A spatially separated double-layer hydrogel and its preparation method and application

Abstract

The present application belongs to the technical field of biomedical antibacterial materials, and specifically relates to a spatially separated double-layer hydrogel, a preparation method thereof, and an application thereof. The spatially separated double-layer hydrogel consists of two layers: the inner layer is agarose gel microsphere loaded with engineered lactic acid bacteria, and the outer layer is methacrylated gelatin loaded with a photosensitizer. The present invention overcomes the problem of mutual interference between active ingredients in the multifunctional hydrogel by designing the loading structure of lactic acid bacteria and AIE photosensitizer in the gel, thereby ensuring the activity of lactic acid bacteria and the photodynamic effect of the photosensitizer. The 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. It also has good biocompatibility, antibacterial activity and the potential to promote the healing of difficult-to-heal diabetic wounds, providing a new idea for the application of multifunctional hydrogels in the treatment of diabetic wounds.

Description

Technical Field

[0001] The present 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 major complications of diabetes, referring to symptoms such as foot infection, suppuration, and ulceration that occur in diabetic patients. DFUs heal slowly, are susceptible to infection, and are prone to recurrence, and in severe cases can lead to amputation. Currently, the main clinical treatments for diabetic foot ulcers include anti-infection, negative pressure suction, improving local blood supply, and thorough local debridement. Although current treatments are effective to a certain extent, due to the difficulty in controlling infection and the body's poor healing ability, gangrene in a significant number of patients remains uncontrolled, and their condition continues to worsen. Therefore, actively exploring more reasonable treatment options and researching and developing new treatments are essential to improving the treatment of DFUs.

[0003] Photodynamic therapy (PDT) is a treatment modality that uses light irradiation of a specific wavelength to induce localized microbial inactivation. Unlike traditional antimicrobial agents, its mechanism is to transfer the excitation energy through a photosensitizer to molecular oxygen, thereby generating reactive oxygen species (ROS) with strong redox activity. ROS can act on bacterial cell walls, membranes, and nucleic acids, effectively killing both antibiotic-sensitive and resistant strains while mitigating the development of resistance. Because ROS are extremely short-lived and their action is limited to local tissues, this treatment approach effectively targets localized infections while avoiding damage to surrounding healthy tissue. However, traditional organic photosensitizers (such as Rose Bengal and methylene blue) are susceptible to aggregation quenching and inefficient ROS generation, limiting their photodynamic antimicrobial efficacy. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide a spatially separated double-layer hydrogel and a preparation method and application thereof, specifically adopting the following technical solutions:

[0005] In a first aspect, the present invention provides a spatially separated double-layer hydrogel, wherein the spatially separated double-layer hydrogel comprises:

[0006] An inner gel layer, wherein the inner gel layer is agarose gel microspheres loaded with engineered lactic acid bacteria;

[0007] an outer gel layer comprising methacryloyl gelatin and a photosensitizer loaded on the methacryloyl gelatin;

[0008] The photosensitizer is TPA-Py + -C4; the TPA-Py+ -C4's structural formula is as follows:

[0009]

[0010] The present invention encapsulates live bacteria (engineered lactic acid bacteria) in a hydrogel, effectively protecting the activity of probiotics and providing a stable microenvironment. The probiotics in the hydrogel are protected from the adverse effects of the wound environment, extending their survival time in the wound. Furthermore, the application of photocurable hydrogels in wound treatment further expands the advantages of hydrogels. These hydrogels rapidly solidify under illumination, providing improved adaptability and precise conformability to wounds of various shapes and sizes. Methacrylamide gelatin (GELMA) is an important biomedical material with properties similar to those of the natural extracellular matrix. Under illumination with a wavelength of 405 nm, GELMA cross-links to form a stable gel structure, which serves the purpose of drug release in this invention.

[0011] Among them, (1) the inner 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 immunity and promote wound healing; the outer gel has photocuring properties and can adapt to different wound shapes, and the loaded photosensitizer TPA-Py + -C4 can provide sustained antibacterial and anti-biofilm effects and promote wound healing.

