Self-healing hydrogel for preventing intrauterine adhesion as well as preparation method and application of self-healing hydrogel

Abstract

The invention relates to self-healing hydrogel for preventing intrauterine adhesion and a preparation method and application thereof. The preparation method comprises the following steps: dissolving hyaluronic acid in ultrapure water, then adding EDC and NHS, adjusting the pH value, then adding cysteamine hydrochloride, and after the reaction is finished, dialyzing and freeze-drying to obtain sulfydryl modified hyaluronic acid marked as HASH; dissolving the HASH in ultrapure water, then adding EDC and NHS, adjusting the pH value, then adding dopamine hydrochloride, and after the reaction is finished, dialyzing and freeze-drying to obtain sulfydryl and dopamine modified hyaluronic acid which is marked as DAHASH; carrying out surface sulfhydrylation treatment on the mEXO to obtain an engineered exosome which is marked as SmEXO; and respectively dissolving the DAHASH, the SmEXO and silver nitrate in ultrapure water, and then mixing to obtain an exosome-hydrogel system which is marked as DAHASH (at) SmEXO. The hydrogel for preventing intrauterine adhesion has a self-healing characteristic, is suitable for a special structure of an intrauterine, can be anchored on a wounded surface of a uterus, is continuously adhered to an endometrium for slowly releasing SmEXO, and creates a good condition for long-term repair of the endometrium.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a self-healing hydrogel for preventing intrauterine adhesions, and a preparation method and application thereof. Background Art

[0002] Intrauterine adhesions (IUA), also known as Asherman syndrome, are caused by endometrial damage leading to partial or complete occlusion of the uterine cavity, resulting in abnormal menstruation, infertility and recurrent miscarriage. Its occurrence is related to trauma, pregnancy, infection and other factors. Patients with moderate to severe intrauterine adhesions have more severe damage to the basal layer of the endometrium, low regenerative capacity of the endometrium and glands, poor endometrial receptivity and poor clinical prognosis.

[0003] Currently, the main treatment for intrauterine adhesions is hysteroscopic intrauterine adhesion separation, but this surgery is controversial because it can only separate fibrous scar tissue, which may cause re-injury to the remaining endometrium. Therefore, how to prevent the occurrence of intrauterine adhesions and restore normal endometrial function is an important challenge facing reproductive doctors.

[0004] Most sustained-release systems are constructed by directly mixing exosomes with bioactive materials. However, nanoscale exosomes mainly rely on diffusion mechanisms and can easily diffuse out of the hydrogel network, resulting in a burst release phenomenon, uncontrollable release time, and reduced retention time. In addition, existing hydrogels are not optimized for the special structure of the uterine cavity and the special environment of postoperative bleeding or exudate, and lack properties such as self-healing and wet tissue adhesion. These problems limit their clinical application and efficacy. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a self-healing hydrogel for preventing intrauterine adhesions and a preparation method and application thereof. The DAHASH side chain SH of the self-healing hydrogel for preventing intrauterine adhesions can react with Ag. + The ionic Ag-S dynamically coordinates and cross-links to form a hydrogel with self-healing properties, which is suitable for the special structure of the uterine cavity. The DA group in the DAHASH hydrogel can form covalent and non-covalent bonds with the endometrial tissue, thereby anchoring the hydrogel on the uterine wound surface and adhering to the endometrium; the thiol (SH) group coordinates and binds with the surface SH group of the engineered exosome SmEXO to achieve controlled release of the engineered SmEXO, creating good conditions for long-term repair of the endometrium.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for preparing a self-healing hydrogel for preventing intrauterine adhesions, which comprises the following steps:

[0008] S1. Dissolve hyaluronic acid (HA) in ultrapure water, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS), adjust the pH, add cysteamine hydrochloride (CSH), dialyze after the reaction, and freeze-dry to obtain thiol-modified hyaluronic acid, denoted as HASH;

[0009] S2. The HASH is dissolved in ultrapure water, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) are added. After adjusting the pH, dopamine hydrochloride (DA) is added. After the reaction is completed, the hyaluronic acid is dialyzed and freeze-dried to obtain thiol- and dopamine-modified hyaluronic acid, which is recorded as DAHASH.

[0010] S3. The surface of mEXO is thiolated to obtain engineered exosomes, which are denoted as SmEXO;

[0011] S4. The DAHASH, the SmEXO, and silver nitrate are dissolved in ultrapure water respectively and then mixed to obtain an exosome-hydrogel system, which is recorded as DAHASH@SmEXO.

