A pH-responsive hydrogel composite dressing as well as a preparation method and application thereof

Abstract

This invention relates to a pH-responsive hydrogel composite dressing, its preparation method, and its application, belonging to the field of biomedical materials and drug delivery technology. The pH-responsive hydrogel composite dressing comprises a hydrogel matrix and engineered exosomes and platelet-rich plasma uniformly loaded within the hydrogel matrix. The hydrogel matrix comprises a three-dimensional dynamic cross-linked network formed by a Schiff base reaction between an amino-containing polysaccharide derivative and an oxidized aldehyde-containing polysaccharide derivative. The engineered exosomes are derived from donor cells subjected to pathogen-associated molecular pattern stress treatment. The dressing provided by this invention possesses both toxin capture and immunometabolic regulation functions, achieving synergistic therapeutic effects against MRSA-infected diabetic wounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and drug delivery technology, specifically relating to a pH-responsive hydrogel composite dressing, its preparation method, and its application. Background Technology

[0002] Diabetic wounds are one of the most common and serious complications of diabetes, especially when complicated by methicillin-resistant Staphylococcus aureus (MRSA) infection. Treatment faces severe challenges, including high bacterial load, local immunosuppression, inhibited angiogenesis, extremely slow healing, and even increased risk of amputation. MRSA-secreted alpha-toxins are key virulence factors leading to tissue necrosis and immune escape. They can bind to the host cell surface receptor ADAM10, inducing cell damage. The high glucose and hypoxic microenvironment of diabetic wounds further exacerbates tissue destruction and inflammatory dysregulation.

[0003] Current first-line clinical treatments mainly include surgical debridement, systemic or local antibiotics, negative pressure drainage, and growth factor preparations. While these methods can control infection or promote repair to some extent, they are mostly single-function and cannot synergistically resolve the core contradiction caused by MRSA infection—the vicious cycle between the continuous attack of bacterial virulence factors and the dysregulation of the host's immune microenvironment. For example, while antibiotics can kill bacteria, they cannot neutralize the secreted alpha-toxins, nor can they reverse the immunosuppression caused by high glucose and hypoxia; growth factors, on the other hand, have limited activity in an uncontrolled inflammatory environment. Therefore, conventional treatments often fall into a dilemma of infection-inflammation-tissue necrosis-delayed healing-reinfection, resulting in poor efficacy.

[0004] Hydrogel dressings have shown great potential in the repair of diabetic wounds due to their high water content, good biocompatibility, and ability to mimic the extracellular matrix. Among them, pH-responsive hydrogels based on Schiff base dynamic covalent bonds offer advantages such as injectability, self-healing, and degradation in response to the weakly acidic microenvironment of the wound, making them suitable for on-demand controlled release of loaded active ingredients. However, most existing hydrogel dressings are limited to loading a single type of drug, making it difficult to simultaneously achieve targeted neutralization of α-toxins and effective remodeling of the wound's immune microenvironment. Therefore, developing a hydrogel composite dressing that integrates both toxin neutralization and immune regulation functions and possesses intelligent controlled-release properties is of significant clinical importance for the treatment of diabetic wounds infected with MRSA. Summary of the Invention

[0005] To address the technical shortcomings of existing diabetic wound dressings in simultaneously neutralizing bacterial toxins and reshaping the wound's immune microenvironment when dealing with methicillin-resistant Staphylococcus aureus (MRSA) infections, this invention provides a pH-responsive hydrogel composite dressing, its preparation method, and its application. Specifically, it provides a smart responsive composite dressing, comprising a hydrogel matrix formed by a Schiff base reaction of carboxymethyl chitosan and oxidized hyaluronic acid, loaded with engineered exosomes highly expressing the ADAM10 protein (induced by infection stress) as toxin decoys, and combined with host-derived platelet-rich plasma. The dressing of this invention forms a dynamic covalently cross-linked network through a Schiff base reaction, integrating engineered exosomes highly expressing the ADAM10 receptor as molecular decoys and loading platelet-rich plasma, thus possessing both toxin capture and immunometabolic regulation functions, achieving synergistic therapeutic effects against MRSA-infected diabetic wounds.

