic acid bacteria (Lactococcus lactis MG1363), which can continuously release CXCL12 and lactic acid in situ, activate the immune system and promote wound healing; the outer-layer gel has photocuring properties, can adapt to different wound shapes, and the loaded photosensitizer TPA-Py + -C4 can provide continuous antibacterial and anti-biofilm effects and promote wound healing.

[0006] (2) The photosensitizer TPA-Py + -C4 has aggregation-induced emission characteristics, with the strongest fluorescence emission in toluene. As the concentration of toluene increases, its fluorescence intensity increases and shows a blue shift; TPA-Py + -C4 can significantly generate reactive oxygen species (ROS), including singlet oxygen (¹O2) and superoxide anion (O2 •- ), and its ROS generation efficiency is superior to that of the traditional photosensitizer Rose Bengal (RB).

[0007] (3) Methacrylated gelatin (GELMA) can transform from a liquid gel to a solid gel structure after being irradiated with 405 nm blue light, and has good rheological properties, water retention rate, swelling rate and stability, which helps to maintain the moist environment of the wound; and the freeze-dried GELMA gel shows obvious structural and morphological characteristics, with a uniform distribution of microspheres inside.

[0008] As a further preferred embodiment, the engineered lactic acid bacteria is Lactococcus lactis MG1363; the Lactococcus lactis MG1363 is obtained by genetic engineering transformation; The synthesis route of the TPA-Py + -C4 is as follows: .

[0009] The above-mentioned Lactococcus lactis MG1363 is genetically engineered to be able to express CXCL12 in situ in the culture medium. After 12 hours of culture, its expression level reaches 1200 pg / mL, and the Lactococcus lactis MG1363 shows a faster growth rate than the wild-type lactic acid bacteria in the initial stage of culture.

[0010] In a second aspect, the present invention provides a method for preparing the above-mentioned spatially separated double-layer hydrogel, comprising the following steps: Mix the engineered lactic acid bacteria, agarose solution, M17 medium, and glucose, then add mineral oil, and stir until the solution becomes a milky white emulsion. Centrifuge and wash to obtain agarose gel microspheres loaded with the engineered lactic acid bacteria. Mix the agarose gel microspheres, methacrylated gelatin solution, photoinitiator, and photosensitizer, then inject them into a mold and cure them by blue light irradiation to obtain the spatially separated double-layer hydrogel.

[0011] In the above preparation process, the preparation method of the agarose gel microspheres (MPS) loaded with the engineered lactic acid bacteria is the emulsion method, and the average particle size of the microspheres is 68.4 μm; among them, the addition of M17 medium and glucose can significantly improve the survival rate of Lactococcus lactis MG1363 in the microspheres.

[0012] As a further preferred embodiment, the concentration of the agarose solution is 0.5%-1%; the concentration of the methacrylated gelatin solution is 100 mg / mL - 150 mg / mL; the concentration of the photoinitiator is 2.5 mg / mL - 3.5 mg / mL; the final concentration of the M17 medium is 10 mg / mL; the final concentration of the glucose is 50 mg / mL. Among them, in the above preparation process, (1) the higher the agarose concentration, the larger the size and the stronger the hardness of the formed microspheres; the reason is that the higher the agarose concentration, the greater the viscosity of the solution, which makes it easier for the polymer to coagulate during the formation of the microspheres, thus forming larger and harder microspheres. (2) The higher the concentration of the methacrylated gelatin solution, the faster the photocuring speed; the reason is that the photosensitive groups in methacrylated gelatin will undergo a cross-linking reaction when irradiated by light, forming a three-dimensional network structure. A high concentration of methacrylated gelatin means more cross-linking points per unit volume, so it will accelerate the photocuring process. (3) The higher the concentration of the photoinitiator, the faster the photocuring speed; the reason is that an increase in the concentration of the photoinitiator will lead to the generation of more free radicals or reactive species, thus promoting the cross-linking reaction of the polymer chains and making the photocuring reaction more rapid. However, too high a concentration of the photoinitiator may lead to an overly fast polymerization reaction, resulting in an uneven cross-linked structure. Therefore, this concentration range is the optimized concentration to obtain an ideal curing speed and uniformity.

