Hydrogel dressing for promoting regeneration and repair of vaginal mucosa and preparation method of hydrogel dressing

Abstract

The invention discloses a hydrogel dressing for promoting vaginal mucosa regeneration and repair and a preparation method, the hydrogel dressing comprises a grape exosome and a ternary polysaccharide response matrix, the grape exosome is a grape cell exosome wrapping estradiol, and the ternary polysaccharide response matrix is formed by stirring and dissolving an OCS solution, an HA solution and an SA solution; the grape exosome and the ternary polysaccharide respond to the matrix, SA is triggered by Ca < 2 + > to perform ionic crosslinking, and the hydrogel dressing for promoting vaginal mucosa regeneration and repair is prepared. According to the preparation method, intelligent response of'antibacterial controlled release in an acid environment and targeted repair of alkaline inflammation parts' is realized, the problems of infection control, inflammation regulation and control, regeneration efficiency and mechanical adaptation are synchronously solved, and the biological safety is improved through an extremely simple preparation process (exosome extraction-polysaccharide blending-ionic crosslinking); and a natural, accurate and efficient repair scheme is provided for vaginal mucosa injuries (such as delivery laceration, operative wounds and chronic inflammation).

Description

Technical Field

[0001] The invention relates to a hydrogel dressing for promoting vaginal mucosal regeneration and repair and a preparation method thereof, belonging to the technical field of medical biomaterials. Background Art

[0002] Vaginal mucosal repair often faces problems such as infection risk and inflammation, low tissue regeneration efficiency, and complex mechanical adaptation and preparation. Traditional repair materials have a single antibacterial component, making it difficult to deal with the various pathogens in the weakly acidic environment of the vagina, and lack source inhibition of the inflammatory cascade reaction. Synthetic materials have poor biocompatibility, and natural plant crude extracts have complex components and unknown active ingredients, making them unable to accurately promote epithelial cell proliferation and collagen synthesis. Existing hydrogels rely on nanofiber spinning or multi-component compounding, which is a cumbersome process and the mechanical properties do not match the dynamic environment of the vagina.

[0003] In recent years, plant-derived exosomes have attracted significant attention as emerging active carriers. Grapevine exosomes (GExo) are enriched with polyphenols such as resveratrol and anthocyanins, as well as pro-repair miRNAs. They offer advantages such as targeted delivery, well-defined composition, and high biosafety. However, existing technologies have yet to utilize grapevine exosomes in vaginal repair.

[0004] Vaginal mucosal repair faces significant challenges in hormone-deficient states (such as menopause, ovarian failure, or a sudden drop in estrogen after childbirth): estrogen deficiency can lead to a prolonged epithelial cell proliferation cycle (a 40% decrease in the proportion of S-phase cells), thinning the mucosal layer and susceptibility to damage; at the same time, increased matrix metalloproteinase (MMP-9) activity exacerbates type I collagen degradation (annual loss rate of 8-10%), leading to loss of mucosal elasticity and weakened barrier function; in addition, an imbalance in the vaginal microecology (a decrease in lactobacilli and an increase in pH to 6.0-7.0) causes excessive proliferation of opportunistic pathogens (such as Escherichia coli and Candida albicans), further delaying repair. In existing technologies, traditional vaginal creams or suppositories rely on frequent administration (1-2 times a day), and free estradiol is easily absorbed through the mucosa into the blood circulation, causing plasma estrogen levels to fluctuate by more than 30%, which may cause side effects such as breast tenderness and endometrial hyperplasia; at the same time, non-targeted delivery exposes healthy tissue to high concentrations of hormones, increasing the risk of local irritation; in addition, existing materials lack the synergistic design of antibacterial and anti-inflammatory functions, and cannot simultaneously respond to infection and oxidative stress after injury, resulting in the repair period being extended to more than 14 days. Summary of the Invention

[0005] To address the aforementioned issues with existing technologies, the present invention provides a hydrogel dressing and preparation method for promoting vaginal mucosal regeneration and repair, enabling precise repair of hormone-deficient vaginal mucosal damage. This invention is suitable for clinical scenarios such as menopausal vaginal atrophy, postpartum mucosal tears, and postoperative wound healing disorders.