[0012] The present invention introduces thiol (SH) and dopamine (DA) groups into hyaluronic acid (HA) to synthesize thiol- and dopamine-modified hyaluronic acid DAHASH, and obtains DAHASH@SmEXO hydrogel by physically mixing with SmEXO. The SH side chain of DAHASH can react with Ag. + The ionic Ag-S dynamically coordinates and crosslinks to form a hydrogel with self-healing properties, which is suitable for the special structure of the uterine cavity; at the same time, the DA group in the DAHASH hydrogel can form covalent and non-covalent bonds with the endometrial tissue, thereby anchoring the hydrogel on the uterine wound surface and adhering to the endometrium; the thiol (SH) group coordinates and binds with the surface SH group of the engineered exosome SmEXO to achieve the controlled release of the engineered SmEXO, creating good conditions for the long-term repair of the endometrium.

[0013] As a further improvement of the above scheme of the present invention, in step S1, the molar ratio of hyaluronic acid, EDC, NHS and hemihydroxyethylamine hydrochloride is 1:0.5:0.5:(0.2-0.4).

[0014] As a further improvement of the above scheme of the present invention, in step S1, the pH adjustment is to adjust the pH of the solution to 5.

[0015] As a further improvement of the above scheme of the present invention, in step S1, the reaction is carried out at room temperature overnight;

[0016] And / or, in step S1, the dialysis is to place the HASH reaction solution into a 3500Da dialysis bag and perform dialysis in a dark environment for 3 days to remove unreacted chemicals and by-products.

[0017] As a further improvement of the above scheme of the present invention, in step S2, the molar ratio of HASH, EDC, NHS and dopamine hydrochloride is 1:0.4:0.4:(0.1-0.2).

[0018] As a further improvement of the above scheme of the present invention, in step S2, the pH adjustment is to adjust the pH of the solution to 5.

[0019] As a further improvement of the above scheme of the present invention, in step S2, the reaction is carried out at room temperature and in an inert atmosphere for 12 hours;

[0020] And / or, in step S2, the dialysis is to place the DAHASH reaction solution into a dialysis bag with a molecular weight of 3500Da and perform dialysis in a dark environment for 3 days to remove unreacted chemicals and by-products.

[0021] As a further improvement of the above scheme of the present invention, in step S3, the method for treating the surface of mEXO with thiols is: dissolving mEXO in tris(2-chloroethyl)phosphate and treating at 37° C. for 30-50 min, wherein the concentration of the tris(2-chloroethyl)phosphate is 0.5-2 mM;

[0022] And / or, in step S4, the concentration of DAHASH is 40 mg / mL, the concentration of SmEXO is 20 μg / mL, and the concentration of silver nitrate is 45 mM.

[0023] The present invention provides a hydrogel for preventing intrauterine adhesions, which is prepared by the above-mentioned method for preparing the hydrogel for preventing intrauterine adhesions.

[0024] The present invention provides an application of a hydrogel for preventing intrauterine adhesions prepared by the preparation method as described above in preparing a biological repair scaffold capable of delivering active substances or an endometrial repair drug or a drug for treating intrauterine adhesions.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention introduces thiol (SH) and dopamine (DA) groups into hyaluronic acid (HA) to synthesize thiol- and dopamine-modified hyaluronic acid DAHASH, and obtains DAHASH@SmEXO hydrogel by physically mixing with SmEXO. The SH side chain of DAHASH can react with Ag. +The ionic Ag-S dynamically coordinates and crosslinks to form a hydrogel with self-healing properties, which is suitable for the special structure of the uterine cavity; at the same time, the DA group in the DAHASH hydrogel can form covalent and non-covalent bonds with the endometrial tissue, thereby anchoring the hydrogel on the uterine wound surface and adhering to the endometrium; the thiol (SH) group coordinates and binds with the surface SH group of the engineered exosome SmEXO to achieve the controlled release of the engineered SmEXO, creating good conditions for the long-term repair of the endometrium. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 HA, HASH, and DAHASH in Test Example 1 of the present invention 1 HNMR characterization diagram;

[0028] Figure 2 FT-IR characterization spectra of HA, HASH and DAHASH in Test Example 2 of the present invention;

[0029] Figure 3 This is a graph showing the gelling ability of DAHASH@SmEXO in Test Example 3 of the present invention;

[0030] Figure 4 The storage modulus (G') and loss modulus (G") variation diagram of DAHASH and DAHASH@SmEXO in Test Example 4 of the present invention;

