A hydrogel dressing for promoting the regeneration and repair of vaginal mucosa and a preparation method thereof

Abstract

The application discloses a hydrogel dressing for promoting regeneration and repair of vaginal mucosa and a preparation method thereof, and comprises grape exosomes and a ternary polysaccharide responsive matrix, wherein the grape exosomes are grape cell exosomes wrapped with estradiol, and the ternary polysaccharide responsive matrix is formed by stirring and dissolving OCS solution, HA solution and SA solution; the grape exosomes and the ternary polysaccharide responsive matrix are combined through Ca 2+ The SA is triggered to carry out ion crosslinking, so that the hydrogel dressing for promoting regeneration and repair of vaginal mucosa is prepared. The application realizes intelligent response of 'acidic environment antibacterial controlled release and alkaline inflammation site targeted repair', synchronously solves the problems of infection control, inflammation regulation, regeneration efficiency and mechanical adaptation, improves biological safety through a simple preparation process (exosome extraction-polysaccharide blending-ion crosslinking), and provides a natural, precise and efficient repair scheme for vaginal mucosa injury (such as childbirth laceration, surgical trauma and chronic inflammation).

Description

Technical Field

[0001] This invention relates to a hydrogel dressing that promotes vaginal mucosal regeneration and repair, and its preparation method, belonging to the field of medical biomaterials technology. Background Technology

[0002] Vaginal mucosal repair often faces challenges such as infection risks and inflammation, low tissue regeneration efficiency, and complex mechanical compatibility and preparation. Traditional repair materials contain only a single antibacterial component, making it difficult to combat the diverse pathogens present in the weakly acidic environment of the vagina, and lacking the ability to inhibit the source of inflammatory cascades. Synthetic materials have poor biocompatibility, and the composition of crude extracts from natural plants is complex with unclear active ingredients, failing to precisely promote epithelial cell proliferation and collagen synthesis. Existing hydrogels rely on nanofiber spinning or multi-component compounding, which involves cumbersome processes and whose mechanical properties are incompatible with the dynamic environment of the vagina.

[0003] In recent years, plant-derived exosomes have attracted much attention as emerging bioactive carriers. Grape cell exosomes (GExo) are enriched with polyphenols such as resveratrol and anthocyanins, as well as repair-promoting miRNAs, and possess advantages such as targeted delivery, well-defined components, and high biocompatibility. However, current technologies have not yet applied grape cell exosomes to the field of vaginal repair.

[0004] Vaginal mucosal repair faces significant challenges under hormone deficiency conditions (such as menopause, ovarian dysfunction, or a sudden drop in estrogen after childbirth): insufficient estrogen leads to a prolonged epithelial cell proliferation cycle (a 40% decrease in the proportion of S-phase cells), resulting in a thinner and more easily damaged mucosal layer; simultaneously, increased activity of matrix metalloproteinase-9 (MMP-9) exacerbates the degradation of type I collagen (an annual loss rate of 8-10%), leading to loss of mucosal elasticity and weakened barrier function; furthermore, vaginal microecological imbalance (reduction of lactobacilli, pH rising 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 daily), and free estradiol is easily absorbed into the bloodstream through the mucosa, leading to fluctuations in plasma estrogen levels of >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 a synergistic design for antibacterial and anti-inflammatory functions, and cannot simultaneously address infection and oxidative stress after injury, resulting in a repair cycle that is extended to more than 14 days. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a hydrogel dressing that promotes vaginal mucosal regeneration and repair, as well as its preparation method, enabling precise repair of hormone-deficient vaginal mucosal damage. This invention is applicable to clinical scenarios such as menopausal vaginal atrophy, postpartum mucosal tears, and postoperative wound healing disorders.

[0006] To achieve the above objectives, the present invention employs a hydrogel dressing that promotes 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 OCS solution, HA solution and SA solution;

[0007] The grape exosomes and ternary polysaccharide responsive matrix, via Ca 2+ The hydrogel dressing that promotes vaginal mucosal regeneration and repair is prepared by triggering ionic cross-linking of SA.

[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 has been damaged due to menopausal vaginal atrophy, postpartum mucosal tearing, or postoperative wound healing disorders.