[0006] To achieve the above objectives, the present invention adopts a hydrogel dressing for promoting vaginal mucosal regeneration and repair, comprising grape exosomes and a ternary polysaccharide responsive matrix, wherein the grape exosomes are grape cell exosomes encapsulating estradiol, and the ternary polysaccharide responsive matrix is ​​formed by stirring and dissolving an OCS solution, a HA solution, and a SA solution;

[0007] The grape exosomes and trivalent polysaccharides respond to the matrix through Ca 2+ SA is triggered to undergo ionic cross-linking to prepare the hydrogel dressing that promotes the regeneration and repair of the vaginal mucosa.

[0008] As an improvement, the particle size of the grape exosomes is 80-120 nm.

[0009] As an improvement, the vaginal mucosa refers to the vaginal mucosa that is damaged due to menopausal vaginal atrophy, mucosal tearing after childbirth, or postoperative wound healing disorder.

[0010] The second aspect of the present invention further provides a method for preparing the hydrogel dressing for promoting vaginal mucosal regeneration and repair, comprising the following steps:

[0011] (1) Fresh grapes were homogenized and centrifuged to remove pulp fragments, and the supernatant was centrifuged to obtain grape cell exosomes;

[0012] (2) adding estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1, and encapsulating estradiol in the lumen of grape cell exosomes by ultrasonic dispersion, controlling the encapsulation rate to be ≥85%, thereby obtaining grape exosomes;

[0013] (3) Dissolve OCS in 1% acetic acid solution to obtain an OCS solution with a concentration of 4-6% w / v, then add HA and SA in sequence to make the HA concentration 1-1.5% w / v and the SA concentration 3-4% w / v, and stir magnetically until completely dissolved to form a uniform and transparent ternary polysaccharide responsive matrix solution;

[0014] (4) Add grape exosomes at a concentration of 10-15 μg / mL to the ternary polysaccharide response matrix solution, disperse them evenly by ultrasonication, add CaCl2 solution, and control the CaCl2 concentration to 0.2-0.25% w / v. Cross-linking reaction is performed to obtain a hydrogel dressing. After passing through a sterile filter membrane, the dressing is dispensed into syringes and stored at 4°C.

[0015] As an improvement, in step (1), the fresh grapes are homogenized and then centrifuged at low speed to remove pulp fragments, and the supernatant is collected by ultracentrifugation to collect grape cell exosomes, wherein the centrifugal force of the low-speed centrifugation is 3000g and the centrifugal force of the ultracentrifugation is 100000g.

[0016] As an improvement, in step (2), 120 Hz ultrasonic dispersion is used for 5 minutes.

[0017] As an improvement, the temperature of the magnetic stirring in step (3) is 37°C.

[0018] As an improvement, the ultrasonic dispersion in step (4) is carried out at a frequency of 40 kHz at 4°C for 5 minutes.

[0019] As an improvement, the hydrogel dressing dispensed into the syringe in step (4) has a shelf life of 2 weeks when stored at 4°C.

[0020] Mechanism of the present invention:

[0021] The natural polysaccharide composite repair hydrogel with grape exosomes as the core active ingredient breaks through the limitations of traditional vaginal mucosal repair materials in antibacterial, anti-inflammatory and regenerative repair. By extracting grape cell exosomes and using their natural membrane structure to target the delivery of grape cell exosomes and encapsulated estradiol, a multi-effect synergistic system of "anti-inflammatory-antioxidant-promoting epithelial proliferation-collagen synthesis" is constructed: resveratrol in grape cell exosomes inhibits the NF-κB inflammatory pathway, anthocyanins scavenge ROS, and estradiol enhances the ability of mucosal tissue remodeling, solving the problems of complex components and poor targeting of traditional plant crude extracts, and increasing the utilization rate of active ingredients by 3 times. At the same time, grape exosomes are embedded in a ternary polysaccharide matrix of oxidized chitosan (OCS)-hyaluronic acid (HA)-sodium alginate (SA), and the pH response characteristics of OCS and SA-Ca are utilized. 2+ The thermosensitive cross-linked network achieves the intelligent response of "antibacterial controlled release in acidic environment and targeted repair in alkaline inflammatory sites", simultaneously solving the problems of infection control, inflammation regulation, regeneration efficiency and mechanical adaptation. Through a minimalist preparation process (exosome extraction-polysaccharide blending-ionic cross-linking), it improves biosafety and provides a natural, precise and efficient repair solution for vaginal mucosal injuries (such as childbirth lacerations, surgical trauma, and chronic inflammation).

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. Natural targeting and multi-functional integration of grape exosomes

[0024] (1) Precision delivery and functional synergy: Existing technologies rely on crude plant extracts or synthetic carriers, which have the disadvantages of complex ingredients and poor targeting (for example, the utilization rate of active ingredients in traditional chitosan-glycyrrhizic acid gel is less than 30%). This invention utilizes the natural membrane structure of grape exosomes (particle size 80-120nm) to achieve active targeted uptake of vaginal epithelial cells through HA mediation. Estradiol encapsulated in grape cell exosomes (promotes collagen synthesis and accelerates mucosal remodeling) forms a natural synergistic system with four functions: anti-inflammatory, anti-oxidative, regenerative, and tissue remodeling, solving the problem of "functional fragmentation" of traditional materials.

[0025] (2) Clear composition and biosafety: Grape cell exosome extracts are purified by ultracentrifugation and have clear composition (including polyphenols, miRNA and estrogen), avoiding the complex composition risks of traditional plant extracts; they have excellent biocompatibility, and their degradation products are natural monosaccharides without the potential toxicity of synthetic materials, which significantly improves clinical safety.

[0026] 2. Accuracy of pH-responsive antibacterial and intelligent drug release

[0027] (1) Dynamic antibacterial barrier: Existing pH-responsive materials (such as chitosan) have an antibacterial rate of less than 30% in acidic environments. This invention directly kills pathogens in the weakly acidic environment of the vagina (pH 4.5-5.5) by protonating the amino groups of oxidized chitosan (OCS). At the same time, it inhibits the premature release of exosomes by tightening the matrix network, preventing drug leakage from healthy tissue. When inflammation causes the pH to rise to alkaline, the OCS amino groups deprotonate, triggering the rapid release of active ingredients from exosomes, achieving an intelligent switch between "acidic antibacterial controlled release and alkaline repair burst release".

[0028] (2) Optimization of drug release kinetics: The traditional single-response hydrogel has a 24-hour release rate of over 60% when the pH is greater than 5.5. The present invention precisely regulates the exosome release rate through the electrostatic interaction between OCS and HA. In the pH 7.4 inflammatory environment, the cumulative release rate reaches 73% in 48 hours, which is 30% higher than that of traditional materials. The drug release curve also matches the dynamic requirements of the "inflammatory phase-proliferation phase" of vaginal repair.

[0029] 3. Thermosensitive mechanical adaptation and preparation process innovation

[0030] (1) Dynamic mechanical matching: Existing hydrogels rely on nanofiber spinning, which is complex in process and lacks mechanical strength. 2+ The ion-crosslinked network gels in situ within 3 minutes at 37°C body temperature, which can resist the mechanical stress in vaginal physiological activities while maintaining viscoelasticity (returning to its original shape within 10 seconds after deformation) to avoid material displacement or premature degradation.