[0012] (2) Photosensitizer TPA-Py + -C4 has aggregation-induced emission characteristics, and its fluorescence emission is strongest in toluene. With the increase of toluene concentration, its fluorescence intensity increases and blue shift occurs; TPA-Py + -C4 can significantly generate reactive oxygen species (ROS), including singlet oxygen (¹O2) and superoxide anion (O2 •- ), whose ROS generation efficiency is better than that of the traditional photosensitizer Rose Bengal (RB).

[0013] (3) Methacryloylated gelatin (GELMA) can be transformed from liquid gel to solid gel structure after irradiation with 405 nm blue light, and has good rheological properties, water retention rate, swelling rate and stability, which helps to maintain a moist environment in the wound; and the GELMA gel after freeze-drying shows obvious structural and morphological characteristics, with uniform microsphere distribution inside.

[0014] As a further preferred embodiment, the engineered lactic acid bacteria is Lactococcus lactis MG1363; the Lactococcus lactis MG1363 is obtained by genetic engineering;

[0015] The TPA-Py +-C4 synthesis route is as follows:

[0016] .

[0017] The above-mentioned Lactococcus lactis MG1363 has been genetically engineered to be able to express CXCL12 in situ in the culture medium, and its expression level reaches 1200 pg / mL after 12 hours of culture. In addition, the Lactococcus lactis MG1363 shows a faster growth rate than wild-type lactic acid bacteria in the initial stage of culture.

[0018] In a second aspect, the present invention provides a method for preparing the above-mentioned spatially separated double-layer hydrogel, comprising the following steps:

[0019] The engineered lactic acid bacteria, agarose solution, M17 medium and glucose were mixed, and then mineral oil was added and stirred until the solution became a milky white emulsion, centrifuged and washed to obtain agarose gel microspheres loaded with the engineered lactic acid bacteria;

[0020] The agarose gel microspheres, methacrylated gelatin solution, photoinitiator and photosensitizer are mixed evenly, then injected into a mold, and cured by blue light irradiation to obtain the spatially separated double-layer hydrogel.

[0021] In the above preparation process, the preparation method of agarose gel microspheres (MPS) loaded with engineered lactic acid bacteria is an emulsion method, and the average particle size of the microspheres is 68.4 μm; among them, the addition of M17 culture medium and glucose can significantly improve the survival rate of Lactococcus lactis MG1363 in the microspheres.

[0022] 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 culture medium is 10 mg / mL; and the final concentration of the glucose is 50 mg / mL. In the above preparation process, (1) the higher the agarose concentration, the larger the size of the microspheres formed and the stronger the hardness. 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 microsphere formation process, thereby 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 the methacrylated gelatin undergo cross-linking reaction after being exposed to light, forming a three-dimensional network structure. A high concentration of methacrylated gelatin means that there are more cross-linking points per unit volume, thereby accelerating 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 active species, thereby promoting the cross-linking reaction of the polymer chain and making the photocuring reaction faster. However, too high a concentration of the photoinitiator may lead to an overly rapid polymerization reaction and produce an uneven cross-linking structure. Therefore, this concentration range is the optimized concentration to obtain the ideal curing speed and uniformity.

[0023] As a further preferred embodiment, the stirring temperature is 45° C.-75° C., the stirring speed is 800 rpm-1200 rpm, the stirring time is 5 min-15 min; and the blue light irradiation curing time is 30 s-90 s.

[0024] 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.

[0025] As a further preferred embodiment, the bacteria include at least one of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus.

[0026] The results of the present invention show that the spatially separated double-layer hydrogel exhibits excellent antibacterial properties and can effectively inhibit and kill Escherichia coli ( Escherichia coli , EC), Staphylococcus aureus ( Staphylococcus aureus , SA), Pseudomonas aeruginosa ( Pseudomonas aeruginosa , PA) and methicillin-resistant Staphylococcus aureus (Methicillin-resistant Staphylococcus aureusIn addition, the hydrogel has a significant inhibitory effect on the formation of bacterial biofilms and can effectively remove existing biofilms, making it suitable for the prevention and treatment of antibacterial and anti-biofilm related diseases.

[0027] In a fourth aspect, the present invention provides an antibacterial material comprising the above-mentioned spatially separated double-layer hydrogel.

