Exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules and preparation method and application thereof

Abstract

This invention discloses a thermosensitive hydrogel loaded with anti-inflammatory small molecules, its preparation method, and its applications, relating to the field of biomedical technology. It comprises mesenchymal stem cell-derived exosomes loaded with anti-inflammatory small molecules, sodium hyaluronate, a thermosensitive hydrogel matrix, and an enzymatic debridement agent. This invention encapsulates papain with enzymatic debridement function, anti-inflammatory small molecules, and mesenchymal stem cell-derived exosomes as the main body of repair activity within a thermosensitive hydrogel matrix composed of a fixed ratio of poloxamer 407 and poloxamer 188. This composition is liquid at low temperatures, making it easy to apply, and transforms in situ into a semi-solid gel upon contact with oral temperature, achieving wound adhesion and long-term residence. Papain is used for initial debridement, creating conditions for subsequent repair; the bioaffinity between the anti-inflammatory small molecules and exosomes enables controlled and sustained release of the active ingredients; and finally, mesenchymal stem cell-derived exosomes promote wound repair.

Description

Technical Field

[0001] This invention relates to a thermosensitive exosome hydrogel loaded with anti-inflammatory small molecules, its preparation method and application, belonging to the field of biomedical technology. Background Technology

[0002] The oral mucosa is a soft tissue lining the interior of the oral cavity. Due to its structural and functional characteristics, it is easily damaged by physical trauma, chemical irritation, or pathological factors, such as oral ulcers and traumatic erosions. Although these injuries are common, they are often accompanied by symptoms such as pain and discomfort when eating, affecting the patient's quality of life. The oral environment is complex, and the continuous flow of saliva, tongue movements, and chewing motions pose significant challenges to topical medication application.

[0003] Recurrent aphthous ulcers (RAU) are the most prevalent oral mucosal disease, affecting approximately 25% of the population. Clinically, they are characterized by periodic recurrences, are self-limiting, and cause significant burning pain. While moderate inflammation is necessary to initiate healing, RAU involves an excessive and persistent inflammatory response, hindering subsequent proliferation and epithelialization. Currently, there is no cure for RAU and other oral ulcer diseases. Treatment for oral mucosal lesions typically involves topical administration in formulations such as solutions, ointments, or gels. However, these conventional preparations have limited adhesion to the moist and dynamic mucosal surface, making it difficult to form a stable drug coating on the wound. The preparations themselves and their active ingredients are easily diluted and rapidly cleared by saliva, resulting in a short residence time and low bioavailability at the target site. Therefore, frequent administration is necessary to maintain a certain local drug concentration.

[0004] Furthermore, the condition of the wound bed directly affects the healing process during the physiological process of wound repair. A pseudomembrane or necrotic tissue composed of necrotic cells, fibrin, and bacterial metabolites often forms on the surface of the injured area. This layer not only triggers a persistent inflammatory response but also constitutes a physical barrier, hindering the penetration and action of exogenous repair factors into the deeper tissues of the wound, thereby delaying the healing process.

[0005] To improve wound healing outcomes, some studies have begun to focus on the application of bioactive factors (such as growth factors and extracellular vesicles). These bioactive factors can participate in and regulate repair processes such as cell proliferation and migration. However, how to effectively deliver these bioactive factors to the wound and ensure their sustained effectiveness throughout the entire repair cycle remains a technical challenge. Simply mixing these bioactive factors with the matrix often results in uncontrolled release behavior, typically manifesting as an initial burst of release that leads to rapid depletion of the active ingredients. This fails to provide stable and lasting biological signals for tissue repair, thus limiting the final therapeutic effect.

[0006] Glycyrrhetinic acid (GA) is the main active triterpenoid aglycone component of glycyrrhizic acid. Its 18β configuration (18β-GA) has been shown to possess significant anti-inflammatory activity, primarily by inhibiting IκB-inhibiting protein degradation, thereby suppressing NF-κB signaling pathway activation and ultimately reducing the expression levels of key pro-inflammatory factors such as TNF-α and IL-6. However, the clinical application of 18β-GA faces significant challenges: its low solubility in water, strong hydrophobicity, and poor membrane permeability limit its bioavailability, making therapeutic efficacy highly dependent on the optimization of delivery systems (such as liposomes). Traditional formulations often suffer from uncontrollable drug release, typically exhibiting an initial burst of release followed by rapid depletion of the active ingredient, failing to maintain an effective therapeutic concentration at the lesion site and severely restricting its full therapeutic efficacy. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide an exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules, its preparation method and application. It is liquid at low temperatures, making it easy to apply, and can be transformed into a semi-solid gel in situ after contact with oral cavity temperature, achieving wound adhesion and long-term residence. It solves the problems of existing oral mucosal repair preparations, such as poor wound adhesion, easy removal of drugs by saliva, insufficient debridement ability of necrotic tissue, and explosive release of active repair factors, which cannot achieve long-term sustained release.

[0008] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0009] In a first aspect, the present invention provides an exosome-loaded thermosensitive hydrogel containing anti-inflammatory small molecules, comprising exosomes, sodium hyaluronate, an enzymatic debridement agent and a thermosensitive hydrogel matrix, wherein the exosomes are loaded with anti-inflammatory small molecules.

[0010] Furthermore, the enzymatic debridement agent is papain; and / or, the thermosensitive hydrogel matrix includes poloxamer 407 and poloxamer 188; and / or, the exosomes are mesenchymal stem cell-derived exosomes.

[0011] Furthermore, by weight percentage, the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules comprises: 0.5~2.7×10 10 One particle / mL of exosomes, 28-32% poloxamer 407, 10-12% poloxamer 188, 0.20-0.26% sodium hyaluronate, and 0.10-0.15% papain.

[0012] Furthermore, the anti-inflammatory small molecule is glycyrrhetinic acid; and / or, the mass percentage of glycyrrhetinic acid in the exosome thermosensitive hydrogel loaded with the anti-inflammatory small molecule is 0.01~0.03%.

[0013] Furthermore, the molecular weight of the sodium hyaluronate is 1500~2000 kDa.

[0014] Secondly, the present invention also provides a method for preparing an exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules, comprising:

[0015] Poloxamer 407, poloxamer 188, sodium hyaluronate and papain were dissolved in a buffer solution to obtain a liquid hydrogel matrix.

[0016] Exosomes were incubated with anti-inflammatory small molecules to obtain an exosome suspension loaded with anti-inflammatory small molecules;

[0017] An exosome suspension loaded with anti-inflammatory small molecules was stirred and mixed with a liquid hydrogel matrix to obtain a thermosensitive exosome hydrogel loaded with anti-inflammatory small molecules.