[0013] As a further preferred embodiment, the temperature of the stirring is 45°C - 75°C, the speed of the stirring is 800 rpm - 1200 rpm, and the time of the stirring is 5 min - 15 min; the time of the blue light irradiation curing is 30 s - 90 s.

[0014] In a third aspect, the present invention provides the use of the above-mentioned spatially separated double-layer hydrogel in the preparation of antibacterial materials.

[0015] As a further preferred embodiment, the above-mentioned bacteria include at least one of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Methicillin-resistant Staphylococcus aureus.

[0016] The results of the examples of the present invention show that the spatially separated double-layer hydrogel exhibits excellent antibacterial properties and can effectively inhibit and kill four common pathogenic bacteria including Escherichia coli ( Escherichia coli , EC), Staphylococcus aureus ( Staphylococcus aureus , SA), Pseudomonas aeruginosa ( Pseudomonas aeruginosa , PA) and Methicillin-resistant Staphylococcus aureus ( Staphylococcus aureus , MRSA). In addition, the hydrogel has a significant inhibitory effect on the formation of bacterial biofilms, can efficiently remove the formed biofilms, and can be used for the prevention and treatment related to antibacterial and anti-biofilm.

[0017] In a fourth aspect, the present invention provides an antibacterial material, which includes the above-mentioned spatially separated double-layer hydrogel.

[0018] In a fifth aspect, the present invention provides the use of the above-mentioned spatially separated double-layer hydrogel in the preparation of products for promoting the healing of diabetic wounds.

[0019] In a sixth aspect, the present invention provides a dressing for promoting the healing of diabetic wounds, and the dressing includes the above-mentioned spatially separated double-layer hydrogel.

[0020] According to the results of the examples of the present invention, the spatially separated double-layer hydrogel can significantly promote the healing of full-thickness skin wounds of type II diabetic mice.

[0021] Compared with the prior art, the beneficial effects of the present invention at least include: (1) The present invention provides a double-layer hydrogel, which can exert therapeutic effects at different stages of wound healing. This dressing synergizes photodynamic antibacterial and wound-healing promotion, and can significantly promote the healing of diabetic wounds. Compared with the prior art, the present invention confines the engineered bacteria inside the hydrogel, reducing the irritation of immunogenic substances on the surface of Lactococcus lactis MG1363 to tissues. The inability of Lactococcus lactis MG1363 to leak out also effectively prevents it from entering the blood circulation, reducing the risks such as sepsis caused by treatment, and further ensuring the safety of treatment; (2) The present invention optimizes the components of Lactococcus lactis gel microspheres, ensuring the activity of probiotics inside the gel microspheres. During the synthesis of the gel microspheres in the present invention, by adding nutrients such as glucose and M17 medium, the internal environment of the gel microspheres is optimized. Compared with the pure hydrogel environment, the osmotic pressure inside the gel after adding nutrients is not only more suitable for the growth of Lactococcus lactis MG1363, but also can provide sufficient nutrients for its growth and metabolism; (3) The present invention has excellent antibacterial effects: the double-layer hydrogel of the present invention shows significant inhibitory effects on a variety of pathogenic bacteria (such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus), and can effectively inhibit and remove the corresponding bacterial biofilms; (4) The double-layer hydrogel of the present invention is designed with a spatially separated structure, which separates Lactococcus lactis and the photosensitizer in different spaces, avoiding direct contact between the two, and thus ensuring that their respective functions are not affected. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 The schematic diagram of the preparation principle of the double-layer hydrogel G / MPS / C4 of the present invention is shown, where a in the figure is the preparation process of the gel microspheres; b in the figure is the preparation process of the double-layer dressing; c in the figure is the animal experiment process.

[0024] Figure 2 The antibacterial and anti-biofilm effects of the double-layer hydrogel G / MPS / C4 on four common pathogenic bacteria are shown, where a in the figure is the antibacterial plate; b in the figure is the live and dead bacteria staining; c in the figure is the removal effect on the mature biofilm; d in the figure is the partial enlarged schematic diagram of the damaged biofilm.

[0025] Figure 3The therapeutic effects of hydrogels with different components on infected diabetic wounds are shown. Among them, a in the figure is the treatment timeline of mice; b in the figure is the photos of the wound at different time points during the treatment and the changes of the wound; c in the figure is the wound healing rate; d in the figure is the change of blood glucose.

