A poloxamer-loaded follicular fluid exosome hydrogel and its application

Abstract

The present invention provides a poloxamer-loaded follicular fluid exosome hydrogel and its application, the hydrogel comprising follicular fluid exosomes, a first gelling agent, and a second gelling agent; wherein the first gelling agent comprises poloxamer 407, and the second gelling agent comprises poloxamer 188; calculated according to the mass ratio, the first gelling agent: the second gelling agent = 16-22: 2-4. The present invention prepares the hydrogel by using poloxamer hydrogel to load follicular fluid exosomes, which can control the degradation time of the poloxamer / FF-Exos hydrogel and promote the retention of follicular fluid exosomes in the endometrium, thereby further repairing the damage of the endometrium. Thus, the poloxamer / FF-Exos hydrogel provided by the present invention can improve the safety and effectiveness of treating endometrial damage or intrauterine adhesions, and has broad medical prospects and economic value.

Description

Technical Field

[0001] The present invention belongs to biomedical engineering, and in particular relates to a poloxamer-loaded follicular fluid exosome hydrogel and application thereof. Background Art

[0002] The endometrium is located in the female uterus and together with the myometrium and exometrium, it forms the uterine wall. The endometrium is composed of a single layer of columnar epithelium and a lamina propria. The epithelium is similar to the fallopian tube epithelium and is also composed of ciliated cells and secretory cells. The lamina propria is composed of connective tissue, uterine glands, and blood vessels. When the endometrium is damaged, it will affect the reproductive health of women.

[0003] Endometrial damage may be caused by a variety of factors, including frequent intrauterine operations, infection, ischemia, etc. Endometrial damage may cause bleeding, pain and other problems in women. In addition, endometrial damage can destroy the integrity of the endometrium, which may cause the endometrium to become thinner and less thick, thus causing endometritis. If the disease is not treated in time, it will cause adhesions between the endometrial layers, causing partial or complete occlusion of the uterine cavity, resulting in intrauterine adhesions, which will affect women's menstruation and fertility.

[0004] At present, the methods for treating endometrial damage or intrauterine adhesions include traditional treatment methods, stem cell therapy and other methods. Among them, traditional treatment methods include hysteroscopy, high-dose estrogen stimulation, and intrauterine perfusion of growth hormone. For intrauterine adhesions, hysteroscopy is the main treatment method, but there is a risk of perforation and bleeding, and the postoperative recurrence rate is high for patients with severe intrauterine adhesions. For thin endometrium, although hormone therapy can improve endometrial receptivity to a certain extent, it has limited effect on severely damaged endometrium.

[0005] Stem cell therapy provides new hope for the treatment of endometrial damage, because mesenchymal stem cells (MSCs) have the potential to repair endometrial damage due to their differentiation ability, paracrine activity and immunomodulatory effects. However, stem cell therapy is easy to be recognized as foreign substances by the host's immune system under certain circumstances, causing rejection reactions, or proliferating indefinitely in the body, increasing the risk of their transformation into tumor cells. Summary of the invention

[0006] In order to improve the safety and effectiveness of treating endometrial damage, the present invention provides a poloxamer-loaded follicular fluid exosome hydrogel and application thereof.

[0007] According to one aspect of the present application, a poloxamer-loaded follicular fluid exosome hydrogel is provided, the hydrogel comprising follicular fluid exosomes, a first gelling agent, and a second gelling agent; wherein the first gelling agent comprises poloxamer 407, and the second gelling agent comprises poloxamer 188; calculated by mass ratio, the first gelling agent: the second gelling agent = 16-22: 2-4.