[0006] A pH-responsive hydrogel composite dressing includes a hydrogel matrix and engineered exosomes and platelet-rich plasma uniformly loaded in the hydrogel matrix. The hydrogel matrix comprises a three-dimensional dynamic cross-linked network formed by a Schiff base reaction between an amino-containing polysaccharide derivative and an oxidized aldehyde-containing polysaccharide derivative. The engineered exosomes are derived from donor cells subjected to pathogen-associated molecular pattern stress treatment.

[0007] Preferably, the stress treatment involves co-incubating donor cells with a pathogen-associated molecular pattern for 6-72 hours.

[0008] In the pH-responsive hydrogel composite dressing of the present invention, the mass ratio of the amino-containing polysaccharide derivative to the oxidized aldehyde-containing polysaccharide derivative is 1:5 to 5:1.

[0009] In the pH-responsive hydrogel composite dressing of the present invention, the loading amount of engineered exosomes in the hydrogel composite dressing is 50~500 μg / mL; and the volume percentage of platelet-rich plasma in the hydrogel composite dressing is 1%~50%.

[0010] In the pH-responsive hydrogel composite dressing of the present invention, the donor cells are selected from one or more of mesenchymal stem cells, dendritic cells, macrophages or fibroblasts.

[0011] In the pH-responsive hydrogel composite dressing of the present invention, the pathogen-associated molecular pattern is selected from one or more of the following: heat-inactivated methicillin-resistant Staphylococcus aureus cells, methicillin-resistant Staphylococcus aureus cell wall components, methicillin-resistant Staphylococcus aureus nucleic acid extracts, or recombinant α-toxin proteins.

[0012] Furthermore, the engineered exosomes of the present invention highly express ADAM10 protein on their surface, and their expression level is higher than that of natural exosomes that have not undergone pathogen-associated molecular pattern stress treatment. They can act as competitive receptor decoys to bind to and neutralize α-toxin secreted by methicillin-resistant Staphylococcus aureus.

[0013] In the pH-responsive hydrogel composite dressing of the present invention, the platelet-rich plasma is derived from the separation and concentration of autologous or allogeneic whole blood.

[0014] Preferably, the platelet-rich plasma is obtained by collecting whole blood from the host, separating and removing red blood cells and excess plasma by gradient centrifugation, and then concentrating the plasma.

[0015] More preferably, the gradient centrifugation includes a first centrifugation to separate red blood cells and a second centrifugation to concentrate platelets, with the first centrifugation force being 500 g to 1000 g and the second centrifugation force being 800 g to 1500 g.

[0016] In the pH-responsive hydrogel composite dressing of the present invention, the amino-containing polysaccharide derivative is selected from one or more of carboxymethyl chitosan, chitosan hydrochloride, or hydroxypropyl chitosan.

[0017] Preferably, the amino-containing polysaccharide derivative is carboxymethyl chitosan.

[0018] In the pH-responsive hydrogel composite dressing of the present invention, the oxidized polysaccharide derivative containing aldehyde groups is selected from one or more of oxidized hyaluronic acid, oxidized sodium alginate, or oxidized chondroitin sulfate.

[0019] Preferably, the oxidized polysaccharide derivative containing an aldehyde group is oxidized hyaluronic acid.

[0020] More preferably, the oxidized hyaluronic acid is obtained by oxidizing hyaluronic acid with sodium periodate.

[0021] Another object of the present invention is to provide a method for preparing the above-mentioned pH-responsive hydrogel composite dressing, comprising the following steps: (1) The donor cells were co-incubated with the molecular model of pathogenic microorganisms to induce stress, and the supernatant was collected. The supernatant was then separated and purified by differential ultracentrifugation or tangential flow filtration to obtain engineered exosomes. (2) Under stirring or shaking conditions, the polysaccharide derivative solution containing aldehyde groups is mixed with the engineered exosomes and platelet-rich plasma obtained in step (1) to obtain precursor solution A; the precursor solution A is mixed evenly with the polysaccharide derivative solution containing amino groups and allowed to stand for crosslinking to obtain the pH-responsive hydrogel composite dressing.

[0022] Preferably, in step (2), the volume percentage of platelet-rich plasma in the precursor solution A is 20%, and the concentration of engineered exosomes is 150 μg / mL; the concentrations of the amino-containing polysaccharide derivative solution and the oxidized aldehyde-containing polysaccharide derivative solution are both 0.5%~10% (w / v), and the volume ratio of the precursor solution A to the amino-containing polysaccharide derivative solution is 2:1.