[0010] A second aspect of the present invention also provides a method for preparing the hydrogel dressing that promotes vaginal mucosal regeneration and repair, comprising the following steps:

[0011] (1) After homogenizing fresh grapes, centrifuge to remove pulp fragments, and obtain grape cell exosomes by centrifuging the supernatant;

[0012] (2) Add estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1, and disperse the estradiol by ultrasonication to encapsulate it in the lumen of the grape cell exosomes, controlling the encapsulation rate to ≥85%, to obtain grape exosomes;

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

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

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

[0016] As an improvement, step (2) involves ultrasonic dispersion at 120 Hz for 5 min.

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

[0018] As an improvement, the ultrasonic dispersion in step (4) is performed at 40 kHz for 5 min at 4 °C.

[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 invention:

[0021] This natural polysaccharide composite repair hydrogel, with grape exosomes as its core active ingredient, overcomes the limitations of traditional vaginal mucosal repair materials in antibacterial, anti-inflammatory, and regenerative repair. By extracting grape cell exosomes and utilizing their natural membrane structure to target and deliver grape cell exosomes and encapsulated estradiol, a multi-effect synergistic system of "anti-inflammatory, anti-oxidative, epithelial proliferation-collagen synthesis" is constructed: resveratrol in grape cell exosomes inhibits the NF-κB inflammatory pathway, anthocyanins scavenge ROS, and estradiol enhances the mucosal tissue remodeling capacity, solving the problems of complex composition and poor targeting of traditional plant crude extracts, and increasing the utilization rate of active ingredients by 3 times. Simultaneously, grape exosomes are embedded in an oxidized chitosan (OCS)-hyaluronic acid (HA)-sodium alginate (SA) ternary polysaccharide matrix, utilizing the pH-responsive characteristics of OCS and the SA-Ca... 2+ The thermosensitive cross-linked network enables intelligent response to "antibacterial controlled release in acidic environments and targeted repair of inflamed sites in alkaline environments," simultaneously solving problems related to infection control, inflammation regulation, regeneration efficiency, and mechanical compatibility. Through a simplified preparation process (exosome extraction - polysaccharide blending - ionic cross-linking), it enhances biocompatibility and provides a natural, precise, and efficient repair solution for vaginal mucosal damage (such as childbirth tears, surgical trauma, and chronic inflammation).

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. Integration of natural targeting and multi-effect activity of grape exosomes

[0024] (1) Precise delivery and functional synergy: Existing technologies rely on crude plant extracts or synthetic carriers, which have drawbacks such as complex composition and poor targeting (e.g., 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. The estradiol (which promotes collagen synthesis and accelerates mucosal remodeling) encapsulated in grape cell exosomes forms a natural synergistic system with four effects: anti-inflammatory, antioxidant, regenerative, and tissue remodeling, solving the problem of "functional fragmentation" in traditional materials.

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

[0026] 2. Precision 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 utilizes the amino protonation mechanism of oxidized chitosan (OCS) to directly kill pathogens in the weakly acidic environment of the vagina (pH 4.5-5.5). At the same time, it inhibits premature release of exosomes through matrix network contraction, avoiding drug leakage from healthy tissues. When inflammation causes the pH to rise to alkalinity, OCS amino groups are deprotonated, triggering the rapid release of active ingredients from exosomes, achieving intelligent switching between "acidic antibacterial controlled release and alkaline repair burst release".

[0028] (2) Optimization of drug release kinetics: Traditional single-response hydrogels have a release rate of over 60% in 24 hours when pH>5.5. This invention precisely regulates the exosome release rate through the electrostatic interaction between OCS and HA. In an inflammatory environment at pH 7.4, the cumulative release rate reaches 73% in 48 hours, which is 30% higher than that of traditional materials. Moreover, the drug release curve matches the dynamic requirements of the "inflammatory phase-proliferative phase" of vaginal repair.

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

[0030] (1) Dynamic mechanical matching: Existing hydrogels rely on nanofiber spinning, which is complex and lacks sufficient mechanical strength. This invention achieves dynamic mechanical matching by combining SA and Ca. 2+ The ion-crosslinked network gels in situ within 3 minutes at a body temperature of 37°C, which can resist mechanical stress during vaginal physiological activities while maintaining viscoelasticity (returning to its original state within 10 seconds after deformation), thus preventing material displacement or premature degradation.