[0018] Furthermore, the liquid hydrogel matrix is ​​obtained by mixing within a temperature range of 2~4℃.

[0019] Furthermore, the incubation conditions include incubation at a temperature range of 35~38℃ for 40~60 minutes.

[0020] Furthermore, the mixing conditions include mixing for 15 to 30 minutes at a speed of 50 to 100 rpm.

[0021] Thirdly, the present invention also provides the application of the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules as described in any of the above claims in the preparation of a drug for oral mucosal repair.

[0022] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0023] When the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules of the present invention is applied to the oral mucosa, it utilizes the thermosensitive properties of poloxamer 407 and poloxamer 188. It is liquid at low temperatures, which is easy to apply. After contacting the oral temperature, it transforms from a liquid in situ into a semi-solid hydrogel, which prolongs the residence time on the wound surface and effectively improves the repair ability of the oral mucosa.

[0024] The oral mucosal repair principle of the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules of the present invention is as follows: First, papain is used to clean the wound surface to create conditions for subsequent repair. Then, the adhesive anchoring effect of sodium hyaluronate and exosomes is used to release exosomes and anti-inflammatory small molecules to the wound for efficient repair of oral mucosal epithelium. Oral adhesion repair targets conditions such as recurrent aphthous ulcers, traumatic ulcers, or oral mucositis. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the average particle size distribution curve of the exosome stock solution in Example 1 of the present invention;

[0026] Figure 2 This is a schematic diagram of the intensity-particle size distribution of the exosome stock solution in Example 1 of the present invention;

[0027] Figure 3 This is a schematic diagram showing the results of DLS (Malvern) measurement of vesicle diameter in the exosome stock solution in Example 1 of the present invention;

[0028] Figure 4 This is a schematic diagram of the CCK8 cytotoxicity test results of the present invention, wherein a is a schematic diagram of the cell biocompatibility under a microscope of the blank control group, and b is a schematic diagram of the cell biocompatibility under a microscope of the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules prepared in Example 1.

[0029] Figure 5 This is a schematic diagram showing the results of the CCK8 in vitro biocompatibility test between the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules prepared in Example 1 of the present invention and the blank control group.

[0030] Figure 6 This is a schematic diagram showing the test results of the gelation phase transition temperature of the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules prepared in Example 1 of the present invention.

[0031] Figure 7 This is a schematic diagram showing the test results of the gelation time of the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules prepared in Example 1 of the present invention.

[0032] Figure 8 This is a schematic diagram showing the test results of the shear thinning properties of the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules prepared in Example 1 of the present invention.

[0033] Figure 9 This is a schematic diagram showing the viscosity-temperature dependence test results of the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules prepared in Example 1 of the present invention.

[0034] Figure 10 This is a schematic diagram of histological sections showing the changes in mouse ulcer wounds over time under different treatment methods in the in vivo mouse experiment of the present invention;

[0035] Figure 11 This is a line graph illustrating the change in the recovery rate of mouse ulcer wounds under different treatment methods with the number of days in the in vivo mouse experiment of the present invention;

[0036] Figure 12 This is a schematic diagram of the histological evaluation results of oral ulcers in mice during in vivo experiments of the present invention. In the diagram, a is a schematic diagram of the histological evaluation results of oral ulcers in mice under the treatment method of Example 1, b is a schematic diagram of the histological evaluation results of oral ulcers in mice under the treatment method of Comparative Example 1, c is a schematic diagram of the histological evaluation results of oral ulcers in mice under the treatment method of Comparative Example 2, d is a schematic diagram of the histological evaluation results of oral ulcers in mice under the treatment method of Comparative Example 3, and e is a schematic diagram of the histological evaluation results of oral ulcers in mice under the treatment method of the blank control group. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0038] Example 1

[0039] This invention provides a method for preparing an exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules, comprising the following steps:

[0040] (1) Preparation of basic hydrogel sol:

[0041] Add 0.23 g of sodium hyaluronate to 1.15 mL of pre-cooled PBS (2-4℃) to obtain a sodium hyaluronate stock solution. Add the sodium hyaluronate stock solution to a centrifuge tube containing 2.85 mL of pre-cooled PBS (2-4℃), stir and mix well to obtain 4 mL of sodium hyaluronate dilution.

[0042] Add 50 mL of poloxamer 188 stock solution (20% mass concentration) to a beaker containing 4 mL of the above-mentioned sodium hyaluronate dilution, and place a magnetic stir bar inside. Place the beaker on a low-temperature magnetic stirrer and stir at 250 rpm for 1 hour at 2-4°C until homogeneous to obtain a poloxamer 188 solution.

[0043] Add 30 g of poloxamer 407 powder to a poloxamer 188 solution and stir continuously overnight at 2–4°C until a clear, transparent poloxamer-based sol is formed. In this example, approximately 80 mL of the poloxamer-based sol was obtained at this point and was stored at 4°C throughout.

[0044] (2) Preparation of a hydrogel solution containing papain:

[0045] Weigh 0.125 g of papain and place it in 1 mL of pre-cooled PBS to obtain 1 mL of papain working solution with a mass percentage of 12.5%.

[0046] The above papain working solution was mixed with 79 mL of basic hydrogel sol and stirred to form a clear and transparent papain-containing hydrogel solution (80 mL).

[0047] (3) Preparation of exosomes loaded with glycyrrhetinic acid

[0048] Human umbilical cord-derived mesenchymal stem cells (hMSCs) were seeded into culture flasks and cultured in DMEM / F12 medium containing 10% fetal bovine serum and penicillin-streptomycin solution at 37°C and 5% CO2. When the hMSCs reached 90% confluence, the cells were washed twice with PBS and the medium was replaced with α-MEM medium containing 0.1% FBS and 2 mM L-glutamine, and cultured for another 48 h. The culture supernatant was collected and centrifuged at 2000 g for 10 min to remove cell debris, yielding the culture medium. The collected culture media were combined and centrifuged at 10000 g at 4°C for 40 min in a refrigerated centrifuge to remove larger vesicles, and stored at 4°C for later use.

[0049] Transfer the supernatant after centrifugation to a Beckman ultracentrifuge tube, balance it, and centrifuge it in an Optima™ XPN ultracentrifuge at 100,000 g and 4 °C for 90 min. Discard the supernatant, add pre-chilled PBS, repeatedly pipette the precipitate, balance it, and centrifuge it again at 100,000 g and 4 °C for 90 min to collect the precipitate. Finally, resuspend the precipitate in a small amount of pre-chilled PBS to obtain the exosome stock solution (hMSC-Exosomes), and store it in a -80 °C freezer for later use.

