Exosome-loaded hydrogel as well as preparation method and application thereof

Abstract

The invention relates to exosome-loaded hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The hydrogel loaded with the exosome is prepared from ginger exosome, tripterine and glycyrrhizic acid hydrogel; the invention provides a colitis treatment platform for cooperatively delivering the tripterine by the ginger exosome-glycyrrhizic acid hydrogel, so that the problems of poor solubility and low bioavailability of the tripterine are obviously improved, and the toxic and side effects of the tripterine are effectively reduced; according to the treatment platform, no extra carrier needs to be introduced, all components have the activity of treating enteritis, and full functionalization of a drug carrying system is achieved; all the active ingredients have a synergistic effect through multiple ways, the treatment effect on ulcerative colitis is remarkably enhanced, and a safe, efficient and pure natural novel drug delivery scheme is provided for clinical treatment of ulcerative colitis.

Description

Technical Field

[0001] This invention relates to a hydrogel loaded with exosomes, its preparation method and application, belonging to the field of biomedical technology. Background Technology

[0002] Celastrol (Cel) is a pentacyclic triterpenoid natural compound extracted and isolated from the roots of *Tripterygium wilfordii* Hook.F., a plant belonging to the Celastraceae family. As a key active ingredient in *Tripterygium wilfordii* that exerts various pharmacological effects, its medicinal value has attracted considerable attention. This compound, along with artemisinin, paclitaxel, camptothecin, and quinine—four classic traditional medicines—is included in the category of "traditional natural compounds with the greatest potential for modern drug development," highlighting its important position in the field of drug research. Extensive preclinical and clinical research data have confirmed that celastrol exhibits significant biological activity in the treatment of various diseases, covering common and intractable conditions such as atherosclerosis, obesity, rheumatoid arthritis, liver fibrosis, and inflammatory bowel disease.

[0003] Despite its numerous excellent pharmacological activities, triptolide's inherent physicochemical properties and biopharmaceutical defects severely limit its widespread clinical application. Specifically, the compound has extremely poor water solubility, resulting in low absorption efficiency and bioavailability after oral administration. Furthermore, its toxic side effects are significant; oral administration easily causes strong irritation to the gastric mucosa, triggering gastrointestinal reactions such as nausea, vomiting, and stomach discomfort. Long-term use or high-dose administration may also damage vital organs such as the liver and kidneys, affecting normal physiological functions. Therefore, researchers have long been committed to the development of triptolide drug delivery carriers, hoping to improve its water solubility, increase bioavailability, and reduce toxic side effects through carrier technology, thereby promoting its clinical translation. However, currently developed synthetic nanodelivery systems, such as liposomes and polymer nanoparticles, generally face problems such as poor biocompatibility and potential toxicity risks. Taking the triptolide nano-suspension and its preparation method disclosed in Chinese invention patent CN106309364B as an example, this formulation effectively improves the efficacy of the drug in vivo by adding amphiphilic stabilizers such as mPEG-PCL, mPEG-DSPE, benzalkonium chloride 78, SPC, and Tween 80. However, the carrier material used may still induce an immune response in the body or produce toxic effects due to accumulation in the body during long-term use, and its safety still needs to be further optimized.

[0004] Exosomes are nanoscale microvesicles secreted by cells, belonging to the extracellular vesicle family. Their structural characteristic is a phospholipid bilayer membrane encapsulating abundant bioactive substances, including proteins, nucleic acids, and lipids. Due to their natural origin, good biocompatibility, low immunogenicity, high safety, and low toxicity, exosomes are considered a promising novel drug delivery carrier with broad application prospects in clinical drug delivery. However, traditional animal-derived exosomes have many limitations, such as complex extraction processes, long preparation cycles, high production costs, and low yields, making it difficult to meet the needs of large-scale clinical applications. In contrast, plant-derived exosomes share similarities in composition with animal-derived exosomes and possess unique advantages such as wide availability, simple extraction steps, short preparation cycles, and low cost, making them more suitable for large-scale production. It is worth noting that plant-derived exosomes also carry a variety of natural active ingredients, which endow them with anti-inflammatory, antioxidant and other pharmacological activities. While serving as drug delivery carriers, they may also have synergistic effects with the drugs they carry, further enhancing the therapeutic effect and providing a new ideal option for the delivery of tripterygium wilfordii.

[0005] Glycyrrhizic acid (GA) is a natural triterpenoid saponin extracted from the rhizome of licorice. It possesses pharmacological effects such as antiviral, antioxidant, antitumor, and anti-inflammatory properties. However, its clinical application is severely limited by drawbacks such as poor gastrointestinal stability and short intestinal retention time when used directly in its free form. Summary of the Invention

[0006] This invention provides a hydrogel loaded with exosomes, its preparation method, and its application to solve the technical problems existing in the prior art as described above.