[0023] In the preparation method of the pH-responsive hydrogel composite dressing of the present invention, the co-incubation time in step (1) is 6~72 h.

[0024] Preferably, in step (1), the co-incubation time is 12~72 h.

[0025] In the preparation method of the pH-responsive hydrogel composite dressing of the present invention, in step (1), the differential ultracentrifugation method specifically involves: the collected supernatant is subjected to centrifugation at 1500 g to 2500 g, centrifugation at 8000 g to 12000 g, and ultracentrifugation at 100000 g to 150000 g in sequence.

[0026] In the preparation method of the pH-responsive hydrogel composite dressing of the present invention, in step (2), the static crosslinking temperature is 4~40℃ and the time is 1~60 min.

[0027] Preferably, in step (2), the static crosslinking temperature is 15~30℃ and the time is 1~15 min.

[0028] Another object of the present invention is to provide the application of the above-mentioned pH-responsive hydrogel composite dressing in the preparation of medicaments or medical dressings for treating diabetic wounds.

[0029] Furthermore, the diabetic wound is a diabetic wound infected with methicillin-resistant Staphylococcus aureus.

[0030] Furthermore, the pH-responsive hydrogel composite dressing exhibits accelerated degradation and responsive release characteristics in a weakly acidic environment of pH 5.0 to 6.5, which is a typical characteristic of the microenvironment of diabetic wounds infected with MRSA.

[0031] Furthermore, the pH-responsive hydrogel composite dressing can effectively promote wound closure, reduce inflammatory response, promote angiogenesis, and induce macrophage polarization toward the M2 repair phenotype in diabetic wounds infected with methicillin-resistant Staphylococcus aureus.

[0032] The beneficial effects of this invention are: 1. The pH-responsive hydrogel composite dressing provided by this invention is a dual-function therapeutic system that targets the pathological mechanism of MRSA damage by upregulating ADAM10 receptors and lactic acid to enhance α-toxin toxicity. Specifically, the engineered exosomes loaded with the dressing act as "molecular decoys" to precisely capture and neutralize α-toxins, blocking the attack of MRSA virulence factors at the source; while the platelet-rich plasma loaded with the dressing promotes angiogenesis and alleviates tissue hypoxia by releasing various growth factors, thereby reducing lactic acid accumulation and synergistically enhancing the therapeutic effect at the metabolic level.

[0033] 2. The hydrogel matrix in the pH-responsive hydrogel composite dressing provided by the present invention adopts a Schiff base dynamic covalent cross-linking strategy, which has pH-responsive degradation capability. It can intelligently respond to the weakly acidic microenvironment of MRSA-infected wounds, realize the on-demand, continuous and controllable release of active ingredients, and significantly prolong the retention time and bioavailability of exosomes and platelet-rich plasma (PRP) in the local wound.

[0034] 3. The engineered exosomes provided by this invention are prepared by using an infection-simulated stress strategy to induce upregulation of ADAM10 expression in donor cells. The resulting exosomes combine the advantages of low immunogenicity and high stability of natural exosomes with the high affinity binding function of specific α-toxins. Moreover, the preparation process is simple and easy to scale up.

[0035] 4. The pH-responsive hydrogel composite dressing provided by this invention integrates three major functions: toxin neutralization, metabolic regulation, and immune remodeling. Through the synergistic effect of engineered exosomes and PRP, it effectively reverses the immunosuppressive state of diabetic infected wounds, promotes macrophage polarization towards a pro-repair phenotype, and achieves the transformation from a "cold wound" to a "warm wound." Furthermore, the composite dressing exhibits excellent biocompatibility, with all components being naturally derived or autologous derivatives, possessing good in vivo degradability and safety, and demonstrating significant clinical translational potential. Attached Figure Description

[0036] Figure 1 Scanning electron microscope image of the pH-responsive hydrogel composite dressing loaded with PRP and engineered exosomes prepared in Example 1 of this invention.

[0037] Figure 2 The in vitro cumulative release curves of PRP and engineered exosomes in the hydrogel composite dressing loaded with PRP and engineered exosomes prepared in Example 1 of the present invention demonstrate its pH-responsive sustained release capability.