[0031] Figure 5 The microstructures of DAHASH hydrogel and DAHASH@SmEXO hydrogel in Test Example 5 of the present invention;

[0032] Figure 6 This is a comparison chart of the sustained release ability of DAHASH@SmEXO hydrogels of SmEXO with different degrees of SH engineering in Test Example 6 of the present invention;

[0033] Figure 7 This is the HE staining evaluation diagram of rats with intrauterine adhesions treated in Test Example 7 of the present invention;

[0034] Figure 8 This is a Masson staining evaluation diagram of rats with intrauterine adhesions treated in Test Example 8 of the present invention;

[0035] Fig. 9 This is a staining evaluation diagram of angiogenesis and endometrial stromal cell proliferation in rats with intrauterine adhesions treated in Test Example 9 of the present invention;

[0036] Fig.10 This is a graph showing the number of embryo implanted in the mouse uterus on the 10th day of pregnancy in Test Example 10 of the present invention. DETAILED DESCRIPTION

[0037] For ease of understanding of the present invention, the present invention will be described more fully below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0039] Example 1

[0040] This embodiment provides a self-healing hydrogel for preventing intrauterine adhesions, and the preparation method thereof comprises the following steps:

[0041] S1. Dissolve HA in ultrapure water, add EDC and NHS, adjust the solution pH to 5, add CSH, react overnight at room temperature, dialyze, and freeze-dry to obtain thiol-modified hyaluronic acid, denoted as HASH. The molar ratio of HA, EDC, NHS, and hemihydroxyamine hydrochloride is 1:0.5:0.5:(0.2-0.4).

[0042] S2. HASH prepared in step S1 is dissolved in ultrapure water, and then EDC and NHS are added. DA is added after adjusting the pH of the solution to 5. After reacting for 12 hours at room temperature and nitrogen atmosphere, hyaluronic acid modified with thiol and dopamine is obtained by dialyzing and freeze drying, which is recorded as DAHASH. The molar ratio of HASH, EDC, NHS and dopamine hydrochloride is 1:0.4:0.4:(0.1-0.2).

[0043] S3. mEXO was dissolved in tris(2-chloroethyl)phosphate at concentrations of 0.5 mM, 1 mM, and 2 mM, respectively, and then purified at 37°C for 30 min to obtain engineered exosomes, which were recorded as SmEXO-1, SmEXO-2, and SmEXO-3.

[0044] S4. The DAHASH, SmEXO-1, SmEXO-2, SmEXO-3 and silver nitrate prepared in step S2 are dissolved in ultrapure water to obtain 40 mg / mL DAHASH solution, 20 μg / mL SmEXO-1 solution, 20 μg / mL SmEXO-2 solution, 20 μg / mL SmEXO-3 solution and 45 mM silver nitrate solution respectively; 40 mg / mL DAHASH solution, 20 μg / mL SmEXO-1 solution and 45 mM silver nitrate solution are stirred. The exosome-hydrogel system was obtained by stirring and mixing, and the system was recorded as DAHASH@SmEXO-1. A DAHASH solution with a concentration of 40 mg / mL, a SmEXO-2 solution with a concentration of 20 μg / mL, and a silver nitrate solution with a concentration of 45 mM were stirred and mixed to obtain an exosome-hydrogel system, which was recorded as DAHASH@SmEXO-2. A DAHASH solution with a concentration of 40 mg / mL, a SmEXO-3 solution with a concentration of 20 μg / mL, and a silver nitrate solution with a concentration of 45 mM were stirred and mixed to obtain an exosome-hydrogel system, which was recorded as DAHASH@SmEXO-3.

[0045] Test Example 1

[0046] 10 mg of HA, 10 mg of HASH prepared in step S1 of Example 1, and 10 mg of DAHASH prepared in step S2 of Example 1 were weighed and dissolved in 600 μL of deuterium oxide to prepare samples A, B, and C, respectively. Samples A, B, and C were transferred to nuclear magnetic resonance tubes, and samples A, B, and C were chromatographed using a Bruker 400 nuclear magnetic resonance instrument. 1 HNMR scan, as shown in Figure 1 HA, HASH, DAHASH shown 1 HNMR characterization diagram.