[0026] Figure 4 The active protection effect of the double-layer hydrogel engineered bacteria is shown. Among them, a in the figure is a schematic diagram of the synthetic structure comparison between the single-layer hydrogel and the double-layer hydrogel; b in the figure is the growth curve of the engineered bacteria measured in different gels; c in the figure is the plate counting result after replacing the photosensitizer with an antibiotic; d and e in the figure are the corresponding plate photos. Specific embodiments

[0027] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the protection scope of the present application.

[0028] Example 1 A spatially separated double-layer hydrogel loaded with engineered lactic acid bacteria and AIE photosensitizer, and its preparation method includes the following steps: Step 1: The process of genetic engineering transformation of engineered lactic acid bacteria (Lactococcus lactis MG1363) is as follows: 1. Construction of pMG36e CXCL12 plasmid (1) Construction of pMG36e-CXCL12 recombinant plasmid: The pUC57-CXCL12 recombinant plasmid was obtained by chemical synthesis of the full sequence of PstI-SPusp45-CXCL12-HindIII; (2) Construction of pMG36e-CXCL12 recombinant plasmid: A: Use the OMEGA plasmid miniprep kit to extract pMG36e and pUC57-CXCL12 plasmids; B: The enzyme digestion system of pMG36e and pUC57-CXCL12 is as follows. The total system is 25 μL, and the enzyme digestion is carried out at 37 °C for 4 h; C: Prepare a 2% agarose gel electrophoresis, and perform electrophoresis at 110 V for 45 min; Cut and recover the gel at about 276 bp and 3600 bp; D: The enzyme ligation system is: the total system is 10 μL, DNA fragment: vector = 5:1 and 10:1, and incubate overnight at 10 °C; E: The transformation consists of the following steps: a: Take 200 μL of MC1061 competent cell suspension from an -80 °C refrigerator and thaw it on ice. b: Add 2 μL of plasmid solution with a plasmid concentration of not less than 200 ng / μL, gently shake well, and place it on ice for 30 min. c: Perform heat shock in a 42 °C water bath for 90 s, and immediately place it on ice to cool for 3 min - 5 min after heat shock. d: Add 800 μL of LB liquid medium to the tube, mix well, and culture it with shaking at 37 °C for 1 h to allow the bacteria to resume normal growth state. Centrifuge at 4000 rpm for 1 min, discard 800 μL of the supernatant, and leave 200 μL of the bacterial solution, then mix well. e: After shaking the above bacterial solution well, take 200 μL and spread it on a screening plate containing 300 ng / μL erythromycin. Place it face up for half an hour. After the bacterial solution is completely absorbed by the medium, invert the culture dish and culture it at 37 °C for 16 h - 24 h, then pick a single clone. F: Sequencing G: After successful sequencing, extract the recombinant plasmid pMG36e-CXCL12 using the OMEGA plasmid miniprep kit and store it for later use. The steps are the same as above.

[0029] 2. Electrotransform the prepared pMG36e-CXCL12 into Lactococcus lactis MG1363. The transformation process consists of the following steps: A: Preparation of Lactobacillus competent cells: a: Inoculate the cryopreserved Lactococcus lactis MG1363 at -80 °C into 5 mL of M17 Lactobacillus liquid medium containing 5% glucose and culture it overnight at 30 °C. b: Inoculate the obtained bacterial solution at 1% into M17 Lactobacillus liquid medium containing 2.5% Gly and 5% glucose, and culture it statically at 30 °C until the OD 600 value is 0.3 - 0.4, then collect and reserve it. c: Ice-bath the above collected bacterial culture solution for 10 min, centrifuge at 5000 rpm at 4 °C for 5 min; collect the bacteria. d: Wash the precipitate twice with a 1 / 10 volume of a mixed solution of ice-cold 10% sucrose and 10% glycerol, centrifuge at 8000 rpm at 4 °C for 5 min, and collect the precipitate. e: Finally, resuspend the precipitate in a 1 / 100 volume of a mixed solution of 10% sucrose and 10% glycerol, and it can be used after ice-bathing for 10 min. B: Electrotransformation of Lactococcus lactis MG1363: a: Take 2 μL of the recombinant plasmid with a concentration of not less than 150 ng / μL. After gently mixing it with 50 μL of the ice-cold Lactobacillus competent cell suspension prepared by the above method, add it to a pre-cooled electroporation cuvette with a spacing of 0.1 cm, place it on ice for 5 min, and set the conditions as 1800 V, 200 Ω, 25 μF; b: After electroporation, quickly aspirate the liquid in the electroporation cuvette into a centrifuge tube, and simultaneously add 800 μL of M17 liquid medium containing 5% glucose. Incubate at 30 °C for 2 h. Take 100 μL of the transformed product and spread it on an M17 plate containing 5% glucose with erythromycin resistance, and incubate statically at 30 °C for 24 h - 72 h to screen for positive clones.