[0038] Figure 3 The rheological properties test diagram of the hydrogel composite dressing loaded with PRP and engineered exosomes prepared in Example 1 of the present invention includes scanning curves of storage modulus and loss modulus as a function of strain.

[0039] Figure 4 Photographs demonstrating the injectability and self-healing properties of the hydrogel composite dressing loaded with PRP and engineered exosomes prepared in Example 1 of this invention.

[0040] Figure 5 Transmission electron micrograph of the engineered exosomes prepared in Example 2 of this invention.

[0041] Figure 6 This is a Western blotting comparison of ADAM10 protein expression in engineered exosomes prepared in Example 2 of the present invention and natural exosomes.

[0042] Figure 7 This is an in vitro ELISA test result of the binding ability of engineered exosomes to MRSA α-toxin in Example 2 of the present invention.

[0043] Figure 8 This is a diagram showing the cytotoxicity results of engineered exosomes neutralizing α-toxins and protecting host cells in Example 2 of the present invention.

[0044] Figure 9 This is a graph showing the cell compatibility test results of the hydrogel composite dressing leachate loaded with PRP and engineered exosomes on fibroblasts in Example 3 of the present invention.

[0045] Figure 10 This is a flow cytometry analysis result of the effect of different treatment groups on the expression of macrophage polarization markers in Example 3 of the present invention, showing the changes in the M1 / M2 phenotype ratio.

[0046] Figure 11 This is a comparison chart of the effects of different treatment groups on the scavenging of intracellular reactive oxygen species in Example 3 of the present invention.

[0047] Figure 12 The figure shows the results of the cell scratch healing experiment in Example 3 of the present invention, illustrating the effect of different treatments on cell migration ability, in order to verify the repair-promoting function of PRP.

[0048] Figure 13 These are representative photographs of wound healing at different time points in the MRSA-infected full-thickness skin defect model of diabetic mice in Example 4 of this invention.

[0049] Figure 14 This is an immunofluorescence co-staining image of macrophage markers in the wound tissue of each group on day 7 in Example 4 of the present invention, showing the distribution of M1 and M2 macrophages.

[0050] Figure 15 This is a statistical chart of plate colony counts of bacterial load in wound tissues of each group in Example 4 of the present invention.

[0051] Figure 16 This is an ELISA detection result of the expression level of inflammatory factors in wound tissues of each group in Example 4 of the present invention. Detailed Implementation

[0052] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0053] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0054] Example 1 This embodiment provides a pH-responsive hydrogel composite dressing loaded with PRP and engineered exosomes formed by crosslinking carboxymethyl chitosan and oxidized hyaluronic acid, and a composite dressing loaded with engineered exosomes and platelet-rich plasma, while characterizing its physicochemical properties.

[0055] A method for preparing a pH-responsive hydrogel composite dressing includes the following steps: (1) Preparation of engineered exosomes Take mouse fibroblasts (L929) in the logarithmic growth phase, and use 1×10 6 Cells were seeded at a density of 10 cm in 10 cm culture dishes and placed in DMEM medium containing 10% exosome-free fetal bovine serum. Heat-inactivated methicillin-resistant Staphylococcus aureus (MRSA) was added to the culture system at a multiplicity of infection (MOI) of 1:50, and the culture was incubated at 37°C and 5% CO2 for 48 hours to induce infection-simulated stress. The cell culture supernatant was collected and centrifuged sequentially at 4°C at 2000 g for 10 min and then at 10000 g for 15 min to remove cells and cell debris. The resulting supernatant was then ultracentrifuged at 120000 g for 90 min, the supernatant was discarded, and the precipitate was resuspended in pre-chilled PBS, washed, and centrifuged again at 120000 g for 90 min. The final precipitate was the engineered exosome with high ADAM10 protein expression on its surface, which was resuspended in a small amount of PBS and stored at -80°C for later use.

[0056] (2) Preparation of platelet-rich plasma Whole blood was collected from BALB / c mice and injected into EDTA-K2 anticoagulant tubes. The tubes were centrifuged at 800 g for 5 minutes at room temperature, and the supernatant plasma and white membrane layers were transferred to new centrifuge tubes. The tubes were then centrifuged again at 1100 g for 10 minutes, and the supernatant platelet-rich plasma was discarded; the bottom precipitate was platelet-rich plasma (PRP). Platelet count analysis showed that the platelet concentration in the obtained PRP was approximately 4–6 times that of whole blood.