[0047] from Figure 1 It can be seen that the characteristic peak of deuterium oxide solvent is located at δ4.7ppm, the characteristic peak of proton hydrogen in the HA and HASH ring structures is roughly located at δ3-4ppm, and the characteristic peak of HASH is 1 HNMR spectrum and HA 1 Compared with the HNMR spectrum, no new characteristic peaks appeared after SH grafting; 1 HNMR spectrum and HASH 1 Compared with the HNMR spectrum of DAHASH 1 The HNMR spectrum showed new characteristic peaks at δ6.5-7.2 ppm, indicating the three hydrogens at the ortho and meta positions on the benzene ring in DA, which confirmed that DA had been successfully grafted onto the HASH side chain.

[0048] Test Example 2

[0049] Take appropriate amounts of HA, HASH obtained in step S1 of Example 1, and DAHASH obtained in step S2 of Example 1, and use Fourier transform infrared spectrometer IRAffinity-1S to detect the infrared absorption spectra of HA, HASH, and DAHASH to obtain the following: Figure 2 FT-IR characterization spectra of HA, HASH and DAHASH are shown.

[0050] from Figure 2 The FT-IR characterization spectrum of HA in HA molecules shows that -CH 2 The stretching vibration peak of the - group appears at 2900cm -1 Nearby; Compared with the FT-IR characterization spectrum of HASH and HA, the stretching vibration band of -SH in HASH is at 2560cm -1 Nearby, the original stretching vibration peaks were found in HASH after SH modification, which indicates that the modification did not change their basic structure; compared with the FT-IR characterization spectrum of HASH, the FT-IR characterization spectrum of DAHASH shows that the unique stretching vibration band of the main structure of the modified HA is retained at 1638cm -1 A new vibration absorption peak appeared around , which represented the grafting of dopamine. This also confirmed that DA had been successfully grafted onto the HASH side chain.

[0051] Test Example 3

[0052] 100ul of DAHASH@SmEXO-1 hydrogel, DAHASH@SmEXO-2 hydrogel, and DAHASH@SmEXO-3 hydrogel with different degrees of SH-engineered SmEXO prepared in Example 1 were respectively soaked in 10ml of ultrapure water, and the sustained-release capacity of SH-engineered SmEXO with different degrees was detected by BCA method within 12 days; Figure 3 The comparison chart of DAHASH@SmEXO hydrogel's sustained-release SmEXO ability is shown.

[0053] from Figure 3 The results show that the surface exposure of exosomes to SH is optimal after 1 mM TCEP, and SmEXO can be controlled by forming dynamic Ag-S bonds with DAHASH.

[0054] Next, taking SmEXO-2 and DAHASH@SmEXO-2 hydrogels as examples, the following tests were performed.

[0055] Test Example 4

[0056] The DAHASH, silver nitrate and SmEXO-2 prepared in Example 1 were dissolved in ultrapure water to prepare a 40 mg / mL DAHASH solution, a 45 mM silver nitrate solution and a 20 μg / mL SmEXO-2 solution, and the three solutions were physically mixed to obtain the following: Figure 4 The gel-forming ability diagram of DAHASH@SmEXO-2 is shown in FIG. Figure 4 a is a physical picture of DAHASH obtained in Example 1, Figure 4 b is DAHASH and AgNO prepared in Example 1 3 The actual picture of the hydrogel formed after physical mixing with SmEXO-2; Figure 4 As shown in b, DAHASH, AgNO 3 The results show that DAHASH@SmEXO-2 can be injected into the uterus in liquid form and spontaneously form a gel in the uterus after a short period of time, filling the interior of the uterus and avoiding postoperative uterine adhesion.

[0057] Test Example 5

[0058] The DAHASH and DAHASH@SmEXO-2 prepared in Example 1 were used to prepare circular hydrogel samples with a diameter of 8 mm. The two were prepared using the same mold and the American TA rheometer was used to obtain the following at 37°C: Figure 5 The storage modulus (G') and loss modulus (G") of DAHASH and DAHASH@SmEXO-2 are shown in the figure.

[0059] Combination Figure 5 a, The time scan results of DAHASH hydrogel and DAHASH@SmEXO-2 hydrogel show that the storage modulus (G') is always higher than the loss modulus (G"), confirming that both have the properties of viscoelastic solids and have formed a relatively stable network structure. Figure 5 b. This example also detects the strain scan of DAHASH hydrogel and DAHASH@SmEXO-2 hydrogel, confirming that the addition of SmEXO does not affect the formation of hydrogel. Figure 5 c It can be seen that DAHASH hydrogel and DAHASH@SmEXO-2 hydrogel have a satisfactory "sol-gel" cyclic conversion process. The SmEXO-loaded DAHASH hydrogel switches to the "sol" state (G″>G′) under a high shear strain of 300%, and quickly recovers to the "gel" state (G′>G″) at a low shear strain of 1%. The above process undergoes multiple cycles, proving that the hydrogel has good self-healing properties, which is due to the dynamic characteristics of the Ag-S cross-linking inside the DAHASH@SmEXO hydrogel.