[0031] (2) Minimal preparation and clinical adaptability: Only a four-step process of "exosome extraction (ultracentrifugation) - polysaccharide blending (OCS / HA / SA) - exosome loading - ionic crosslinking" is required. No chemical modification or nanospinning is required. It supports clinical instant preparation and in situ injection (such as sterile syringe packaging). The gelation time is less than 3 minutes, which significantly improves the operational convenience and reduces the preparation cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the hydrated particle size and zeta potential of GExo and GExo-E2 according to Example 1 of the present invention; Figure (a) is a diagram of the hydrated particle size of GExo and GExo-E2, and (b) is a diagram of the zeta potential of GExo and GExo-E2;

[0033] Figure 2 TEM images of GExo-E2 and GExo-E2 / OCS / SA / HA according to Example 1 of the present invention; Figure (a) is a TEM image of GExo-E2, and Figure (b) is a TEM image of GExo-E2 / OCS / SA / HA;

[0034] Figure 3 This is a rheological test graph of GExo-E2 / OCS / SA / HA in Example 1 of the present invention;

[0035] Figure 4 Schematic diagram of pH (4.0, 5.5, 7.4) responsive drug release of the hydrogel dressing of Example 1 of the present invention;

[0036] Figure 5 Schematic diagram of the inflammation inhibition effect of Example 1 of the present invention; (a) is the gene expression level of the pro-inflammatory factor IL-6, and (b) is the gene expression level of the pro-inflammatory factor TNF-α;

[0037] Figure 6 Schematic diagram of the regeneration-promoting ability of Example 1 of the present invention. DETAILED DESCRIPTION

[0038] 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.

[0039] Explanation of some relevant terms in this invention:

[0040] Grape exosomes (GExo-E2): A composite delivery system of grape cell exosomes loaded with estradiol, with a particle size of 80-120 nm.

[0041] Estrogen receptors (ERs): including ERα and ERβ, are widely expressed in vaginal epithelial cells and activate downstream pro-proliferation signaling pathways after binding to estradiol.

[0042] Thermosensitive ionic crosslinking: Sodium alginate (SA) and calcium chloride (CaCl2) are ion-linked at 37°C. 2+ Ionic bonds quickly form a gel network with a response time of ≤3 minutes.

[0043] Example 1

[0044] A method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair comprises the following steps:

[0045] (1) Fresh grapes were homogenized (using a homogenizer at 120 Hz for 10 min) and then centrifuged at low speed to remove pulp debris. The supernatant was collected by ultracentrifugation at a centrifugal force of 3000 g for low speed centrifugation and 100,000 g for ultracentrifugation. After resuspending in PBS, the particle size (80-120 nm) was verified by nanoparticle size analysis (DLS), and the protein concentration was determined by the BCA method.

[0046] (2) Adding estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1, and encapsulating estradiol in the lumen of grape cell exosomes by ultrasonic dispersion (120 Hz, 5 min), controlling the encapsulation rate to be ≥85% (after encapsulation, centrifuge and dilute the supernatant, and the residual unencapsulated estradiol in the supernatant is calculated by comparing the absorbance value at 278 nm with the standard curve using an ultraviolet spectrophotometer), to obtain grape exosomes;

[0047] (3) OCS was dissolved in 1% acetic acid solution to obtain a 5% w / v OCS solution, and HA and SA were added in sequence to make the HA concentration 1.2% w / v and SA concentration 3.5% w / v, and magnetic stirring was performed at 37°C until completely dissolved to form a uniform and transparent ternary polysaccharide response matrix solution;

[0048] (4) Add grape exosomes at a concentration of 12 μg / mL to the ternary polysaccharide response matrix solution. After uniform dispersion by ultrasonication (40 kHz, 5 min) at 4°C, add CaCl2 solution and control the CaCl2 concentration to 0.25% w / v. Cross-linking reaction is performed to obtain a hydrogel dressing. After passing through a sterile filter membrane, dispense into syringes and store at 4°C (valid for 2 weeks). The hydrogel dressing can be used directly as a dressing.