[0030] Step 2: Synthesis of the AIE photosensitizer C4 (TPA-Py + -C4), the specific steps are as follows: 4-(Diphenylamino)benzaldehyde and 1-butyl-4-methylpyridinium iodide are used to synthesize TPA-Py + -C4 through an aldol condensation reaction in a methanol solvent. The specific process is as follows: First, dissolve 1.5 g of 4-(diphenylamino)benzaldehyde and 1.38 g of 1-butyl-4-methylpyridinium iodide in 20 mL of methanol. Then add 3 drops of piperidine as a catalyst and stir the reaction at 50 °C for 4 h; during the reaction, piperidine first abstracts the proton on the methyl group of the pyridinium salt to form a carbanion, and this carbanion nucleophilically attacks the carbonyl carbon of the aldehyde group to generate a β-hydroxy intermediate. Subsequently, dehydration occurs under heating conditions to form a stable C=C double bond, constructing a conjugated system with a D-π-A structure. After the reaction is completed, the methanol solvent is removed by vacuum distillation to obtain the crude product, and then column chromatography purification is carried out using dichloromethane / methanol (30:1) as the eluent. The reaction formula of this reaction is as follows: Step 3: Preparation of agarose gel microspheres loaded with engineered Lactobacillus Select 1 mL of Lactococcus lactis MG1363 in the logarithmic growth phase obtained in step (1), use a centrifuge to centrifuge at 5000 rpm for 10 min to remove the culture medium, and retain the precipitate part; Prepare an agarose solution with a concentration of 0.5%. Dissolve the agarose in deionized water and heat it in a microwave oven to 90 °C until the agarose is completely dissolved, ensuring that the solution is clear and free of particulate matter. When the agarose solution cools to 45 °C, add 500 μL of the agarose solution to the precipitate of Lactococcus lactis MG1363, then add 5 mg of M17 medium powder and 25 mg of glucose powder, and mix well to obtain a mixture. Dropwise add the mixture into 3 mL of mineral oil containing 2% Span, set the stirring rate to 1000 rpm, and stir for 3 min. At this time, the mixture changes from a clear and transparent state to a milky white emulsion state. During the stirring process, the agarose solution gradually condenses to form gel microspheres. Centrifuge at 2000 rpm for 1 min to remove the mineral oil supernatant, wash the gel microspheres three times with petroleum ether, then wash them three times with deionized water, and finally transfer the gel microspheres to the aqueous phase, quantify to 1 mL, and store at 4 °C for later use.

[0031] Step 4: Prepare a spatially separated double-layer hydrogel First, prepare a methacrylated gelatin (GELMA) stock solution: Take 120 mg of GELMA freeze-dried product, add 1 mL of ultrapure water, and shake several times to ensure complete wetting. Place the solution in a water bath at 60 °C - 70 °C and heat for 30 min until completely dissolved. After the solution is completed, store it in a 4 °C dark environment for later use. Prepare a photoinitiator stock solution: Take 3 mg of photoinitiator DMA (dimethyl acrylate) powder, add it to 1 mL of phosphate buffer solution (PBS), heat it in a water bath at 40 °C - 50 °C for 15 min, and shake until completely dissolved to ensure that no precipitate forms in the solution. Prepare an AIE photosensitizer C4 (TPA-Py + -C4) stock solution: Dissolve 10 mg of TPA-Py + -C4 in 1 mL of DMSO (dimethyl sulfoxide), and shake until completely dissolved to obtain an AIE photosensitizer C4 (TPA-Py + -C4) stock solution. Mix 60 μL of the GELMA stock solution, 30 μL of the gel microspheres, 9 μL of the photoinitiator stock solution, and 1 μL of the TPA-Py + -C4 stock solution. After ensuring uniform stirring, inject the mixture into a cylindrical mold with a diameter of 5 mm. Finally, irradiate with 405 nm blue light for 30 s to complete the photocuring reaction and obtain a spatially separated double-layer hydrogel (G / MPS / C4).