[0007] The technical solution provided by this invention is as follows: One objective of this invention is to provide a hydrogel loaded with exosomes, including ginger exosomes (GDNPs), tripterygium oleoresin (Cel), and glycyrrhizic acid hydrogel.

[0008] The effect of adopting the above technical solution is that glycyrrhizic acid hydrogel is an amphiphilic triterpenoid saponin composed of hydrophobic glycyrrhetinic acid and hydrophilic glucuronic acid. Under the combined drive of non-covalent bonds, hydrogen bonds, hydrophobic interactions and electrostatic interactions, glycyrrhizic acid can self-assemble into a three-dimensional network hydrogel in aqueous solution. This not only improves its poor gastrointestinal stability and prolongs intestinal retention time, but also has the potential to serve as a drug carrier for synergistic delivery.

[0009] A second objective of this invention is to provide a method for preparing a hydrogel loaded with exosomes as described above, comprising the following steps: (1) Dissolve Cel in anhydrous ethanol to prepare a Cel solution; (2) Add Cel solution to GDNPs solution and incubate in a water bath shaker; (3) The unencapsulated free Cel was removed by ultracentrifugation and the precipitate after centrifugation was Cel-GDNPs; (4) Take GA powder, add pure water to dissolve it, and obtain GA aqueous solution; (5) Heat the GA aqueous solution in a water bath, stir until completely dissolved, and cool at room temperature to obtain glycyrrhizic acid hydrogel; (6) Add the Cel-GDNPs obtained in step (3) to the glycyrrhizic acid hydrogel, stir evenly, and let it stand to solidify to obtain Cel-GDNPs@Gel.

[0010] Further, the preparation of the GDNPs solution includes the following steps: extracting ginger juice, centrifuging it sequentially at 800-1200g for 8-12 min, 2000-4000g for 10-30 min, and 80000-120000g for 50-70 min to remove impurities, filtering, centrifuging the supernatant at 80000-120000g for 50-70 min, then resuspending the precipitate in PBS solution, placing it on the upper layer of a 15%, 30%, 45%, and 60% sucrose density gradient solution, centrifuging at 80000-120000g for 90-140 min, taking the 30-45% layer solution, centrifuging at 80000-120000g for 50-70 min to wash away the sucrose solution, and then resuspending the precipitate in PBS solution to obtain the GDNPs solution.

[0011] Furthermore, the preparation of the GDNPs solution includes the following steps: extracting ginger juice, centrifuging it sequentially at 1000g for 10 min, 3000g for 20 min, and 100000g for 60 min to remove impurities, filtering, centrifuging the supernatant at 100000g for 60 min, then resuspending the precipitate in PBS solution, placing it on the upper layer of a 15%, 30%, 45%, and 60% sucrose density gradient solution, centrifuging at 100000g for 120 min, taking the 30-45% layer solution, centrifuging at 100000g for 60 min to wash away the sucrose solution, and then resuspending the precipitate in PBS solution to obtain the GDNPs solution.

[0012] Furthermore, in step (1), the concentration of the Cel solution is 0.5~2 mg / mL.

[0013] Furthermore, in step (1), the concentration of the Cel solution is 1 mg / mL.

[0014] Further, in step (2), the volume ratio of Cel solution to GDNPs solution is 1:8~10; the protein concentration of GDNPs solution is 0.05~0.5 mg / mL; the temperature of the water bath is 35~40℃; and the incubation time is 1~3h.

[0015] Furthermore, in step (2), the volume ratio of Cel solution to GDNPs solution is 1:9; the protein concentration of the GDNPs solution is 0.1 mg / mL; the water bath temperature is 37°C; and the incubation time is 2 hours.

[0016] Furthermore, in step (3), the parameters for ultracentrifugation are: temperature 4℃, centrifugation at 80000~120000g for 50~70min.

[0017] Furthermore, in step (3), the parameters for ultracentrifugation are: temperature 4℃, centrifugation at 100000 g for 60 min.

[0018] Furthermore, in step (4), the concentration of the GA aqueous solution is 20~50 mg / mL.

[0019] Furthermore, in step (4), the concentration of the GA aqueous solution is 40 mg / mL.

[0020] Furthermore, in step (5), the temperature of the water bath is 60~100℃ and the heating time is 20~50min.

[0021] Furthermore, in step (5), the temperature of the water bath is 80°C and the heating time is 30 minutes.

[0022] Further, in step (6), the volume ratio of glycyrrhizic acid hydrogel to Cel-GDNPs is 0.5~1.5:1; the static solidification is to stand in a refrigerator at 4°C for 24~48 hours.

[0023] Furthermore, in step (6), the volume ratio of glycyrrhizic acid hydrogel to Cel-GDNPs is 1:1.

[0024] A third objective of this invention is to provide the application of the hydrogel loaded with exosomes as described above in the preparation of drugs for treating colitis.

