Injectable hydrogel for preventing esophageal benign stenosis as well as preparation method and application of injectable hydrogel

Abstract

The invention discloses injectable hydrogel for preventing esophageal benign stenosis as well as a preparation method and application of the injectable hydrogel, and belongs to the technical field of biological medicines. The hydrogel comprises an exosome and a gel packaging material used for packaging the exosome, the exosome comprises overexpressed miRNA34a, and the gel packaging material comprises polydopamine and chitosan. And the gel packing material also contains an antioxidant. And the antioxidant is cerium oxide CeO2. Animal experiments find that the hydrogel preparation prepared by the invention can effectively improve the bioavailability of exosomes, improve the stability and residence rate of the exosomes in vivo, realize drug sustained release, promote the synergistic operation of wound tissue cells and cell factors, and prevent esophageal ESD postoperative stenosis. The hydrogel provided by the invention is simple and convenient to use, has a good stenosis prevention effect, and can avoid systemic side effects and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically, to an injectable hydrogel for preventing benign esophageal stenosis, and a preparation method and application thereof. Background Art

[0002] Esophageal stenosis often occurs in patients who undergo mucosal resection for esophageal cancer, causing dysphagia or local discomfort, significantly reducing the patient's quality of life. With the development of endoscopic technology, most early esophageal cancers can now be diagnosed and removed en bloc through endoscopic mucosal dissection (ESD). ESD uses a high-frequency electric scalpel under endoscopy to gradually peel off the submucosal layer, completely remove the lesion, and reduce local recurrence and metastasis. It is widely used because it is minimally invasive and has a safe and reliable treatment effect. However, due to the tubular structure of the esophagus, if a large area of ​​mucosal defects occurs during ESD, especially those that accumulate the entire circumference, complications such as esophageal stenosis are prone to occur. ESD that removes more than 3 / 4 of the esophagus often causes complications such as bleeding and esophageal stenosis. The incidence of esophageal stenosis reaches 70-90%, and the stenosis rate of those who undergo full circumferential resection is almost 100%. Once esophageal stenosis occurs, the treatment risk and cost will increase significantly, greatly reducing the patient's quality of life. So far, there is no specific preventive measure or treatment method to fundamentally solve this serious complication.

[0003] After ESD, the epithelial barrier function is lost, triggering the recruitment of immune cells, leading to infiltrative inflammation. In the later stage, fibroblasts and pericytes transform into myofibroblasts that cause fibrosis. The mucosal gap is gradually covered by squamous epithelial cells, and collagen fibers in the submucosal layer proliferate massively to form scar tissue, which further leads to esophageal stenosis. Therefore, inhibiting the inflammatory response at the surgical gap is an effective way to prevent esophageal stenosis.

[0004] At present, the main methods used in clinical treatment or prevention of postoperative esophageal stenosis include drug therapy, balloon dilatation and stents. These methods are effective to a certain extent, but still bring complications of varying degrees. With the extensive research and application of hydrogel biomedical materials, some studies have proposed methods for preventing esophageal stenosis. Based on conventional drug-free PEG hydrogels, the anti-inflammatory effect is poor, only plays a barrier function, and the anti-stenosis effect is limited. Cross-linked polymer hydrogels prepared based on polypeptides or polypeptide derivatives have good biocompatibility, but poor mechanical properties and high preparation costs. Based on three-dimensional culture and in situ delivery of esophageal epithelial cells and esophageal interstitial fibroblasts in hydrogels, the operation is complicated, there are many influencing factors, and it is limited to experimental research. At present, injection of anti-inflammatory drugs is still used clinically to reduce the inflammatory response and fibrosis of damaged mucosal tissues. However, drug treatment can cause systemic toxicity and have drug side effects. Summary of the invention

[0005] To solve the above problems, the present invention provides an injectable hydrogel for preventing benign esophageal stenosis, and its preparation method and application. The hydrogel of the present invention is an in situ injectable hydrogel CeO2-miRNA34a@PCHG formed by polydopamine and chitosan, which encapsulates the antioxidant cerium oxide CeO2 and exosomes overexpressing miRNA34a. The hydrogel prepared by the present invention can stay on the surface of the defective mucosa, deliver exosomes and drugs to the defective mucosa in situ, which can not only effectively improve the bioavailability of exosomes, improve their stability and residence rate in the body, and achieve sustained release of drugs, but also promote the coordinated operation of wound tissue cells and cytokines to prevent esophageal ESD postoperative stenosis.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides an injectable hydrogel for preventing benign esophageal stenosis. The hydrogel comprises exosomes and a gel packaging material for encapsulating the exosomes. The exosomes comprise overexpressed miRNA34a.