[0032] During the curing process, the lactic acid bacteria gel microspheres are effectively embedded in the gel, and at the same time, TPA-Py +-C4 is evenly distributed in the gel structure, ensuring that the dressing has the functions of photodynamic antibacterial and promoting wound healing.

[0033] Example 2 The space-separated double-layer hydrogel prepared in Example 1 was subjected to an antibacterial experiment test. The specific process is as follows: Escherichia coli (EC), Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and methicillin-resistant Staphylococcus aureus (MRSA) were selected for antibacterial testing. The experimental steps are as follows: First, the four bacteria were respectively cultured to the logarithmic growth phase and diluted to 1×10 4 CFU / mL using fresh LB medium. Subsequently, 5 mL of the diluted bacterial solution was transferred to a test tube, and then the G / MPS / C4 gel dressing was added and subjected to light treatment. The light treatment conditions were white light irradiation at 50 W / m² for 15 min. After the light treatment was completed, at regular intervals, 100 μL of the treated bacterial solution was evenly spread on a plate containing LB agar and placed in an incubator at 37°C for 24 hours, and the number of bacterial colonies in each group was recorded.

[0034] Calculate the relative bacterial viability: Relative bacterial viability = (N b / N a )×100%, where N a is the number of bacterial colonies in the control group, and N b is the number of bacterial colonies in the experimental group.

[0035] Subsequently, a BB / cell live-dead bacterial staining kit was used to evaluate the bacterial survival rate. The staining conditions of the bacteria were observed through a fluorescence microscope before and after staining, so as to visually evaluate the inhibitory effect of the hydrogel on bacteria.

[0036] Finally, in order to further evaluate the anti-biofilm ability of the G / MPS / C4 gel dressing, 500 µL of a Staphylococcus aureus (1×10 5 CFU / mL) suspension was added to a 24-well plate and allowed to stand in an incubator at 37°C for 48 h to form a bacterial biofilm. The free bacteria were removed by washing with 0.01 M PBS, and then the pre-prepared liquid gel was added and treated at 37°C for 20 min, followed by light treatment. After treatment, staining was performed and the destruction of the bacterial biofilm was observed.

[0037] The results are shown in Figure 2 in a- Figure 2As shown in d of [reference], the experimental results indicate that G / MPS / C4 has significant antibacterial effects against four pathogenic bacteria (EC, PA, SA, MRSA), and the antibacterial effect is enhanced by light treatment. The results of live / dead bacteria staining show that the bacteria in the G / MPS / C4 group are hardly stained, indicating that the bacteria in these groups are completely killed (as shown in Figure 2 b of [reference]). The results of the anti-biofilm experiment show that the bacterial biofilm in the G / MPS / C4 group is the most significantly damaged (as shown in Figure 2 c of [reference]). In contrast, the structure of the bacterial biofilm in the PBS group remains intact. Further microscopic observations (as shown in Figure 2 d of [reference]) show that the structure of the bacterial biofilm in the G / MPS / C4 group is significantly damaged, with fragments of bacterial communities appearing, while the PBS group maintains an intact membrane structure. It is shown that under light treatment, the double-layer hydrogel G / MPS / C4 can effectively kill bacteria and remove bacterial biofilms.