[0025] The technical solution provided by this invention has the following advantages compared with the prior art: 1. This invention provides a colitis treatment platform for the synergistic delivery of triptolide by ginger exosomes and glycyrrhizic acid hydrogel, which significantly improves the problems of poor solubility and low bioavailability of triptolide and effectively reduces its toxic side effects. The treatment platform does not require the introduction of an additional carrier, and all components have the activity of treating enteritis, realizing the full functionality of the drug delivery system. The active ingredients work synergistically through multiple pathways, significantly enhancing the therapeutic effect on ulcerative colitis, and providing a safe, efficient, and all-natural drug delivery solution for the clinical treatment of ulcerative colitis.

[0026] 2. The exosome-loaded hydrogel prepared in this invention maintains structural stability within the temperature range of 25-55℃, effectively preventing premature drug release in the gastric environment and ensuring targeted drug delivery to the intestine. It prolongs drug retention time in the intestine, promoting drug penetration and absorption in the colon, thereby increasing local drug concentration and efficacy. It also possesses significant antioxidant capacity, helping to scavenge reactive oxygen species in the intestinal inflammatory environment and alleviate oxidative stress damage. Animal experiments have confirmed that it can significantly reduce the levels of inflammatory factors in colitis model mice, increase the activity of intestinal antioxidant enzymes, and promote the expression of tight junction proteins, thereby effectively improving the pathological state and inflammatory symptoms of colonic tissue. Furthermore, no significant toxicity was observed in vital organs, demonstrating good safety for clinical application. Attached Figure Description

[0027] Figure 1 Frequency scan diagram of Cel-GDNPs@Gel.

[0028] Figure 2 Temperature scan of Cel-GDNPs@Gel.

[0029] Figure 3 The in vitro release curves of Cel, Cel-GDNPs, and Cel-GDNPs@Gel are shown.

[0030] Figure 4 Plots showing the apparent permeability coefficients of IR780, IR780-GDNPs, and IR780-GDNPs@Gel. P<0.01.

[0031] Figure 5 Laser scanning confocal microscopy (CLSM) images of ROS removal by Cel, Cel-GDNPs, and Cel-GDNPs@Gel.

[0032] Figure 6 In vivo tissue distribution map of IR780, IR780-GDNPs and IR780-GDNPs@Gel.

[0033] Figure 7 Gastrointestinal distribution map of IR780, IR780-GDNPs and IR780-GDNPs@Gel.

[0034] Figure 8 The DAI score charts for each group of mice are shown.

[0035] Figure 9 This is a graph showing the expression levels of the inflammatory factor TNF-α in the colon tissue of mice in each group. P<0.01, P<0.001, P<0.0001.

[0036] Figure 10 This is a graph showing the expression levels of the inflammatory cytokine IL-1β in the colon tissue of mice in each group. P<0.01, P<0.001, P<0.0001.

[0037] Figure 11 This is a graph showing the expression levels of the inflammatory cytokine IL-6 in the colon tissue of mice in each group. P<0.01, P<0.001, P<0.0001.

[0038] Figure 12 The content of catalase (CAT) in the colon tissue of mice in each group was measured. P<0.05, P<0.01, P<0.001, P<0.0001.

[0039] Figure 13The content of glutathione peroxidase (GSH-Px) in the colon tissue of mice in each group was measured. P<0.05, P<0.01, P<0.001, P<0.0001.

[0040] Figure 14 The content of superoxide dismutase (SOD) in the colon tissue of mice in each group was determined. P<0.05, P<0.01, P<0.001, P<0.0001.

[0041] Figure 15 The content of myeloperoxidase (MPO) in the colon tissue of mice in each group was measured. P<0.05, P<0.01, P<0.001, P<0.0001.

[0042] Figure 16 Immunohistochemical results of the tight junction-related proteins ZO-1, Claudin-1, and Occludin in the colon tissue of mice in each group.

[0043] Figure 17 The Chao index represents the gut microbiota of mice in each group.

[0044] Figure 18 The Shannon index represents the gut microbiota of mice in each group.

[0045] Figure 19 The Simpson index represents the gut microbiota of mice in each group.