[0028] In a fifth aspect, the present invention provides the use of the above-mentioned spatially separated double-layer hydrogel in the preparation of a product that promotes diabetic wound healing.

[0029] In a sixth aspect, the present invention provides a dressing for promoting diabetic wound healing, wherein the dressing comprises the spatially separated double-layer hydrogel described above.

[0030] 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 in type II diabetic mice.

[0031] Compared with the prior art, the beneficial effects of the present invention include at least:

[0032] (1) The present invention provides a double-layer hydrogel that can exert therapeutic effects at different stages of wound healing. The dressing synergizes photodynamic antibacterial and healing promotion, and can significantly promote the healing of diabetic wounds. Compared with the existing technology, the present invention confines the engineered bacteria inside the hydrogel, reducing the stimulation of the immunogenic substances on the surface of Lactococcus lactis MG1363 to the tissue. The inability of Lactococcus lactis MG1363 to leak out also effectively prevents it from entering the blood circulation, reducing the risk of sepsis caused by treatment, and further ensuring the safety of treatment;

[0033] (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, the present invention optimizes the internal environment of the gel microspheres by adding nutrients such as glucose and M17 culture medium. Compared with the pure hydrogel environment, the internal osmotic pressure of the gel after adding nutrients is not only more suitable for the growth of Lactococcus lactis MG1363, but also provides sufficient nutrients for its growth and metabolism;

[0034] (3) The present invention has excellent antibacterial effects: the double-layer hydrogel of the present invention exhibits significant inhibitory effects on a variety of pathogenic bacteria (such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus), and can effectively inhibit and remove the corresponding bacterial biofilms;

[0035] (4) The double-layer hydrogel of the present invention is designed with a spatial separation structure, which separates Lactococcus lactis and the photosensitizer in different spaces, avoiding direct contact between the two, thereby ensuring that their respective functions are not affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 The figure shows the principle diagram of the preparation of the double-layer hydrogel G / MPS / C4 of the present invention, wherein a in the figure shows the preparation process of gel microspheres; b in the figure shows the preparation process of the double-layer dressing; and c in the figure shows the animal experiment process.

[0038] Figure 2 Figure 4 shows the antibacterial and anti-biofilm effects of the double-layer hydrogel G / MPS / C4 against four common pathogens. Figure a is an antibacterial plate; Figure b is a staining of dead and live bacteria; Figure c is the removal effect on mature biofilms; and Figure d is a partially enlarged schematic diagram of a damaged biofilm.

[0039] Figure 3 Figure 4 shows the therapeutic effects of hydrogels with different components on infected diabetic wounds. Figure a shows the treatment timeline for mice; Figure b shows photos of the wound at different time points during the treatment process and the changes in the wound; Figure c shows the wound healing rate; and Figure d shows the changes in blood sugar.

[0040] Figure 4 Figure 1 shows the active protective effect of engineered bacteria in double-layer hydrogels. Figure a shows a schematic diagram comparing the synthetic structures of single-layer and double-layer hydrogels; Figure b shows the growth curves of engineered bacteria in different hydrogels; Figure c shows the plate count results after replacing the photosensitizer with an antibiotic; Figures d and e show the corresponding plate photographs. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0042] Example 1

[0043] A spatially separated double-layer hydrogel loaded with engineered lactic acid bacteria and an AIE photosensitizer, the preparation method of which comprises the following steps:

[0044] Step 1: The process of genetic engineering of engineered lactic acid bacteria (Lactococcus lactis MG1363) is as follows:

[0045] 1. Construction of pMG36e CXCL12 Plasmid

[0046] (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;

[0047] (2) Construction of pMG36e-CXCL12 recombinant plasmid:

[0048] A: Use OMEGA plasmid extraction kit to extract pMG36e and pUC57-CXCL12 plasmids;

[0049] B: Enzyme digestion system for pMG36e and pUC57-CXCL12 is as follows: total system volume is 25 μL, digestion is carried out at 37°C for 4 h;

[0050] C: Prepare 2% agarose gel for electrophoresis at 110V for 45 min; cut the gel at around 276 bp and 3600 bp for recovery;