[0057] (3) Preparation of pH-responsive hydrogel composite dressings loaded with PRP and engineered exosomes Weigh 1 g of high molecular weight hyaluronic acid (400-800 kDa) and dissolve it in 1 L of distilled water with stirring. Weigh 535 mg of sodium periodate and dissolve it in 5 mL of distilled water, then add this solution dropwise to the hyaluronic acid solution. Continue stirring the reaction system at room temperature in the dark for 6 hours. Then add 1 mL of ethylene glycol to quench the reaction and continue stirring for 1 hour. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8-14 kDa and dialyze with distilled water for 3 days, changing the dialysate every 8 hours. After dialysis, freeze-dry to obtain oxidized hyaluronic acid powder, and store at 2-4℃ for later use. Weigh carboxymethyl chitosan and dissolve it in PBS buffer to prepare a 5% (w / v) solution, adjusting the pH to 7.4. Weigh oxidized hyaluronic acid and dissolve it in PBS buffer to prepare a 2% (w / v) solution. Oxidized hyaluronic acid solution, platelet-rich plasma (PRP), and engineered exosomes were mixed in a specific ratio to achieve a final PRP volume percentage of 20% and an engineered exosome concentration of 150 μg / mL. This mixture was then used as precursor solution A. Precursor solution A was rapidly mixed with carboxymethyl chitosan solution at a volume ratio of 2:1 at room temperature. After thorough mixing, the mixture was transferred to a mold and allowed to stand at room temperature for 3 minutes to form a pH-responsive hydrogel composite dressing loaded with PRP and engineered exosomes, denoted as P / A Gel.

[0058] The hydrogel composite dressing containing PRP and engineered exosomes prepared in Example 1 was flash-frozen in liquid nitrogen, freeze-dried, sectioned, and sputter-coated with gold. The cross-sectional morphology was observed using scanning electron microscopy. The results are as follows: Figure 1 As shown, the internal structure exhibits a highly interconnected three-dimensional porous network structure with a pore size ranging from 20 to 200 μm. This structure is beneficial for cell migration and nutrient exchange.

[0059] The hydrogel composite dressing prepared in Example 1 was placed in PBS buffer at pH 6.5 and pH 7.4, respectively, and incubated at 37°C with constant shaking. Protein concentration was measured at predetermined time points. The resulting release curves are shown below. Figure 2 As shown, it can be seen that under the simulated acidic microenvironment of infected wound at pH 6.5, the cumulative release rate of engineered exosomes and PRP is significantly higher than that under pH 7.4, indicating that they have obvious pH-responsive release characteristics.

[0060] The strain scanning test of the hydrogel composite dressing containing PRP and engineered exosomes prepared in Example 1 was performed using a rotational rheometer. The test conditions were: fixed angular frequency of 1 rad / s, strain range of 1%–1000%, and temperature of 25°C. The results are as follows: Figure 3As shown, the storage modulus of the hydrogel is higher than the loss modulus under low strain conditions, exhibiting solid gel characteristics. As the strain increases, the gel network structure is destroyed, the modulus reverses, and it exhibits shear-thinning behavior, indicating that it has good injectability.

[0061] The hydrogel composite dressings prepared in Example 1, loaded with PRP and engineered exosomes, were labeled with two different colors of dye. The dressings were extruded into a culture dish using a syringe, allowing the two colored gel blocks to come into contact and stand. Their injectability and self-healing properties were then evaluated. Figure 4 As shown, the two colored gel blocks gradually merge after contact and can form a complete whole when picked up again, with no obvious cracks at the contact interface, indicating that they have excellent injectability and self-healing properties.

[0062] Example 2 Functional characterization and toxin neutralization verification of engineered exosomes prepared in Example 1 This embodiment describes the morphology, protein expression, and α-toxin neutralization function of the engineered exosomes prepared in Example 1.

[0063] (1) Morphological observation by transmission electron microscopy 10 μL of engineered exosome suspension was dropped onto a copper mesh carbon support membrane. After standing at room temperature for 2 minutes for adsorption, excess liquid was absorbed with filter paper. Then, 2% phosphotungstic acid solution was added for negative staining for 1 minute. After absorbing the stain, the membrane was allowed to air dry. The images obtained by transmission electron microscopy are shown below. Figure 5 As shown, engineered exosomes exhibit typical saucer-shaped or spherical vesicle structures with clear boundaries and uniform size.