[0031] (2) Simplified preparation and clinical adaptation: Only four steps are required: “exosome extraction (ultracentrifugation) - polysaccharide blending (OCS / HA / SA) - exosome loading - ion crosslinking”. No chemical modification or nanospinning is required. It supports immediate clinical preparation and in-situ injection (such as sterile syringe dispensing). The gelation time is <3 minutes, which significantly improves the convenience of operation and reduces the preparation cost. Attached Figure Description

[0032] Figure 1 Figure 1 shows the hydrated particle size and Zeta potential of GExo and GExo-E2 in Embodiment 1 of the present invention; Figure (a) shows the hydrated particle size of GExo and GExo-E2, and Figure (b) shows the Zeta potential of GExo and GExo-E2.

[0033] Figure 2 Figure 1 shows TEM images of GExo-E2 and GExo-E2 / OCS / SA / HA in Embodiment 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 The rheological test diagram of GExo-E2 / OCS / SA / HA in Embodiment 1 of the present invention is shown below.

[0035] Figure 4 This is a schematic diagram illustrating the pH (4.0, 5.5, 7.4) response drug release of the hydrogel dressing in Example 1 of the present invention;

[0036] Figure 5 This is a schematic diagram of the anti-inflammatory 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 This is a schematic diagram illustrating the regeneration-promoting capacity of Embodiment 1 of the present invention. Detailed Implementation

[0038] The following embodiments are further illustrations of the present invention and serve as explanations of the technical content of the present invention. However, the essence of the present invention is not limited to the embodiments described below. Those skilled in the art can and should know that any simple changes or substitutions based on the spirit of the present invention should fall within the protection scope claimed by the present invention.

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

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

[0041] Estrogen receptors (ERs): including ERα and ERβ, are widely expressed in vaginal epithelial cells. After binding with estradiol, they activate downstream proliferative signaling pathways.

[0042] Thermosensitive ionic crosslinking: Sodium alginate (SA) and calcium chloride (CaCl2) crosslink at a physiological temperature of 37°C via Ca... 2+ Ionic bonds rapidly form a gel network with a response time of ≤3 minutes.

[0043] Example 1

[0044] A method for preparing a hydrogel dressing that promotes vaginal mucosal regeneration and repair includes the following steps:

[0045] (1) Fresh grapes were homogenized (using a homogenizer at 120 Hz for 10 min) and centrifuged at low speed to remove pulp fragments. The supernatant was used to collect grape cell exosomes by ultracentrifugation. The centrifugal force for low speed centrifugation was 3000 g, and the centrifugal force for ultracentrifugation was 100000 g. 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.

[0046] (2) Add estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1. Disperse the estradiol by ultrasonication (120Hz, 5min) to encapsulate it in the lumen of the grape cell exosomes, and control the encapsulation rate to be ≥85% (after encapsulation, centrifuge and dilute the supernatant. There is residual unencapsulated estradiol in the supernatant. Use a UV spectrophotometer to measure the absorbance at 278nm and compare it with the standard curve to calculate the concentration of free estradiol in the supernatant). Grape exosomes are obtained.

[0047] (3) Dissolve OCS in 1% acetic acid solution to obtain OCS solution with a concentration of 5% w / v, add HA and SA in sequence to make the concentration of HA 1.2% w / v and the concentration of SA 3.5% w / v, and stir magnetically at 37℃ until completely dissolved to form a uniform and transparent ternary polysaccharide response matrix solution.

[0048] (4) Grape exosomes at a concentration of 12 μg / mL were added to the ternary polysaccharide responsive matrix solution and ultrasonically dispersed at 4°C (40 kHz, 5 min) until homogeneous. Then, CaCl2 solution was added, and the concentration of CaCl2 was controlled at 0.25% w / v. The cross-linking reaction was carried out to obtain the hydrogel dressing. After passing through a sterile filter membrane, the dressing was dispensed into syringes and stored at 4°C (shelf life 2 weeks). This hydrogel dressing can be used directly as a dressing.

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

[0050] 1. Characterization of exosomes and mechanical adaptation properties of hydrogels

[0051] Figure 1 Hydrated particle size and zeta potential of GExo and GExo-E2 (grape cell exosomes encapsulating estradiol, i.e., grape exosomes): DLS analysis showed that the hydrated particle size of GExo (91.5±3.1 nm) and GExo-E2 (88.8±2.3 nm) was significantly different. Figure 1 (a) No significant difference was observed, both falling within the 80-120 nm targeting range, indicating that estradiol loading did not disrupt the exosome membrane structure; regarding zeta potential ( Figure 1 (b) GExo (-25.1±0.2 mV) and GExo-E2 (-23.5±0.3 mV) are significantly different. GExo-E2 has a weaker negative potential but still maintains its negative electrical properties.