[0051] D: Enzyme ligation system: total system 10 μL, DNA fragment: vector = 5:1 and 10:1, 10℃ overnight;

[0052] E: The transformation consists of the following steps:

[0053] a: Take 200 μL of MC1061 competent cell suspension from the -80℃ freezer and thaw on ice;

[0054] b: Add 2 μL of plasmid solution (the plasmid concentration should be no less than 200 ng / μL), shake gently, and place on ice for 30 min;

[0055] c: Heat shock in a 42°C water bath for 90 s, then quickly cool on ice for 3-5 min;

[0056] d: Add 800 μL of LB liquid medium to the tube, mix well, and culture at 37°C with shaking for 1 h to allow the bacteria to recover to normal growth state. Centrifuge at 4000 rpm for 1 min, discard 800 μL of the supernatant, retain 200 μL of the bacterial solution, and mix well.

[0057] e) After shaking the above bacterial solution, spread 200 μL onto a screening plate containing 300 ng / μL erythromycin. Place the plate face up for half an hour. After the bacterial solution is completely absorbed by the culture medium, invert the plate and incubate at 37°C for 16-24 hours. Pick a single colony.

[0058] F: sequencing;

[0059] G: After successful sequencing, the recombinant plasmid pMG36e-CXCL12 was extracted using the OMEGA plasmid extraction kit and stored for future use. The steps are the same as above.

[0060] 2. The prepared pMG36e-CXCL12 was transformed into Lactococcus lactis MG1363 by electroporation. The transformation process consisted of the following steps:

[0061] A: Preparation of competent lactic acid bacteria:

[0062] a: Take Lactococcus lactis MG1363 stored at -80℃ and inoculate it into 5 mL of M17 lactic acid bacteria liquid culture medium containing 5% glucose, and culture it at 30℃ overnight;

[0063] b: The obtained bacterial solution was inoculated into M17 lactic acid bacteria liquid culture medium containing 2.5% Gly and 5% glucose at 1% and cultured at 30°C until the bacterial OD 600 The value is 0.3-0.4, collect for later use;

[0064] c. Place the collected bacterial culture solution in an ice-water bath for 10 min, centrifuge at 5000 rpm for 5 min at 4°C, and collect the bacterial cells.

[0065] d: The precipitate was washed twice with 1 / 10 volume of ice-cold 10% sucrose and 10% glycerol mixed solution, centrifuged at 8000 rpm for 5 min at 4°C, and the precipitate was collected;

[0066] e: Finally, resuspend the pellet in a 1 / 100 volume mixture of 10% sucrose and 10% glycerol and place in an ice-water bath for 10 minutes before use.

[0067] B: Electrotransformation of Lactococcus lactis MG1363:

[0068] a: Take 2 μL of recombinant plasmid (at a concentration of not less than 150 ng / μL) and gently mix with 50 μL of ice-cold lactic acid bacteria competent cell suspension prepared as described above. Add to a pre-cooled 0.1 cm gap electroporation cuvette and place on ice for 5 min. Set the conditions to 1800 V, 200 Ω, and 25 μF.

[0069] b: After the electroporation is completed, quickly aspirate the liquid in the electroporation cup into a centrifuge tube and add 800 μL of M17 liquid culture 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 containing erythromycin resistance. Incubate at 30°C for 24-72 h to screen for positive clones.

[0070] Step 2: AIE photosensitizer C4 (TPA-Py + -C4), the specific steps are as follows:

[0071] TPA-Py was synthesized by aldehyde-ketone condensation reaction of 4-(diphenylamino)benzaldehyde and 1-butyl-4-methylpyridinium iodide in methanol solvent. + -C4, the specific process is as follows:

[0072] First, 1.5 g of 4-(diphenylamino)benzaldehyde and 1.38 g of 1-butyl-4-methylpyridinium iodide were dissolved in 20 mL of methanol. Three drops of piperidine were then added as a catalyst, and the reaction was stirred at 50°C for 4 h. During the reaction, piperidine first abstracted the proton from the methyl group of the pyridinium salt to form a carbanion, which then nucleophilically attacked the carbonyl carbon of the aldehyde group to form a β-hydroxy intermediate. This intermediate then dehydrated under heating to form a stable C=C double bond, constructing a conjugated system with a D-π-A structure. After completion of the reaction, the methanol solvent was removed by vacuum distillation to obtain the crude product, which was then purified by column chromatography using dichloromethane / methanol (30:1) as the eluent. The reaction equation is as follows:

[0073]

[0074] Step 3: Preparation of agarose gel microspheres loaded with engineered lactic acid bacteria

[0075] Select 1 mL of Lactococcus lactis MG1363 in the logarithmic growth phase obtained in step (1), centrifuge at 5000 rpm for 10 min to remove the culture medium, and retain the precipitate;

[0076] A 0.5% agarose solution was prepared by dissolving agarose in deionized water and heating it to 90°C in a microwave oven until the agarose was completely dissolved, ensuring that the solution was clear and free of particulate matter. When the agarose solution cooled to 45°C, 500 μL of agarose solution was added to the Lactococcus lactis MG1363 pellet, followed by 5 mg of M17 medium powder and 25 mg of glucose powder, and the mixture was thoroughly mixed to obtain a mixture. The mixture was then added dropwise to 3 mL of mineral oil containing 2% Span at a stirring rate of 1000 rpm for 3 min. At this point, the mixture changed from a clear and transparent state to a milky white emulsion. During stirring, the agarose solution gradually condensed to form gel microspheres. The mixture was centrifuged at 2000 rpm for 1 min, the mineral oil supernatant was removed, and the gel microspheres were washed three times with petroleum ether and then three times with deionized water. Finally, the gel microspheres were transferred to the aqueous phase, quantified to 1 mL, and stored at 4°C until use.

[0077] Step 4: Preparation of spatially separated bilayer hydrogels

[0078] First, prepare the methacryloyl gelatin (GELMA) stock solution: take 120 mg of lyophilized GELMA, add 1 mL of ultrapure water, and shake several times to ensure complete infiltration. Heat the solution in a 60-70°C water bath for 30 minutes until completely dissolved. Once the solution is complete, store it at 4°C in a dark environment until used.

[0079] Prepare the photoinitiator stock solution: Take 3 mg of photoinitiator DMA (dimethacrylate) powder and add it to 1 mL of phosphate buffered saline (PBS). Heat it in a 40-50°C water bath for 15 min and shake until it is completely dissolved. Ensure that no precipitate forms in the solution.

[0080] Preparation of AIE photosensitizer C4 (TPA-Py + -C4) mother solution: 10 mg TPA-Py + -C4 was dissolved in 1 mL DMSO (dimethyl sulfoxide) and shaken until completely dissolved to obtain the AIE photosensitizer C4 (TPA-Py + -C4) mother liquor:

[0081] 60 μL GELMA stock solution, 30 μL gel microspheres, 9 μL photoinitiator stock solution and 1 μL TPA-Py + The G / MPS / C4 mother liquor was mixed and stirred evenly before being poured into a cylindrical mold with a diameter of 5 mm. Finally, the mixture was irradiated with 405 nm blue light for 30 seconds to complete the photocuring reaction and obtain a spatially separated bilayer hydrogel (G / MPS / C4).

[0082] During the solidification process, the lactic acid bacteria gel microspheres were effectively embedded in the gel, and TPA-Py + -C4 is evenly distributed in the gel structure, ensuring that the dressing has the functions of photodynamic antibacterial and promoting wound healing.

[0083] Example 2

[0084] The spatially separated double-layer hydrogel prepared in Example 1 was subjected to an antibacterial test, and the specific process is as follows:

[0085] 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:

[0086] First, the four bacteria were cultured to the logarithmic growth phase and diluted to 1×10 4CFU / mL. Subsequently, 5 mL of the diluted bacterial solution was transferred to a test tube, followed by the addition of the G / MPS / C4 gel dressing and exposure to light at 50 W / m² of white light for 15 minutes. After the illumination treatment, 100 μL of the treated bacterial solution was evenly spread on a plate containing LB agar at regular intervals and incubated in a 37°C incubator for 24 hours. The bacterial colony counts in each group were recorded.