[0050] The mass concentration of the exosome stock solution was determined to be 0.01142% using a Micro-BCA kit. 20 mL of this exosome stock solution was taken, and 20 mg of solid glycyrrhetinic acid was added. The solution was incubated at 37°C for 60 minutes to allow the glycyrrhetinic acid to be fully loaded, resulting in 20 mL of exosome suspension loaded with glycyrrhetinic acid (at which point the mass concentration of glycyrrhetinic acid was 0.1%). This suspension was then stored at -80°C for later use.

[0051] (4) Preparation of exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules:

[0052] In a low-temperature environment of 0~4℃, the exosome suspension loaded with glycyrrhetinic acid from step (3) was slowly added to the papain-containing hydrogel solution obtained in step (2). The mixture was stirred at 50 rpm for 30 minutes to ensure homogeneity, ultimately yielding 100 mL of temperature-sensitive exosome hydrogel loaded with anti-inflammatory small molecules. The mass percentage of each component was 1.38 × 10⁻⁶ exosomes loaded with glycyrrhetinic acid. 10 Particles / mL, 30% poloxamer 407, 10% poloxamer 188, sodium hyaluronate 0.23%, papain 0.125%, glycyrrhetinic acid 0.02%.

[0053] The thermosensitive hydrogel of exosomes loaded with anti-inflammatory small molecules should be stored in a sealed container at 4°C.

[0054] Example 2

[0055] This invention provides a method for preparing an exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules, comprising the following steps:

[0056] (1) Preparation of basic hydrogel sol:

[0057] Add 0.26 g of sodium hyaluronate to 1.3 mL of pre-cooled PBS (2-4℃) to obtain a sodium hyaluronate stock solution. Add the sodium hyaluronate stock solution to a centrifuge tube containing 27 mL of pre-cooled PBS (2-4℃), stir and mix well to obtain 28.3 mL of sodium hyaluronate dilution.

[0058] Add 32 g of poloxamer 407 powder and 12 g of poloxamer 188 powder to a beaker containing the above-mentioned sodium hyaluronate dilution, and place a magnetic stir bar inside. Place the beaker on a low-temperature magnetic stirrer and stir continuously overnight at 2–4°C until a clear, transparent poloxamer-based sol is formed. In this example, approximately 72 mL of the poloxamer-based sol was obtained at this point and was stored at 4°C throughout.

[0059] (2) Preparation of a hydrogel solution containing papain:

[0060] Weigh 0.15 g of papain and place it in 1 mL of pre-cooled PBS to obtain 1 mL of papain working solution with a mass percentage of 15%.

[0061] The above papain working solution was mixed with 71 mL of basic hydrogel sol and stirred to form a clear and transparent papain-containing hydrogel solution (approximately 72 mL).

[0062] (3) Preparation of exosomes loaded with glycyrrhetinic acid

[0063] Human umbilical cord-derived mesenchymal stem cells were seeded into culture flasks and cultured in DMEM / F12 medium containing 10% fetal bovine serum and penicillin-streptomycin solution at 37°C and 5% CO2. When confluence reached 90%, the cells were washed twice with PBS and the medium was replaced with α-MEM medium containing 0.1% FBS and 2 mM L-glutamine, and cultured for another 48 h. The culture supernatant was collected and centrifuged at 2000 g for 10 min to remove cell debris, yielding the culture medium. The collected culture media were combined and centrifuged at 10000 g at 4°C for 40 min in a refrigerated centrifuge to remove larger vesicles, and stored at 4°C for later use.

[0064] Transfer the supernatant after centrifugation to a Beckman ultracentrifuge tube, balance it, and centrifuge at 100,000 g, 4 °C for 90 min. Discard the supernatant, add pre-cooled PBS, repeatedly pipette the precipitate, balance it again, and centrifuge at 100,000 g, 4 °C for 90 min to collect the precipitate. Finally, resuspend the precipitate in a small amount of pre-cooled PBS to obtain exosomes, and store them in a -80 °C freezer for later use.

[0065] The mass concentration of the exosome stock solution was determined to be 0.01142% using a Micro-BCA kit. 26.7 mL of this exosome stock solution was taken, and 20 mg of solid glycyrrhetinic acid was added. The solution was incubated at 37°C for 60 minutes to allow the glycyrrhetinic acid to fully load, resulting in 26.7 mL of exosome suspension loaded with glycyrrhetinic acid (at which point the mass concentration of glycyrrhetinic acid was approximately 0.075%). This suspension was then stored at -80°C for later use.

[0066] (4) Preparation of exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules:

[0067] In a low-temperature environment of 0~4℃, the exosome suspension loaded with glycyrrhetinic acid from step (3) was slowly added to the papain-containing hydrogel solution obtained in step (2). The mixture was stirred at 50 rpm for 30 minutes to ensure homogeneity, ultimately yielding approximately 100 mL of thermosensitive exosome hydrogel loaded with anti-inflammatory small molecules. The mass percentage of each component was 1.84 × 10⁻⁶ exosomes loaded with glycyrrhetinic acid. 10 Particles / mL, 32% poloxamer 407, 12% poloxamer 188, sodium hyaluronate 0.26%, papain 0.15%, glycyrrhetinic acid 0.02%.

[0068] The thermosensitive hydrogel of exosomes loaded with anti-inflammatory small molecules should be stored in a sealed container at 4°C.

[0069] Comparative Example 1:

[0070] The only difference between this comparative example and Example 1 is that glycyrrhetinic acid is not added in step (3).

[0071] Comparative Example 2:

[0072] The only difference between this comparative example and Example 1 is that exosomes are not added in step (4), but glycyrrhetinic acid is still added at the same concentration.

[0073] Comparative Example 3:

[0074] The only difference between this comparative example and Example 1 is that in step (4), instead of adding an exosome suspension loaded with glycyrrhetinic acid, an equal volume of PBS buffer solution is added.

[0075] Comparative Example 4:

[0076] The only difference between this comparative example and Example 1 is that sodium hyaluronate is not added in step (1), but an equal volume of PBS buffer solution is added instead.

[0077] Comparative Example 5:

[0078] This comparative example provides a sample, which is the exosome suspension loaded with glycyrrhetinic acid prepared in step (3) of Example 1, which is not mixed with the basic hydrogel matrix prepared in step (1).

[0079] The performance of the hydrogels prepared in Example 1 and Comparative Examples 1-4 was analyzed and tested below.

[0080] First, the exosome stock solution in Example 1 was characterized and tested.