[0008] In general, follicular fluid (FF) provides a microenvironment for follicular development and is secreted by follicular cells and theca cells. The follicles are located in the ovaries of mammals. With the deepening of research, people have found that follicular fluid, as a microenvironment for oocyte development, contains plasma components, follicular secretions, and a variety of cytokines secreted by granulosa cells, which can promote follicular development and oocyte maturation. Follicular fluid exosomes contain a variety of functional molecules, such as vascular endothelial growth factor, insulin-like growth factor, transforming growth factor-β, miRNA, etc., which have potential repair and regeneration functions. Exosomes refer to small membrane vesicles (30-150nm) containing complex RNA and proteins. A variety of cells can secrete exosomes under normal and pathological conditions. Exosomes exist naturally in body fluids, including blood, saliva, urine, cerebrospinal fluid and breast milk. They are mainly derived from multivesicular bodies formed by the invagination of intracellular lysosomal microparticles. After the multivesicular outer membrane fuses with the cell membrane, it is released into the extracellular matrix and is responsible for transporting biologically active proteins, lipids and nucleic acids. It also acts as a carrier of intercellular communication to transfer these molecules from one cell to another, thereby achieving information exchange and functional regulation between cells. In general, follicular fluid exosomes (FF-Exos) are involved in the regulation of follicular cell function.

[0009] The present invention uses poloxamer hydrogel to load follicular fluid exosomes to prepare hydrogel (poloxamer / FF-Exos hydrogel), and unexpectedly finds that the hydrogel can promote the migration and proliferation ability of human endometrial stromal cells (HESCs), and can also reduce the relative expression level of mRNA of its fibrosis gene, thereby significantly repairing endometrial damage or intrauterine adhesion. In addition, the hydrogel can repair the mouse endometrium by promoting cell proliferation and angiogenesis in the endometrium of intrauterine adhesion model mice.

[0010] Among them, the chemical formula of poloxamer 407 is HO(C2H4O) a (C3H6O) b (C2H4O) a H, where a and b represent the number of repeating units of polyoxyethylene (PEO) and polyoxypropylene (PPO), respectively; and the chemical formula of poloxamer 188 is H(C2H4O) a (C3H6O) b (C2H4O)a OH, Poloxamer 407 is generally used as a dispersant, emulsifier, solubilizer, etc., while Poloxamer 188 is a commonly used pharmaceutical excipient.

[0011] The present invention finds that by limiting the ratio of the first gel and the second gel, the degradation time of the poloxamer / FF-Exos hydrogel can be controlled, and the retention of follicular fluid exosomes in the endometrium can be promoted, thereby further repairing endometrial damage. Therefore, the poloxamer / FF-Exos hydrogel provided by the present invention can improve the safety and effectiveness of treating endometrial damage or intrauterine adhesions, and has broad medical prospects and economic value.

[0012] Preferably, in the hydrogel, the ratio of the first gelling agent to the second gelling agent is 18:2.

[0013] Preferably, in the hydrogel, the concentration of follicular fluid exosomes is 200-400 μg / mL.

[0014] Preferably, the method for preparing a hydrogel comprises the following steps: S1. configuring a first gelling agent and an aqueous solvent into a first solution in an ice bath environment; S2. subsequently, adding a second gelling agent to the first solution in an ice bath environment to obtain a second solution; S3. mixing the second solution with follicular fluid exosomes, performing a gelation reaction at 37°C, and obtaining a hydrogel after 5 minutes.

[0015] Preferably, the mass volume concentration of the first gelling agent in the first solution is 16-22%; and / or the mass volume concentration of the second gelling agent in the second solution is 2-4%.

[0016] The second aspect of the present application provides a use of the above-mentioned hydrogel in the preparation of a composition for reducing the expression level of cell fibrosis genes.

[0017] Preferably, the above cells include human endometrial stromal cells.

[0018] Preferably, the fibrosis gene includes at least one of Col IA1, Col IA2, fibronectin, and α-SMA.

[0019] The third aspect of the present application provides a use of the above-mentioned hydrogel in the preparation of a composition for medical or general surgical use for treating endometrial damage or intrauterine adhesions.