[0008] Preferably, the gel packaging material comprises yeast extract, collagen, carbomer, sodium hyaluronate, trehalose, polydopamine, chitosan and glycerol.

[0009] Preferably, the gel packaging material is mainly made of one or more of polydopamine and chitosan.

[0010] Preferably, the hydrogel further contains an antioxidant, which is cerium oxide CeO2.

[0011] The present invention also provides a method for preparing the hydrogel preparation, comprising the following steps: encapsulating the exosomes in a gel packaging material.

[0012] Preferably, the preparation of the gel packaging material is by first preparing a gel factor and then gelling, wherein the gel factor is prepared by mixing liquid A and liquid B, wherein the liquid A is chitosan dissolved in 0.1M HCl, 3-(3,4-dihydroxyphenyl) propionic acid is added and completely dissolved; the liquid B is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride dissolved in ethanol-water (v / v=1 / 1) solution.

[0013] Preferably, the pH of solution A is adjusted to 3.86, and an equal volume of solution A is mixed with solution B, the pH is adjusted to 4.6, and the solution is dialyzed after vigorous stirring.

[0014] The gelling agent comprises:

[0015] ① The prepared gel factor is dissolved in an aqueous solution, and a cerium oxide solution is added to form a hydrogel-loaded nanoparticle dispersion;

[0016] ② Add 0.1M hydrogen peroxide aqueous solution, then add appropriate amount of horseradish peroxidase (HRP), stir evenly, and let stand to form a gel.

[0017] Preferably, the gel packing material is prepared by the following method:

[0018] 1) Preparation of gel factor:

[0019] ① Weigh chitosan and dissolve it in 0.1M HCl. Stir evenly until the chitosan is completely dissolved to obtain a transparent light yellow liquid.

[0020] ② Adjust the pH of the above liquid to 5.4, add 3-(3,4-dihydroxyphenyl)propionic acid, stir until completely dissolved, adjust the pH to 3.86, the solution is brown (translucent), which is liquid A.

[0021] ③ Mix equal volumes of solution A and solution B [1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) dissolved in ethanol-water (v / v=1 / 1) solution], adjust the pH to 4.6, and stir vigorously for 1 h.

[0022] ④ Dialysis: Dialyze against 100 mM NaCl solution for 72 h, changing the water every 12 h.

[0023] 2) Hydrogel formation:

[0024] ① The prepared gel factor is dissolved in aqueous solution, and 10 μl of cerium oxide solution (containing 50 μg) is added to each 1 ml to form a hydrogel-loaded nanoparticle dispersion;

[0025] ② Add 0.1M hydrogen peroxide aqueous solution to the above solution, then add appropriate amount of horseradish peroxidase (HRP), stir evenly, and let stand to form a gel.

[0026] 3) Encapsulating exosomes overexpressing miRNA34a therein.

[0027] The present invention also provides an application of the hydrogel in preparing a medicine for preventing benign esophageal stenosis.

[0028] The present invention also provides an application of the hydrogel in preparing a drug for preventing stenosis after esophageal cancer resection and a submucosal injection solution before ESD surgery.

[0029] Beneficial effects

[0030] The in situ injectable hydrogel provided by the present invention can not only effectively improve the bioavailability of exosomes, improve their stability and retention rate in the body, and achieve sustained drug release, but also promote the coordinated operation of wound tissue cells and cytokines to prevent esophageal stenosis after ESD surgery.

[0031] The hydrogel provided by the present invention is simple and convenient to use, has a good effect in preventing stenosis, and can avoid causing systemic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Variation of rheological properties of hydrogel systems with frequency.

[0033] Figure 2 Changes in the rheological properties of hydrogel systems with temperature.

[0034] Figure 3 Variation of rheological properties of hydrogel systems with strain.