[0087] Calculate relative bacterial activity: relative bacterial activity = (N b / N a )×100%, where N a is the bacterial colony count of the control group, N b is the bacterial colony count of the experimental group.

[0088] Subsequently, bacterial survival was assessed using the BB / cell live / dead bacterial staining kit. Fluorescence microscopy was used to observe bacterial staining before and after staining to visually assess the hydrogel's inhibitory effect on bacteria.

[0089] Finally, to further evaluate the anti-biofilm ability of G / MPS / C4 gel dressing, 500 μL of Staphylococcus aureus (1×10 5 A suspension of 100 CFU / mL (0.17 mmol / mL) was added to a 24-well plate and incubated at 37°C for 48 hours to allow bacterial biofilm formation. Free bacteria were removed by washing with 0.01 M PBS. The plates were then added with the prepared liquid gel and incubated at 37°C for 20 minutes. The plates were then exposed to light. After treatment, the plates were stained and observed for biofilm disruption.

[0090] See the results Figure 2 Middle a- Figure 2 As shown in Figure d, the experimental results show that G / MPS / C4 has a significant antibacterial effect on four pathogenic bacteria (EC, PA, SA, and MRSA), and light treatment enhances its antibacterial effect. The results of live and dead bacteria staining showed that the bacteria in the G / MPS / C4 group were almost not stained, indicating that the bacteria in these groups were completely killed (e.g. Figure 2 The anti-biofilm test results showed that the bacterial biofilm in the G / MPS / C4 group was most significantly destroyed (e.g. Figure 2 In contrast, the bacterial biofilm structure of the PBS group remained intact. Further microscopic observation (e.g. Figure 2 (d) shows that the bacterial biofilm structure in the G / MPS / C4 group was significantly damaged, with bacterial community fragments appearing, while the PBS group maintained an intact membrane structure. This indicates that the bilayer hydrogel G / MPS / C4 can effectively kill bacteria and remove bacterial biofilms under light treatment.

[0091] Example 3

[0092] The spatially separated double-layer hydrogel prepared in Example 1 was used to measure the therapeutic effect of infected diabetic wounds. The specific process is as follows:

[0093] To comprehensively evaluate the effects of G / MPS / C4 on diabetic wound healing in vivo, this study used 6-8 week-old SPF-grade BALB / c male mice. Diabetes was induced using STZ combined with a high-sugar, high-fat diet. A full-thickness diabetic wound model with MRSA infection was then established. The specific experimental steps are as follows:

[0094] First, a diabetic mouse model was established. Solution A and solution B were prepared. Solution A consisted of 2.1 g of citric acid and solution B consisted 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 accurately adjusted to a pH of 4.2-4.5 before use. Sterile STZ solution was prepared by dissolving 100 mg of streptozotocin powder in 10 mL of freshly prepared sodium citrate buffer, filtering the solution with a 0.22 μm filter, and storing it at 4°C in the dark. Seven-week-old male BALB / c mice were purchased and fed a week of adaptive feeding before the experiment began. After fasting for 12 hours, the mice were intraperitoneally injected with the prepared streptozotocin solution at a dose of 50 mg per kg of body weight. Injections were continued for 5 days, while the mice were fed a high-sugar, high-fat diet. After the injection, 5 mm of the tail was clipped and 2 μL of blood was collected. Blood glucose concentration was measured using a glucometer. Mice whose blood glucose levels reached or exceeded 16.7 mmol / L were considered to have achieved glycemic control and were placed in the diabetic group. Mice that did not meet the target received further injections of streptozotocin solution until their blood glucose levels met the target. Blood glucose levels were measured every three days. Mice that maintained blood glucose levels above 16.7 mmol / L for two consecutive weeks were considered to have successfully established a diabetic model and maintained a stable hyperglycemic state.

[0095] Subsequently, a diabetic full-thickness infected wound model was constructed. A 30 mg / mL sodium pentobarbital solution was prepared using normal saline. After thorough mixing, 1 mL / kg of sodium pentobarbital solution was injected intraperitoneally according to the mouse's weight for general anesthesia. The anesthetized mouse was fixed on the operating table, and a 6 mm diameter full-thickness skin wound was constructed using sterile ophthalmic scissors. 1×10 6 CFU / mL of MRSA solution was inoculated into the wound to construct MRSA bacterial-infected diabetic wounds.