[0060] Test Example 6

[0061] The microstructures of DAHASH hydrogel and DAHASH@SmEXO-2 hydrogel prepared in Example 1 were observed by SEM, and the following results were obtained: Figure 6 The microstructure of DAHASH hydrogel and DAHASH@SmEXO-2 hydrogel is shown in Figure 2. Figure 6 It can be seen that the DAHASH hydrogel and DAHASH@SmEXO-2 hydrogel prepared in Example 1 both have a highly interconnected network porous structure, which is conducive to the diffusion of SmEXO. Compared with the DAHASH hydrogel, the loading of SmEXO in the DAHASH@SmEXO-2 hydrogel does not affect the microstructure of the hydrogel.

[0062] Test Example 7

[0063] The thickness and number of glands of the endometrium are crucial to the successful implantation and development of the embryo. In this test example, HE staining was used to evaluate the effects of DAHASH@SmEXO-2 hydrogel prepared in Example 1, DAHASH hydrogel prepared in Example 1, and SmEXO-2 on the thickness and number of glands of the endometrium of rats. The specific steps are as follows:

[0064] ① Use 2% sodium pentobarbital injection to anesthetize SD female rats. After sufficient anesthesia, make a 3-cm long longitudinal incision in the midline of the abdomen in the lower abdomen of the rats, and separate the abdominal wall structures layer by layer until the entire uterus is exposed;

[0065] ② Use sterile forceps to lift the uterus, cut a 2mm transverse wound on the surface of the uterine wall, and insert a homemade 1.5mm uterine curette into the 2mm transverse wound, repeatedly scrape the endometrium to produce endometrial damage, until the entire uterine wall becomes rough and granular. Then use 4-0 absorbable sutures to suture the transverse wound of the uterine wall;

[0066] ③Use vascular clamps to clamp the upper and lower ends of the uterus, and inject each group of therapeutic agents into the uterine cavity according to the prescribed grouping. After the treatment, use 4-0 non-absorbable sutures to suture the longitudinal incision of the abdomen layer by layer. Among them, each group of therapeutic agents is:

[0067] (A) Model group: 200 μL PBS buffer solution was injected into the uterus after physical injury of the uterus;

[0068] (B) SmEXO-2 group: after physical injury of the uterus, intrauterine injection of PBS buffer containing SmEXO-2 (100 μg / 100 μL, 200 μL);

[0069] (C) DAHASH hydrogel group: 200 μL DAHASH hydrogel therapeutic agent was injected into the uterine cavity after physical injury of the uterus;

[0070] (D) DAHASH@SmEXO-2 hydrogel group: DAHASH hydrogel therapeutic agent containing SmEXO-2 (100 μg / 100 μL, 200 μL) was injected into the uterine cavity after physical injury of the uterus.

[0071] HE histological staining was performed on the 30th day after treatment to obtain the following results: Figure 7 Shown is the HE staining evaluation of rats treated for intrauterine adhesions.

[0072] Figure 7 The results showed that the normal uterine cavity structure disappeared in the Model group, accompanied by thinning of the endometrium and disappearance of glands. Compared with the Model group, the DAHASH group, SmEXO-2 group and DAHASH@SmEXO-2 hydrogel group all showed a certain degree of increase in endometrial thickness and regeneration of gland number. Among them, the transplantation effect of DAHASH@SmEXO-2 hydrogel was the best, which could make the recovery of the endometrium closer to normal.

[0073] Test Example 8

[0074] The effects of DAHASH@SmEXO-2 hydrogel prepared in Example 1, DAHASH hydrogel prepared in Example 1, and SmEXO-2 on endometrial collagen reconstruction in IUA rats were evaluated by Masson staining. The specific method is as follows:

[0075] ① Use 2% sodium pentobarbital injection to anesthetize SD female rats. After sufficient anesthesia, make a 3-cm long longitudinal incision in the midline of the abdomen in the lower abdomen of the rats, and separate the abdominal wall structures layer by layer until the entire uterus is exposed;

[0076] ② Use sterile forceps to lift the uterus, cut a 2mm transverse wound on the surface of the uterine wall, and insert a homemade 1.5mm uterine curette into the 2mm transverse wound, repeatedly scrape the endometrium to produce endometrial damage, until the entire uterine wall becomes rough and granular. Then use 4-0 absorbable sutures to suture the transverse wound of the uterine wall;