[0064] (2) Detection of ADAM10 protein expression Equal volumes of engineered exosomes and untreated natural exosomes were taken and lysed on ice for 15 minutes with RIPA lysis buffer. The supernatant was collected by centrifugation, quantified using the BCA method, and then subjected to SDS-PAGE electrophoresis. After transfer, the membrane was blocked with 5% skim milk powder for 1 hour, incubated overnight at 4°C with anti-ADAM10 primary antibody, washed with TBST, and incubated at room temperature for 1 hour with HRP-labeled secondary antibody. ECL staining was then performed. Western blot results are shown below. Figure 6 As shown, the gray value of the ADAM10 protein band in the engineered exosome group was significantly higher than that in the natural exosome group, indicating that infection-simulated stress treatment effectively induced the upregulation of ADAM10 expression in donor cells and enriched it on the exosome membrane surface.

[0065] (3) ELISA verification of α-toxin binding capacity The purified α-toxin solution was mixed with engineered exosomes and incubated at 37°C for 24 hours. Subsequently, the mixture was ultracentrifuged at 120,000g for 70 minutes, and the supernatant was collected. The free α-toxin content was determined by ELISA, with absorbance measured at 450 nm after TMB colorimetric development. Results are as follows: Figure 7 As shown, engineered exosomes exhibit a binding affinity of up to 69.96% for α-toxin.

[0066] (4) α-Toxin neutralization and cell protection experiment The protective effect of engineered exosomes against host cells under α-toxin attack was evaluated using a live / dead cell staining method. L929 cells were seeded in 24-well plates and cultured for 24 hours, followed by incubation for another 24 hours with extracts from different treatment groups. Subsequently, the cells were incubated at 37°C in the dark with AM / PI mixed staining solution for 20 minutes. Fluorescence images were acquired using laser scanning confocal microscopy. Results are shown below. Figure 8 As shown in the figure, the treatment groups are: a blank hydrogel formed by carboxymethyl chitosan and oxidized hyaluronic acid (Blank Gel), a hydrogel loaded with engineered exosomes (Exos Gel), a hydrogel loaded with platelet-rich plasma (PRP Gel), and the P / A Gel obtained in Example 1. Compared with the control group, the number of dead cells in the Exos Gel group and the PRP Gel group was significantly reduced, indicating that engineered exosomes can effectively bind and neutralize α-toxins, protecting host cells from toxin attack.

[0067] Example 3 In vitro biological function verification of the hydrogel composite dressing prepared in Example 1 This embodiment verifies the biocompatibility, immunomodulatory function, and repair-promoting ability of the hydrogel composite dressing prepared in Example 1 in vitro.

[0068] (1) Cell compatibility test The hydrogel composite dressing loaded with PRP and engineered exosomes was prepared into an extract according to ISO 10993-12 standard. L929 mouse fibroblasts were used at a concentration of 5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 hours until cell attachment. Different concentrations of extraction buffer were then added, and the plates were cultured for another 24 hours. Cell viability was assessed using the MTT assay, with absorbance measured at 490 nm. Results are as follows: Figure 9 As shown, the relative cell survival rate of each concentration of extract group was greater than 95%, indicating that it has good cell compatibility and no obvious cytotoxicity.

[0069] (2) Macrophage polarization regulation experiment Mouse RAW264.7 macrophages were seeded in 6-well plates and M1 polarization was induced with 100 ng / mL lipopolysaccharide. Different treatment extracts were added, and the cells were co-cultured for 24 hours. Cells were collected and analyzed by flow cytometry using anti-CD86 and anti-CD206 antibodies. Results are as follows: Figure 10 As shown, compared with the model group, the proportion of CD86-positive M1 macrophages in the PRP-loaded and engineered exosome-treated hydrogel composite dressing group was significantly reduced, while the proportion of CD206-positive M2 macrophages was significantly increased. The M2 / M1 ratio was about 3.6 times higher than that in the model group, indicating that it can effectively regulate macrophage polarization towards the repair-promoting M2 phenotype.