[0052] The results showed that, regarding particle size stability, the particle size of grape cell exosomes remained unchanged after loading with estradiol, ensuring that the exosomes retained their targeted delivery capability (80-120 nm can be endocytosed by vaginal epithelial cells). The active ingredient (estradiol) was stably embedded in the exosome lumen through an ultrasonic encapsulation process (ultrasonic treatment caused cell membrane rupture and reorganization into vesicles, with lipid-soluble drugs embedded in the lipid bilayer of the vesicles, and some drugs adsorbed onto the vesicle surface through electrostatic interactions), maintaining the integrity of the membrane structure. The negative potential of GExo-E2 decreased (related to the reduction of membrane negative charge exposure due to estradiol encapsulation) but remained positively charged (-NH3) under the acidic conditions of oxidized chitosan (OCS). + Electrostatic attraction is formed, which allows exosomes to be stably embedded in an acidic environment (healthy vagina) (acid-stable); in an alkaline inflammatory environment, OCS deprotonates (-NH2, negatively charged), the electrostatic effect is released, and exosomes are released (alkaline release), achieving pH-responsive controlled release and precisely matching the dynamic changes of 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 in shape, with a particle size of around 100 nm. High electron density substances are visible inside, possibly encapsulated estradiol and other components. Figure 2 As shown in (a). Figure 2 As shown in (b), GExo-E2 / OCS / SA / HA (grape exosomes and ternary polysaccharide matrix crosslinked composite system, i.e. the hydrogel dressing of the present invention) has a three-dimensional porous structure with pore size in the micrometer range. The ternary polysaccharide matrix forms interwoven sheet or block structures, in which exosomes may be dispersed or attached to the matrix surface.

[0054] like Figure 3As shown, the energy storage modulus (G') of GExo-E2 / OCS / SA / HA is 10 5 - 10 6 The Pa range increases with frequency from 0.1 Hz to 100 Hz, and the loss modulus (G'') is at 10 4 - 10 5 The Pa range shows a slow decreasing 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 secondary viscosity, and the elasticity increases with increasing frequency. Sodium alginate (SA) and Ca 2+ The ionic crosslinking of oxidized chitosan (OCS) and the electrostatic crosslinking of SA under acidic conditions form a three-dimensional rigid network, endowing the hydrogel with a high storage modulus (elasticity). With increasing frequency, the movement of the crosslinked network segments is restricted, enhancing elasticity. Its low viscous loss (G'') stems from the network's effective resistance to viscous flow. In the dynamic physiological and mechanical environment of the vagina (peristalsis, friction, etc.), the hydrogel's predominantly 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 and Candida albicans in the control group were 3.28 × 10⁻⁶. 12 and 5.64×10 11 The GExo-E2 group showed limited antibacterial effects, with log reduction values ​​of 0.19±0.25 and 0.29±0.14 for both groups. The present invention group (GExo-E2 / OCS / SA / HA) showed a significant decrease in CFU / mL, with E. coli at 4.13 × 10⁻⁶. 11 The number of Candida albicans was 4.17 × 10⁻⁶. 10 The Log Reduction values ​​were 0.89 ± 0.31 and 1.12 ± 0.43, respectively, indicating that the antibacterial efficacy was significantly better than that of the GExo-E2 group.

[0057] Oxidized chitosan (OCS) undergoes amino protonation (-NH3) in an acidic environment (pH 4.0-5.5). + This method utilizes electrostatic adsorption to disrupt the bacterial membrane structure, achieving broad-spectrum antibacterial activity (effective against both E. coli and C. albicans); sodium alginate (SA) and Ca...2+ The resulting thermosensitive gel provides a carrier for OCS, ensuring continuous antibacterial activity in an acidic environment. Estradiol loaded on GExo-E2 is released in an alkaline inflammatory environment (pH 7.4), inhibiting pathogen biofilm formation and forming a spatiotemporal synergy with the acidic antibacterial effect of OCS: "acid-stable, alkaline-release, and end-to-end antibacterial." Hyaluronic acid (HA) mediates the targeting of exosomes to mucosal injury sites, increasing the local concentration of antibacterial components and further enhancing the antibacterial effect (the Log Reduction in this invention group was higher than that in the GExo-E2 group, especially for C. albicans, increasing by 1.12, verifying the synergistic mechanism).