[0038] Example 3 The space-separated double-layer hydrogel prepared in Example 1 was used to measure the therapeutic effect on infected diabetic wounds. The specific process is as follows: To comprehensively evaluate the effect of G / MPS / C4 on the healing efficiency of diabetic wounds in vivo, in this study, SPF-grade male BALB / c mice aged 6 - 8 weeks were selected. A method of combining STZ with a high-sugar and high-fat diet was used to induce diabetes, and on this basis, a full-thickness diabetic wound model infected with MRSA was constructed. The specific experimental steps are as follows: First, a diabetic mouse model was constructed. Prepare solution A and solution B. Solution A consists of 2.1 g of citric acid and solution B consists of 2.94 g of sodium citrate dissolved in 100 mL of deionized water. Then, solution A and solution B were mixed in a ratio of 1:1.32 and precisely adjusted to a pH of 4.2 - 4.5 for standby. 100 mg of streptozotocin powder was dissolved in 10 mL of freshly prepared sodium citrate buffer solution, and the solution was filtered through a 0.22 μm filter to prepare a sterile STZ solution, which was stored in the dark at 4°C. Male BALB / c mice at 7 weeks of age were purchased and started the experiment after one week of adaptive feeding. After the mice were fasted for 12 hours, the prepared streptozotocin solution was injected intraperitoneally at a dose of 50 mg per kilogram of body weight. The injection continued for 5 days, and a high-sugar and high-fat diet was provided simultaneously. After the injection, 5 mm of the mouse's tail was cut off and 2 μL of blood was collected, and the blood glucose concentration was measured using a blood glucose meter. Mice with a blood glucose concentration reaching or exceeding 16.7 mmol / L were considered to have reached the blood glucose standard and entered the diabetes group. Mice that did not meet the standard continued to be injected with the streptozotocin solution until the blood glucose level met the standard. The blood glucose concentration was measured every three days, and mice with a blood glucose concentration higher than 16.7 mmol / L for two consecutive weeks were confirmed to have successfully established a diabetes model and maintained a stable hyperglycemic state.

[0039] Subsequently, a full-thickness infected diabetic wound model was constructed. A pentobarbital sodium solution with a concentration of 30 mg / mL was prepared using normal saline. After thorough mixing, according to the body weight of the mice, 1 mL / kg of the pentobarbital sodium solution was intraperitoneally injected for general anesthesia. The anesthetized mice were fixed on the operating table, and a full-thickness skin wound with a diameter of 6 mm was constructed using sterile ophthalmic scissors. A solution of MRSA at a concentration of 1×10 6 CFU / mL was inoculated onto the wound to construct an MRSA-infected diabetic wound.

[0040] Next, the full-thickness skin infected diabetic wounds were treated: The 6 groups of mice with successfully established diabetic wounds were randomly divided into six groups, including a control group (PBS group) and five treatment groups (G group, G / MPS group, G / C4 / L group, G / MPS / C4 / D group, and G / MPS / C4 / L group). There were six mice in each group. The specific treatment groups were as follows: PBS group, G group: treated with a single GELMA hydrogel (wherein, G / MPS group: treated with GELMA hydrogel loaded with engineered probiotic gel microspheres; G / C4 / L group: treated with GELMA hydrogel loaded with photosensitizer C4 and combined with light treatment; G / MPS / C4 / D group: treated with GELMA hydrogel loaded with engineered probiotic gel microspheres and C4 without light treatment; G / MPS / C4 / L group: treated with GELMA hydrogel loaded with engineered probiotic gel microspheres and C4 and combined with light treatment). The treatment process was as follows: First, the liquid gels of the five different treatment groups were precisely dropped onto the wound surface. Then, blue light irradiation was performed for 30 seconds to promote the cross-linking reaction of the liquid hydrogel and convert it into a solid gel. Once the solid gel was formed, the wound surface was covered with a sterile transparent surgical patch. The new hydrogel dressing was changed every 24 hours, and the changes in the wound surface were recorded every two days using a digital camera. Then, the wound surface area was quantitatively analyzed using Image J software to calculate the wound healing rate. The wound healing rate was calculated using the following formula: Healing rate = ((Initial wound area - Unhealed wound area) / Initial wound area) × 100%.