[0020] Preferably, the composition is a topical composition, an implantable composition, or a controlled release composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The experimental data related to the preparation and characterization of FF-Exos provided in Test Example 1 of the present invention, wherein: Figure 1 The reference numerals have the following meanings:-

[0022] Figure 1 A is the TEM image of FF-Exos;

[0023] Figure 1 B is the test result of Western blot analysis;

[0024] Figure 1 C is the DLC particle size distribution analysis of FF-Exos;

[0025] Figure 1 D is a scratch test to study the effect of FF-Exos on the migration ability of HESCs, where (i) is a direct view of the migration effect and (ii) is the quantification of the closed area (n=6);

[0026] Figure 1 E is the effect of FF-Exos on HESCs proliferation detected by CCK 8 method at different times (n=3);

[0027] Figure 1 F is the relative mRNA levels of fibrotic genes (Col IA1, Col IA2, fibronectin, and α-SMA) after HESCs were incubated with FF-Exos for 24 h;

[0028] Figure 2 The poloxamer / FF-Exos hydrogel provided in Test Example 2 of the present invention promotes the retention of FF-Exos in the endometrium of IUA model mice, wherein: Figure 2 The meanings of the reference numerals are as follows:

[0029] Figure 2 A shows the gel-sol-gel transition of poloxamer / FF-Exos hydrogel at different temperatures;

[0030] Figure 2 B is the release curve of FF-Exos from poloxamer / FF-Exos hydrogel in PBS buffer (pH = 7.4) at 37°C;

[0031] Figure 2 C is a graph showing the degradation test data of the hydrogels prepared with 2% poloxamer 188 and different concentrations of poloxamer 407;

[0032] Figure 2 D is a graph showing the degradation test data of the hydrogels prepared with 4% poloxamer 188 and different concentrations of poloxamer 407;

[0033] Figure 2 E is the rheological characterization of poloxamer / FF-Exos hydrogel;

[0034] Figure 2 F is the in vivo fluorescence imaging of mice in the control group and the treatment group on days 0, 1, 3, 5, 7, and 10; the mice on the right are the untreated control group to exclude the interference of autofluorescence, and the mice on the left are the treatment group, in which the same amount of CM-DiD-labeled FF-Exos and poloxamer / FF-Exos hydrogel were implanted in the left and right uteri of the treatment group, respectively, n=3;

[0035] Figure 2 G is in vitro fluorescence imaging of mouse uterus on days 0, 1, 3, 5, 7, and 10 after injection, n = 3;

[0036] Figure 2 H is serial frozen sections of isolated uterus showing exosome retention, labeled with CM-Di D (red), and cell nuclei labeled with DAPI (blue);

[0037] Figure 3 The characterization of the poloxamer / FF-Exos hydrogel provided in Test Example 3 of the present invention and the experimental data related to its effect on endometrial regeneration in a mouse acute endometrial injury model, wherein: Figure 3 The meanings of the reference numerals are as follows:

[0038] Figure 3 A is a schematic diagram of the in vivo study of the mouse acute endometrial injury model;

[0039] Figure 3 B is a representative image of the uterus under different treatments (7 days) examined using hematoxylin and eosin (H&E) staining, and the insets outlined by the black dashed boxes are shown at higher magnification in the following rows;

[0040] Figure 3 C is the quantitative endometrial thickness (n=8);

[0041] Figure 3 D is endometrial gland quantification (n=8), *p<0.05, **p<0.01;

[0042] Figure 4 The experimental data related to the restoration of functional endometrium in mice with intrauterine adhesion model by poloxamer / FF-Exos treatment provided in Test Example 3 of the present invention, wherein: Figure 4 The meanings of the reference numerals are as follows:

[0043] Figure 4 A is the Masson staining results (blue) used to evaluate the endometrial fibrosis area in 4 groups;

[0044] Figure 4 B: Immunohistochemistry to detect the expression of Ki67;

[0045] Figure 4 C is the expression of CD31 detected by immunohistochemistry;

[0046] Figure 4 D is the statistical analysis of uterine fibrosis area, fibrosis area = ratio of endometrial fibrosis area / endometrial area;

[0047] Figure 4 E is the IRS statistical analysis of endometrial Ki67;

[0048] Figure 4 F is the IRS statistical analysis of endometrial CD31, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in this application will be clearly and completely described below in conjunction with the embodiments of this application and the drawings in the embodiments. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application.