[0046] Figure 20 This is a graph showing the Firmicutes / Bacteroidota ratio (F / B ratio) of the gut microbiota in each group of mice. P<0.05, P<0.01, P<0.001, P<0.0001. Detailed Implementation

[0047] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0048] Example 1 Preparation of Cel-GDNPs@Gel (1) Weigh 10 mg of Cel, add 10 mL of anhydrous ethanol to dissolve it, and prepare a Cel solution with a concentration of 1 mg / mL; (2) Add Cel solution to ginger exosome (GDNPs) solution (protein concentration of 0.1 mg / mL) (Cel solution: GDNPs solution = 1:9, v / v), and incubate in a constant temperature water bath shaker at 37℃ for 2 h; (3) After incubation, centrifuge at 100,000 g for 60 min at 4 °C in an ultra-high speed centrifuge, discard the supernatant, and disperse the precipitate with 1 mL of PBS (pH 7.2-7.4) solution to obtain Cel-GDNPs; (4) Weigh 400 mg of glycyrrhizic acid powder (GA), place it in a tall beaker, add 10 mL of pure water to obtain glycyrrhizic acid aqueous solution; (5) Heat the glycyrrhizic acid aqueous solution in an 80°C water bath for 30 min and stir slowly until it is completely dissolved. Remove it and cool it to 40°C at room temperature to obtain glycyrrhizic acid hydrogel. (6) Add the Cel-GDNPs prepared in step (3) to the glycyrrhizic acid hydrogel (the volume ratio of glycyrrhizic acid hydrogel to Cel-GDNPs is 0.5~1.5), stir evenly, place in a refrigerator at 4℃ and let stand for 24 hours, and solidify to obtain Cel-GDNPs@Gel.

[0049] In step (2), the ginger exosome solution is prepared by the following steps: squeezing ginger juice, sequentially performing gradient centrifugation for impurity removal at 1000 g for 10 min, 3000 g for 20 min, and 100000 g for 60 min, filtering, centrifuging the supernatant at 100000 g for 60 min, then resuspending the precipitate with PBS (pH 7.2 - 7.4) solution, placing it on the upper layer of 15%, 30%, 45%, 60% sucrose density gradient solution, centrifuging at 100000 g for 120 min, taking the 30% - 45% layer solution and centrifuging at 100000 g for 60 min to wash away the sucrose solution, and finally resuspending the precipitate with PBS (pH 7.2 - 7.4) solution to obtain the ginger exosome solution.

[0050] Example 2 Rheology of Cel - GDNPs@Gel The viscoelastic properties of the hydrogel were analyzed by a rotational rheology method.

[0051] The results are as Figure 1 shown. In the frequency sweep test (0.1 - 200 rad / s), the fixed stress was 2.8%, and the temperatures were 25°C and 37°C respectively. At 25°C, G'>G'', indicating that Cel - GDNPs@Gel exhibits a solid - like property dominated by elasticity at 25°C, with a stable structure and strong anti - deformation ability, facilitating storage and transportation. At 37°C (the temperature of the gel after oral administration), when the angular frequency is 0.1 - 15 rad / s, G'>G'', and the gel still has a certain elasticity. When the angular frequency > 15 rad / s, G'<G'', and at this time the gel exhibits a liquid - like property dominated by viscosity, indicating that after oral administration, the gel becomes a viscous fluid under the shearing action of the digestive tract such as peristalsis during swallowing, facilitating oral administration and absorption.

[0052] As Figure 2 shown, in the temperature sweep test, the fixed stress was 2.8% and the angular frequency was 10 rad / s. When the temperature reached 58°C, the phenomenon that the storage modulus G′ was less than the loss modulus G′′ indicates that 58°C is the phase transition temperature of the hydrogel. The results show that Cel - GDNPs@Gel is stable within the temperature range of 25°C - 55°C.

[0053] Example 3 In vitro release of Cel - GDNPs@Gel The drug release behavior of Cel-GDNPs@Gel in different media was studied by dialysis method. The standard curve of Cel was generated by HPLC method. 1 mL of Cel, Cel-GDNPs and Cel-GDNPs@Gel solutions with the same concentration were taken respectively, placed in dialysis bags (MWCO 3000 Da), the two ends of the dialysis bags were clamped and then immersed in 30 mL of PBS buffer (pH 1.2) respectively, and then placed in a constant temperature shaker, and oscillated at a constant temperature of 75 rpm at 37°C. 1 mL of the release medium was taken out at 0.5 h, 1 h and 2 h respectively, and the same volume of dialysis fluid was supplemented. After taking out the release medium at 2 h, the dialysis bag was transferred to 30 mL of PBS buffer (pH 6.8), and 1 mL of the release medium was taken at 3 h, 4 h and 6 h respectively, and the same volume of dialysis fluid was supplemented. After taking out the release medium at 6 h, the dialysis bag was transferred to 30 mL of PBS buffer (pH 7.4), and 1 mL of the release medium was taken at 8 h, 10 h, 12 h, 24 h, 48 h and 72 h respectively, and the same volume of dialysis fluid was supplemented.

[0054] The taken dialysis fluid was measured by HPLC injection and the content of Cel was calculated according to the standard curve equation. The concentration of Cel in the samples at different time points was calculated according to the following formula, and the cumulative release rate Q was calculated. The in vitro release curves of Cel, Cel-GDNPs and Cel-GDNPs@Gel were plotted with the cumulative release rate Q against time T.