[0049] The performance of the hydrogel dressing prepared in Example 1 was characterized:

[0050] 1. Exosome characterization and hydrogel mechanical adaptability

[0051] Figure 1 The hydrated particle size and Zeta potential of GExo and GExo-E2 (grape cell exosomes encapsulating estradiol, i.e. grape exosomes) were as follows: DLS detection showed that the hydrated particle size of GExo (91.5±3.1 nm) and GExo-E2 (88.8±2.3 nm) was Figure 1 (a) There was no significant difference, both were within the 80-120 nm target range, indicating that the loaded estradiol did not destroy the exosome membrane structure; Zeta potential ( Figure 1 (b) ), GExo (-25.1±0.2 mV) and GExo-E2 (-23.5±0.3 mV) showed significant differences, and the negative potential of GExo-E2 was weakened but still maintained its negative electrical characteristics.

[0052] The results showed that in terms of particle size stability, the particle size of grape cell exosomes remained unchanged after loading with estradiol, ensuring that the exosomes retained their targeted delivery ability (80-120 nm can be internalized by vaginal epithelial cells). The active ingredient (estradiol) was stably embedded in the exosome cavity through the ultrasonic encapsulation process (ultrasonic treatment caused the cell membrane to rupture and then reorganize into vesicles, and the fat-soluble drugs were embedded in the lipid bilayer of the vesicles, and some drugs could be adsorbed on the surface of the vesicles through electrostatic interaction), and the membrane structure was intact. The negative potential of GExo-E2 was weakened (related to the reduction of negative charge exposure of the membrane by estradiol encapsulation), and it still had a positive charge (-NH3 + ) forms electrostatic attraction, allowing exosomes to be stably embedded (acid-stable) in an acidic environment (healthy vagina); in an alkaline inflammatory environment, OCS is deprotonated (-NH2, negatively charged), the electrostatic effect is released, and the exosomes are released (alkaline release), achieving pH-responsive controlled release and accurately matching the dynamic changes in the vaginal microenvironment.

[0053] Figure 2 TEM images of GExo-E2 and GExo-E2 / OCS / SA / HA show that GExo-E2 exhibits a typical exosome morphology, approximately elliptical, with a particle size of about 100 nm. Substances with high electron density can be seen inside, which may be the encapsulated estradiol and other components, such as Figure 2 (a). Figure 2 As shown in (b), GExo-E2 / OCS / SA / HA (a cross-linked composite system of grape exosomes and a ternary polysaccharide matrix, i.e., the hydrogel dressing of the present invention) has a three-dimensional porous structure with a pore size at the micron level. The lamellar or block structures formed by the ternary polysaccharide matrix are interwoven with each other, and exosomes may be dispersed in them or attached to the surface of the matrix.

[0054] like Figure 3As shown, the storage modulus (G') of GExo-E2 / OCS / SA / HA is 10 5 - 10 6 The Pa range increases with the frequency from 0.1 Hz to 100 Hz, and the loss modulus (G'') is 4 - 10 5 The Pa range shows a slow downward trend. For example, at 100 Hz, G' is 224000 Pa and G'' is 64700 Pa; at 0.1 Hz, G' is 130000 Pa and G'' is 123000 Pa, indicating that the hydrogel dressing is mainly elastic with viscosity as a supplement, and the elasticity increases with increasing frequency. 2+ Ionic crosslinking and electrostatic crosslinking of oxidized chitosan (OCS) and SA under acidic conditions form a three-dimensional rigid network, endowing the hydrogel with a high storage modulus (elasticity). As frequency increases, segmental motion in the crosslinked network is restricted, enhancing elasticity. Its low viscous loss (G'') stems from the network's effective resistance to viscous flow. In the physiological dynamic mechanical environment of the vagina (peristalsis, friction, etc.), the hydrogel's primarily elastic properties enable it to resist external forces, maintain structural stability, prevent displacement, achieve mechanical adaptation, and provide stable support for vaginal mucosal repair.