[0077] ③Use vascular clamps to clamp the upper and lower ends of the uterus, and inject each group of therapeutic agents into the uterine cavity according to the prescribed grouping. After the treatment, use 4-0 non-absorbable sutures to suture the longitudinal incision of the abdomen layer by layer. Among them, each group of therapeutic agents is:

[0078] (A) Model group: 200 μL PBS buffer solution was injected into the uterus after physical injury of the uterus;

[0079] (B) SmEXO-2 group: after physical injury of the uterus, intrauterine injection of PBS buffer containing SmEXO-2 (100 μg / 100 μL, 200 μL);

[0080] (C) DAHASH hydrogel group: 200 μL DAHASH hydrogel therapeutic agent was injected into the uterine cavity after physical injury of the uterus;

[0081] (D) DAHASH@SmEXO-2 hydrogel group: DAHASH hydrogel therapeutic agent containing SmEXO-2 (100 μg / 100 μL, 200 μL) was injected into the uterine cavity after physical injury of the uterus.

[0082] Masson's histological staining was performed on the 30th day after treatment to obtain the following results: Figure 8 Shown is the Masson staining evaluation of rats treated for intrauterine adhesions. Figure 8 The results showed that compared with the Model group, the Masson staining scores of the DAHASH group, SmEXO-2 group and DAHASH@SmEXO-2 hydrogel group decreased to varying degrees; among them, the Masson staining score of the DAHASH@SmEXO-2 hydrogel group decreased the most, followed by the SmEXO-2 hydrogel group. Compared with the Model group, all groups were statistically significant. The transplantation of SmEXO-2 and DAHASH@SmEXO-2 hydrogels was beneficial to the reconstruction of rat endometrial collagen, among which DAHASH@SmEXO hydrogel had the best effect.

[0083] Test Example 9

[0084] Angiogenesis is crucial in the repair of uterine damage. CD34 immunohistochemical staining was used to evaluate the angiogenesis of the uterus of rats with intrauterine adhesions by DAHASH@SmEXO-2 hydrogel, DAHASH hydrogel, and SmEXO-2 prepared in Example 1; and Vimentin immunohistochemistry was used to evaluate the proliferation of rat uterine stromal cells by DAHASH@SmEXO-2 hydrogel, DAHASH hydrogel, and SmEXO-2 prepared in Example 1. The specific method is as follows:

[0085] ① Use 2% sodium pentobarbital injection to anesthetize SD female rats. After sufficient anesthesia, make a 3-cm long longitudinal incision in the midline of the abdomen in the lower abdomen of the rats, and separate the abdominal wall structures layer by layer until the entire uterus is exposed;

[0086] ② Use sterile forceps to lift the uterus, cut a 2mm transverse wound on the surface of the uterine wall, and insert a homemade 1.5mm uterine curette into the 2mm transverse wound, repeatedly scrape the endometrium to produce endometrial damage, until the entire uterine wall becomes rough and granular. Then use 4-0 absorbable sutures to suture the transverse wound of the uterine wall;

[0087] ③ Use vascular clamps to clamp the upper and lower ends of the uterus, and inject each group of therapeutic agents into the uterine cavity according to the prescribed grouping. After the treatment, use 4-0 non-absorbable sutures to suture the longitudinal incision of the abdomen layer by layer. Among them, the therapeutic agents in each group are: (A) Model group: 200μL PBS buffer therapeutic agent was injected into the uterine cavity after physical injury to the uterus; (B) SmEXO-2 group: PBS buffer therapeutic agent containing SmEXO (100μg / 100μL, 200μL) was injected into the uterine cavity after physical injury to the uterus; (C) DAHASH hydrogel group: 200μL DAHASH hydrogel therapeutic agent was injected into the uterine cavity after physical injury to the uterus; (D) DAHASH@SmEXO-2 hydrogel group: DAHASH hydrogel therapeutic agent containing SmEXO-2 (100μg / 100μL, 200μL) was injected into the uterine cavity after physical injury to the uterus. IHC (CD34 and Vimentin) histological staining was performed on the 30th day after treatment, and the following results were obtained: Fig. 9 Shown are staining assessments of angiogenesis and endometrial stromal cell proliferation in rats treated for intrauterine adhesions.