[0096] Next, the diabetic full-thickness skin wounds were treated: the six 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), with six mice in each group. The treatment groups were PBS, G group (treated with a single GELMA hydrogel) (G / MPS group: treated with GELMA hydrogel loaded with engineered probiotic hydrogel 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 hydrogel microspheres and C4 without light treatment; and G / MPS / C4 / L group: treated with GELMA hydrogel loaded with engineered probiotic hydrogel microspheres and C4 and combined with light treatment). The treatment process involved precisely dropping the liquid gel from each of the five different treatment groups onto the wound surface. Next, blue light was irradiated for 30 seconds to induce cross-linking of the liquid hydrogel and convert it into a solid gel. Once the solid gel was formed, the wound was covered with a sterile transparent surgical patch. The hydrogel dressing was changed every 24 hours, and wound surface changes were recorded every two days using a digital camera. Wound surface area was then quantified using Image J software to calculate wound healing rates. The wound healing rate was calculated using the following formula: healing rate = (initial wound area − unhealed wound area × initial wound area) × 100%.

[0097] The experimental results show that Figure 3 a- Figure 3 As shown in Figure (b), wounds in all treatment groups showed a trend of healing over time. Specifically, the wounds in the PBS group healed the slowest, while the wounds in the other treatment groups (including the G, G / MPS, G / C4 / L, G / MPS / C4 / D, and G / MPS / C4 / L) contracted faster. In particular, the healing effect of the G / MPS / C4 / L group was significantly superior to that of the other gel-treated groups. On the second day of treatment, all wounds showed significant suppuration and a yellowish-white biofilm, with no significant differences between the groups. This is due to the early acute inflammatory phase and the presence of a severe inflammatory response. However, by the fourth day, the wounds in the PBS group still contained significant yellowish pus, while the wounds in the other gel-treated groups were relatively dry. This is primarily due to the swelling of the gel dressing, which absorbs exudate from the wound.

[0098] On the 6th day, the wound healing began to show significant differences. According to the healing rate statistics ( Figure 3c in the figure), the healing rate of the G / MPS / C4 / L group was the highest, while the healing rates of the G / C4 / L group and the G / MPS / C4 / D group were slightly improved. This is because the bactericidal effect of C4 helps to eliminate the pathogenic bacteria of the infection and relieve inflammation during the transition period of the wound. By the 8th day, the healing rate of the G / MPS / C4 / L group reached 89.4%, which was significantly higher than that of the other groups (such as 76.6% in the G / C4 / L group, 72.5% in the G / MPS group, 66.6% in the G group, and 62.4% in the PBS group). At this time, the wound surface of the G / MPS / C4 / L group was completely covered with epithelium and almost completely healed, leaving only tiny scars. At the same time, blood glucose monitoring during the treatment process proved that the experimental model was stable ( Figure 3 The above research results show that G / MPS / C4 / L exhibits excellent effects in promoting wound healing.

[0099] Comparative Example 1

[0100] In order to evaluate the spatial separation effect of the double-layer hydrogel, especially the protective effect on probiotics, this study set up a single-layer gel ( Figure 4 For comparison, the specific operations are as follows:

[0101] First, take 30 μL of 10 8 CFU / mL MG1363 culture medium, 60 μL GELMA stock solution and 1 μL TPA-Py + -C4 were mixed evenly, and then 9 μL of DMA was added and irradiated under blue light for 30 seconds to form a gel to prepare a single-layer gel.

[0102] The single-layer gel prepared in this comparative example and the double-layer gel (G / MPS / C4) prepared in Example 1 were added to 50 mL of LB culture medium and cultured under white light. 200 μL samples were taken every hour and the culture was continuously monitored for 36 hours, and the absorbance at 600 nm was measured. Finally, the OD value was calculated with the culture time as the horizontal axis. 600 The bacterial growth curve was drawn with nm as the vertical axis to evaluate the activity of bacteria inside the gel dressing.

