Vaginal repair hydrogel dressing based on microsphere-matrix synergistic response and preparation method

Abstract

The present invention discloses a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response and a preparation method thereof, comprising GA@CC microspheres and SA-CC dynamic cross-linking matrix, wherein the GA@CC microspheres have glycyrrhizic acid as an active core and aldehyde chitosan as a shell; the SA-CC dynamic cross-linking matrix serves as a supporting framework and presents a porous honeycomb network structure, which is synergistically formed by SA solution, CC solution and calcium ion cross-linking agent; the GA@CC microspheres and SA-CC dynamic cross-linking matrix are cross-linked by the Ca bond between SA carboxyl groups and GA@CC microsphere surface hydroxyl groups. 2+ The present invention overcomes the bottlenecks of traditional technologies in response mechanism compatibility, mechanical-response balance, and spatiotemporal drug release control through the molecular design of a dual-response gating mechanism, the construction of a multiple network of dynamic cross-linking matrices, and the spatiotemporal coordinated loading of functional components. This achieves comprehensive improvements in dual-response accuracy, mechanical stability, and functional synergy.

Description

Technical Field

[0001] The present invention relates to a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response and a preparation method thereof, belonging to the technical field of vaginal mucosal damage repair. Background Art

[0002] Repair of vaginal mucosal damage is a major challenge in the field of gynecology. Its repair process has significant dynamic microenvironmental characteristics: the inflammatory phase (0-3 days after injury) is accompanied by pathogen invasion and immune cell infiltration, resulting in a surge in local ROS concentration (such as H2O2 up to 1-5mM) and an increase in pH from normal weak acidity (4.0-5.5) to neutral / weak alkalinity (6.0-7.4); during the proliferative phase (3-14 days), inflammation subsides and the pH gradually returns to the normal range, requiring the promotion of epithelial cell and fibroblast proliferation to rebuild the mucosal tissue.

[0003] Existing repair technologies have the following defects:

[0004] ① The single response mechanism leads to a narrow therapeutic window. Traditional materials can only sense a single signal, pH or ROS, and cannot simultaneously identify the dual characteristics of "pH increase + ROS surge" during the inflammatory period, resulting in premature drug release in an acidic environment (such as a release rate exceeding 40% when pH < 5.5) or insufficient release when ROS concentration is high (such as a 24-hour release rate increase of only 20% under 1 mM H2O2).

[0005] ② There is an imbalance between mechanical properties and biological functions. In pursuit of environmental responsiveness, existing hydrogels often use weak cross-linked networks, resulting in insufficient mechanical strength. For example, although a certain ROS-responsive hydrogel can release drugs quickly, its tensile strength is only 0.2 MPa. After implantation, it is easily decomposed by tissue fluid and cannot maintain the three-dimensional spatial structure required for repair.

[0006] ③ The synergistic effect of active ingredients is missing. Antibacterial, anti-inflammatory and pro-regenerative ingredients are mostly loaded through physical mixing, lacking a dynamic synergistic mechanism. For example, in traditional chitosan-glycyrrhizic acid gel, the anti-inflammatory effect of glycyrrhizic acid and the antibacterial function of chitosan are exerted independently, and a chain reaction of "inflammatory perception-antibacterial response-tissue regeneration" is not formed. Summary of the Invention

[0007] In response to the above problems in the prior art, the present invention provides a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response and a preparation method.

[0008] To achieve the above objectives, the present invention adopts a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response, comprising GA@CC microspheres and SA-CC dynamic cross-linked matrix. The GA@CC microspheres have glycyrrhizic acid as the active core and aldehyde chitosan as the shell.

[0009] The SA-CC dynamic cross-linking matrix serves as a supporting framework and presents a porous honeycomb network structure, which is formed by the SA solution, CC solution and calcium ion cross-linking agent;

[0010] The GA@CC microspheres and SA-CC dynamic cross-linking matrix are fixed by ionic bonds, which are formed by the SA carboxyl groups and the hydroxyl groups on the surface of the GA@CC microspheres through Ca 2+ Bridge formation.

[0011] The second aspect of the present invention further provides a method for preparing the vaginal repair hydrogel dressing based on microsphere-matrix synergistic response, comprising the following steps:

[0012] S1. Preparation of GA@CC microspheres

[0013] CC was dissolved in deionized water to prepare a solution, and GA was added and stirred to form an aqueous phase;

[0014] Take liquid paraffin as the oil phase, add emulsifier, and make the emulsifier evenly dispersed in the oil phase;

[0015] The water phase is slowly added dropwise to the oil phase, stirred and emulsified to form a stable oil-in-water emulsion;

[0016] Add a crosslinker to the emulsion and stir the mixture at room temperature to crosslink the aldehyde group of CC with the hydroxyl group of GA via Schiff base bonds to form a microsphere shell.

[0017] After the reaction is completed, petroleum ether is added to break the emulsion, centrifuged, the supernatant is discarded, the precipitate is washed with anhydrous ethanol, and freeze-dried to obtain microsphere powder with a particle size of 1-5 μm, which is sealed and stored for later use;

[0018] S2. Preparation of SA-CC dynamic cross-linking matrix

[0019] Weigh SA powder, add deionized water, and stir to dissolve to form a clear and transparent colloidal SA solution;

[0020] Take aldehyde chitosan, dissolve it in deionized water, stir to dissolve, and filter to obtain a uniform CC solution;

[0021] SA solution was mixed with CC solution, CaCl2 powder was added, and then freeze-dried microsphere powder was added, and ultrasonic treatment was performed to uniformly disperse the microspheres to form a milky white suspension;

[0022] S3. Preparation of hydrogel dressing

[0023] The milky white suspension was injected into the vaginal injury model and placed in a constant temperature environment of 36-38°C to solidify into a hydrogel dressing, which was light yellow and translucent.