[0081] Characterization was performed using nanoparticle tracking analysis (NTA): the particle size distribution and concentration of extracted exosomes were determined using a NanoSight NS300 (Malvern) nanoparticle tracking analyzer, with NTA 3.4 Build 3.4.4 as the software version. The prepared exosome samples were appropriately diluted with PBS before analysis. The instrument recorded Brownian motion using laser scattering and a camera, and calculated the particle size and concentration. The characterization results are as follows:

[0082] Uniform particle size and height: such as Figure 1As shown, the particle size distribution curve of the exosome sample exhibits a distinct single-peak distribution, with a sharp main peak and a narrow base. According to the data, the mode particle size (Mode) is 126.6 nm, and the median particle size (D50) is 150.9 nm. This is consistent with the characteristics of exosomes: the vast majority of particles are concentrated in the 100–200 nm range (D10 is 92.1 nm, D90 is 345.3 nm), which matches the typical physical size characteristics of small extracellular vesicles (sEVs), demonstrating the good selectivity of the extraction process.

[0083] Good dispersibility: such as Figure 2 As shown, the particles are densely and uniformly distributed, and no obvious particle aggregation was observed.

[0084] High abundance and concentration: The results showed that the concentration of the exosome stock solution was 6.89 × 10⁻⁶. 10 ±5.33×10 9 Particles / mL.

[0085] Stability and repeatability: Figure 1 The high degree of overlap of the three independent measurement curves demonstrates the stability of the preparation process of this invention and ensures the accuracy of the dosage of active ingredients in subsequent hydrogel products.

[0086] Therefore, based on the above NTA results, Example 1 contains 1.38 × 10 10 Hydrogels with 1 particle / mL exosomes have the following significant characteristics:

[0087] ① Highly efficient delivery of bioactive substances: Experiments have shown that the exosomes used have a particle size of approximately 126.6 nm. This nanoscale size advantage allows the exosomes to penetrate the interstitial space more easily after being applied to mucosal wounds, and be efficiently internalized by wound cells (such as epithelial cells and fibroblasts), thereby rapidly releasing biological factors that promote healing.

[0088] ② Precise adaptation to the mucosal environment: The unimodal distribution shown in the NTA results proves that the exosomes are free of aggregation and impurities. This high-purity biological component has good compatibility with the hydrogel matrix of this invention, enabling the slow and uniform release of exosomes in a moist mucosal environment, thereby providing a lasting microenvironment regulation for wound healing.

[0089] Next, the exosome stock solution in Example 1 was measured using a Malvern dynamic light scattering particle size analyzer.

[0090] The hydrodynamic diameter can reflect the true state of exosomes under physiological conditions, as shown in the DLS intensity map. Figure 3As shown, the Z-average particle size is 160.9 nm. The main peak of the particle size distribution (peak 1) is located near 259.7 nm, and its intensity accounts for as much as 90.2%. This indicates that the majority of the components in the sample are uniformly sized nanoscale vesicles. This particle size allows them to maintain good diffusivity after being applied to mucosal wounds, which is conducive to the exosomes penetrating the mucus layer and interacting efficiently with damaged epithelial cells, thereby achieving a highly efficient healing effect. The main peak accounts for more than 90%, proving that the preparation process of this invention can effectively remove cell debris and large particulate impurities. High-purity exosome vesicles, as active ingredients, can significantly reduce the inflammatory response that may occur in hydrogel dressings in clinical applications, improving the safety of mucosal repair.

[0091] Then, in order to evaluate the safety of the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules prepared in Example 1, CCK8 cytotoxicity test and CCK8 in vitro biocompatibility test were performed.

[0092] The CCK8 cytotoxicity assay procedure is as follows:

[0093] The thermosensitive exosome hydrogel loaded with anti-inflammatory small molecules prepared in Example 1 was mixed with a culture medium containing 10% serum as the experimental group, and an equal volume of the culture medium containing 10% serum was used as the blank control group. Human MSCs cells were co-cultured with the two solutions for 24-48 hours, and the cell growth status was observed using an inverted microscope. Figure 4 As shown:

[0094] Blank control group: Cells adhered well, exhibiting a typical long spindle or fibroblast-like morphology, with clear cytoplasm, prominent nucleoli, and tightly packed cells, showing healthy fish-like growth characteristics.

[0095] Experimental group (Example 1 treatment group): Cells treated with exosome hydrogel in Example 1 maintained a highly consistent morphology with the control group. No typical cytotoxic reactions such as cell shrinkage, rounding, detachment, or the production of large amounts of granular debris were observed. This indicates that the high-purity exosomes and hydrogel matrix in Example 1 had no significant damaging effect on the cells. Furthermore, under a microscope, the cells in Example 1 showed high confluence and uniform distribution, indicating that Example 1 not only did not inhibit normal cell metabolism but also provided a favorable microenvironment to support cell adhesion and proliferation.

[0096] The steps for the CCK8 in vitro biocompatibility test are as follows:

[0097] The in vitro biocompatibility of the gel of Example 1 was evaluated using the CCK-8 assay. MSCs were seeded at a density of 5000 cells / well in 96-well plates and cultured for 24 hours. Then, culture medium containing the gel of Example 1 was added. After another 24 hours of culture, 100 µL of CCK-8 reagent was added to each well and incubated for 1 hour. The absorbance (OD value) was measured at 450 nm using a microplate reader.

[0098] Experimental results are as follows Figure 5 As shown, the net OD value of the blank control group (culture medium containing 10% serum) was calculated to be 0.266 ± 0.028 (n=6), and the net OD value of Example 1 was 0.244 ± 0.021 (n=6). With the cell viability of the blank control group as 100.0%, the cell survival rate after treatment in Example 1 was 91.61%. Independent samples t-test analysis using Graphpad Prism software showed that there was no statistically significant difference between the experimental group and the blank control group (P=0.328>0.05, labeled as ns). According to ISO 10993-5 standard (cell viability > 70% is considered non-toxic), the experimental results confirm that the gel described in this invention is non-cytotoxic, has excellent biocompatibility, and meets the safety requirements for clinical medical devices.

[0099] The performance of the samples obtained in Examples 1-2 and Comparative Examples 1-4 is compared and analyzed below.

[0100] 1. Determination of hydrogel gelation phase transition temperature, viscoelasticity, shear thinning behavior, and time-dependent gelation kinetics.

[0101] The gelation properties and mechanical behavior of exosome-containing hydrogels were systematically evaluated using a TA Instruments rotational rheometer (Discovery HR series). The specific steps are as follows:

[0102] Take 3 mL each of Example 1, Example 2, Comparative Example 4, and Comparative Example 5 (all in a refrigerated sol-liquid state) and place them in the center of the lower clamp of the rheometer. Using a parallel plate rotor with a diameter of 40 mm, slowly lower the upper rotor, setting the gap to 500~1000 μm. Remove any excess sample overflowing from the edges of the parallel plates. Apply a ring of low-viscosity silicone oil to the outer edge of the parallel plates to prevent edge hardening and data distortion caused by moisture evaporation during the test.