[0070] (3) Detection of intracellular reactive oxygen species levels L929 cells were seeded in 6-well plates and stimulated with 1 μg / mL lipopolysaccharide to induce intracellular reactive oxygen species (ROS) generation. Extracts from different treatment groups were added simultaneously, and the cells were co-cultured for 12 hours. The culture medium was discarded, and a final concentration of 10 μM DCFH-DA fluorescent probe was added. The cells were incubated at 37°C in the dark for 30 minutes. After washing with PBS, intracellular ROS levels were detected by flow cytometry. Results are as follows: Figure 11 As shown, the intracellular ROS fluorescence intensity of the hydrogel composite dressing group loaded with PRP and engineered exosomes decreased by about 88% compared with the model group, indicating that it has a strong reactive oxygen species scavenging ability.

[0071] (4) Cell scratch healing experiment L929 cells were seeded into 6-well plates and cultured until cell confluence reached over 90%. Using a 200 μL sterile pipette tip, a uniform scratch was made perpendicular to the bottom of the plate, and detached cells were washed away with PBS. Extracts from different treatment groups were added, and the scratch width was recorded at 0 and 24 hours post-scratching using an inverted microscope. The scratch healing rate was calculated using ImageJ software. Results are shown below. Figure 12 As shown, the 24-hour scratch healing rate of the hydrogel composite dressing group loaded with PRP and engineered exosomes was significantly higher than that of the other groups, indicating that the PRP component contained therein can effectively promote cell migration and accelerate the wound repair process.

[0072] Example 4 MRSA infection wound healing experiment in diabetic mice This embodiment verifies the therapeutic effect of the hydrogel composite dressing loaded with PRP and engineered exosomes obtained in Example 1 on diabetic wounds infected with MRSA at the animal level.

[0073] (1) Animal model establishment and grouping Male BALB / c diabetic mice aged 4-6 weeks were anesthetized by intraperitoneal injection of sodium pentobarbital. Symmetrical full-thickness skin defects with a diameter of 8 mm were created on both sides of the midline of the back. Each wound was inoculated with 1×10⁻⁶ mg of sodium pentobarbital. 7 A diabetic wound model of MRSA infection was established using CFU-containing MRSA bacterial suspension. Mice were randomly divided into four groups: a blank control group, a blank gel group, a free drug group, and the gel group of this invention, with five mice in each group.

[0074] (2) Dosing regimen Dressings were changed or medications were administered on postoperative days 1, 4, and 7. The blank control group received no treatment; the blank gel group received CMCS-OHA blank hydrogel without active ingredients; the free drug group received a mixed solution of PRP and engineered exosomes; and the hydrogel composite dressing group loaded with PRP and engineered exosomes of the present invention received the hydrogel composite dressing prepared in Example 1.

[0075] (3) Observation of wound healing Postoperatively, the wound was photographed on days 0, 2, 4, 6, 8, 10, and 12. ImageJ software was used to measure the wound area and calculate the wound closure rate. Representative photographs are shown below. Figure 13 As shown, the hydrogel composite dressing group loaded with PRP and engineered exosomes of the present invention had the fastest wound healing speed. The wound area had significantly shrunk by the 6th day after surgery and was basically completely closed by the 12th day. In contrast, the wound healing of the blank control group and the free drug group was significantly delayed, with a large area still not healed.

[0076] (4) Macrophage polarization immunofluorescence analysis Mice were sacrificed on postoperative day 12, and sections of the wound and surrounding tissue were prepared for CD86 and CD206 immunofluorescence co-staining. Results are as follows: Figure 14 As shown, the fluorescence intensity of CD206-positive M2 macrophages in the wound tissue of the hydrogel composite dressing loaded with PRP and engineered exosomes in this invention was significantly enhanced, while the fluorescence intensity of CD86-positive M1 macrophages was significantly weakened. This indicates that the hydrogel composite dressing loaded with PRP and engineered exosomes can effectively induce macrophages to polarize towards the pro-repair M2 phenotype in vivo, thereby reshaping the immune microenvironment of the wound.

[0077] (5) Bacterial load detection of wound On the 7th day post-surgery, wound tissue was harvested, weighed, homogenized, serially diluted, and spread onto mannitol high-salt agar plates. The plates were incubated upside down at 37°C for 24 hours, and colony-forming units were counted. Results are as follows: Figure 15 As shown, the MRSA load on the wound of the hydrogel composite dressing group loaded with PRP and engineered exosomes of the present invention was significantly reduced compared with the blank control group, indicating that it effectively weakens the pathogenic colonization ability of bacteria through a toxin neutralization strategy.