[0055] 2. pH-responsive antimicrobial barrier function

[0056] Antibacterial rate test: In the antibacterial experiment (as shown in Table 1), the CFU / mL of Escherichia coli (E. coli) and Candida albicans (C. albicans) in the control group were 3.28×10 12 and 5.64×10 11 The LogReduction (logarithmic reduction value) of the GExo-E2 group against the two bacteria was 0.19±0.25 and 0.29±0.14, respectively, with limited antibacterial effect. The CFU / mL of the present invention group (GExo-E2 / OCS / SA / HA) was significantly reduced, with the CFU / mL of Escherichia coli being 4.13×10 11 , Candida albicans was 4.17×10 10 , Log Reduction reached 0.89 ± 0.31 and 1.12 ± 0.43, respectively, and the antibacterial efficacy was significantly better than that of the GExo-E2 group.

[0057] Oxidized chitosan (OCS) undergoes amino protonation (-NH3 + ), destroying the membrane structure of pathogens by electrostatic adsorption, achieving broad-spectrum antibacterial effect (effective against E. coli and C. albicans); sodium alginate (SA) and Ca2+ The resulting thermosensitive gel provides a carrier for OCS, ensuring sustained antibacterial activity in an acidic environment. Estradiol loaded into GExo-E2 is released in the alkaline inflammatory environment (pH 7.4), inhibiting pathogen biofilm formation. This, combined with the acidic antibacterial properties of OCS, creates a spatiotemporal synergy of "acid-stable alkaline release and comprehensive antibacterial activity." Hyaluronic acid (HA) mediates the targeting of exosomes to sites of mucosal damage, increasing the local concentration of antibacterial components and further enhancing the antibacterial effect. (The Log Reduction of the inventive group was higher than that of the GExo-E2 group, particularly against C. albicans, increasing by 1.12, confirming the synergistic mechanism.)

[0058] Table 1 Antibacterial ability data

[0059]

[0060] Under pH 4.0 (healthy acidic), 5.5 (mild inflammation), and 7.4 (severe inflammation alkaline) environments, the release rate of grape exosomes (GExo-E2) from GExo-E2 / OCS / SA / HA showed significant pH response characteristics. Figure 4 As shown in Figure 2 , the release rate was ≤14.2% in an acidic environment (pH 4.0-5.5) from 2 to 48 hours (e.g., 14.2% in 48 hours at pH 4.0), indicating low release. In an alkaline environment (pH 7.4), the release rate increased significantly over time, reaching 32.4% in 48 hours. This indicates that the hydrogel dressing of this invention provides low drug release in healthy / mildly inflammatory conditions, avoiding interference with the normal mucosa; while it provides efficient drug release in an alkaline environment of severe inflammation, enabling targeted repair of damaged tissue. The pH-sensitive charge change of oxidized chitosan (OCS) is central to this process: in acidic conditions, the OCS amino groups protonate, electrostatically crosslinking with the carboxylate groups of sodium alginate (SA), forming a dense network that inhibits exosome release. In alkaline conditions, the OCS deprotonates, loosening the network and triggering the rapid release of exosomes (loaded with estradiol and grape polyphenols). The three work together to achieve "acid-stable alkaline release and targeted repair", accurately matching the dynamic changes of the vaginal microenvironment, solving the drawbacks of inaccurate drug release of traditional materials, and ensuring low drug exposure to healthy tissues and efficient repair of inflamed areas.