[0024] As an improvement, the preparation process of CC in step S1 is as follows:

[0025] Chitosan was added to a glacial acetic acid solution and stirred until completely dissolved, and then sodium periodate was weighed and added to the solution, wherein the mass ratio of sodium periodate to chitosan was 0.4:1-1:1;

[0026] The reaction was stirred in the dark at room temperature and a pH of 3.5-4.5. Ethylene glycol was then added and stirred for a certain period of time to terminate the reaction. The pH was then adjusted to 7.0-8.0. Anhydrous ethanol was added dropwise to precipitate. After centrifugation, the precipitate was washed with ethanol. The precipitate was dissolved in deionized water and transferred to a dialysis bag for dialysis. After filtration, it was freeze-dried to obtain a white flocculent CC powder.

[0027] As an improvement, in step S1, CC is dissolved in deionized water to prepare a 10-15% w / v solution, wherein the mass percentage of CC and GA is 60-70% by mass, and the balance is GA;

[0028] Span 80 is used as the emulsifier, and the mass of the emulsifier is 3-5% of the mass of the oil phase.

[0029] As an improvement, in step S1, the volume ratio of the water phase to the oil phase is 1:10, and the mixture is stirred and emulsified at a speed of 800-1200 rpm for 30-45 minutes.

[0030] As an improvement, in step S1, EDC and NHS are used as cross-linking agents, and the mass ratio of EDC, NHS and CC is (0.5-1.5): (0.5-1):5.

[0031] As an improvement, in step S1, petroleum ether twice the volume of the emulsion is added to break the emulsion, the mixture is transferred to a centrifuge tube and centrifuged at 8000 rpm for 10-15 minutes, the supernatant is discarded, the precipitate is washed with anhydrous ethanol to remove the residual oil phase, and finally freeze-dried in a freeze dryer at -80°C and a vacuum degree of ≤10 Pa for 24 hours to obtain microsphere powder.

[0032] As an improvement, in step S2, SA powder with a molecular weight of 100-200 kDa is used, deionized water is added to prepare a 1-3% w / v solution, and the solution is magnetically stirred in a 40°C water bath until completely dissolved;

[0033] Take aldehyde chitosan and add deionized water to make a 2-5% w / v solution. Stir at room temperature until dissolved. Filter through a 0.45 μm filter membrane to remove insoluble matter to obtain a uniform CC solution.

[0034] As an improvement, in step S2, the SA solution and the CC solution are mixed in a volume ratio of 1:1, CaCl2 powder is added to make the calcium ion concentration reach 0.05-0.1 M, and then the freeze-dried microsphere powder is added to make the microsphere powder 1-3% w / v, and the mixture is treated by an ultrasonic disperser for 10-15 minutes.

[0035] As an improvement, the hydrogel dressing has viscoelasticity, a tensile strength of 0.5-1.3 MPa, and an elongation at break of 180-350%.

[0036] The mechanism of the present invention: Through a dual-response gating mechanism, the dual-response functions of pH and ROS are integrated into the shell of GA@CC microspheres, wherein the shell realizes dual controlled release through Schiff base bond and oxidation cleavage of catechol groups. During the inflammatory period (pH>5.5 and ROS>1mM), the increase in pH enhances the polarity of the Schiff base bond and increases the hydrolysis cleavage rate. At the same time, the increase in ROS concentration promotes the oxidation of catechol groups to form quinone structures, resulting in the rupture of the shell cross-linked network. The dual action precisely triggers drug release, avoiding the hysteresis or excessive release problem caused by microenvironment fluctuations in traditional single response. Through the design of an interpenetrating network dynamic cross-linking matrix of sodium alginate (SA) and aldehyde chitosan (CC), the carboxylate groups of SA and Ca 2+ The hydrogel dressing forms ionic bonds, enabling rapid initial solidification and providing foundational mechanical strength. The aldehyde groups of CC and the hydroxyl groups of SA react via a Schiff base reaction to form dynamic covalent bonds, endowing the network with reversible breakage and recombination capabilities. This allows the hydrogel dressing to possess both tensile strength and elongation at break, adapting to the dynamic mechanical environment of the vaginal mucosa. Using repair-promoting microspheres (GA@CC), during the inflammatory phase (0-3 days), GA inhibits the NF-κB pathway to reduce the secretion of proinflammatory cytokines. After degradation of the CC shell, the amino groups with positive charges adsorb on bacterial membranes, achieving antibacterial effects. Simultaneously, ROS triggers a cascade effect of CC antimicrobial and GA anti-inflammatory effects, reducing inflammatory cytokine levels. During the proliferation phase (3-14 days), as the pH returns to acidic, CC amino groups protonate, enhancing cell adhesion. Its degradation product, chitosan oligosaccharide, activates the AKT signaling pathway, promoting fibroblast proliferation and inducing collagen synthesis. This achieves a spatiotemporal synergistic effect of anti-inflammatory, antimicrobial, and pro-regenerative functions, overcoming the bottlenecks of traditional technologies characterized by a single response, an imbalance between mechanical and functional properties, and a lack of synergistic effects among active ingredients.

[0037] The main difficulties that traditional single-response (pH or ROS) hydrogels face in achieving dual-response are: first, the chemical compatibility bottleneck of the response mechanism. The chemical environment required for pH response and ROS response may conflict. For example, ROS stability decreases under acidic conditions and ROS response groups are easily inactivated in alkaline environments, making it difficult for the two to be compatible in the same system; second, the problem of balancing mechanical properties and response efficiency. Traditional single-response hydrogels often use weak cross-linked networks to achieve sensitive properties, which lack mechanical strength, while dual-response requires complex network design, which is prone to the contradiction of "sensitive response but easy to break" or "sufficient strength but delayed response"; third, the obstacle of precise control of spatiotemporal coordinated drug release. Traditional single mechanisms cannot distinguish between microenvironmental changes in the inflammatory and proliferative phases, which can easily lead to insufficient or excessive drug release.