[0078] (6) Detection of inflammatory factor expression On postoperative day 7, wound tissue homogenate was collected, and the expression levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α were detected using an ELISA kit. Results are as follows: Figure 16 As shown, compared with the model group, the levels of IL-1β, IL-6 and TNF-α in the hydrogel composite dressing group loaded with PRP and engineered exosomes decreased by 80.58%, 59.02% and 85.60% respectively, while the levels increased by about 4.5 times. This indicates that the hydrogel composite dressing loaded with PRP and engineered exosomes can effectively inhibit local inflammatory response and promote the formation of anti-inflammatory microenvironment.

Claims

1. A pH-responsive hydrogel composite dressing, characterized in that: The pH-responsive hydrogel composite dressing includes a hydrogel matrix and engineered exosomes and platelet-rich plasma uniformly loaded in the hydrogel matrix. The hydrogel matrix comprises a three-dimensional dynamic cross-linked network formed by a Schiff base reaction between an amino-containing polysaccharide derivative and an oxidized aldehyde-containing polysaccharide derivative. The engineered exosomes are derived from donor cells subjected to pathogen-associated molecular pattern stress treatment.

2. The pH-responsive hydrogel composite dressing according to claim 1, wherein: The mass ratio of the amino-containing polysaccharide derivative to the oxidized aldehyde-containing polysaccharide derivative is 1:5 to 5:1; the loading amount of the engineered exosomes in the hydrogel composite dressing is 50 to 500 μg / mL; and the volume percentage of the platelet-rich plasma in the hydrogel composite dressing is 1% to 50%.

3. The pH-responsive hydrogel composite dressing according to claim 1, wherein: The donor cells are selected from one or more of mesenchymal stem cells, dendritic cells, macrophages, or fibroblasts; the pathogen-associated molecular pattern is selected from one or more of heat-inactivated methicillin-resistant Staphylococcus aureus cells, methicillin-resistant Staphylococcus aureus cell wall components, methicillin-resistant Staphylococcus aureus nucleic acid extracts, or recombinant α-toxin proteins.

4. The pH-responsive hydrogel composite dressing of claim 1, wherein: The amino-containing polysaccharide derivative is selected from one or more of carboxymethyl chitosan, chitosan hydrochloride, or hydroxypropyl chitosan; the oxidized aldehyde-containing polysaccharide derivative is selected from one or more of oxidized hyaluronic acid, oxidized sodium alginate, or oxidized chondroitin sulfate.

5. The method for preparing the pH-responsive hydrogel composite dressing according to any one of claims 1 to 4, characterized in that: Includes the following steps: (1) The donor cells were co-incubated with the molecular model of pathogenic microorganisms to induce stress, and the supernatant was collected. The supernatant was then separated and purified by differential ultracentrifugation or tangential flow filtration to obtain engineered exosomes. (2) Under stirring or shaking conditions, the oxidized polysaccharide derivative solution containing aldehyde groups is mixed with the engineered exosomes and platelet-rich plasma obtained in step (1) to obtain precursor solution A; the precursor solution A is mixed evenly with the polysaccharide derivative solution containing amino groups and allowed to stand for cross-linking to obtain pH-responsive hydrogel composite dressing.

6. The method for preparing the pH-responsive hydrogel composite dressing according to claim 5, characterized in that: In step (1), the co-incubation time is 6~72 h.

7. The method of claim 5, wherein the pH-responsive hydrogel composite dressing is prepared by the steps of: In step (1), the differential ultracentrifugation method specifically involves: the collected supernatant is subjected to centrifugation at 1500 g to 2500 g, centrifugation at 8000 g to 12000 g, and ultracentrifugation at 100000 g to 150000 g in sequence.

8. The method of claim 5, wherein the pH-responsive hydrogel composite dressing is prepared by the steps of: In step (2), the static crosslinking temperature is 4~40℃ and the time is 1~60 min.

9. The use of the pH-responsive hydrogel composite dressing according to any one of claims 1 to 4 in the preparation of a medicament or medical dressing for treating diabetic wounds.

10. Use according to claim 9, characterized in that: The diabetic wound in question was a diabetic wound infected with methicillin-resistant Staphylococcus aureus.

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