[0035] Figure 4 Behavior of hydrogel systems when subjected to compression.

[0036] Figure 5 Behavior of a hydrogel system when subjected to stretching.

[0037] Figure 6 Degradation curve of hydrogel system.

[0038] Figure 7 Changes of CeO2-miRNA34a@PCHG hydrogel after 1, 3, 5, and 7 days of degradation.

[0039] Figure 8 Study on the safety of CeO2-miRNA34a@PCHG hydrogel on organ tissues.

[0040] Fig. 9 Effect of CeO2-miRNA34a@PCHG hydrogel on hemolysis.

[0041] Fig.10 Effect of CeO2-miRNA34a@PCHG hydrogel on liver function.

[0042] Fig.11 Effect of CeO2-miRNA34a@PCHG hydrogel on blood routine.

[0043] Fig.12 The dysphagia scores of the five groups of animals on days 7, 14, 21, and 28 after surgery (* indicates P < 0.05).

[0044] Fig.13 The weight loss and proportion of the five groups of animals on days 7 and 28 after surgery (* indicates P < 0.05).

[0045] Fig.14 Wound recovery and specimen conditions of the five groups of animals at 0 week, 1 week, 2 weeks, 3 weeks and 4 weeks after surgery.

[0046] Fig.15The mucosal contraction rates of the five groups of animals on days 7 and 28 after surgery (* indicates P < 0.05).

[0047] Fig.16 Representative histopathological images of H&E staining of specimens from the five groups on days 7 and 28 (×40 / ×100).

[0048] Fig.17 Comparison of inflammatory cell counts among the five groups of specimens on days 7 and 28 (* indicates P<0.05, **** indicates P<0.0001). DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] Example 1: Preparation method of CeO2-miRNA34a@PCHG hydrogel

[0052] 1) Preparation of gel factor:

[0053] ① Weigh chitosan (3 g) and dissolve it in 0.1 M HCl. Stir evenly until the chitosan is completely dissolved to obtain a transparent light yellow liquid.

[0054] ② Adjust the pH of the above liquid to 5.4, add 3-(3,4-dihydroxyphenyl)propionic acid (3 g), stir until completely dissolved, adjust the pH to 3.86, the solution is brown (translucent), and is liquid A.

[0055] ③ Mix equal volumes of solution A and solution B [1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) dissolved in 50 ml of ethanol-water (v / v=1 / 1) solution], adjust the pH to 4.6, and stir vigorously for 1 h.

[0056] ④ Dialysis: Dialyze against 100 mM NaCl solution for 72 h, changing the water every 12 h.

[0057] 2) Hydrogel formation:

[0058] ① The prepared gel factor is dissolved in aqueous solution, and 10 μl of cerium oxide solution (containing 50 μg) is added to each 1 ml to form a hydrogel-loaded nanoparticle dispersion;

[0059] ② Add 0.1M hydrogen peroxide aqueous solution to the above solution, then add appropriate amount of horseradish peroxidase (HRP), stir evenly, and let stand to form a gel.

[0060] 3) Encapsulating exosomes overexpressing miRNA34a therein.

[0061] Example 2: Characterization of CeO2-miRNA34a@PCHG hydrogel

[0062] The rheometer tested the storage modulus and loss modulus of the hydrogel material of the present invention with frequency, temperature and strain. When the scanning frequency changes, the storage modulus of the CeO2-miRNA34a@PCHG hydrogel material does not fluctuate significantly, while the loss modulus fluctuates significantly with the scanning frequency, which ultimately manifests as a decrease in the viscosity of the hydrogel with an increase in the scanning frequency ( Figure 1 We also determined the relationship between the rheological characteristics of the hydrogel and temperature changes. The rheometer test results showed that in the range of 10 to 50°C, the storage modulus and loss modulus of the hydrogel did not change significantly with temperature. When the temperature continued to rise, the loss modulus of the hydrogel showed an obvious positive correlation with temperature when the temperature rose to above 50°C, while the storage modulus was less sensitive to temperature than the loss modulus, and only showed an increasing trend with increasing temperature at about 60°C ( Figure 2 ).