[0038] From the traditional single response to the realization of dual response, the core technical point of this invention lies in the molecular design of the dual-response gating mechanism (synergistic triggering of Schiff base bond and catechol oxidation to form a logical "AND" gating), the multiple network construction of the dynamic cross-linked matrix (ionic bond + covalent bond double cross-linking of SA-CC interpenetrating network, combined with the microsphere-matrix cascade effect), and the spatiotemporal coordinated loading of functional components (GA@CC microsphere core-shell division of labor realizes rapid drug release in the inflammatory period and sustained release repair in the proliferation period). It systematically breaks through the bottlenecks of traditional technologies in response mechanism compatibility, mechanical-response balance and spatiotemporal drug release control, and achieves a comprehensive improvement in the accuracy, mechanical stability and functional synergy of dual response.

[0039] Therefore, the vaginal repair hydrogel dressing of the present invention has the following advantages:

[0040] ① In terms of dual-response accuracy, at pH = 7.4 (simulated environment during the inflammatory period) and ROS = 1 mM, the release rate of GA@CC in the first 12 hours was higher than that of traditional pH-responsive hydrogels, and its ROS response performance was stronger, indicating that the present invention has significantly improved the synergistic response ability to dual microenvironmental signals.

[0041] ②The swelling rate of the SA-CC network structure is 530% at pH>5.5, which is more than twice that of the single SA hydrogel (swelling rate 250%), ensuring a dynamic balance between drug release and mechanical stability.

[0042] ③ In terms of multifunctional timing synergy, during the inflammatory period (0-3 days), ROS triggered the antibacterial activity of CC and the anti-inflammatory effect of GA, reducing the level of inflammatory factors by 68%. During the proliferation period (3-14 days), as the pH dropped, the CC-promoting cell proliferation effect was enhanced (72h proliferation rate 145%), which was more than 30% higher than traditional materials (72h proliferation rate 110%), realizing the staged precision treatment of "inflammation control-tissue regeneration".

[0043] ④ In terms of biosafety, the core ingredients SA, CC, and GA are all natural biomaterials. SA is widely used in the food and pharmaceutical fields. CC and GA have been toxicologically verified to be non-cytotoxic. The preparation process does not require the use of harmful cross-linking agents such as glutaraldehyde, avoiding the potential irritation risks of traditional synthetic materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Microscopic morphology of GA@CC microspheres and hydrogel dressings according to Example 1 of the present invention; (a) GA@CC microsphere structure, (b) hydrogel dressing network;

[0045] Figure 2Schematic diagram of the response of the hydrogel dressing of Example 1 of the present invention to pH and ROS; (a) Response diagram of the hydrogel dressing to pH (4.0, 6.0, 7.4); (b) Response diagram of the hydrogel dressing to ROS (0.1mM, 1mM, 5mM);

[0046] Figure 3 Schematic diagram of pH (4.0, 5.5, 7.4) responsive drug release of the hydrogel dressing of Example 1 of the present invention;

[0047] Figure 4 Schematic diagram of the degradation of the hydrogel dressing in Example 1 of the present invention in response to ROS (H2O2 concentrations of 0.1mM, 0.5mM, 1mM, and 5mM);

[0048] Figure 5 Schematic diagram of the anti-inflammatory ability of the hydrogel dressing of Example 1 of the present invention;

[0049] Figure 6 Schematic diagram of the repair-promoting function of the hydrogel dressing of Example 1 of the present invention. DETAILED DESCRIPTION

[0050] The following embodiments are further descriptions of the present invention to illustrate the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0051] Explanation of some relevant terms in this invention:

[0052] ROS: Reactive Oxygen Species, whose concentration can rise to 1-5mM in an inflammatory environment (<0.1mM in normal tissues), triggering oxidative stress damage.

[0053] SA: Sodium Alginate, a natural polysaccharide, whose carboxylate dissociates at pH>5.5 and reacts with Ca 2+ Forming an "egg box" shaped ion cross-linked structure.

[0054] CC: Carboxymethyl Chitosan Aldehyde. Chitosan is modified by oxidation to introduce aldehyde groups, which has pH-responsive antibacterial activity (inhibition rate <30% when pH < 5.5, inhibition rate > 99% when pH > 5.5).

[0055] Example 1

[0056] A method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response, comprising the following steps:

[0057] S1. Preparation of GA@CC microspheres

[0058] Preparation method of CC: First, 5 g of chitosan with a deacetylation degree of ≥90% was added to 100 mL of 1% glacial acetic acid solution and stirred until completely dissolved. Then, 4 g of sodium periodate was weighed and added to the solution. The mixture was stirred in the dark at room temperature and pH 4 for 3 hours. Then, 5 mL of ethylene glycol was added and stirred for 30 minutes to terminate the reaction. The pH was then adjusted to 7.5 with 1 M NaOH. Four volumes of anhydrous ethanol were added dropwise to precipitate. After centrifugation, the precipitate was washed with ethanol. The precipitate was dissolved in deionized water and transferred to a dialysis bag for 72 hours. Finally, it was filtered through a filter membrane and freeze-dried for 48 hours to obtain a white flocculent CC powder.

[0059] Preparation of aqueous phase: Dissolve 6.5g CC in deionized water to prepare a 10% w / v solution, add 3.5g GA (glycyrrhizic acid), and stir magnetically at room temperature for 30 minutes until it is completely dissolved to form a homogeneous transparent aqueous phase;

[0060] Preparation of oil phase: Liquid paraffin was used as the oil phase, and 4% of the oil phase weight of Span 80 was added as an emulsifier. The emulsifier was evenly dispersed in the oil phase by vortex oscillation.

[0061] Emulsification: The aqueous phase was slowly added dropwise to the oil phase (aqueous phase:oil phase volume ratio of 1:10) while stirring at 1000 rpm for 35 minutes to form a stable oil-in-water (O / W) emulsion. Optical microscopy confirmed uniform particle size distribution (average particle size 1-5 μm).

[0062] Chemical crosslinking: Add crosslinking agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the emulsion at a mass ratio of EDC:NHS:CC of 1:0.8:5. Stir the mixture at room temperature for 5 hours to allow the CC aldehyde groups to crosslink with the GA hydroxyl groups via Schiff base bonds to form the microsphere shell.