[0050] Example 1

[0051] The follicular fluid exosomes (FF-Exos) required in this example were extracted in the following manner.

[0052] First, human follicular fluid was collected, wherein the human follicular fluid in this embodiment was collected from normal female patients who received ICSI assisted pregnancy due to male illness at the Reproductive Medicine Center of Luohu Hospital Affiliated to Shenzhen University from November 2023 to January 2024.

[0053] Subsequently, exosomes were extracted by ultracentrifugation, and the specific steps were as follows: centrifugation at 4°C and 300×g for 10 min to remove live and dead cells, retaining the follicular fluid supernatant, changing the centrifuge tube, centrifuging at 2,000×g for 10 min to remove cell debris, retaining the supernatant, changing the centrifuge tube, centrifuging at 10,000×g for 10 min to remove larger vesicles, transferring the cell supernatant to an ultracentrifuge tube, and then ultracentrifuging at 4°C and 100,000×g for 90 min, washing the precipitate with PBS, and then filtering with a 0.22μm filter to remove bacteria and microvesicles, ultracentrifuging again at 100,000×g for 90 min to purify the exosomes, resuspending the precipitate with an appropriate amount of PBS, and finally determining the concentration of exosome protein by the BCA method, with a mass concentration of 0.8μg / μL, and storing it in a -80°C refrigerator.

[0054] Test Example 1

[0055] 1. Test subjects

[0056] The FF-Exos group was prepared by adding the follicular fluid exosomes (FF-Exos) provided in Example 1, and the control group was prepared by adding PBS.

[0057] 2. Test Method

[0058] (1) TEM observation of exosomes: Use a pipette to draw up the prepared suspension sample and drop it onto a copper mesh with a support film. Depending on the sample concentration in the suspension, use filter paper to absorb excess liquid from the edge of the liquid droplet immediately or after a few minutes, and then drop the staining solution on it. The staining time is 3 to 5 minutes. Then use filter paper to absorb the staining solution, dry it, and observe it by TEM.

[0059] (2) Western blotting identification: Identification of exosome-specific marker proteins: The exosomes were placed on ice and lysed for 20 min. 5× SDS loading buffer was added and boiled at 100°C for 10 min. The proteins were separated by 10% SDS-PAGE electrophoresis. Electrophoresis was performed at 80 V for 30 min and 120 V for 60 min. The membrane was wet-transferred and transferred at 250 mA for 120 min. The proteins were transferred to PVDF membrane and blocked with 5% skim milk at room temperature for 1 h. Primary antibodies (CD9, dilution ratio of 1:1000; CD81, dilution ratio of 1:1000; TSG101, dilution ratio of 1:1000; Alix, dilution ratio of 1:1000) were added and incubated at 4°C overnight. The membrane was washed with TBST three times for 10 min each time. Secondary antibodies (1:3000) were added and incubated at room temperature for 1 h. The membrane was washed with TBST and developed with ECL. The developed bands were photographed using a gel electrophoresis system.

[0060] (3) Nanoparticle Tracking Analysis (NTA): The particle size distribution and concentration of ADSCs-Exos were analyzed using the NTA method: First, the sample pool was cleaned with deionized water, the instrument was calibrated with polystyrene microspheres (100 nm), and then the sample pool was cleaned with PBS buffer. Finally, the sample was diluted with PBS buffer and injected to detect the particle size distribution and concentration of the exosome sample.