[0041] The experimental results showed that as Figure 3 in a- Figure 3As shown in b of [Figure 0], over time, the wounds in all treatment groups showed a healing trend. Specifically, the wound healing rate of the PBS group was the slowest, while the wound contraction in other treatment groups (including the G group, G / MPS group, G / C4 / L group, G / MPS / C4 / D group, and G / MPS / C4 / L group) was relatively fast. In particular, the healing effect of the G / MPS / C4 / L group was significantly better than that of other gel treatment groups. On the 2nd day of treatment, obvious suppuration and yellowish-white biofilms appeared in all wounds, and there was no significant difference among the groups. This was because the wounds were in the early acute inflammatory phase with severe inflammatory reactions. However, by the 4th day, there was still severe yellow pus in the wounds of the PBS group, while the wounds in other groups treated with gels were relatively drier. This was mainly because the swelling of the gel dressing could absorb the exudates from the wounds.

[0042] On the 6th day, the healing conditions of the wounds began to be significantly different. According to the healing rate statistics ( Figure 3 shown in c of [Figure 0]), the G / MPS / C4 / L group had the highest healing rate, while the healing rates of the G / C4 / L group and the G / MPS / C4 / D group increased slightly. This was because the bactericidal effect of C4 helped eliminate the pathogenic bacteria causing infection and relieve the inflammation during the wound transition period. By the 8th day, the healing rate of the G / MPS / C4 / L group reached 89.4%, which was significantly higher than that of other groups (such as 76.6% for the G / C4 / L group, 72.5% for the G / MPS group, 66.6% for the G group, and 62.4% for the PBS group). At this time, the wounds in the G / MPS / C4 / L group had been completely covered with epithelium and were almost completely healed, leaving only tiny scars. Meanwhile, blood glucose monitoring during the treatment proved that the experimental model was stable ( Figure 3 shown in d of [Figure 0]). The above research results indicate that G / MPS / C4 / L shows excellent effects in promoting wound healing.

[0043] Comparative Example 1 To evaluate the spatial separation effect of the double-layer hydrogel, especially the protective effect on probiotics, a single-layer gel ( Figure 4 shown in a of [Figure 0]) was set as a control in this study, and the specific operation was as follows: First, 30 μL of 10 8 CFU / mL MG1363 culture solution, 60 μL of GELMA mother solution, and 1 μL of TPA-Py + -C4 were mixed evenly, and then 9 μL of DMA was added, and the mixture was irradiated with blue light for 30 seconds to form a gel, thus preparing the single-layer gel.

[0044] The monolayer gel prepared in this comparative example and the bilayer gel (G / MPS / C4) prepared in Example 1 were separately added to 50 mL of LB medium and cultured under white light irradiation. 200 μL of samples were taken every 1 hour, and the culture was continuously monitored for 36 hours to measure the absorbance value at 600 nm. Finally, with the culture time as the abscissa and OD 600 nm as the ordinate, a bacterial growth curve was plotted to evaluate the activity of bacteria inside the gel dressing.

[0045] The results are as shown in Figure 4 b in it. The growth of Lactococcus lactis MG1363 slowed down after the monolayer gel was treated with light. This may be because a large amount of ROS was generated by the photosensitizer C4 under light, which had an obvious killing effect on the surrounding Lactococcus lactis MG1363, thereby resulting in a weakened growth activity. However, when Lactococcus lactis MG1363 was embedded in the gel microspheres, the spatial isolation effect of the microspheres effectively avoided the influence of ROS on the growth activity of Lactococcus lactis MG1363 under light treatment, laying a foundation for its subsequent better exertion of functional activity.

[0046] To further verify the protective effect of the spatial isolation strategy on active microorganisms, in this study, TPA-Py + -C4 in the bilayer gel was replaced with small molecule antibiotics (including ampicillin, kanamycin, and amoxicillin), and the growth activities of microorganisms in the monolayer gel and the bilayer gel were studied by the plate growth method. The results are as shown in Figure 4 c- Figure 4 e in it. There were obvious differences in the plate coating results of the monolayer gel and the bilayer gel. The Lactococcus lactis MG1363 embedded in the bilayer gel maintained a high growth activity, while the bacteria loaded in the monolayer gel were almost completely killed, and only a few scattered colonies remained on the plate.

[0047] The above results indicate that the bilayer gel can effectively avoid the non-specific killing of loaded drugs on active microorganisms through the spatial isolation effect, so that the active microorganisms can better exert their functional activities.