[0063] Finally, the changes in the rheological properties of the CeO2-miRNA34a@PCHG hydrogel system when strain occurs were tested. The results showed that the storage modulus hardly changed with the occurrence of material strain, while the loss modulus increased with the increase of strain when the strain reached about 10% ( Figure 3 ).

[0064] In order to further predict the behavior of the hydrogel system in vivo, its behavior after being subjected to tension and compression was studied. When subjected to pressure, CeO2-miRNA34a@PCHG produced a maximum deformation of 80% ( Figure 4 ), and in the tensile test, it withstood a maximum tensile force of 30kpa before drug loading and produced a 100% deformation. After encapsulating Exo and CeO2 particles, the elastic modulus increased, but it broke after producing about 55% deformation ( Figure 5 ). At the same time, the swelling rate of CeO2-miRNA34a@PCHG was measured to be 83.4% at 37°C.

[0065] Example 3: In vivo degradation ability of CeO2-miRNA34a@PCHG hydrogel

[0066] In order to clarify the degradation ability of CeO2-miRNA34a@PCHG in vivo and determine its drug release ability. The changes of the hydrogel under a scanning electron microscope after 1, 3, 5, and 7 days of degradation were observed, and its degradation curve was drawn according to the residual mass and degradation rate of the hydrogel at each stage. CeO2-miRNA34a@PCHG began to degrade at a gradually slowing rate immediately after gelation, reaching the degradation endpoint in about 7 days, and about 40% of the hydrogel remained at the degradation endpoint ( Figure 6 ).

[0067] After 1 day of treatment, the hydrogel began to degrade slightly, and a small amount of fragments separated from the lamellar structure; after 3 days, the hydrogel structure degraded to a large extent, the internal pores increased, and the network cross-linked structure began to be destroyed; after 5 days of degradation, the network cross-linked structure of the hydrogel had basically disappeared, forming many relatively independent smaller units. After 7 days, the hydrogel almost lost its original structure, leaving many discrete and broken units. At this time, the hydrogel reached the end of degradation ( Figure 7 ).

[0068] Example 4: Safety of CeO2-miRNA34a@PCHG hydrogel

[0069] The results of H&E staining of histopathology showed that the injection of CeO2-miRNA34a@PCHG did not cause obvious damage to the heart, liver, spleen, lung, kidney and other important organs of pigs ( Figure 8 ). Meanwhile, exosomes, hydrogels and CeO2-miRNA34a@PCHG overexpressing miRNA-34a did not cause hemolysis ( Fig. 9 ).

[0070] In addition, since the degradation of CeO2-miRNA34a@PCHG in the body is mainly carried out through the liver, the effect on liver function was tested. Compared with the saline group, the injection of composite hydrogel had no significant effect on common liver function indicators such as ALT, AST, ALP, γ-GT, LDH, and CK ( Fig.10 ).

[0071] Finally, the blood routine test results showed that the composite hydrogel had no significant effect on the levels of RBC, WBC, HGB, PLT, MCHC, and HCT ( Fig.11 ).

[0072] Example 5: Effectiveness of CeO2-miRNA34a@PCHG hydrogel

[0073] Twenty Bama miniature pigs with the same or similar body weight and no dysphagia before surgery were randomly divided into 5 groups, 4 pigs in each group, set as:

[0074] Physiological saline group (blank group), hormone group (triamcinolone acetonide group), exosome group (Exo group), hydrogel group without exosomes (Gel group) and CeO2-miRNA34a@PCHG hydrogel of the present invention (hydrogel + exosome group, Exo + Gel group).

[0075] After the pigs were anesthetized and injected with the same amount of CeO2-miRNA34a@PCHG hydrogel prepared in Example 1 of the present invention and each control substance, two esophageal ESDs with a length of about 5 cm and involving 3 / 4 of the circumference were successfully performed, with a total of 40 wounds, ensuring that there was no statistical difference in the operation time and specimen size, and the esophageal stenosis was checked regularly. Wound treatment: Through the gastroscope channel, the four groups of simple hydrogel solution, simple exosome solution, exosome-loaded hydrogel solution and triamcinolone acetonide solution were injected and sprayed into the postoperative wound defect mucosa using a submucosal injection needle, and the control group was treated with saline as the fifth group. The dosage of hydrogel, etc. is 5ml (injection) and 5ml (spraying), the dosage of exosome injection is: 3mg, and the clinical dosage is 80mg of triamcinolone acetonide hormone.