[0061] (4) Cell scratch assay: Mouse endometrial epithelial cells (HESCs) were seeded in 6-well plates at a cell density of 1×10 6 / well. After 18-24h of cell culture, the cells covered the bottom of the plate, and a 200μL pipette tip was used to scratch the cells perpendicular to the well plate. The cell culture medium was aspirated and the plate was washed 3 times with PBS. Among them, the control group was PBS; the FF-Exos group continued to culture cells by adding F12K culture medium containing FF-Exos. Cell migration was recorded by taking photos at different time points, and data was collected and analyzed based on the photos.

[0062] (5) CCK 8 method to detect cell proliferation activity: HESCs in the logarithmic growth phase were collected and 8×10 3 CFU / well was inoculated in a 96-well plate, with a volume of 100 μL per well. After 24 hours, the cells were completely attached to the wall, the medium was aspirated, and 100 μL of F12K medium containing 0, 50, 100 and 200 μg / mL FF-Exos (FF-Exos group) and PBS control group were added, with at least 5 replicates per group. After the action time was reached, 10 μL / well of CCK8 (5 mg / mL) was added and incubated in a 37°C incubator for 4 hours. The absorbance value (OD value) of each well at a wavelength of 450 nm was read by an ELISA reader to calculate the cell survival rate.

[0063] (6) Real-time PCR detection of related gene expression levels: RNA from HESCs in the control group and FF-Exos group was extracted using RNAisoPlus, and RNA was reverse transcribed into cDNA using the EasyScript cDNA first-strand synthesis kit. cDNA was used as a template and amplified using TransStart Top Green qPCR SuperMix (+DyeⅡ). The amplification conditions were: 94℃ pre-denaturation for 34s, 94℃ denaturation for 5s, 60℃ annealing and extension for 30s, for a total of 40 cycles. The relative expression of each gene was calculated using 2 -ΔΔCt The data were quantified by the PCR method, with β-actin as the internal reference, at least 4 replicates each time, and the experiment was repeated 3 times. The primer sequences are shown in Table 1.

[0064] Table 1. Primer sequences used for real-time quantitative PCR

[0065]

[0066]

[0067] 3. Test results and analysis

[0068] The test results of this test case are as follows Figure 1 As shown, the test results identified by TEM observation and Western blotting (such as Figure 1 A to 1B), it can be confirmed that the present embodiment successfully prepared follicular fluid exosomes (FF-Exos), wherein Western blot analysis showed that the FF-Exos prepared in the present embodiment expressed exosome surface markers CD9, CD81, TSG101 and Alix. In addition, combined with nanoparticle tracking analysis (NTA) (such as Figure 1 C), the particle size range of the prepared FF-Exos is 90-300nm.

[0069] Furthermore, the cell scratch test (such as Figure 1 D) confirmed that FF-Exoss can promote the migration ability of human endometrial stromal cells (HESCs); CCK 8 method was used to detect cell proliferation activity (as shown in Figure 1 E) confirmed that FF-Exoss can promote the proliferation of HESCs; Real-time PCR was used to detect the expression levels of related genes (as shown in Figure 1 (F) confirmed that FF-Exoss can inhibit the expression of relative mRNA levels of fibrosis genes.

[0070] Example 2

[0071] 1. Preparation of Poloxamer Hydrogel

[0072] Step 1. Weigh 18 g of poloxamer 407 and stir evenly with 60 mL of pre-cooled ultrapure water until the powder is completely dissolved to obtain a first solution;

[0073] S2. Subsequently, 2 g of poloxamer 188 was added to the first solution in an ice bath environment, stirred, and then diluted to 100 mL with pre-cooled ultrapure water, and stirred again to obtain a second solution;

[0074] S3. The second solution was subjected to gel reaction at 37°C to obtain poloxamer hydrogel after 5 minutes.