[0103] 1.1 Determination of gelation phase transition temperature: Experimental procedure: Set the oscillation mode to a fixed frequency of 1 Hz and the strain to 1% (within the linear viscoelastic region). Set the temperature scan range to 25~45℃ and the heating rate to 2℃ / min. Record the changes in storage modulus (G'), loss modulus (G''), and loss factor (tanδ) with temperature. Perform three measurements and take the average value.

[0104] Experimental results:

[0105] The gelation phase transition temperature test results of the samples prepared in Examples 1, 2 and Comparative Example 5 are shown in Table 1.

[0106] Table 1: Measurement results of gelation phase transition temperature of samples prepared in Examples 1, 2 and Comparative Example 5

[0107]

[0108] Combination Figure 6 As shown in Table 1, the sample of Example 1 already possessed a preliminary elastic modulus at room temperature (25°C), exhibiting a "pre-gel" state, which enhanced initial adhesion and ensured that it did not easily drip upon application. With increasing temperature, the storage modulus (G') and loss modulus (G'') increased sharply in the range of 32~35°C, and the mechanical strength rapidly increased after contact with body temperature (37°C). 2 Increased sharply to 10 4 The temperature range is defined as the rapid curing zone. Furthermore, the loss factor (tanδ) peaks at 37.5℃ and then sharply decreases, indicating that the cross-linked network within the system reaches dynamic equilibrium and solidifies at simulated body temperature. In contrast, Comparative Example 5, which does not contain poloxamer or other matrix components, remains in a liquid state throughout the test temperature range without undergoing a gelation transition. This suggests that the hydrogel matrix composed of poloxamer 407, poloxamer 188, sodium hyaluronate, etc., is the basis for the system's thermosensitive phase transition.

[0109] This gelation behavior originates from the physical properties of the molecules of poloxamer 407 and 188 in the composition. At lower temperatures, the hydrophilic polyoxyethylene (PEO) segments of poloxamer 407 are fully bound to water molecules, allowing the polymer chains to dissolve in the aqueous solution as monomers, resulting in a low-viscosity liquid state. As the temperature increases, the dehydration of its hydrophobic polyoxypropylene (PPO) segments intensifies, leading to the spontaneous aggregation of polymer monomers to form micelles. With further increases in temperature towards the gelation temperature, these micelles pack tightly together, forming an ordered three-dimensional lattice network structure spanning the entire system.

[0110] This physical network structure can encapsulate and immobilize water, exosomes loaded with anti-inflammatory small molecules, and other active components, transforming the macroscopic rheological properties of the system from liquid to semi-solid, making it less susceptible to direct rinsing by saliva in the oral cavity. Since the measured rapid gel solidification range (approximately 32–35°C) is lower than human body temperature, this composition, when applied to oral mucosal wounds, can rapidly gel in situ upon contact with body temperature, transitioning from its initial liquid state at administration. This characteristic is a prerequisite for prolonging the composition's residence time on the wound and achieving subsequent programmed repair functions.

[0111] 1.2 Time-dependent gelation kinetics experiment:

[0112] Experimental Procedure: The program was started to rapidly raise the temperature of the splint from room temperature to 37°C and maintain it at a constant temperature, simulating the heat conduction process during the instant the dressing contacts the mucosa. Data acquisition was initiated simultaneously with the heating process, recording the storage modulus (G'), loss modulus (G''), and loss factor (tanδ) every 7 seconds. Monitoring continued until the G' and G'' curves intersected and G' entered a stable plateau. The point at which the G' and G'' curves first intersected (i.e., tanδ = 1) was recorded; this point was defined as the sol-gel transition point of the system. Three measurements were taken, and the average value was recorded.

[0113] Experimental results:

[0114] The gelation times of the samples prepared in Examples 1, 2 and Comparative Example 5 are shown in Table 2.

[0115] Table 2: Results of gelation time tests for samples prepared in Examples 1, 2 and Comparative Example 5

[0116]

[0117] Combination Figure 7 As shown in Table 2, during the period from 0 to 25 seconds, tanδ > 1 and G'' > G', indicating that the material is in a sol state, possessing fluidity and easy to apply or spray. After 25 seconds, tanδ < 1 and G' > G'', indicating that the material has transformed into a gel state, possessing elasticity and shape retention. This crossover point at approximately 24-25 seconds is defined as the gelation time.

[0118] In Examples 1 and 2, the gelation time was controlled at around 25 seconds. This time period not only allows sufficient time for clinical operation (to facilitate medical staff to apply the drug solution evenly), but also ensures that curing is completed in a very short time. Furthermore, rapid gelation reduces the foreign body sensation and flow of the drug on the mucosal surface, thereby improving patient medication compliance.

[0119] 1.3 Shear rate scan experiment:

[0120] Experimental procedure: Steady-state shear tests were conducted at a simulated human body temperature (37℃). The shear rate scan range was set to 0.1 ~ 100 s. -1 Record the response curves of complex viscosity (η*) and shear stress as a function of shear rate for each group of samples, and calculate the shear thinning coefficient (defined as at a low shear rate of 0.1 s⁻¹). -1 With a high shear rate of 100 s -1 The viscosity ratio (below) was used to evaluate its injectability and sprayability.

[0121] Experimental results:

[0122] The complex viscosity of the samples prepared in Examples 1, 2 and Comparative Examples 4, 5 as a function of shear rate is shown in Table 3.

[0123] Table 3: Complex viscosity of samples prepared in Examples 1 and 2 and Comparative Examples 4 and 5 as a function of shear rate

[0124]

[0125] Combination Figure 8 As shown in Table 3, Examples 1 and 2 both exhibit significant pseudoplastic (shear-thinning) behavior. With increasing shear rate, the viscosity of the system decreases sharply by nearly two orders of magnitude. Furthermore, a larger shear-thinning coefficient indicates a better balance between the material's "handleability" and "retention." This characteristic ensures that the composition exhibits low resistance and excellent handleability when subjected to high shear forces (such as through needle injection or spray pump extrusion).

[0126] Compared to Example 1, Comparative Example 4, which does not contain sodium hyaluronate, exhibits a significantly lower viscosity under low shear. This indicates that the addition of sodium hyaluronate not only enhances the static modulus of the system but, more importantly, significantly increases its zero-shear viscosity, meaning that Example 1 possesses stronger resistance to gravitational collapse under the absence of external forces. Observing the green shear stress curve, the stress rises steadily and tends to plateau with increasing rate, without a significant hysteresis loop, proving that the system can quickly recover its physical structure after the shear force disappears.