[0076] 1. Changes in swallowing and eating

[0077] According to the Mellow-Pinkas standard, the dysphagia is divided into 5 levels: 0 point: no dysphagia; 1 point: occasional dysphagia when eating solid food; 2 points: only semi-liquid food and soft food can be taken; 3 points: only liquid food can be taken; 4 points: completely unable to eat.

[0078] Two pigs in each group were killed on the 7th day after surgery. There were 2 pigs and 4 wound surfaces left in each group, for a total of 10 pigs. Two pigs in the blank group vomited after being fed solid feed on the 10th and 13th days after surgery, respectively. The incidence of dysphagia was 100% (2 / 2). By the 4th week, one of the pigs was basically unable to eat at all. There was no significant difference in the eating conditions of the other groups at 2 and 3 weeks after surgery; in the 4th week after surgery, the eating conditions of the Exo+Gel group were significantly better than those of the other groups, and the eating conditions of the Exo group alone were slightly worse. The specific eating conditions are shown in Table 1. Fig.12 .

[0079] Table 1: Dysphagia scores of different groups sacrificed 4 weeks after surgery

[0080]

[0081] Exo: exosomes; Gel: hydrogel; Exo+Gel: exosomes+hydrogel

[0082] (II) Weight and changes

[0083] Among the 10 animals killed on the 7th day after surgery, the average weight loss of the saline group, hormone group, Exo group, Gel group and Exo+Gel group was 0.95±0.49kg, 0.30±0.14kg, 0.40±0kg, 0.20±0kg and 0.35±0.07kg, respectively, and the weight loss ratio was 3.87±2.03%, 1.27±0.56%, 1.21±0.56%, 1.24±0.52 and 0.85±0.05, respectively (P<0.05); among the 10 animals killed on the 28th day, the average weight loss of the five groups was 2.45± The weight loss rates were 0.21kg, 1.80±0.42kg, 1.60±0.42kg, 1.55±0.07kg, 1.45±0.07kg (P<0.05), and the weight loss rates were 10.25±0.83%, 7.55±1.91%, 5.95±0.82%, 7.07±1.05, 5.85±0.68 (P<0.05), respectively. The differences in weight loss and weight loss rate were statistically significant. The weight loss of hormone group, Exo group, Gel group and Exo+Gel group on the 7th and 28th day after surgery was milder than that of the control group ( Fig.13 ).

[0084] 3. Postoperative endoscopic review

[0085] The results of endoscopic review in the first 4 weeks after surgery are shown in Fig.14 At 3 weeks after surgery, the esophagus in the saline group was severely narrowed and deformed, with a pinhole-like change of about 2-3 mm in diameter, and the endoscope could not pass through. The wounds in the Gel group and Exo+Gel group had basically healed after surgery, with scar formation and a slightly stiff esophagus. In the Exo group alone, one of the wounds was severely contracted, and there was resistance to the passage of the endoscope. The endoscope passed smoothly through the other wounds. The wounds in the hormone group healed slowly, and the surface was still covered with ulcers and white fur.

[0086] Endoscopic review at 4 weeks after surgery showed that the esophageal mucosal contracture in the saline group continued to worsen, and the stenosis diameter further decreased. The wounds in the Exo group, Gel group, and Exo+Gel group were all healed, especially in the Exo+Gel group, where the lumen was the most unobstructed and the endoscope passed smoothly. The wounds in the hormone group were smaller than before, but not completely healed, with a little granulation tissue and white fur still attached to the surface.

[0087] From the results of postoperative endoscopic review, it can be seen that the CeO2-miRNA34a@PCHG hydrogel (hydrogel+exosome group) of the present invention can not only effectively improve the bioavailability of exosomes, but also promote the coordinated operation of wound tissue cells and cytokines to prevent esophageal stenosis after ESD surgery.