[0055] Q n =[C n ×V+∑ i=1 n 1 (C i ×v)] / M total ×100% Q n : Cumulative release rate at the nth time point (%).

[0056] C n : Drug concentration measured from the sample taken at the nth time point.

[0057] V: Total volume of the release medium (30 mL).

[0058] v: Volume of each sampling (1 mL).

[0059] C i : Drug concentration measured from the sample taken at the i (i < n)th time point.

[0060] ∑ i=1 n 1 (Ci ×v): The sum of all the drugs taken from the first to the (n-1)th sampling.

[0061] M total The initial total amount of drug loaded in the formulation (0.45 mg).

[0062] The results are as follows Figure 3 As shown, under highly acidic conditions, Cel-GDNPs and Cel-GDNPs@Gel remained largely undissociated, effectively preventing premature drug release from the stomach and providing an important basis for oral administration. In the small intestine, compared to Cel-GDNPs, the release rate of Cel-GDNPs@Gel was lower. The cumulative release rate of Cel in Cel-GDNPs was 7.37±0.03%, while that in Cel-GDNPs@Gel was 2.08±0.03%, indicating that glycyrrhizic acid hydrogel delayed Cel release and increased its accumulation in the colonic environment.

[0063] Example 4 Preparation of IR780 solution: Weigh 10 mg of IR780 fluorescent dye powder in the dark and place it in a 10 mL volumetric flask. Dissolve it by sonication with a small amount of anhydrous ethanol. After returning to room temperature, bring the volume up to 10 mL with anhydrous ethanol to obtain an IR780 ethanol solution with a concentration of 1 mg / mL. Dilute with PBS (pH 7.2-7.4) to the required concentration.

[0064] Preparation of IR780-GDNPs: Dilute 1.0 mg / mL GDNPs solution to 0.1 mg / mL, and dilute 1.0 mg / mL IR780 ethanol solution to 0.25 mg / mL. Take 9 mL of 0.1 mg / mL GDNPs solution and slowly add 1 mL of the above 0.25 mg / mL IR780 ethanol solution dropwise under gentle vortex stirring (i.e., GDNPs solution:IR780 solution = 9:1, v / v). Place the mixture in a 37℃ constant temperature water bath shaker and incubate for 2 hours. After incubation, transfer the mixture to a centrifuge tube and centrifuge at 10000 × g for 10 min at 4℃. Transfer the supernatant after centrifugation to an ultracentrifuge tube and centrifuge at 100000 × g for 60 min at 4℃. Discard the supernatant and resuspend the resulting precipitate in 1 ml of PBS (pH 7.2-7.4), i.e., IR780-GDNPs solution, and dilute with PBS to the required concentration.

[0065] Preparation of IR780-GDNPs@Gel: Same as the preparation of Cel-GDNPs@Gel in Example 1. Take the above IR780-GDNPs and add them to a glycyrrhizic acid solution cooled to 40°C. Stir well and place in a refrigerator for refrigeration. Once solidified, IR780-GDNPs@Gel is obtained. Dilute with PBS (pH 7.2-7.4) to the required concentration.

[0066] In vitro diffusion evaluation of Cel-GDNPs@Gel Weigh 25 mg of porcine small intestinal mucus and add it to the center of the polycarbonate support membrane (3 µm pore size) of the Transwell chamber. Gently spread the mucus with a micropipette to ensure it covers the membrane surface as evenly as possible. Place the Transwell chamber in a 12-well plate and incubate at 37 °C for 15 min to allow the mucus to fully hydrate and stabilize. Gently add 200 µL each of IR780, IR780-GDNPs, and IR780-GDNPs@Gel to the surface of the mucus layer (IR780 concentration: 250 µg / mL). Add 500 µL of preheated PBS buffer (pH 7.4) to the receiving chamber. Place the Transwell cell culture plate on a shaker and adjust the shaking frequency to 150 rpm and the temperature to 37 °C. At 0h, 0.25h, 0.5h, 1h, 2h, 3h, and 4h, 200µL of PBS buffer was collected from the receiving cell, and the same volume of fresh PBS buffer (pH 7.4) was added simultaneously to maintain a constant volume of the receiving cell. The collected 200µL sample was mixed with 800µL of anhydrous ethanol (ethanol:sample = 4:1, v / v) and vortexed until homogeneous. The resulting solution was sonicated for 10 min to completely disrupt the lipid structure of the nanoparticles and allow for the complete release of the IR780 dye.

[0067] The absorbance (Abs) of the processed sample solution at λ785 nm was measured using a UV spectrophotometer. The measured absorbance (Abs) was then substituted into the linear equation of the standard curve to calculate the concentration of IR780 in the sample. The apparent permeability coefficient (Papp) of the particles was calculated using the following formula: Papp=dQ / (A×C0×d t ) Wherein dQ / d t This represents the transmittance of IR780 in the receiving cell per unit time (dQ / d). t (Units µg / h converted to µg / s), C0 is the initial concentration of IR780 in the supply cell (C0 = 250 µg / mL = 250 µg / cm³), and A is the surface area of ​​the supporting membrane (1.12 cm³). 2 ).