[0063] Microsphere collection: After the reaction is completed, petroleum ether (twice the volume of the emulsion) is added to break the emulsion. The mixture is transferred to a centrifuge tube and centrifuged at 8000 rpm for 12 minutes. The supernatant is discarded, and the precipitate is washed three times with anhydrous ethanol to remove the residual oil phase. Finally, it is freeze-dried in a freeze dryer (-80°C, vacuum degree 10Pa) for 24 hours to obtain dry microsphere powder, which is sealed and stored at 4°C until use. Microsphere particle size control: By adjusting the emulsification speed (800-1200 rpm) and the aqueous phase concentration (10-15% CC), the microsphere particle size can be controlled within the range of 1-5 μm.

[0064] S2. Preparation of SA-CC dynamic cross-linking matrix

[0065] Preparation of SA solution: Weigh sodium alginate powder (molecular weight 100-200 kDa) and add deionized water to make a 2% w / v solution. Stir magnetically in a 40°C water bath until completely dissolved (approximately 2 hours). Allow to stand for 30 minutes to defoam, forming a clear, transparent colloidal SA solution.

[0066] Preparation of CC solution: Take aldehyde chitosan and add deionized water to make a 4% w / v solution. Stir at room temperature until dissolved (about 1 hour). Filter through a 0.45 μm filter membrane to remove insoluble matter to obtain a homogeneous CC solution.

[0067] Preparation of the precursor solution: SA solution and CC solution were mixed in a volume ratio of 1:1. CaCl2 powder was added to make the calcium ion concentration reach 0.08 M. Then, freeze-dried microsphere powder was added to make the microsphere concentration 2% w / v. The microspheres were evenly dispersed in the matrix precursor using an ultrasonic disperser (power 200 W, frequency 40 kHz) for 12 minutes to form a milky white suspension.

[0068] S3. Preparation of hydrogel dressing

[0069] The milky white suspension was injected into the vaginal injury model (physical injury model) and placed in a constant temperature environment of 37°C. 2+ The carboxylate group (-COO - ) rapidly form ionic bonds, while the aldehyde groups of CC and the hydroxyl groups of SA react via a Schiff base to form dynamic covalent bonds. This dual crosslinking effect allows the hydrogel dressing to cure within 10 minutes. The cross-linked hydrogel dressing exhibits a light yellow, translucent appearance and exhibits excellent viscoelasticity, a tensile strength of 0.8 MPa, and an elongation at break of 250%. Its self-healing properties can be verified by a finger compression test (recovering to its original shape within 10 seconds after deformation). The hydrogel dressing prepared by this invention can be used directly as a dressing.

[0070] The performance of the GA@CC microspheres and hydrogel dressing prepared in Example 1 was analyzed:

[0071] Among them, the appearance is Figure 1 As shown, Figure 1 (a) GA@CC microsphere structure: regular spherical shape, uniform particle size distribution (1-5 μm), smooth surface and no obvious agglomeration; Figure 1 (b) Hydrogel dressing network: The SA-CC interpenetrating network exhibits a porous honeycomb structure with pore sizes of approximately 50-200 nm. The microspheres are uniformly dispersed within the matrix pores and bonded to the matrix through ionic bonds. The regular structure of the GA@CC microspheres ensures stable drug loading, while the porous network of the hydrogel facilitates cell adhesion (such as fibroblasts) and nutrient diffusion, while also providing pathways for drug release.

[0072] The dual-responsive swelling behavior is reflected in the fact that under different pH and ROS conditions, VEGF-hydrogel can respond to release the required active ingredients. The hydrogel dressing samples were immersed in phosphate buffers with pH 4.0 (simulating a healthy acidic environment), pH 6.0 (simulating the injury transition period), and pH 7.4 (simulating the alkaline environment of the inflammatory period), respectively. The samples were shaken at a constant temperature of 37°C, and the wet weight was measured at regular intervals to calculate the swelling rate. Figure 2 (a) pH response: At pH 4.0 (acidic), the swelling rate was 122.3±1.3%, and the matrix was tightly curled, inhibiting drug release. At pH 7.4 (simulating the inflammatory phase), the swelling rate was 530.8±1.7%, and the SA carboxyl group dissociated, causing the network to stretch and triggering the opening of the microsphere release channel. The hydrogel dressing samples were placed in PBS solutions containing 0.1mM (normal level) and 5mM (high concentration during inflammatory phase) H2O2 and incubated at 37°C. The swelling rate of the material was measured. Figure 2 (b) shows the ROS response: At 0.1 mM H2O2 (normal concentration), the swelling rate is 158.2 ± 0.7%; at 5 mM H2O2 (high concentration during the inflammatory phase), the swelling rate is 620.3 ± 1.0%. Oxidative cleavage of the CC catechol groups causes local degradation of the matrix, accelerating swelling. This dual-responsive swelling behavior dynamically matches the injury microenvironment. The inflammation phase (increased pH and ROS concentration) significantly increases the swelling rate, providing a physical basis for efficient drug release.

[0073] pH-responsive drug release: The hydrogel dressing samples were placed in 0.1M phosphate buffer at pH 4.0 (healthy acidic), pH 5.5 (injury transition phase), and pH 7.4 (inflammatory phase alkaline) respectively, and kept at 37°C with constant temperature and oscillation. The supernatant of the solution was taken at regular intervals and the GA release rate was measured and calculated using a UV spectrophotometer. In an acidic environment (pH < 5.5), the CC amino group was protonated (-NH3 + ) and SA carboxylate (-COO - ) Electrostatic interaction forms a dense layer, inhibiting drug diffusion; when pH>5.5, the amino group is deprotonated to release the electrostatic interaction, and the SA network swells, triggering the breakage of the Schiff base bonds in the microsphere shell, releasing GA and CC. Figure 3The 48-hour drug release profile of VEGF-GA@CC / SA-CC microspheres at different pH values ​​is shown. At pH 4.0 (simulating the inflammatory phase), the 48-hour release rate was 13.1±1.1%, with electrostatic interactions inhibiting drug diffusion. At pH 5.5 (transition phase), the 48-hour release rate was 49.6±4.4%, indicating weakening electrostatic interactions and the onset of Schiff-base bond breakage. At pH 7.4 (inflammatory phase), the 48-hour release rate was 73.4±4.8%, indicating rapid drug release due to the dual effects of matrix swelling and Schiff-base bond breakage. This indicates that VEGF-GA@CC / SA-CC precisely matches the pH changes of vaginal lesions from the proliferative (acidic) to the inflammatory (alkaline) phase, preventing drug leakage from healthy tissue (pH < 5.5) and ensuring effective drug delivery to the inflammatory site.