[0088] Fig. 9 The results showed that compared with the Model group, the CD34-positive area in the SmEXO-2 group and the DAHASH@SmEXO-2 hydrogel group increased significantly, and both were statistically significant. This shows that both SmEXO-2 and DAHASH@SmEXO-2 hydrogels can effectively promote endometrial angiogenesis. Among them, the DAHASH@SmEXO-2 hydrogel has the best angiogenesis effect. Similar to CD34 staining, the DAHASH group, the SmEXO-2 group, and the DAHASH@SmEXO-2 hydrogel group can promote the proliferation of rat endometrial stromal cells. Among them, the DAHASH@SmEXO-2 hydrogel group had the best transplantation effect, and the strong SmEXO-2 group and DAHASH hydrogel group.

[0089] Test Example 10

[0090] Pregnancy test is considered the gold standard for evaluating the recovery of endometrial function. The specific method is:

[0091] ① On the 30th day after treatment, SD female rats were housed together with SD male rats in a 1:1 ratio;

[0092] ② At 6 a.m. the next day after the cage is put together, check for vaginal suppositories. When vaginal suppositories appear, it is confirmed that this is day 0 of pregnancy.

[0093] ③ The pregnant uterus of SD female rats was collected from the 20th to 21st day of pregnancy, and the pregnancy rate and number of fetuses in the uterus were recorded. Fig.10 The number of embryo implanted in the mouse uterus at day 20-21 of gestation is shown.

[0094] Fig.10 The results showed that 40% of the uterine pregnancies in the DAHASH group were accompanied by embryos, 80% of the uterine pregnancies in the SmEXO-2 group were accompanied by embryos, and the DAHASH@SmEXO-2 group increased the pregnancy rate to 100%. In contrast, the pregnancy rate in the Model group was only 30%. Among all pregnancy groups, the DAHASH@SmEXO-2 group had the largest number of fetal pregnancies, indicating that DAHASH@SmEXO-2 transplantation can promote the recovery of endometrial function in rats.

[0095] Example 2

[0096] This embodiment provides a self-healing hydrogel for preventing intrauterine adhesions, and the preparation method thereof comprises the following steps:

[0097] S1. Dissolve HA in ultrapure water, add EDC and NHS, adjust the solution pH to 5, add CSH, react overnight at room temperature, dialyze, and freeze-dry to obtain thiol-modified hyaluronic acid, denoted as HASH. The molar ratio of HA, EDC, NHS, and hemihydroxyamine hydrochloride is 1:0.4:0.4:0.3.

[0098] S2. The HASH prepared in step S1 is dissolved in ultrapure water, and then EDC and NHS are added. After adjusting the pH of the solution to 5, DA is added, and the mixture is reacted for 12 hours at room temperature and in a nitrogen atmosphere, and then dialyzed and freeze-dried to obtain hyaluronic acid modified with thiol and dopamine, which is referred to as DAHASH. The molar ratio of HASH, EDC, NHS, and dopamine hydrochloride is 1:0.3:0.3:0.2.

[0099] S3. mEXO was dissolved in tris(2-chloroethyl)phosphate at a concentration of 1 mM and purified at 37°C for 30 min to obtain engineered exosomes, which were recorded as SmEXO-2.

[0100] S4. The DAHASH prepared in step S2, the SmEXO-2 prepared in step S3, and silver nitrate were dissolved in ultrapure water, respectively, to obtain a DAHASH solution with a concentration of 40 mg / mL, a SmEXO-2 solution with a concentration of 20 μg / mL, and a silver nitrate solution with a concentration of 45 mM, respectively. The three solutions were stirred and mixed to obtain an exosome-hydrogel system, recorded as DAHASH@SmEXO-2.

[0101] Example 3

[0102] This embodiment provides a self-healing hydrogel for preventing intrauterine adhesions, and the preparation method thereof comprises the following steps:

[0103] S1. Dissolve HA in ultrapure water, add EDC and NHS, adjust the solution pH to 5, add CSH, react overnight at room temperature, dialyze, and freeze-dry to obtain thiol-modified hyaluronic acid, denoted as HASH. The molar ratio of HA, EDC, NHS, and hemihydroxyamine hydrochloride is 1:0.4:0.4:0.3.

[0104] S2. The HASH prepared in step S1 is dissolved in ultrapure water, and then EDC and NHS are added. After adjusting the pH of the solution to 5, DA is added, and the mixture is reacted for 12 hours at room temperature and in a nitrogen atmosphere, and then dialyzed and freeze-dried to obtain hyaluronic acid modified with thiol and dopamine, which is referred to as DAHASH. The molar ratio of HASH, EDC, NHS, and dopamine hydrochloride is 1:0.3:0.3:0.2.