[0061] 3. Targeted repair function of exosomes

[0062] Inflammation inhibition effect: In the LPS-induced inflammation model, the gene expression levels of pro-inflammatory factors IL-6 and TNF-α were detected by qPCR. The results are as follows Figure 5 As shown in (a), the relative expression of IL-6 in the control group was the highest (average value 1.026), which was significantly decreased in the GExo-E2 group (0.607) and the present invention group (0.567), and the present invention group was lower than the GExo-E2 group. Figure 5As shown in (b), TNF-α expression was highest in the control group (average 1.377), while significantly decreased in the GExo-E2 group (0.646) and the present invention group (0.708). Through the dual mechanism of "exosome-targeted delivery of active ingredients combined with pH-responsive antibacterial action by a polysaccharide matrix," the present invention significantly outperformed grape cell exosome-loaded E2 alone (GExo-E2 group). Both IL-6 and TNF-α expression levels decreased by ≥40% compared to the control group, demonstrating the present hydrogel dressing's ability to regulate the vaginal inflammatory microenvironment and providing direct evidence for clinical anti-inflammatory and repair effects.

[0063] Regeneration ability: Figure 6 As shown in the CCK-8 experiment, the mean cell viability of the control group was 100.94%, which increased to 119.55% in the GExo-E2 group. The group of the present invention (GExo-E2 / OCS / HA / SA) further increased to 123.96%, representing increases of 19% and 23% respectively compared to the control group, significantly outperforming the control group. This demonstrates that the hydrogel dressing of the present invention is non-toxic and potently promotes cell proliferation. MiR-159 and resveratrol carried by grape cell exosomes directly promote vaginal epithelial cell proliferation by activating the PI3K-AKT pathway (GExo-E2 group effect). In the ternary polysaccharide matrix, OCS inhibits bacteria in acidic environments (reducing pathogen damage) and releases exosomes in alkaline environments. HA mediates cell adhesion and nutrient uptake, while SA's Ca 2+ The thermosensitive gel provides three-dimensional support, which, in synergy with exosomes, further enhances the proliferation-promoting effect (an additional benefit of the inventive group). Through the combination of bioactive molecule delivery and microenvironmental regulation, the material safely and efficiently enhances cell viability, directly supporting the technical solution of promoting vaginal mucosal regeneration.

[0064] Example 2

[0065] A method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair comprises the following steps:

[0066] (1) Fresh grapes were homogenized and centrifuged at low speed to remove pulp debris. The supernatant was collected by ultracentrifugation at a centrifugal force of 3000 g for low speed centrifugation and 100,000 g for ultracentrifugation. After resuspending in PBS, the particle size (80-120 nm) was verified by nanoparticle size analysis (DLS), and the protein concentration was determined by BCA method.

[0067] (2) Adding estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1, and encapsulating estradiol in the lumen of grape cell exosomes by ultrasonic dispersion (120 Hz, 5 min), controlling the encapsulation rate to be ≥85%, thereby obtaining grape exosomes;

[0068] (3) OCS was dissolved in 1% acetic acid solution to obtain a 4% w / v OCS solution, and HA and SA were added in sequence to make the HA concentration 1% w / v and SA concentration 3% w / v, and magnetic stirring was performed at 37°C until completely dissolved to form a uniform and transparent ternary polysaccharide response matrix solution;

[0069] (4) Add grape exosomes at a concentration of 10 μg / mL to the ternary polysaccharide response matrix solution, disperse evenly by ultrasonication (40 kHz, 5 min) at 4°C, add CaCl2 solution, and control the CaCl2 concentration to 0.2% w / v. Cross-linking reaction is performed to obtain a hydrogel dressing. After passing through a sterile filter membrane, dispense into syringes and store at 4°C (valid for 2 weeks).

[0070] Example 3

[0071] A method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair comprises the following steps:

[0072] (1) Fresh grapes were homogenized and centrifuged at low speed to remove pulp debris. The supernatant was collected by ultracentrifugation at a centrifugal force of 3000 g for low speed centrifugation and 100,000 g for ultracentrifugation. After resuspending in PBS, the particle size (80-120 nm) was verified by nanoparticle size analysis (DLS), and the protein concentration was determined by BCA method.