[0127] The microscopic mechanism of this shear-thinning behavior originates from the unique interpenetrating network structure of this invention. Under low shear conditions, poloxamer micelles and long chains of sodium hyaluronate form a highly ordered, high-friction-resistance lattice network through physical entanglement. When the shear force increases, the originally disordered polymer chains align along the flow direction, and the physical constraints between micelles temporarily dissociate, resulting in a sharp decrease in macroscopic viscosity. In clinical applications, shear-thinning performance is the core advantage of this invention in achieving "precise repair," such as injectability and sprayability, and a "apply and stop" retention mechanism. A shear-thinning coefficient of over 80 times allows the composition containing exosomes to be uniformly atomized through a micro-spray nozzle, covering the complex anatomical folds of the oral mucosa, achieving uniform and complete drug delivery. The composition is a low-viscosity liquid at the moment of spraying (high shear), but once sprayed and adhered to the wound surface (low shear), the viscosity rapidly recovers to the hundreds of Pa·s level. This characteristic ensures that the drug film is quickly fixed to the wound substrate, avoiding dripping or running. Combined with the thermal response properties of poloxamer, this shear recovery capability ensures the long-term and stable retention of exosome active components in a moist mucosal environment, providing physical protection for subsequent programmed repair of damaged mucosa.

[0128] 1.4 Viscosity-Temperature Dependence Test:

[0129] Experimental procedure: The test mode was set to oscillating scan, with a constant angular frequency of 6.28 rad / s (equivalent frequency 1 Hz) and a strain of 1%. The temperature scan range was set to 25℃ to 45℃, with a constant heating rate of 2℃ / min. The kinetic change curve of the complex viscosity (η*, Pa·s) of the system as a function of temperature was recorded to evaluate its thermal response gelation rate and gel strength.

[0130] Experimental results:

[0131] The complex viscosity results of the samples prepared in Examples 1 and 2 and Comparative Examples 4 and 5 are shown in Table 4.

[0132] Table 4: Results of gel complex viscosity as a function of temperature

[0133]

[0134] Combination Figure 9 As shown in Table 4, although Comparative Example 4 (without sodium hyaluronate) still exhibits temperature-sensitive phase transition characteristics due to the presence of poloxamer components, its plateau viscosity at 37°C is significantly lower than that of Example 1. This indicates that the addition of sodium hyaluronate not only acts as a lubricant, but also significantly enhances the cohesion and mechanical strength of the three-dimensional lattice network through the physical entanglement and interpenetration of its long polymer chains with the poloxamer micelle network.

[0135] As the temperature increased from 26°C to 35°C, the viscosity of Example 1 jumped across three orders of magnitude, and its final gel strength was increased by approximately 60-80% compared to Comparative Example 4. This difference in explosive curing strength determines the performance of the composition in the complex oral mucosal environment.

[0136] The microscopic mechanism underlying this difference in physical properties lies in the fact that sodium hyaluronate (HA-Na), as a high-molecular-weight polyelectrolyte, acts as a reinforcing "molecular backbone" in the micelle array formed by poloxamer. During the temperature-sensitive gelation process, the poloxamer micelles are tightly packed, while the long chains of HA-Na form an interpenetrating network structure in their gaps, greatly increasing the frictional resistance and structural stability of the system.

[0137] In clinical applications, although Comparative Example 4 can also form a gel, the ultra-high viscosity platform of Examples 1 and 2 has irreplaceable advantages. The oral mucosa is constantly subjected to saliva rinsing and mechanical movements such as chewing and swallowing. The "strong cohesion and high adhesion" characteristics of Examples 1 and 2 ensure that the gel membrane containing a certain amount of exosome active components can be more firmly anchored to the wound surface, and is not easily torn or diluted by physical forces. This optimized viscosity profile is a key physical guarantee for achieving the long-term local residence of exosomes on the mucosa and the formation of a stable bioactive barrier at the damaged tissue, thereby ensuring efficient repair of mucosal injuries such as oral ulcers.

[0138] 2. Determination of the ulcer repair performance of hydrogel

[0139] 2.1 In vivo experiments in mice

[0140] Experimental grouping: Eighteen mice were used and divided into groups 1# to 6#, with n=3 in each group. Each mouse in each group had one sampling point on each cheek. The same wound modeling method was used, but different treatment methods were applied. The recovery of the mice was observed under the specific implementation and comparative methods to reduce the influence of individual differences. In the table, "gel" represents a mixture of poloxamer 407 and poloxamer 188 containing sodium hyaluronate, "exos" represents exosomes, and "GA" represents glycyrrhetinic acid. Specific treatment methods are shown in Table 5.

[0141] Table 5: Results of Treatment Design in In Vivo Mouse Experiments

[0142]

[0143] The testing procedure included: anesthetizing mice with 1% sodium pentobarbital (0.4~0.5 mL / 100g), fixing their mouths with a simple mouth opener to fully expose the buccal mucosa; cutting out a 3 mm diameter circular piece of filter paper, soaking it in 50% glacial acetic acid for 5 seconds, and quickly pressing it onto the buccal mucosa for 40 seconds; wiping away excess glacial acetic acid with a cotton swab dipped in physiological saline; observing that the mucosa at the pressed site was damaged and turned bright red. After 24 hours, observing local redness, swelling, and ulceration at the treated buccal mucosa indicated successful model establishment. After successful model establishment, the drug was administered once daily (10 µL of Example 1 was applied to the ulcer wound), with a 12-hour fast after administration. The first day of administration was recorded as day 0, and 24 hours after the first administration was day 1, and so on. Administration continued for one week, with daily observation of the recovery of the oral mucosa.

[0144] Starting from day 0, photographs of mouse ulcers were taken daily. During photography, a standard reference disc (a 3 mm diameter circular filter paper) was placed parallel to the edge of the ulcer for comparison. Figure 10 As shown:

[0145] Blank control group (1# left, 2# left)

[0146] Wound characteristics: The ulcer presents as a typical depressed defect with sharp edges, and is accompanied by obvious congestion of surrounding tissues in the early stage.

[0147] Evolution process: Insufficient wound shrinkage. From day 0 to day 3, the change in wound diameter was the smallest compared to other groups. By day 7, a distinct red granulation bed was still visible in the center of the wound, indicating that complete centripetal contraction had not been achieved, suggesting a significantly prolonged natural repair cycle.

[0148] Pure gel group (5# left, 6# left)

[0149] Wound characteristics: In the early stages, a thin, translucent biofilm can be seen covering the ulcer surface.