[0074] ROS-responsive degradation: Hydrogel dressing samples were placed in PBS solutions containing 0.1 mM (normal level) and 5 mM (high concentration during inflammation) H₂O₂ and incubated at 37°C. The supernatant was periodically collected and measured using a UV spectrophotometer to calculate the GA release rate. When the H₂O₂ concentration is >1 mM, the CC catechol groups oxidize to form quinone structures, resulting in a break in the shell cross-linking network and a decrease in the GA release rate. Furthermore, low-molecular-weight fragments produced by CC degradation can enhance fibroblast proliferation. Figure 4 The release rate of the microspheres under different H2O2 concentrations is shown. At a H2O2 concentration of 0.1mM, the 12-hour release rate is 16.4±1.4%, indicating slow release in a low ROS environment. At a H2O2 concentration of 5mM, the 12-hour release rate reaches 67.8±2.5%, indicating that ROS triggers the degradation of the CC shell, and the release in the first 3 hours increases by approximately 3 times compared to the ROS-free condition. This ROS-responsive release characteristic can specifically activate microsphere drug release when high ROS concentrations are present during the inflammatory phase, achieving an intelligent release mode of "targeted burst release at the site of injury and low leakage in healthy tissue," thereby reducing systemic side effects.

[0075] Anti-inflammatory ability: The synergistic group builds a "low inflammation-high repair" microenvironment to solve the problem of insufficient anti-inflammatory of traditional materials. THP-1 macrophages in the logarithmic growth phase were taken at 1×10 6Cells / well were seeded on a 6-well plate, and after phorbol ester was added to adhere to the wall, the cells were divided into groups for treatment: the control group was added with complete culture medium, the SA-CC group was added with culture medium containing SA-CC matrix, and the VEGF-GA@CC / SA-CC group was added with culture medium containing microsphere-matrix (i.e., hydrogel dressing). Each group used 1μg / mL LPS to induce M1 macrophages for 24 hours, and after washing, they were cultured with the corresponding culture medium for another 24 hours; flow cytometry was performed to detect the cells, using FITC-labeled CD86 (M1 marker) and APC-labeled CD206 (M2 marker) staining to promote the conversion of M1 pro-inflammatory macrophages to M2 anti-inflammatory macrophages. M2 cells secreted anti-inflammatory factors (such as IL-10) to inhibit excessive immune responses. At the same time, CC degradation products induced collagen synthesis and accelerated vaginal mucosal regeneration. The results are as follows Figure 5 As shown, in the control group, Q1 (M1 macrophages, pro-inflammatory) accounted for 34.5%, while Q3 (M2 macrophages, anti-inflammatory) accounted for 8.77%, indicating a predominant pro-inflammatory response. In the SA-CC group (stromal group), Q1 decreased to 17.6% (a decrease of 16.9%), while Q3 increased to 20.5% (an increase of 11.73%), indicating a preliminary anti-inflammatory response. In the VEGF-GA@CC / SA-CC group (synergistic group), Q1 further decreased to 11.5% (a decrease of 23% compared to the control group and 6.1% compared to the stroma group), while Q3 reached 19.9% ​​(an increase of 11.13%). In the synergistic group, GA inhibited NF-κB, CC modulated the immune phenotype (pH-responsive cell adhesion and M1 to M2 conversion), and VEGF promoted angiogenesis (providing an energy-enhancing anti-inflammatory microenvironment), achieving a triple synergistic effect: direct inhibition of M1 (Q1↓), activation of M2 (Q3↑), SA-CC matrix responsive to the microenvironment for precise drug release, and VEGF maintained and repaired the microenvironment. This synergy enables M2 to secrete anti-inflammatory factors and induce collagen synthesis, establishing an "anti-inflammatory-repair" balance, resolving the defects of traditional materials, and providing an efficient solution for multi-stage repair of vaginal mucosal injuries. It reflects the enhanced anti-inflammatory function of the microsphere-matrix synergy and meets the needs of dynamic repair.

[0076] Antibacterial ability: The antibacterial experiment used Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus) as test strains, and the bacterial solution concentration was adjusted to OD 600 =0.5 (approximately 1×10 8 CFU / mL), and the synergistic group of SA-CC, VEGF-GA@CC microspheres and the mixture of the two were incubated with shaking at pH 7.4 (simulating the inflammatory period) and 37°C for 2 hours; the colonies were counted on LB plates by the gradient dilution method, and the Log Reduction value (i.e., the logarithmic reduction value, Log Reduction = log 10 Control group CFU / mL – log 10CFU / mL in the treatment group), CC had an antibacterial rate of >99% at pH>5.5, and GA inhibited the inflammatory factor IL-6 by inhibiting the NF-κB pathway. The two formed a cascade effect of "clearing bacteria and reducing inflammation" during the inflammatory period. Subsequently, CC promoted cell adhesion through positively charged groups, induced collagen deposition, and achieved tissue regeneration. The results are shown in Table 1: The CFU / mL of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in the control group were both 10 11 The LogReduction of the SA-CC group against the two bacteria was 0.77±0.12 (E. coli) and 0.80±0.07 (S. aureus), respectively, indicating that the matrix has a certain antibacterial ability. The LogReduction of the VEGF-GA@CC group was 0.78±0.34 (E. coli) and 0.97±0.25 (S. aureus), with antibacterial effects comparable to those of the matrix group. The LogReduction of the VEGF-GA@CC / SA-CC synergistic group reached 1.68±0.26 for E. coli (approximately 1.1-fold higher than that of the matrix group) and 2.10±1.04 for S. aureus (approximately 1.6-fold higher than that of the matrix group), indicating that the synergistic effect between the microspheres and the matrix significantly enhanced the antibacterial properties. This synergy stems from dual-responsive drug release (matrix swelling triggers the directional release of CC antibacterial components in the microspheres) and structural complementarity (matrix porous network loads microspheres, and the microsphere core-shell structure breaks through the barrier to effectively inhibit bacteria). It has significant inhibition on both types of bacteria, covering common vaginal pathogens, and achieving "high antibacterial effect on damaged areas and low interference with healthy tissues", verifying the advantages of the "microsphere-matrix" collaborative design in antibacterial function, and providing support for infection control and tissue regeneration during the inflammatory period.