[0105] S3. mEXO was dissolved in tris(2-chloroethyl)phosphate at a concentration of 1 mM and purified at 37°C for 30 min to obtain engineered exosomes, which were recorded as SmEXO-2.

[0106] S4. The DAHASH prepared in step S2, the SmEXO-2 prepared in step S3, and silver nitrate were dissolved in ultrapure water, respectively, to obtain a DAHASH solution with a concentration of 40 mg / mL, a SmEXO-2 solution with a concentration of 20 μg / mL, and a silver nitrate solution with a concentration of 45 mM, respectively. The three solutions were stirred and mixed to obtain an exosome-hydrogel system, recorded as DAHASH@SmEXO-2.

[0107] The results of testing Example 2 and Example 3 were consistent with those of Example 1.

[0108] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0109] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a self-healing hydrogel for preventing intrauterine adhesions, characterized in that: It includes the following steps: S1. Dissolve hyaluronic acid in ultrapure water, add EDC and NHS, adjust the pH, add cysteamine hydrochloride, dialyze after the reaction, and freeze-dry to obtain thiol-modified hyaluronic acid, denoted as HASH; S2. The HASH is dissolved in ultrapure water, and then EDC and NHS are added. After adjusting the pH, dopamine hydrochloride is added. After the reaction is completed, the reaction is dialyzed and freeze-dried to obtain thiol- and dopamine-modified hyaluronic acid, which is referred to as DAHASH. S3. The surface of mEXO is thiolated to obtain engineered exosomes, which are denoted as SmEXO; S4. The DAHASH, the SmEXO, and silver nitrate are dissolved in ultrapure water respectively and then mixed to obtain an exosome-hydrogel system, which is recorded as DAHASH@SmEXO.

2. The method for preparing the self-healing hydrogel for preventing intrauterine adhesion according to claim 1, characterized in that: In step S1, the molar ratio of hyaluronic acid, EDC, NHS and hemihydroxyethylamine hydrochloride is 1:0.5:0.5:(0.2-0.4).

3. The method for preparing the self-healing hydrogel for preventing intrauterine adhesion according to claim 1, characterized in that: In step S1, the pH is adjusted to stabilize the pH of the solution to 5.

4. The method for preparing the self-healing hydrogel for preventing intrauterine adhesion according to claim 1, characterized in that: In step S1, the reaction is carried out at room temperature overnight; And / or, in step S1, the dialysis is performed by placing the reaction solution in a 3500Da dialysis bag and performing light-proof dialysis for 3 days.

5. The method for preparing the self-healing hydrogel for preventing intrauterine adhesion according to claim 1, characterized in that: In step S2, the molar ratio of HASH, EDC, NHS and dopamine hydrochloride is 1:0.4:0.4:(0.1-0.2).

6. The method for preparing a hydrogel for preventing intrauterine adhesion according to claim 1, characterized in that: In step S2, the pH is adjusted to stabilize the pH of the solution to 5.

7. The method for preparing the self-healing hydrogel for preventing intrauterine adhesion according to claim 1, characterized in that: In step S2, the reaction is carried out at room temperature and in an inert atmosphere for 12 hours; And / or, in step S2, the dialysis is performed by placing the reaction solution into a dialysis bag with a molecular weight of 3500 Da and performing light-proof dialysis for 3 days.

8. The method for preparing the self-healing hydrogel for preventing intrauterine adhesion according to claim 1, characterized in that: In step S3, the method for treating the surface of mEXO with thiols is as follows: dissolving mEXO in tris(2-chloroethyl)phosphate and treating at 37° C. for 30-50 min, wherein the concentration of the tris(2-chloroethyl)phosphate is 0.5-2 mM; And / or, in step S4, the concentration of DAHASH is 40 mg / mL, the concentration of SmEXO is 20 μg / mL, and the concentration of silver nitrate is 45 mM.

9. A self-healing hydrogel for preventing intrauterine adhesions, characterized in that: The self-healing hydrogel for preventing intrauterine adhesion is prepared by the method for preparing the self-healing hydrogel for preventing intrauterine adhesion as described in any one of claims 1 to 8.

10. Use of a self-healing hydrogel for preventing intrauterine adhesions prepared by the preparation method according to any one of claims 1 to 8 in preparing a biorepair scaffold capable of delivering active substances or an endometrial repair drug or a drug for treating intrauterine adhesions.

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