[0150] Evolution process: Compared with the blank control group, the peripheral edema was slightly reduced. The wound shrinkage rate was relatively slow. Although there was some shrinkage on the 7th day, the wound was still visible, showing general characteristics of delayed healing.

[0151] Single component group (1# right, 2# right, 3# right, 4# right, 5# right, 6# right)

[0152] Wound characteristics: The wound is relatively dry with relatively little inflammatory exudate.

[0153] Evolution: Both groups showed a certain tendency to promote wound closure. Starting from day 3, the wound edges began to become blurred, indicating epithelial migration. By day 7, the ulcer area had significantly decreased, but superficial red marks remained locally, and the ulcer had not yet fully recovered to the color and smoothness of the surrounding normal mucosa.

[0154] GA+exos composite group (3# left, 4# left)

[0155] Wound characteristics: The healing process is rapid, and the wound exhibits highly efficient contraction properties in the early stages.

[0156] Evolution process: From day 1 to day 3, the wound area shrank significantly faster than in other groups, the redness and swelling around the wound subsided rapidly, and 24 hours after administration, the exudate on the wound surface decreased, and the ulcer base was clearer and more rosy than in the control group. By day 5, the ulcer had basically closed, leaving only pinpoint marks. By day 7, the continuity of the mucosa in the original ulcer area was completely restored, and its color and texture were indistinguishable from the surrounding normal tissue to the naked eye.

[0157] The photographs were analyzed using image analysis software (Image J). Ulcer morphology and relative area were measured through visual inspection and macroscopic quantitative analysis. The degree of ulcer healing was calculated using the following formula: the raw data were averaged within each group to obtain the relative area of ​​each side for each group, and the recovery rate was calculated: Ulcer healing degree (recovery rate) = (A0 - An) / A0 × 100%, where A0 is the ulcer area on day 0, and An is the area of ​​unhealed ulcers on each subsequent day.

[0158] Based on the above data, the results of the change in the recovery rate of oral ulcer wounds in mice with different treatment methods and the evaluation of repair efficiency are shown in Table 6.

[0159] Table 6: Changes in oral ulcer healing rate and repair efficiency in mice under different treatment methods over time

[0160]

[0161] Combined with Table 6 and Figure 11 It can be seen that Embodiment 1 of the present invention has the following advantages:

[0162] (1) Significant early-stage induction of repair (advantage during initiation):

[0163] Example 1 showed a recovery rate of 46.5% on day 1, significantly higher than Comparative Example 3 (39.9%) and the control group (33.2%). This was partly due to the enzymatic debridement effect of papain in the system. By non-specifically hydrolyzing necrotic tissue and fibrin pseudomembrane on the wound surface, the system effectively activated the wound bed, eliminating physical barriers that hindered drug penetration, and creating ideal microenvironmental conditions for subsequent direct action of exosomes and glycyrrhetinic acid on living cells to initiate efficient repair. More innovatively, although the single components (gel + GA and gel + exos) showed significant delayed responses on day 1 (recovery rates of only 8.6% and 15.4%, respectively), this invention, through the combination of the three components, not only overcame the early adaptive stagnation that might be caused by a single bioactive substance, but also produced a significant synergistic accelerating effect.

[0164] (2) Highly efficient synergistic effect:

[0165] On day 3, Comparative Example 2 (57.0%) and Comparative Example 1 (52.6%) showed similar repair rates. However, Example 1 (56.2%) maintained a high level of repair on top of its early lead. This demonstrates that the antioxidant microenvironment provided by GA and the proliferation signal provided by exos achieve spatiotemporal coupling in the gel medium, making the repair process more stable after crossing the inflammatory phase.

[0166] (3) High endpoint repair quality (complete healing ability): By day 7, the recovery rate of Example 1 reached 92.7%, the highest among all experimental groups. This effectively demonstrates that compared with the blank group (87.7%) and Comparative Example 3 (89.5%), Example 1 showed stronger tissue remodeling ability in the later stage of repair. The gross observation of the experiment showed that the wound basically disappeared and the epithelial layer basically recovered to a smooth state, achieving clinical healing.

[0167] Next, histological evaluation of the oral ulcers in mice was performed:

[0168] The testing method included: on day 7 after modeling, tissue samples were collected from four original ulceration sites on both sides of the cheek in each group. Mucosal tissue from two sites was immediately fixed with 10% formaldehyde at room temperature for 24 hours. The specimens were then paraffin-embedded, fixed, sectioned, and stained with eosin (HE) on glass slides. Five fields of view were selected from each slide and photographed at 10× magnification. A healthy mucosal structure should have a neat and continuous epithelial layer (n), tightly packed basal cells, and no inflammation in the underlying connective tissue. The focus of this analysis was to assess epithelial continuity, epithelial layer thickness and structure, basal layer germinal status, and the quality of reconstruction of the underlying connective tissue (lamina propria and muscularis propria). The results are as follows:

[0169] Example 1 ( Figure 12 a)

[0170] The wound surface has achieved complete and continuous epithelial coverage. The newly formed epithelial layer exhibits ideal thickness and density (the best among all groups). The functional stratification of stratified squamous epithelium is clearly visible. Epithelial nails are well-developed and deeply embedded in the connective tissue, exhibiting a regular wavy pattern, indicating a strong mechanical connection between the epithelium and the underlying lamina propria, demonstrating a strong barrier function. Basal cells are neatly and densely arranged, showing an active germinal state. The connective tissue of the lamina propria is dense, and the underlying skeletal muscle (muscle bundles) is arranged in an orderly manner, with no obvious inflammatory cell infiltration or tissue edema, proving that the tissue anatomy has been basically restored. This high-quality tissue remodeling indirectly confirms the thoroughness of early debridement: because papain effectively removed inflammatory debris from the bottom of the wound, newly formed cells could undergo programmed migration and differentiation on a clean base, avoiding tissue disorder (such as the structural disorder shown in Comparative Example 1).

[0171] Conclusion: Example 1 achieved higher quality and more functional mucosal regeneration compared to the blank group and each comparative example.

[0172] Comparative Example 1 ( Figure 12 b)

[0173] The epithelium achieved continuous coverage, indicating wound closure. Although the epithelium was continuous, the density of its epithelial nails was significantly less than in Example 1, and the cell stratification was not obvious. The tissue sections showed a rather chaotic repair phenomenon. Although there was epithelium on the surface, the underlying tissue (lamina propria and muscle layer) was disordered and unclear, and there were still tissue gaps inside. Connective tissue was mixed with deep muscle fibers, lacking the well-defined and orderly arrangement of Example 1.