[0088] 4. Esophageal mucosal contraction rate (MCR)

[0089] Some pigs were euthanized on the 7th and 28th days after surgery, and gross esophageal specimens were obtained and measured. According to previous relevant standards, the short axis length of the shortest part of the ESD site, the short axis length of the normal esophageal mucosa on the oral and anal sides of the wound surface, and the esophageal MCR of each group was calculated by the formula. On the 7th day, the esophageal MCR of the normal saline group, hormone group, Exo group, Gel group and Exo+Gel group were 2.11±0.64%, 1.92±0.56%, 1.91±0.42%, 1.97±0.45% and 1.61±0.29%, respectively (P=0.694), and the difference was not statistically significant; on the 28th day, the esophageal MCR of the blank group, hormone group, Exo group, Gel group and Exo+Gel group were 7.92±0.38%, 3.78±0.52%, 5.53±0.85%, 2.79±0.26% and 1.96±0.59%, respectively (P<0.05). On the 28th day, the esophageal MCR of the Exo+Gel group was lower than that of the other four groups, and the difference was statistically significant (see details). Fig.15 ).

[0090] (V) H&E staining

[0091] (1) Histopathological examination results

[0092] Normal saline group: The local wound surface was still ulcerated and in a state without epithelial coverage. The fibroblasts in the submucosal layer proliferated significantly, with new granulation tissue and inflammatory cell infiltration, and the thickness of the submucosal layer increased significantly. The normal structure of the muscularis propria and muscularis mucosa was unclear or disappeared, with a large amount of fibrous tissue and inflammatory cell infiltration.

[0093] Hormone group: The surface of the local wound was still shallow ulcer, without epithelial coverage. The submucosal fibroblasts proliferated, with new granulation tissue and inflammatory cell infiltration, and the thickness of the submucosal layer increased. The normal structure of the muscularis propria and muscularis mucosa was unclear or disappeared, with a large amount of fibrous tissue and inflammatory cell infiltration.

[0094] Exo group: The surface of the local wound was still shallow ulcer, without epithelial coverage. There was a small amount of proliferation of fibroblasts in the submucosal layer, and the thickness of the submucosal layer increased. The normal structure of the muscularis propria and muscularis mucosa was unclear, with a large amount of fibrous tissue and inflammatory cell infiltration.

[0095] Group Gel: The wound surface was hyperplastic fibrous tissue. There was a small amount of hyperplasia of fibroblasts in the submucosal layer, and the thickness of the submucosal layer increased slightly. The normal structure of the muscularis propria and muscularis mucosa was clear, with a small amount of fibrous tissue and inflammatory cell infiltration.

[0096] Exo+Gel group: The wound surface was not covered by epithelium, but by proliferating fibrous tissue. There was a small amount of proliferative fibroblasts in the submucosal layer, and the thickness of the submucosal layer increased slightly. The normal structure of the muscularis propria and the muscularis mucosa was clear, with a small amount of fibrous tissue (see Fig.16 ).

[0097] (2) Inflammatory cell count results

[0098] Inflammatory cell infiltration was observed in the submucosal layer of the five groups of specimens at different times, which was most obvious in the saline group, followed by the Exo group, hormone group, and Gel group, and the least in the Exo+Gel group. The cells were mainly mononuclear leukocytes, and 12 HPFs were randomly selected for observation and counting (×200) in each specimen to count the number of inflammatory cells. The results showed that the numbers of inflammatory cells in the saline group, hormone group, Exo group, Gel group and Exo+Gel group on the 7th and 28th days were: 622.6±9.1 / HPF, 58.3±5.2 / HPF, 131.3±15.8 / HPF, 39.1±3.1 / HPF, 33.0±2.7 / HPF on the 7th day (P<0.0001); 310.8±22.5 / HPF, 51.7±6.4 / HPF, 89.1±11.7 / HPF, 31.7±2.0 / HPF, 20.4±2.1 / HPF on the 28th day (P<0.0001). On the 7th and 28th days, the numbers of inflammatory cell infiltration in the Exo+Gel group were less than those in the other four groups (see details). Fig.17 ).