[0068] The results are as follows Figure 4As shown, the Papp of the IR780-GDNPs@Gel group was significantly greater than that of the IR780 group, indicating that GDNPs and glycyrrhizic acid hydrogel can promote drug penetration.

[0069] Example 5 In vitro antioxidant activity of Cel-GDNPs@Gel Excessive production of reactive oxygen species (ROS) can cause oxidative stress, which in turn damages lipids, proteins, and nucleic acids, alters the immune balance, and leads to mucosal damage, potentially resulting in ulcerative colitis (UC).

[0070] RAW264.7 cells induced by LPS were co-incubated with Cel, Cel-GDNPs, and Cel-GDNPs@Gel, and their antioxidant capacity was evaluated by detecting the ROS scavenging effect using confocal laser scanning microscopy (CLSM).

[0071] RAW264.7 cells in the logarithmic growth phase were divided into two groups of 2 × 10⁻⁶ cells. 5 Seeds were planted at a density of cells / well in 24-well plates, complete culture medium was added, and the plates were incubated overnight at 37°C with 5% CO2. Then, the plates were switched to serum-free medium and starved for 12 hours. The old medium was discarded, and medium containing Cel, Cel-GDNPs, and Cel-GDNPs@Gel were added to bring the final Cel concentration to 1 μg / mL, and incubated for 2 hours. Then, LPS was added to bring the final concentration to 1 μg / mL, and incubation continued for 6 hours. The control group received no treatment. After incubation, the medium was aspirated and washed twice with PBS (pH 7.2-7.4). 1 mL of 10 μM 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining solution was added to each well, and the plates were incubated at 37°C in the dark for 30 minutes. After staining, the plates were washed three times with PBS (pH 7.2-7.4) to remove excess fluorescent dye. 1 mL of Hoechst 33342 staining solution (5 μg / mL) was added to each well and incubated at 37°C in the dark for 5 min to label cell nuclei. The antioxidant capacity was then assessed by detecting ROS scavenging effects using confocal laser scanning microscopy (CLSM). Results are as follows: Figure 5As shown, green represents DCFH-DA entering the cell and being hydrolyzed by esterases to generate DCFH. DCFH reacts with intracellular ROS and is oxidized to DCF, a substance with strong green fluorescence. Blue represents the nuclei of RAW264.7 cells stained with Hoechst 33342. The results showed that compared with the Control group, the LPS group exhibited significant green fluorescence, while the green fluorescence in the treatment groups was significantly reduced, especially in the LPS+Cel-GDNPs@Gel group, where almost no green fluorescence was observed. This indicates that LPS-induced RAW264.7 cells treated with Cel, Cel-GDNPs, or Cel-GDNPs@Gel had significantly lower ROS levels compared to untreated LPS-induced positive RAW264.7 cells, and Cel-GDNPs@Gel exhibited the strongest antioxidant activity.

[0072] Example 6 Tissue distribution of Cel-GDNPs@Gel Accumulation of orally administered drugs in the colon is crucial for their therapeutic effect on ulcerative colitis (UC). To determine the in vivo accumulation of Cel-GDNPs@Gel, mice were orally administered near-infrared dyes IR780, IR780-GDNPs, and IR780-GDNPs@Gel at 2h, 8h, 24h, and 48h. Their distribution throughout the mice was recorded using a small animal in vivo imaging system. Results are as follows: Figure 6 As shown, the fluorescence in mice was strongest in the IR780 group at 8 hours, and almost undetectable at 24 hours. In contrast, fluorescence intensity was still observable in the IR780-GDNPs and IR780-GDNPs@Gel groups at 48 hours, with the IR780-GDNPs@Gel group showing stronger fluorescence intensity than the IR780-GDNPs group. This indicates that Ga-Gel can increase the retention time of IR780-GDNPs in vivo, showing great potential in the treatment of UC. To verify the accumulation of Cel-GDNPs@Gel in colonic tissue, mice were sacrificed, and fluorescence in the colonic tissue was detected. The results are as follows... Figure 7 As shown, the fluorescence intensity of the IR780-GDNPs@Gel group was stronger than that of the IR780 group and the IR780-GDNPs group at all time points (2h, 8h, 24h and 48h), which confirms that glycyrrhizic acid hydrogel can increase the retention time of IR780-GDNPs in the intestine.