[0075] 2. Preparation of Poloxamer-loaded Follicular Fluid Exosomes Hydrogel

[0076] S1. Weigh 18 g of poloxamer 407 and stir evenly with 60 mL of pre-cooled ultrapure water until the powder is completely dissolved to obtain a first solution;

[0077] S2. Subsequently, 2 g of poloxamer 188 was added to the first solution in an ice bath environment, stirred, and then diluted to 100 mL with pre-cooled ultrapure water, and stirred again to obtain a second solution;

[0078] S3. The second solution was mixed with the follicular fluid exosomes, and a gel reaction was performed at 37°C. After 5 minutes, a poloxamer-loaded follicular fluid exosome hydrogel (poloxamer / FF-Exos hydrogel) was obtained, wherein the concentration of the follicular fluid exosomes in the hydrogel was 200 ug / mL (for in vitro cell experiments) or 400 ug / mL (for in vivo animal experiments).

[0079] Test Example 2

[0080] 1. Test subjects

[0081] The poloxamer-loaded follicular fluid exosomes hydrogel provided in Example 2 was used as the poloxamer / FF-Exos hydrogel group; the poloxamer hydrogel provided in Example 2 was used as the poloxamer group; the follicular fluid exosomes (FF-Exos) were used as the FF-Exos group; and PBS was added as the control group.

[0082] 2. Test Method

[0083] (1) Gel-sol-gel experiment: The mixture of the second solution and follicular fluid exosomes was placed in an environment of 4°C, then placed in an environment of 37°C for 24 hours, and then placed in an environment of 4°C again.

[0084] (2) In vitro release of FF-Exos: Take 700uL of the prepared poloxamer / FF-Exos hydrogel at 4°C and put it into a dry and clear 1.5mL EP tube, and then detect it according to the following steps: ① Add 10 times the volume of 37°C PBS to the prepared poloxamer / FF-Exos hydrogel; ② Collect the supernatant at different time points (0, 0.5, 1, 2, 4, 8, 12, 24, 48, 72h) and add an equal amount of fresh 37°C PBS; ③ Detect the content of exosomes in the supernatant by fluorescence quantitative method.

[0085] (3) Degradation of poloxamer / FF-Exos hydrogel: To study the biodegradability of the hydrogel, 0.5 mL of the hydrogel was weighed, and then 5 mL of PBS was added to the hydrogel and incubated at 37°C with stirring (150 rpm). The remaining hydrogel was weighed once every 2 days (Wt).

[0086] (4) Rheological characterization of poloxamer / FF-Exos hydrogels: The rheological properties of the hydrogels were studied using a Hybrid Rheometer-2 (TA Instruments, USA) to evaluate the gelation kinetics of the poloxamer / FF-Exos hydrogels from 4°C to 37°C by performing an oscillating temperature sweep test at a heating rate of 5°C / min.

[0087] (5) In vivo tracing of CM-DID-labeled poloxamer / FF-Exos: The commercial red fluorescent probe CM-DiD (Everbright, Jiangsu, China) was diluted with DMSO to a storage concentration of 2 mg / mL. FF-Exos at a concentration of 1000 μg / mL were suspended in 5 mL of PBS containing 2 μg / mL CM-DiD (final working concentration). After incubation at 37°C for 20 min, the labeled exosomes were spun at 120,000 g for 70 min and then washed with 5 mL of PBS at 120,000 g for 70 min. CM-DiD-labeled FF-Exos were then mixed with the prepared poloxamer. Mouse models were injected with CM-DiD-labeled FF-Exos and poloxamer / FF-Exos hydrogels and tested at 0, 1, 3, 5, 7, and 10 days after transplantation. Small animal live imaging was also used to evaluate the retention time of follicular fluid exosomes in vivo.

[0088] 3. Test results and analysis

[0089] The test results of this test case are as follows Figure 2 As shown, it can be confirmed by 2A that the poloxamer / FF-Exos hydrogel provided by the present invention is a thermosensitive hydrogel, which can gel at 37°C and return to a sol state at 4°C. Figure 2 B shows that after 96 hours, FF-Exos are basically released.