[0077] Table 1 Antibacterial ability data

[0078]

[0079] Evaluation of repair-promoting function: Using fibroblasts as a model, cells were cultured at a rate of 5×10 3 Cells / well were seeded in a 96-well plate, and SA-CC matrix, VEGF-GA@CC microspheres and serum-free culture medium (10% w / v) of the two were added respectively. The control group was serum-free culture medium. The proliferation rate was detected by CCK-8 method after 72 hours of culture. Figure 6Figure 2 shows the 72-hour proliferation rate of fibroblasts on the hydrogel. The control group had a cell survival rate of approximately 100%, serving as a baseline. There was no significant difference between the SA-CC group (matrix group) and the control group, indicating that the matrix is ​​non-cytotoxic and does not inhibit proliferation. The VEGF-GA@CC group (microsphere group) showed significantly higher cell survival than the control group, as VEGF directly promotes cell growth, while GA indirectly supports proliferation by modulating the inflammatory microenvironment. The VEGF-GA@CC / SA-CC group (synergistic group) showed a slightly lower survival rate than the microsphere group, but still significantly higher than the control group, demonstrating a synergistic effect between the microspheres and the matrix: SA-CC provides three-dimensional support, synergizing with VEGF and CC degradation products (such as chitosan oligosaccharides) in the microspheres, and promoting a sustained release effect. Mechanistically, GA provides anti-inflammatory effects during the first three days, VEGF continues to promote proliferation from days 3 to 14, and CC induces collagen synthesis, achieving a phased regulation of "anti-inflammation first, then proliferation," which meets the physiological needs of vaginal repair. The high proliferation rate of the microsphere group verified the efficacy of VEGF, and the synergistic group could optimize the SA / CC ratio to enhance proliferation. The experimental data demonstrated the "functional synergy-temporal repair" design of the present invention, providing reliable evidence for clinical application.

[0080] Example 2

[0081] A method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response, comprising the following steps:

[0082] S1. Preparation of GA@CC microspheres

[0083] Preparation method of CC: First, 5 g of chitosan with a deacetylation degree of ≥90% was added to 100 mL of 1% glacial acetic acid solution and stirred until completely dissolved. Then, 5 g of sodium periodate was weighed and added to the solution. The mixture was stirred in the dark at room temperature and pH 3.5 for 2 hours. Then, 5 mL of ethylene glycol was added and stirred for 30 minutes to terminate the reaction. The pH was then adjusted to 7.0 with 1 M NaOH. Four volumes of anhydrous ethanol were added dropwise to precipitate. After centrifugation, the precipitate was washed with ethanol. The precipitate was dissolved in deionized water and transferred to a dialysis bag for 72 hours. Finally, it was filtered through a filter membrane and freeze-dried for 48 hours to obtain a white flocculent CC powder.

[0084] Preparation of aqueous phase: Dissolve 7g CC (aldehyde chitosan) in deionized water to prepare a 10% w / v solution, add 3g GA (glycyrrhizic acid), and stir magnetically at room temperature for 30 minutes until completely dissolved to form a homogeneous transparent aqueous phase;

[0085] Preparation of oil phase: Liquid paraffin was used as the oil phase, and 3% of the oil phase weight of Span 80 was added as an emulsifier. The emulsifier was evenly dispersed in the oil phase by vortex oscillation.

[0086] Emulsification: The aqueous phase was slowly added dropwise to the oil phase (aqueous phase:oil phase volume ratio of 1:10) while stirring at 800 rpm for 45 minutes to form a stable oil-in-water (O / W) emulsion. Optical microscopy confirmed uniform particle size distribution (average particle size 1-5 μm).

[0087] Chemical crosslinking: Add crosslinking agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the emulsion, where the mass ratio of EDC:NHS:CC is 1:0.8:5. The mixture is stirred at room temperature for 4 hours to crosslink the CC aldehyde group with the GA hydroxyl group through Schiff base bonds to form the microsphere shell.

[0088] Microsphere collection: After the reaction is completed, petroleum ether (2 times the volume of the emulsion) is added to break the emulsion, and the mixture is transferred to a centrifuge tube and centrifuged at 8000 rpm for 10 minutes. The supernatant is discarded, and the precipitate is washed three times with anhydrous ethanol to remove the residual oil phase. Finally, the precipitate is freeze-dried in a freeze dryer (-80°C, vacuum degree 9 Pa) for 24 hours to obtain dry microsphere powder, which is sealed and stored at 4°C until use;

[0089] S2. Preparation of SA-CC dynamic cross-linking matrix

[0090] Preparation of SA solution: Weigh sodium alginate powder (molecular weight 100-200 kDa) and add deionized water to make a 1% w / v solution. Stir magnetically in a 40°C water bath until completely dissolved (approximately 2 hours). Allow to stand for 30 minutes to defoam, forming a clear, transparent colloidal SA solution.

[0091] Preparation of CC solution: Take aldehyde chitosan and add deionized water to make a 2% w / v solution. Stir at room temperature until dissolved (about 1 hour). Filter through a 0.45 μm filter membrane to remove insoluble matter to obtain a homogeneous CC solution.

[0092] Preparation of precursor solution: SA solution and CC solution were mixed in a volume ratio of 1:1. CaCl2 powder was added to make the calcium ion concentration reach 0.05 M. Then, freeze-dried microsphere powder was added to make the microsphere concentration 1% w / v. Ultrasonic dispersion was performed (power 200 W, frequency 40 kHz) for 10 minutes to uniformly disperse the microspheres in the matrix precursor to form a milky white suspension.