[0103] The results are as follows Figure 4 As shown in Figure (b), the growth of Lactococcus lactis MG1363 slowed down after light treatment on the monolayer gel. This is likely due to the large amount of ROS produced by the photosensitizer C4 under light, which had a significant killing effect on the surrounding Lactococcus lactis MG1363, thereby weakening its growth activity. However, when Lactococcus lactis MG1363 was embedded in the gel microspheres, the spatial isolation effect of the microspheres effectively avoided the impact of ROS on the growth activity of Lactococcus lactis MG1363 under light treatment, laying the foundation for its subsequent better functional activity.

[0104] In order to further verify the protective effect of spatial isolation strategy on active microorganisms, this study used TPA-Py + -C4 was replaced with small molecule antibiotics (including ampicillin, kanamycin and amoxicillin), and the microbial growth activity in single-layer gel and double-layer gel was studied by plate growth method. Figure 4 c- Figure 4 As shown in Figure (e), the results of single-layer and double-layer gel plate coatings show significant differences. Lactococcus lactis MG1363 embedded in the double-layer gel maintained high growth activity, while the bacteria loaded in the single-layer gel were almost completely killed, with only a few scattered colonies remaining on the plate.

[0105] The above results show that the double-layer gel can effectively avoid the nonspecific killing of active microorganisms by loaded drugs through the spatial isolation effect, thereby allowing the active microorganisms to better exert their functional activity.

[0106] In summary, the present invention discloses a method for preparing a spatially separated bilayer hydrogel loaded with functionally active lactic acid bacteria and an aggregation-induced emission (AIE) photosensitizer, and introduces its application in the treatment of diabetic wounds. The prepared hydrogel has an inner and outer layer structure: the inner layer is agarose gel microspheres (MPS) loaded with active lactic acid bacteria, and the outer layer is methacrylic acid gel loaded with the AIE photosensitizer. Due to the separated design of the inner and outer gel layers, the loaded lactic acid bacteria and AIE photosensitizer can independently perform their respective functions. The lactic acid bacteria in the inner agarose microspheres continuously produce chemokines and lactic acid, regulating the wound microenvironment and promoting wound healing. The photodynamic properties of the AIE photosensitizer in the outer GELMA gel provide antibacterial effects, eliminating pathogens and activating the immune response to accelerate wound healing.

[0107] By designing the loading structure of lactic acid bacteria and AIE photosensitizer in the gel, the present invention overcomes the problem of mutual interference between the 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, thereby promoting 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.

[0108] The embodiments of the present application are described above in conjunction with the accompanying drawings. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of the present application, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present application and the claims, all of which are within the protection of the present application.

Claims

1. A spatially separated double-layer hydrogel, characterized in that The spatially separated double-layer hydrogel comprises: An inner gel layer, wherein the inner gel layer is agarose gel microspheres loaded with engineered lactic acid bacteria; an outer gel layer comprising methacryloyl gelatin and a photosensitizer loaded on the methacryloyl gelatin; The photosensitizer is TPA-Py + -C4; the TPA-Py + -C4's structural formula is as follows: ； The engineered lactic acid bacteria is Lactococcus lactis MG1363; the Lactococcus lactis MG1363 is obtained by genetic engineering and can continuously release CXCL12 and lactic acid in situ.

2. The spatially separated double-layer hydrogel according to claim 1, characterized in that The TPA-Py + -C4 synthesis route is as follows: 。 3. The method for preparing the spatially separated double-layer hydrogel according to any one of claims 1 to 2, characterized in that: The following steps are involved: The engineered lactic acid bacteria, agarose solution, M17 medium and glucose were mixed, and then mineral oil was added and stirred until the solution became a milky white emulsion, centrifuged and washed to obtain agarose gel microspheres loaded with the engineered lactic acid bacteria; The agarose gel microspheres, methacrylated gelatin solution, photoinitiator and photosensitizer are mixed evenly, then injected into a mold, and cured 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; and 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 stirring temperature is 45° C.-75° C., the stirring speed is 800 rpm-1200 rpm, and the stirring time is 5 min-15 min; and the blue light irradiation curing time is 30 s-90 s.

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

7. The use according to claim 6, characterized in that 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 comprises the spatially separated double-layer hydrogel according to any one of claims 1 to 2.

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

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

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