[0090] In addition, experiments have shown that if poloxamer 407 is used alone, the gel will not achieve the ideal strength and mucosal adhesion properties, and the sol-gel transition temperature range is very narrow, which limits the delivery route of exosomes. However, using poloxamer 188 alone cannot improve the stability of exosomes because the gel is not temperature-sensitive and remains in a liquid state.

[0091] Furthermore, since FF-Exos will degrade at 37°C and poloxamer will have certain toxicity that affects human health, this test example prepared hydrogels with different concentrations of poloxamer 188 and poloxamer 407, and tested the degradation of the above hydrogels. Figure 2 As shown in the data of C and 2D, in order to increase the residence time and stability of FF-Exos in the uterus and reduce the impact of poloxamer on human health, the optimal concentrations of poloxamer 188 and poloxamer 407 in the poloxamer / FF-Exos hydrogel provided by the present invention are 2% and 18%, respectively. Therefore, it can be seen from the above that when poloxamer 407 and poloxamer 188 are used at the same time and a certain ratio is maintained between the two, the release of follicular fluid exosomes can be more accurately regulated to meet different needs.

[0092] In addition, through Figure 2 From the rheological test data of E, it can be seen that poloxamer / FF-Exos hydrogel can undergo gelation reaction at 18-40°C.

[0093] The in vivo fluorescence imaging of mice on days 0, 1, 3, 5, 7, and 10 after injection was as follows: Figure 2 F. Figure 2 G. Figure 2 H. By Figure 2 The data shown in F to 2H confirm that poloxamer / FF-Exos hydrogel can promote the retention of follicular fluid exosomes in the endometrium.

[0094] Test Example 3

[0095] 1. Test subjects

[0096] Refer to the test object provided in Test Example 2.

[0097] 2. Test Method

[0098] (1) Endometrial injury animal model (IUA) Mouse modeling: Sexually mature healthy female C57BL mice with normal estrous cycle (SPF grade, 32 mice, 8-10 weeks old, body weight 20-25g) were provided by the Experimental Animal Center of Luohu Hospital Medical Center. Breeding conditions: barrier environment animal laboratory of Shenzhen Luohu Hospital Medical Center, room temperature 23-27℃, daily temperature difference does not exceed 3℃, humidity 50%-60%, light and dark time of 12h each, free access to food and water. The estrous cycle of mice was observed by vaginal smear method, and the animal model was established in the diestrus period. The IUA mouse model was established with reference to the research method of Jungho Ahn et al. [8] . Thirty-six mice were randomly divided into four groups: control group, 20 μL of PBS was injected into each of the two uterine horns immediately after modeling to induce intrauterine adhesions in mice; poloxamer group, 20 μL of poloxamer hydrogel was injected into each of the two uterine horns immediately after modeling to induce intrauterine adhesions in mice; FF-Exos group, 20 μL of follicular fluid exosomes were injected into each of the two uterine horns immediately after modeling to induce intrauterine adhesions in mice; poloxamer / FF-Exos hydrogel group, 20 μL of poloxamer / FF-Exos hydrogel was injected into each of the two uterine horns immediately after modeling to induce intrauterine adhesions in mice. One week after treatment, 32 mice (n=8 in each group) were killed during estrus, and uterine tissues were collected for the next experiment.

[0099] (2) Observation and detection of mouse uterus indicators:

[0100] ① Changes in endometrial morphology: 1 hour after the last administration, the uterus was removed by opening the abdomen and fixed in 4% paraformaldehyde for 24 hours. The long axis of the mouse uterus was paraffin-embedded vertically to ensure the integrity of the cross section. 4 μm sections were made and dewaxed to water, and HE staining was performed. The thickness of the endometrium, the distribution of glands and blood vessels were observed under an optical microscope, and the thickness of the endometrium, the number of glands and blood vessels were measured using ImageJ image analysis software.