[0093] S3. Preparation of hydrogel dressing

[0094] The milky white suspension was injected into the vaginal injury model and placed in a constant temperature environment of 37°C. 2+ The carboxylate group (-COO -) rapidly form ionic bonds, while the aldehyde groups of CC and the hydroxyl groups of SA react via a Schiff base reaction to form dynamic covalent bonds. This dual crosslinking effect allows the hydrogel dressing to cure within 12 minutes. The cross-linked hydrogel dressing is light yellow and translucent, exhibiting excellent viscoelasticity, a tensile strength of 0.7 MPa, and an elongation at break of 250%. Its self-healing properties can be verified by a finger pressure test (recovering to its original shape within 10 seconds after deformation).

[0095] Example 3

[0096] A method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response, comprising the following steps:

[0097] S1. Preparation of GA@CC microspheres

[0098] Preparation method of CC: First, 5 g of chitosan with a deacetylation degree of ≥90% was added to 100 mL of 1% glacial acetic acid solution and stirred until completely dissolved. Subsequently, 2 g of sodium periodate was weighed and added to the solution. The mixture was stirred in the dark at room temperature and pH 4.5 for 4 hours. Then, 5 mL of ethylene glycol was added and stirred for 30 minutes to terminate the reaction. The pH was then adjusted to 8.0 with 1 M NaOH. Four volumes of anhydrous ethanol were added dropwise to precipitate. After centrifugation, the precipitate was washed with ethanol. The precipitate was dissolved in deionized water and transferred to a dialysis bag for 72 hours. Finally, it was filtered through a filter membrane and freeze-dried for 48 hours to obtain a white flocculent CC powder.

[0099] Preparation of aqueous phase: Dissolve 6g CC in deionized water to prepare a 15% w / v solution, add 4g GA (glycyrrhizic acid), and stir magnetically at room temperature for 30 minutes until it is completely dissolved to form a homogeneous transparent aqueous phase;

[0100] Preparation of oil phase: Take liquid paraffin as the oil phase, add 5% of the oil phase weight of Span 80 as an emulsifier, and vortex oscillate to evenly disperse the emulsifier in the oil phase;

[0101] Emulsification: The aqueous phase was slowly added dropwise to the oil phase (aqueous phase:oil phase volume ratio of 1:10) while stirring at 1200 rpm for 30 minutes to form a stable oil-in-water (O / W) emulsion. Optical microscopy confirmed uniform particle size distribution (average particle size 1-5 μm).

[0102] Chemical crosslinking: Add crosslinking agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the emulsion, where the mass ratio of EDC:NHS:CC is 1:0.8:5. The mixture is stirred at room temperature for 6 hours to form a microsphere shell by crosslinking the CC aldehyde group with the GA hydroxyl group through Schiff base bonds.

[0103] Microsphere collection: After the reaction is completed, petroleum ether (2 times the volume of the emulsion) is added to break the emulsion, and the mixture is transferred to a centrifuge tube and centrifuged at 8000 rpm for 15 minutes. The supernatant is discarded, and the precipitate is washed three times with anhydrous ethanol to remove the residual oil phase. Finally, the precipitate is freeze-dried in a freeze dryer (-80°C, vacuum degree 7 Pa) for 24 hours to obtain dry microsphere powder, which is sealed and stored at 4°C until use;

[0104] S2. Preparation of SA-CC dynamic cross-linking matrix

[0105] Preparation of SA solution: Weigh sodium alginate powder (molecular weight 100-200 kDa) and add deionized water to make a 3% w / v solution. Stir magnetically in a 40°C water bath until completely dissolved (approximately 2 hours). Allow to stand for 30 minutes to defoam, forming a clear and transparent SA solution.

[0106] Preparation of CC solution: Take aldehyde chitosan and add deionized water to make a 5% w / v solution. Stir at room temperature until dissolved (about 1 hour). Filter through a 0.45 μm filter membrane to remove insoluble matter to obtain a uniform CC solution.

[0107] Preparation of precursor solution: SA solution and CC solution were mixed in a volume ratio of 1:1. CaCl2 powder was added to make the calcium ion concentration reach 0.1 M. Then, freeze-dried microsphere powder was added to make the microsphere concentration 3% w / v. Ultrasonic dispersion was performed (power 200 W, frequency 40 kHz) for 15 minutes to uniformly disperse the microspheres in the matrix precursor to form a milky white suspension.

[0108] S3. Preparation of hydrogel dressing

[0109] The milky white suspension was injected into the vaginal injury model and placed in a constant temperature environment of 37°C. 2+ The carboxylate group (-COO - ) quickly forms an ionic bond, and at the same time, the aldehyde group of CC and the hydroxyl group of SA react to form a dynamic covalent bond through the Schiff base reaction. The double cross-linking effect enables the hydrogel dressing to be cured within 8 minutes; the cross-linked hydrogel dressing is light yellow and translucent, has good viscoelasticity, a tensile strength of up to 1MPa, an elongation at break of 260%, and can pass the finger press test (recovering to its original shape within 10 seconds after deformation) to verify its self-healing performance.

[0110] Example 4

[0111] A vaginal repair hydrogel dressing based on microsphere-matrix synergistic response, which can be used directly as a dressing, is prepared using the preparation method of Example 1 and includes GA@CC microspheres and a SA-CC dynamic cross-linked matrix;

[0112] The GA@CC microspheres (particle size 1-5 μm) serve as the functional core, with an aldehyde-chitosan (CC) cross-linked network as the shell, encapsulating the glycyrrhizic acid (GA) active core, and achieving dual controlled release through pH-sensitive Schiff base bonds and ROS-sensitive catechol oxidative cleavage;

[0113] The SA-CC dynamic cross-linked matrix (SA-CC interpenetrating network) serves as a supporting framework, in which sodium alginate (SA) is connected to the surface of the SA-CC matrix by Ca 2+ Providing mechanical strength, CC imparts self-healing ability through Schiff base dynamic bonds and regulates antibacterial activity and cell adhesion efficiency through amino protonation / deprotonation;

[0114] The GA@CC microspheres and SA-CC dynamic cross-linking matrix are fixed by ionic bonds, which are formed by the SA carboxyl groups and the hydroxyl groups on the surface of the GA@CC microspheres through Ca 2+ Bridge formation realizes the cascade effect of "matrix-responsive regulated microsphere release".