[0058] Table 1 Antibacterial ability data

[0059]

[0060] Under pH conditions of 4.0 (healthy acidity), 5.5 (mild inflammation), and 7.4 (severe inflammation alkalinity), the release rate of grape exosomes (GExo-E2) from the GExo-E2 / OCS / SA / HA combination exhibited a significant pH-responsive characteristic, as shown in the results below. Figure 4 As shown, in acidic environments (pH 4.0-5.5), the release rate is ≤14.2% from 2 to 48 hours (e.g., 14.2% release rate at pH 4.0 after 48 hours), indicating low release volume. In alkaline environments (pH 7.4), the release rate increases significantly over time, reaching 32.4% after 48 hours. This indicates that the hydrogel dressing of the present invention releases drugs at low levels in healthy / mildly inflamed conditions, avoiding interference with normal mucosa; and releases drugs efficiently in alkaline environments during severe inflammation, targeting and repairing damage. The pH-sensitive charge change of oxidized chitosan (OCS) is the core: in acidic conditions, OCS is protonated, electrostatically crosslinking with sodium alginate (SA) carboxylate to form a dense network, inhibiting exosome release; in alkaline conditions, OCS is deprotonated, the network loosens, triggering rapid release of exosomes (loaded with estradiol and grape polyphenols). The three components work together to achieve "acid-stabilized and alkali-released, targeted repair," precisely matching the dynamic changes in the vaginal microenvironment, solving the shortcomings of inaccurate drug release in traditional materials, and ensuring low drug exposure in healthy tissues and efficient repair of inflamed areas.

[0061] 3. Exosome-targeted repair function

[0062] Inflammation suppression effect: In an 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 level of IL-6 was highest in the control group (mean 1.026), significantly lower 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), the control group had the highest TNF-α expression level (mean 1.377), while the GExo-E2 group (0.646) and the present invention group (0.708) showed a highly significant decrease. The present invention, through a dual mechanism of "exosome-targeted delivery of active ingredients + pH-responsive antibacterial action of the polysaccharide matrix," is significantly superior to grape cell exosomes loaded with E2 alone (GExo-E2 group). The expression levels of both IL-6 and TNF-α decreased by ≥40% compared to the control group, demonstrating the regulatory ability of the hydrogel dressing of the present invention on the vaginal inflammatory microenvironment and providing direct evidence for clinical anti-inflammatory repair.

[0063] Regeneration capacity: such as 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, and further increased to 123.96% in the present invention group (GExo-E2 / OCS / HA / SA), representing increases of 19% and 23% respectively compared to the control group, significantly superior to the control group. This demonstrates that the hydrogel dressing of the present invention is non-toxic and effectively promotes cell proliferation. The 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 an acidic environment (reducing pathogen damage) and releases exosomes in an alkaline environment, HA mediates cell adhesion and nutrient uptake, and SA's Ca... 2+ The thermosensitive gel provides three-dimensional support, and the three components work synergistically with exosomes to further enhance the proliferation-promoting effect (an additional gain in this invention). Through "delivery of bioactive molecules + microenvironment regulation," the material achieves safe and efficient enhancement of cell viability, directly supporting the technical solution of "promoting vaginal mucosal regeneration."

[0064] Example 2

[0065] A method for preparing a hydrogel dressing that promotes vaginal mucosal regeneration and repair includes the following steps:

[0066] (1) After homogenizing fresh grapes, centrifuge at low speed to remove pulp fragments. Collect grape cell exosomes from the supernatant by ultracentrifugation. The centrifugal force for low-speed centrifugation is 3000g, and the centrifugal force for ultracentrifugation is 100000g. After resuspending in PBS, verify the particle size (80-120 nm) by nanoparticle size analysis (DLS), and determine the protein concentration by BCA method.

[0067] (2) Add estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1, and disperse by ultrasonication (120Hz, 5min) to encapsulate estradiol in the lumen of grape cell exosomes, controlling the encapsulation rate to ≥85%, to obtain grape exosomes;

[0068] (3) Dissolve OCS in 1% acetic acid solution to obtain OCS solution with a concentration of 4% w / v, add HA and SA in sequence to make the concentration of HA 1% w / v and the concentration of SA 3% w / v, and stir magnetically at 37℃ until completely dissolved to form a uniform and transparent ternary polysaccharide response matrix solution.