[0048] In summary, the present invention discloses a preparation method of a spatially separated double-layer hydrogel loaded with functional active lactic acid bacteria and aggregation-induced emission (AIE) photosensitizers, and introduces its application in the treatment of diabetic wounds. The prepared hydrogel has an inner and outer double-layer structure: the inner layer is agarose gel microspheres (MPS) loaded with active lactic acid bacteria, and the outer layer is a gelma gel loaded with AIE photosensitizers. Due to the separated design of the inner and outer layer gels, the loaded lactic acid bacteria and AIE photosensitizers can independently perform their respective functions. The lactic acid bacteria in the inner agarose microspheres continuously produce chemotactic factors and lactic acid, regulating the wound microenvironment and promoting wound healing. The photodynamic properties of the AIE photosensitizers in the outer gelma gel provide antibacterial effects, clearing pathogenic bacteria and activating immune responses to accelerate wound healing.

[0049] By designing the loading structure of lactic acid bacteria and AIE photosensitizers in the hydrogel, the present invention overcomes the problem of mutual interference of active ingredients in the multifunctional hydrogel, ensuring the activity of lactic acid bacteria and the photodynamic effect of the photosensitizer. This double-layer hydrogel can not only stably release bioactive substances at the wound site, but also effectively reduce the risk of wound infection to promote the healing of diabetic wounds. Through the characterization of material properties, antibacterial properties and immunomodulatory effects, it is verified that the hydrogel of the present invention has good biocompatibility, antibacterial activity and the potential to promote the healing of difficult-to-heal diabetic wounds, providing new ideas for the application of multifunctional hydrogels in the treatment of diabetic wounds.

[0050] The embodiments of the present application have been described above in conjunction with the accompanying drawings. Specific examples are used herein to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only for helping to understand the core idea of the present application. However, the present application is not limited to the above specific implementation manners. The above specific implementation manners are merely illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all belong to the protection scope of the present application.

Claims

1. A spatially separated double-layer hydrogel, characterized in that, The spatially separated double-layer hydrogel includes: An inner gel, which is an agarose gel microsphere loaded with engineered lactic acid bacteria. An outer gel, which includes methacrylated gelatin and a photosensitizer loaded on the methacrylated gelatin. The photosensitizer is TPA-Py + -C4; The TPA-Py + -C4 has the following structural formula: 。 2. The spatially separated double-layer hydrogel according to claim 1, characterized in that The engineered lactic acid bacteria is Lactococcus lactis MG1363; the Lactococcus lactis MG1363 is obtained by genetic engineering modification. The TPA-Py + -C4 synthesis route is as follows: 。 3. The preparation method of the spatially separated double-layer hydrogel according to any one of claims 1-2, characterized in that, It includes the following steps: Mix the engineered lactic acid bacteria, agarose solution, M17 medium, and glucose, then add mineral oil, stir until the solution becomes a milky white emulsion, centrifuge, and wash to obtain agarose gel microspheres loaded with engineered lactic acid bacteria. Mix the agarose gel microspheres, methacrylated gelatin solution, photoinitiator, and photosensitizer, then inject them into a mold and cure them by blue light irradiation to obtain the spatially separated double-layer hydrogel.

4. The preparation method according to claim 3, characterized in that, The concentration of the agarose solution is 0.5%-1%; the concentration of the methacrylated gelatin solution is 100 mg / mL - 150 mg / mL; the concentration of the photoinitiator is 2.5 mg / mL - 3.5 mg / mL.

5. The preparation method according to claim 3, characterized in that, The temperature of the stirring is 45°C - 75°C, the speed of the stirring is 800 rpm - 1200 rpm, the time of the stirring is 5 min - 15 min; the time of the blue light irradiation curing is 30 s - 90 s.

6. Use of the spatially separated double-layer hydrogel according to any one of claims 1-2 in the preparation of an antibacterial material.

7. The application according to claim 6, wherein The bacteria include at least one of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus.

8. An antibacterial material, characterized in that, The antibacterial material includes the spatially separated double-layer hydrogel according to any one of claims 1-2.

9. Use of the spatially separated double-layer hydrogel according to any one of claims 1-2 in the preparation of a product for promoting diabetic wound healing.

10. A dressing for promoting the healing of diabetic wounds, characterized in that, The dressing includes the spatially separated double-layer hydrogel according to any one of claims 1-2.

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