[0073] Example 7 Evaluation of the therapeutic effect of Cel-GDNPs@Gel on colitis mice (1) Establishment of enteritis model: Mice after one week of adaptive feeding were randomly divided into 5 groups, namely control group, DSS group, Cel group, Cel-GDNPs group and Cel-GDNPs@Gel group, with 5 mice in each group. The control group received normal drinking water, followed by gavage of 200 μL of sterile water every other day. The DSS group received drinking water with 1.5% DSS for 4 days of induction, then normal drinking water for 7 days, then drinking water with 1.5% DSS for 4 days of induction, followed by normal drinking water for 7 days, and this cycle was repeated for 37 days to induce chronic colitis. Simultaneously, 200 μL of sterile water was administered by gavage every other day. The Cel, Cel-GDNPs, and Cel-GDNPs@Gel groups received drinking water with 1.5% DSS for 4 days of induction, then normal drinking water for 7 days, then drinking water with 1.5% DSS for 4 days of induction, followed by normal drinking water for 7 days, and this cycle was repeated for 37 days to induce chronic colitis. Simultaneously, 200 μL of Cel, Cel-GDNPs, and Cel-GDNPs@Gel were administered every other day at a dose of 3 mg / kg. After 37 days of intervention, all mice were anesthetized and sacrificed, and colon tissue was collected for subsequent experiments. Ulcers (UC) are characterized by weight loss, diarrhea, and bloody stools; therefore, we observed changes in mouse weight, fecal condition, and degree of bloody stools daily, and recorded the Disease Activity Index (DAI). Higher DAI scores indicated more severe UC. Figure 8 It was found that the DAI in the treatment group was significantly lower than that in the DSS group, and the DAI in the Cel-GDNPs@Gel treatment group was the lowest among all treatment groups. This indicates that it significantly improved the inflammatory response induced by DSS in UC mice.

[0074] (2) Determination of inflammatory factor content in colon tissue of UC mice Colon tissue was collected from each group of mice, homogenized, and then the levels of inflammatory factors (TNF-α, IL-1β, and IL-6) in the colon tissue were measured using enzyme-linked immunosorbent assay (ELISA). Results are as follows: Figures 9-11 As shown, the results indicate that the expression of pro-inflammatory factors TNF-α, IL-1β, and IL-6 in the colon of mice treated with DSS was significantly increased, and the expression levels decreased after drug administration. The DSS+Cel-GDNPs@Gel group showed the most significant decrease, indicating that it can better alleviate colonic inflammation.

[0075] (3) Determination of the content of antioxidant enzymes SOD, CAT and GSH-Px in colon tissue of UC mice Colon tissue was collected from each group of mice, homogenized, and the levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in the mouse colon tissue were measured according to the kit instructions. Results are as follows: Figures 12-14As shown, Cel, Cel-GDNPs, and Cel-GDNPs@Gel can all exert different degrees of preventive effect on UC mice by mediating oxidative stress response, while Cel-GDNPs@Gel has a more effective effect than Cel-GDNPs in alleviating DSS-induced oxidative stress.

[0076] (4) MPO activity in colon tissue of UC mice Myeloperoxidase (MPO) is a heme protease produced by myeloid cells (such as neutrophils and monocytes), primarily stored in neutrophils, and can participate in immune defense as a lysosomal enzyme. During colitis, neutrophils can release MPO at sites of inflammation; excessive release of MPO can lead to oxidative stress, damage colonic epithelial cells, and exacerbate the inflammatory response. Therefore, MPO activity was measured to evaluate the therapeutic effect of Cel-GDNPs@Gel. Results are as follows: Figure 15 The results showed that DSS triggered a large release of MPO in the colonic tissue. Cel, Cel-GDNPs and Cel-GDNPs@Gel all alleviated the increase in MPO activity induced by DSS, thereby reducing its damage to colonic epithelial cells and producing a better therapeutic effect. Among them, Cel-GDNPs@Gel had the most significant effect.

[0077] (5) Expression of tight junction protein in colon tissue of UC mice In UC mice, DSS directly affects epithelial cells, leading to complete epithelial cell loss and disruption of the mucosal barrier. Immunohistochemistry was used to analyze the expression of tight junction-related proteins ZO-1, Claudin-1, and Occludin in the colon tissue of mice from each group to investigate the effect of Cel-GDNPs@Gel on the intestinal barrier. Results are as follows: Figure 16 As shown, DSS stimulation significantly reduced the expression of ZO-1, Claudin-1, and Occludin, while Cel-GDNPs@Gel treatment increased the expression of these tight junction proteins. This indicates that oral administration of Cel-GDNPs@Gel can enhance the expression of tight junction proteins induced by DSS in UC mice, which helps to improve colitis, and its effect is stronger than that of Cel-GDNPs.