[0115] The above description is only a preferred embodiment and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A vaginal repair hydrogel dressing based on microsphere-matrix synergistic response, characterized in that: The invention comprises GA@CC microspheres and SA-CC dynamic cross-linking matrix, wherein the GA@CC microspheres have glycyrrhizic acid GA as the active core and aldehyde chitosan CC as the shell; The SA-CC dynamic cross-linking matrix serves as a supporting framework and presents a porous honeycomb network structure, which is formed by the synergistic combination of sodium alginate SA solution, CC solution and calcium ion cross-linking agent; The GA@CC microspheres and SA-CC dynamic cross-linking matrix are fixed by ionic bonds, which are formed by the SA carboxyl groups and the hydroxyl groups on the surface of the GA@CC microspheres through Ca 2+ Bridge formation.

2. A method for preparing the vaginal repair hydrogel dressing based on microsphere-matrix synergistic response according to claim 1, characterized in that: The following steps are involved: S1. Preparation of GA@CC microspheres CC was dissolved in deionized water to prepare a solution, and GA was added and stirred to form an aqueous phase; Take liquid paraffin as the oil phase, add emulsifier, and make the emulsifier evenly dispersed in the oil phase; The water phase is slowly added dropwise to the oil phase, stirred and emulsified to form a stable oil-in-water emulsion; Add a crosslinking agent to the emulsion and stir the reaction at room temperature to crosslink the CC aldehyde group and the GA hydroxyl group through Schiff base bonds to form a microsphere shell layer; After the reaction is completed, petroleum ether is added to break the emulsion, centrifuged, the supernatant is discarded, the precipitate is washed with anhydrous ethanol, and freeze-dried to obtain microsphere powder with a particle size of 1-5 μm, which is sealed and stored for later use; S2. Preparation of SA-CC dynamic cross-linking matrix Weigh SA powder, add deionized water, and stir to dissolve to form a clear and transparent colloidal SA solution; Take aldehyde chitosan, dissolve it in deionized water, stir to dissolve, and filter to obtain a uniform CC solution; SA solution was mixed with CC solution, CaCl2 powder was added, and then freeze-dried microsphere powder was added, and ultrasonic treatment was performed to uniformly disperse the microspheres to form a milky white suspension; S3. Preparation of hydrogel dressing The milky white suspension was injected into the vaginal injury model and placed in a constant temperature environment of 36-38°C to solidify into a hydrogel dressing, which was light yellow and translucent.

3. The method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response according to claim 2, characterized in that: The preparation process of CC in step S1 is as follows: Chitosan was added to a glacial acetic acid solution and stirred until completely dissolved, and then sodium periodate was weighed and added to the solution, wherein the mass ratio of sodium periodate to chitosan was 0.4:1-1:1; The reaction was stirred in the dark at room temperature and a pH of 3.5-4.

5. Ethylene glycol was then added and stirred for a certain period of time to terminate the reaction. The pH was then adjusted to 7.0-8.

0. Anhydrous ethanol was added dropwise to precipitate. After centrifugation, the precipitate was washed with ethanol. The precipitate was dissolved in deionized water and transferred to a dialysis bag for dialysis. After filtration, it was freeze-dried to obtain a white flocculent CC powder.

4. The method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response according to claim 2, characterized in that: In step S1, CC is dissolved in deionized water to prepare a 10-15% w / v solution, wherein the mass percentage of CC and GA is 60-70% by mass, and the balance is GA; Span 80 is used as the emulsifier, and the mass of the emulsifier is 3-5% of the mass of the oil phase.

5. The method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response according to claim 2, characterized in that: In step S1, the volume ratio of the water phase to the oil phase is 1:10, and the mixture is stirred and emulsified at a speed of 800-1200 rpm for 30-45 minutes.

6. The method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response according to claim 2, characterized in that: In step S1, EDC and NHS are used as cross-linking agents, and the mass ratio of EDC, NHS and CC is (0.5-1.5): (0.5-1):

5.

7. The method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response according to claim 2, characterized in that: In step S1, petroleum ether twice the volume of the emulsion is added to break the emulsion, and the mixture is transferred to a centrifuge tube and centrifuged at 8000 rpm for 10-15 minutes. The supernatant is discarded, and the precipitate is washed with anhydrous ethanol to remove the residual oil phase. Finally, the precipitate is freeze-dried in a freeze dryer at -80°C and a vacuum degree of ≤10 Pa for 24 hours to obtain a microsphere powder.

8. The method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response according to claim 2, characterized in that: In step S2, SA powder with a molecular weight of 100-200 kDa is added to deionized water to prepare a 1-3% w / v solution, and magnetic stirring is performed in a 40° C. water bath until completely dissolved; Take aldehyde chitosan and add deionized water to make a 2-5% w / v solution. Stir at room temperature until dissolved. Filter through a 0.45 μm filter membrane to remove insoluble matter to obtain a uniform CC solution.

9. The method for preparing a vaginal repair hydrogel dressing based on microsphere-matrix synergistic response according to claim 2, characterized in that: In step S2, the SA solution and the CC solution are mixed in a volume ratio of 1:1, CaCl2 powder is added to make the calcium ion concentration reach 0.05-0.1 M, and then the freeze-dried microsphere powder is added to make the microsphere powder 1-3% w / v, and the mixture is treated by ultrasonic dispersion for 10-15 minutes.

10. Use of the hydrogel dressing according to claim 1 in preparing a medicine or medical gel dressing for repairing vaginal mucosal damage, characterized in that: The hydrogel dressing has viscoelasticity, a tensile strength of 0.5-1.3 MPa, and an elongation at break of 180-350%.

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