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

[0070] Example 3

[0071] A method for preparing a hydrogel dressing that promotes vaginal mucosal regeneration and repair includes the following steps:

[0072] (1) After homogenizing fresh grapes, centrifuge at low speed to remove pulp fragments. Collect grape cell exosomes from the supernatant by ultracentrifugation. The centrifugal force for low-speed centrifugation is 3000g, and the centrifugal force for ultracentrifugation is 100000g. After resuspending in PBS, verify the particle size (80-120 nm) by nanoparticle size analysis (DLS), and determine the protein concentration by BCA method.

[0073] (2) Add estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1, and disperse by ultrasonication (120Hz, 5min) to encapsulate estradiol in the lumen of grape cell exosomes, controlling the encapsulation rate to ≥85%, to obtain grape exosomes;

[0074] (3) Dissolve OCS in 1% acetic acid solution to obtain OCS solution with a concentration of 6% w / v, add HA and SA in sequence to make the concentration of HA 1.5% w / v and the concentration of SA 4% w / v, and stir magnetically at 37℃ until completely dissolved to form a uniform and transparent ternary polysaccharide response matrix solution.

[0075] (4) Add grape exosomes at a concentration of 12 μg / mL to the ternary polysaccharide response matrix solution, and sonicate at 4℃ (40 kHz, 5 min) until uniform. Then add CaCl2 solution and control the concentration of CaCl2 to 0.25% w / v. Perform cross-linking reaction to obtain hydrogel dressing. After passing through a sterile filter membrane, dispense into syringes and store at 4℃ (shelf life 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 within the protection scope of the present invention.

Claims

1. A hydrogel dressing that promotes vaginal mucosal regeneration and repair, characterized in that, The product includes grape exosomes and a ternary polysaccharide response matrix. The grape exosomes are grape cell exosomes encapsulating estradiol. The ternary polysaccharide response matrix is ​​formed by stirring and dissolving oxidized chitosan (OCS) solution, hyaluronic acid (HA) solution, and sodium alginate (SA) solution. The grape exosomes and ternary polysaccharide responsive matrix, via Ca 2+ The hydrogel dressing that promotes vaginal mucosal regeneration and repair is prepared by triggering sodium alginate to undergo ionic cross-linking.

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 has been damaged due to menopausal vaginal atrophy, postpartum mucosal tearing, or postoperative wound healing disorders.

4. A method for preparing a hydrogel dressing for promoting vaginal mucosal regeneration and repair as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) After homogenizing fresh grapes, centrifuge to remove pulp fragments, and obtain grape cell exosomes by centrifuging the supernatant; (2) Add estradiol to grape cell exosomes, wherein the mass ratio of grape cell exosomes to estradiol is 2:1, and disperse the estradiol by ultrasonication to encapsulate it in the lumen of the grape cell exosomes, controlling the encapsulation rate to ≥85%, to obtain grape exosomes; (3) Dissolve OCS in 1% acetic acid solution to obtain OCS solution with a concentration of 4-6% w / v, add HA and SA in sequence to make the concentration of HA 1-1.5% w / v and the concentration of SA 3-4% w / v, stir magnetically until completely dissolved to form a uniform and transparent ternary polysaccharide response matrix solution. (4) Add grape exosomes at a concentration of 10-15 μg / mL to the ternary polysaccharide response matrix solution, disperse evenly by ultrasonication, add CaCl2 solution, control the concentration of CaCl2 to 0.2-0.25% w / v, cross-linking reaction, and obtain hydrogel dressing. After passing through a sterile filter membrane, dispense into syringes and store at 4℃.

5. The method for preparing a hydrogel dressing to promote vaginal mucosal regeneration and repair according to claim 4, characterized in that, In step (1), the fresh grapes are homogenized and centrifuged at low speed to remove pulp fragments. The supernatant is then centrifuged at high speed to collect grape cell exosomes. The centrifugal force of the low-speed centrifugation is 3000g, and the centrifugal force of the high-speed centrifugation 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 step (2), ultrasonic dispersion at 120 Hz is used for 5 min.

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 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 performed at 40 kHz for 5 min 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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