[0078] (6) 16S rRNA gene sequencing analysis of gut microbiota After collecting cecal contents from each group of mice, the contents were stored at -80℃. The gut microbiota of the mice was analyzed using 16S rRNA gene sequencing technology by Sangon Biotech (Shanghai) Co., Ltd. Alpha diversity reflects the richness and diversity of the microbial community within a sample and is often used to analyze and assess this richness and diversity. The Chao index is a commonly used index for community distribution richness; a higher value indicates higher species richness in the corresponding sample. The Shannon and Simpson indices are primarily used to measure community distribution diversity. A higher Shannon index indicates higher community diversity, while a higher Simpson index indicates lower community diversity. Results are as follows: Figures 17-19 As shown, compared with the normal control group, the DSS group significantly reduced species diversity and richness (Chao index, Shannon index, and Simpson index). In contrast, the Cel-GDNPs@Ga-Gel administration group increased species diversity and richness compared with the DSS group.

[0079] To further clarify the abnormal changes in the gut microbiota structure of mice with DSS-induced chronic ulcerative colitis and the regulatory effect of Cel-GDNPs@Gel treatment on the gut microbiota, we analyzed the gut microbiota composition of each group of mice at the phylum level. In the colitis mouse model, an increased Firmicutes to Bacteroidetes ratio (F / B ratio) is an important marker of dysbiosis during the development of colitis; therefore, changes in this ratio can be used to assess the therapeutic effect of Cel-GDNPs@Gel on colitis. Results are as follows... Figure 20 As shown, compared with the blank control group, the F / B ratio in the DSS group was increased, indicating that the gut microbiota structure was disordered. The F / B ratio in the Cel-GDNPs@Gel treatment group was significantly decreased, indicating that Cel-GDNPs@Gel administration helps to improve gut microbiota imbalance, thereby alleviating intestinal inflammation.

[0080] 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, improvements, etc., 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 loaded with exosomes, characterized in that, It includes ginger exosomes, tripterygium sorbitol, and glycyrrhizic acid hydrogel.

2. A method for preparing a hydrogel loaded with exosomes as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve Cel in anhydrous ethanol to prepare a Cel solution; (2) Add Cel solution to GDNPs solution and incubate in a water bath shaker; (3) The unencapsulated free Cel was removed by ultracentrifugation and the precipitate after centrifugation was Cel-GDNPs; (4) Take GA powder, add pure water to dissolve it, and obtain GA aqueous solution; (5) Heat the GA aqueous solution in a water bath, stir until completely dissolved, and cool at room temperature to obtain glycyrrhizic acid hydrogel; (6) Add the Cel-GDNPs obtained in step (3) to the glycyrrhizic acid hydrogel, stir evenly, and let it stand to solidify to obtain Cel-GDNPs@Gel.

3. The method for preparing a hydrogel loaded with exosomes according to claim 2, characterized in that, The preparation of the GDNPs solution includes the following steps: extracting ginger juice, centrifuging it sequentially at 800-1200g for 8-12 min, 2000-4000g for 10-30 min, and 80000-120000g for 50-70 min to remove impurities, filtering, centrifuging the supernatant at 80000-120000g for 50-70 min, then resuspending the precipitate in PBS solution, placing it on the upper layer of a 15%, 30%, 45%, and 60% sucrose density gradient solution, centrifuging at 80000-120000g for 90-140 min, taking the 30-45% layer solution, centrifuging at 80000-120000g for 50-70 min to wash away the sucrose solution, and then resuspending the precipitate in PBS solution to obtain the GDNPs solution.

4. The method for preparing a hydrogel loaded with exosomes according to claim 2, characterized in that, In step (1), the concentration of the Cel solution is 0.5~2 mg / mL.

5. The method for preparing a hydrogel loaded with exosomes according to claim 2, characterized in that, In step (2), the volume ratio of Cel solution to GDNPs solution is 1:8~10; the protein concentration of GDNPs solution is 0.05~0.5 mg / mL; the water bath temperature is 35~40℃; and the incubation time is 1~3h.

6. The method for preparing a hydrogel loaded with exosomes according to claim 2, characterized in that, In step (3), the parameters for ultracentrifugation are: temperature 4℃, centrifugation at 80000~120000g for 50~70min.

7. The method for preparing a hydrogel loaded with exosomes according to claim 2, characterized in that, In step (4), the concentration of the GA aqueous solution is 20~50 mg / mL.

8. The method for preparing a hydrogel loaded with exosomes according to claim 2, characterized in that, In step (5), the temperature of the water bath is 60~100℃ and the heating time is 20~50min.

9. The method for preparing a hydrogel loaded with exosomes according to claim 2, characterized in that, In step (6), the volume ratio of glycyrrhizic acid hydrogel to Cel-GDNPs is 0.5~1.5:1; the static solidification is to stand in a refrigerator at 4℃ for 24~48h.

10. The use of the hydrogel loaded with exosomes as described in claim 1 in the preparation of a drug for treating colitis.

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