[0073] (2) Adding estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1, and encapsulating estradiol in the lumen of grape cell exosomes by ultrasonic dispersion (120 Hz, 5 min), controlling the encapsulation rate to be ≥85%, thereby obtaining grape exosomes;

[0074] (3) OCS was dissolved in 1% acetic acid solution to obtain a 6% w / v OCS solution, and HA and SA were added in sequence to make the HA concentration 1.5% w / v and SA concentration 4% w / v. The mixture was stirred magnetically at 37°C until completely dissolved to form a uniform and transparent ternary polysaccharide responsive matrix solution.

[0075] (4) Add grape exosomes at a concentration of 12 μg / mL to the ternary polysaccharide response matrix solution, disperse evenly by ultrasonication (40 kHz, 5 min) at 4°C, add CaCl2 solution, and control the CaCl2 concentration to 0.25% w / v. Cross-linking reaction is performed to obtain a hydrogel dressing. After passing through a sterile filter membrane, dispense into syringes and store at 4°C (valid for 2 weeks).

[0076] The above description is only a preferred embodiment of the present invention 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 hydrogel dressing for promoting regeneration and repair of vaginal mucosa, characterized in that: It includes grape exosomes and a ternary polysaccharide response matrix, wherein the grape exosomes are grape cell exosomes encapsulating estradiol, and the ternary polysaccharide response matrix is ​​formed by stirring and dissolving an OCS solution, a HA solution, and a SA solution; The grape exosomes and trivalent polysaccharides respond to the matrix through Ca 2+ SA is triggered to undergo ionic cross-linking to prepare the hydrogel dressing that promotes the regeneration and repair of the vaginal mucosa.

2. The hydrogel dressing for promoting vaginal mucosal regeneration and repair according to claim 1, characterized in that: The particle size of the grape exosomes is 80-120 nm.

3. The hydrogel dressing for promoting vaginal mucosal regeneration and repair according to claim 1, characterized in that: The vaginal mucosa refers to the vaginal mucosa that is damaged due to menopausal vaginal atrophy, mucosal tearing after childbirth, or postoperative wound healing disorder.

4. A method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Fresh grapes were homogenized and centrifuged to remove pulp fragments, and the supernatant was centrifuged to obtain grape cell exosomes; (2) adding estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1, and encapsulating estradiol in the lumen of grape cell exosomes by ultrasonic dispersion, controlling the encapsulation rate to be ≥85%, thereby obtaining grape exosomes; (3) Dissolve OCS in 1% acetic acid solution to obtain an OCS solution with a concentration of 4-6% w / v, then add HA and SA in sequence to make the HA concentration 1-1.5% w / v and the SA concentration 3-4% w / v, and stir magnetically until completely dissolved to form a uniform and transparent ternary polysaccharide responsive matrix solution; (4) Add grape exosomes at a concentration of 10-15 μg / mL to the ternary polysaccharide response matrix solution, disperse them evenly by ultrasonication, add CaCl2 solution, and control the CaCl2 concentration to 0.2-0.25% w / v. Cross-linking reaction is performed to obtain a hydrogel dressing. After passing through a sterile filter membrane, the dressing is dispensed into syringes and stored at 4°C.

5. The method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair according to claim 4, characterized in that: In step (1), the fresh grapes are homogenized and then centrifuged at low speed to remove pulp fragments, and the supernatant is subjected to ultracentrifugation to collect grape cell exosomes, wherein the centrifugal force of the low-speed centrifugation is 3000g and the centrifugal force of the ultracentrifugation is 100000g.

6. The method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair according to claim 4, characterized in that: In the step (2), 120 Hz ultrasonic dispersion is used for 5 minutes.

7. The method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair according to claim 4, characterized in that: The temperature of the magnetic stirring in step (3) is 37°C.

8. The method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair according to claim 4, characterized in that: The ultrasonic dispersion in step (4) is carried out at a frequency of 40 kHz for 5 minutes at 4°C.

9. The method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair according to claim 4, characterized in that: The hydrogel dressing dispensed into the syringe in step (4) has a shelf life of 2 weeks when stored at 4°C.

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