[0099] Masson staining showed that the collagen volume fractions in the Exo+Gel group were higher than those in the other groups on days 7 and 28 (P<0.0001), indicating that the degree of wound fibrosis was low;

[0100] Sirius red staining showed that the collagen fibers (COL) in the Exo+Ge l group on day 28 were mainly type III, with less type I and more regular arrangement;

[0101] CD34 and VEGF staining showed that the Exo+Ge l group had stronger angiogenesis on day 7 (P<0.0001);

[0102] The expression level of α-smooth muscle actin (α-SMA) showed that the degree of submucosal fibrosis was the lowest (P<0.0001). The expression level of α-SMA is usually used to evaluate the activation state of smooth muscle cells.

[0103] From the above, it can be seen that the hydrogel of the present invention can not only effectively improve the bioavailability of exosomes, improve their stability and retention rate in the body, and achieve sustained drug release, but also promote the coordinated operation of wound tissue cells and cytokines to prevent esophageal stenosis after ESD surgery.

[0104] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A hydrogel, characterized in that: The hydrogel includes exosomes and a gel packaging material for encapsulating the exosomes, and the exosomes include overexpressed miRNA34a.

2. The hydrogel according to claim 1, characterized in that The gel packaging material comprises yeast extract, collagen, carbomer, sodium hyaluronate, trehalose, polydopamine, chitosan and glycerol; Preferably, the gel packaging material is mainly made of one or more of polydopamine and chitosan.

3. The hydrogel according to claim 2, characterized in that The gel packaging material also contains an antioxidant, which is cerium oxide CeO2.

4. A method for preparing a hydrogel according to any one of claims 1 to 3, characterized in that: The exosomes were encapsulated in a gel packaging material.

5. A preparation method according to claim 4, characterized in that: The gel packaging material is prepared by gelling a gel factor, wherein the gel factor is prepared by mixing liquid A and liquid B, wherein the liquid A is chitosan dissolved in 0.1M HCl, and 3-(3,4-dihydroxyphenyl) propionic acid is added and completely dissolved; the liquid B is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride dissolved in ethanol-water (v / v=1 / 1) solution; Preferably, the pH of solution A is adjusted to 3.86, and an equal volume of solution A is mixed with solution B, the pH is adjusted to 4.6, and the solution is dialyzed after vigorous stirring.

6. A preparation method according to claim 5, characterized in that: The gelling process includes: ① The prepared gel factor is dissolved in an aqueous solution, and a cerium oxide solution is added to form a hydrogel-loaded nanoparticle dispersion; ② Add 0.1M hydrogen peroxide aqueous solution, then add appropriate amount of horseradish peroxidase (HRP), stir evenly, and let stand to form a gel.

7. The preparation method according to any one of claims 4 to 6, characterized in that: The gel packaging material is prepared by the following method: 1) Preparation of gel factor: ① Weigh chitosan and dissolve it in 0.1M HCl, stirring evenly until the chitosan is completely dissolved to obtain a transparent light yellow liquid; ② Adjust the pH of the above liquid to 5.4, add 3-(3,4-dihydroxyphenyl)propionic acid, stir until completely dissolved, adjust the pH to 3.86, the solution is brown (semi-transparent), which is liquid A; ③ Mix equal volumes of solution A and solution B [1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) dissolved in ethanol-water (v / v=1 / 1) solution], adjust the pH to 4.6, and stir vigorously for 1 h; ④ Dialysis: 100mM NaCl solution was dialyzed for 72h, and the water was changed every 12h; 2) Hydrogel formation: ① The prepared gel factor is dissolved in aqueous solution, and 10 μl of cerium oxide solution (containing 50 μg) is added to each 1 ml to form a hydrogel-loaded nanoparticle dispersion; ② Add 0.1M hydrogen peroxide solution to the above solution, then add appropriate amount of horseradish peroxidase (HRP), stir evenly, and let stand to form a gel; 3) Encapsulating exosomes overexpressing miRNA34a therein.

8. The hydrogel prepared by the preparation method according to any one of claims 4 to 7.

9. Use of the hydrogel according to any one of claims 1 to 3 or the hydrogel prepared by the preparation method according to any one of claims 4 to 8 in preparing a drug for preventing esophageal stenosis.

10. Use of the hydrogel according to any one of claims 1 to 3 or the hydrogel prepared by the preparation method according to any one of claims 4 to 8 in the preparation of drugs for preventing stenosis after esophageal cancer resection and submucosal injection before ESD surgery.

Images