[0174] Conclusion: Although surface coverage was achieved in Comparative Example 1, the deep tissues were not ideally remodeled, which may lead to local hardening and decreased elasticity of the repaired mucosa.

[0175] Comparative Example 2 ( Figure 12 c)

[0176] The epithelial layer is largely continuous, but with noticeable depressions. The epithelial nail structures are flat and lack downward protrusions, indicating weak adhesion of the newly formed epithelium, making it prone to detachment or blistering under external physical stimuli (such as chewing). The connective tissue is relatively loose and irregular; compared to Example 1, the tissue reconstruction is not dense enough, and there are numerous gaps in the muscle layer, which may indicate residual edema or mild inflammatory reactions.

[0177] Conclusion: Comparative Example 2 only achieved a simple physical closure, and its barrier function and mechanical strength were inferior to those of Example 1.

[0178] Comparative Example 3 ( Figure 12 d)

[0179] Epithelial characteristics: The epithelium achieved complete physical coverage. The epithelial layer had a moderate thickness, significantly better than Comparative Example 2 and the control group, but still significantly thinner than Example 1. The peg structure was relatively dense, but not as deep or active as in Example 1. The underlying lamina propria and muscularis propria structures were well reconstructed, but not as stable or ordered as in Example 1.

[0180] Conclusion: Compared with other comparative examples and the control group, Comparative Example 3 (pure gel group) has certain advantages, providing better physical protection and an environment conducive to cell migration, achieving moderate-quality coverage. However, due to the lack of bioactive kinetics, it could not induce high-quality regeneration as in Example 1.

[0181] Blank control group ( Figure 12 e)

[0182] The epithelial continuity is poor, with visible wound breaks. The epithelial layer is thin, consisting of only a few layers of epithelial cells, lacking typical layered structure and maturity. The peg structures are almost completely absent, presenting as a smooth straight line. This suggests that the epithelium may have only managed to cover the wound through peripheral centripetal creep and contraction. The basal cells are in poor condition. The subepithelial tissue structure is disordered, with connective tissue and muscle layer mixed together, and numerous gaps present.

[0183] Conclusion: The blank control group was in a basic natural healing state, with the slowest repair and the lowest quality. The newly formed barrier was extremely fragile and could not withstand the washing away of saliva and mechanical forces, making it prone to ulcer recurrence in clinical practice.

[0184] Exosomes, as natural nanocarriers, possess unique biological advantages: their phospholipid bilayer structure not only effectively encapsulates hydrophobic drugs but also delivers them directly to the cytoplasm via membrane fusion, significantly improving bioavailability. More importantly, the specific ligands on the exosome surface enable active targeted delivery, greatly enhancing drug accumulation efficiency at lesion sites. Existing research has confirmed the superior performance of drug-loaded exosomes; for example, paclitaxel-loaded exosomes exhibit stronger antiproliferative activity and biocompatibility than free drugs. Thermosensitive hydrogels, serving as drug reservoirs and controlled-release carriers, enable long-term retention and stable release of glycyrrhetinic acid-loaded exosomes at oral ulcer sites. This type of hydrogel is fluid at low temperatures, which facilitates uniform mixing with drug-loaded exosomes and local drug delivery. When the temperature rises to physiological temperature (37°C), it undergoes a rapid phase transition, forming a gel barrier with a three-dimensional network structure in situ on the surface of oral ulcers. Through its pore size regulation, it achieves continuous release of drug-loaded exosomes, effectively avoiding the burst release phenomenon common in traditional dosage forms, prolonging the duration of drug action at the lesion site, and improving the therapeutic effect.

[0185] To further improve drug absorption and bioavailability, papain with enzymatic debridement function was introduced to enhance the removal of necrotic tissue from the ulcer surface.

[0186] In summary, this invention addresses the problems of abnormal immune function, significant drug delivery loss, and short duration of action associated with recurrent oral ulcers. It innovatively introduces MSC-exos, a naturally occurring drug with immunomodulatory, angiogenesis-promoting, and tissue repair properties, loaded with the anti-inflammatory therapeutic drug GA. A thermosensitive hydrogel is used for local delivery and sustained release of the drug, combined with papain debridement to promote absorption of the MSC-Exos-GA drug by living cells on the ulcer surface. This thermosensitive hydrogel-exosome composite delivery system promises to achieve highly efficient repair of the oral mucosa while reducing drug dosage and enhancing safety, providing an innovative solution for the local treatment of oral ulcers.

[0187] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A thermosensitive exosome hydrogel loaded with anti-inflammatory small molecules, characterized in that, The product comprises exosomes, sodium hyaluronate, an enzymatic debridement agent, and a thermosensitive hydrogel matrix, wherein the exosomes are loaded with anti-inflammatory small molecules; The enzymatic wound-cleansing agent is papain; The exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules comprises, by weight percentage: 0.5~2.7×10⁻⁶. 10 The formula contains exosomes at a concentration of 1 particle / mL, 28-32% poloxamer 407, 10-12% poloxamer 188, 0.20-0.26% sodium hyaluronate, and 0.10-0.15% papain. The anti-inflammatory small molecule is glycyrrhetinic acid, and the mass percentage of glycyrrhetinic acid in the exosome thermosensitive hydrogel loaded with the anti-inflammatory small molecule is 0.01~0.03%. The molecular weight of the sodium hyaluronate is 1500~2000 kDa.

2. The exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules according to claim 1, characterized in that, The exosomes are mesenchymal stem cell-derived exosomes.

3. A method for preparing an exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules as described in claim 1, characterized in that, include: Poloxamer 407, poloxamer 188, sodium hyaluronate and papain were dissolved in a buffer solution to obtain a liquid hydrogel matrix. Exosomes were incubated with anti-inflammatory small molecules to obtain an exosome suspension loaded with anti-inflammatory small molecules; An exosome suspension loaded with anti-inflammatory small molecules was stirred and mixed with a liquid hydrogel matrix to obtain a thermosensitive exosome hydrogel loaded with anti-inflammatory small molecules.

4. The method for preparing the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules according to claim 3, characterized in that, The liquid hydrogel matrix was obtained by mixing within a temperature range of 2~4℃.

5. The method for preparing the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules according to claim 3, characterized in that, The incubation conditions include incubation at a temperature range of 35~38℃ for 40~60 minutes.

6. The method for preparing the exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules according to claim 3, characterized in that, The mixing conditions include mixing for 15 to 30 minutes at a speed of 50 to 100 rpm.

7. The use of an exosome thermosensitive hydrogel loaded with anti-inflammatory small molecules as described in claim 1 in the preparation of a medicament for oral ulcers.

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