[0101] ② Immunohistochemical detection of endometrial Ki67, CD31, OPN and α-SMA protein expression: 4 μm paraffin sections were dewaxed to water, and 3% hydrogen peroxide solution was added to the surface of the section tissue and reacted at room temperature for 10 minutes. The sections were taken out and immersed in pre-boiled sodium citrate antigen repair solution and reacted in a boiling water bath for 15 minutes. 5% BSA solution was added and blocked at room temperature for 30 minutes. Ki67 Rabbit mAb, CD31 Rabbit mAb, OPN Rabbit mAb and α-SMA Rabbit mAb (dilution ratios of 1:200, 1:200, 1:300 and 1:500, respectively) were added and incubated at 4°C overnight. The next day, biotin-labeled Goat Anti-Rabbit IgG (dilution ratio of 1:1000) was added and incubated at room temperature for 2 hours, and then SABC solution was added and reacted at room temperature for 30 minutes. DAB kit was used for development, hematoxylin was used for counterstaining, and the sections were sealed. The expression of the target protein was observed and photographed under an optical microscope, and semi-quantitative analysis and statistics were performed using Image J image analysis software.

[0102] (3) Statistical analysis: IBM SPSS 25.0 was used for statistical analysis. GraphPad Prism 9.0 software was used for graphical analysis. Quantitative data were expressed as mean ± standard deviation. The normality test and variance homogeneity test were performed first. If the data were normal and the variance was homogeneous, one-way ANOVA was used for comparison among multiple groups, and LSD-t test was used for comparison within each group. If the variance was not homogeneous, Dunnett's T3 one-way ANOVA was used. P<0.05 indicated that the difference was statistically significant.

[0103] 3. Test results and analysis

[0104] The results of this test case are as follows Figure 3 , Figure 4 As shown. Among them, through Figure 3 The effect of poloxamer / FF-Exos hydrogel on endometrial regeneration in the mouse acute endometrial injury model can be confirmed. The schematic diagram of the in vivo study of the mouse acute endometrial injury model in this test example is shown in FIG. Figure 3 As shown in A.

[0105] Specifically, through Figure 3 B and Figure 3 C shows that FF-Exos can promote endometrial regeneration, while poloxamer / FF-Exos hydrogel can further enhance the repair of endometrial damage. This is because the appropriate ratio of poloxamer 407 to poloxamer 188 can significantly increase the retention time of FF-Exos in the uterus, thereby significantly improving acute endometrial damage in mice. Figure 4 From the data of A to 4D, it can be seen that poloxamer / FF-Exos hydrogel can also restore the functional endometrium of mice with intrauterine adhesion model.

[0106] The above embodiments are only used to illustrate the technical solution of the present application rather than to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, a person skilled in the art should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A poloxamer-loaded follicular fluid exosome hydrogel, characterized in that: The hydrogel comprises follicular fluid exosomes, a first gelling agent, and a second gelling agent; Wherein, the first gelling agent includes poloxamer 407, and the second gelling agent includes poloxamer 188; calculated by mass ratio, the first gelling agent: the second gelling agent = 16-22: 2-4.

2. The hydrogel according to claim 1, characterized in that In the hydrogel, the ratio of the first gelling agent to the second gelling agent is 18:

2.

3. The hydrogel according to claim 1, characterized in that In the hydrogel, the concentration of the follicular fluid exosomes is 200-400 μg / mL.

4. The hydrogel according to claim 1, characterized in that The hydrogel further comprises at least one of an antioxidant, an anti-inflammatory agent, an antiseptic, an antibacterial agent, an antifungal agent, an anticancer agent, and a local anesthetic.

5. The hydrogel according to any one of claims 1 to 4, characterized in that: The method for preparing the hydrogel comprises the following steps: S1. configuring the first gelling agent and the aqueous solvent into a first solution in an ice bath environment; S2. Subsequently, a second gelling agent is added to the first solution in an ice bath environment to obtain a second solution; S3. The second solution is mixed with the follicular fluid exosomes, and a gel reaction is performed at 37° C. to obtain the hydrogel after 5 minutes.

6. The hydrogel according to claim 5, characterized in that The mass volume concentration of the first gelling agent in the first solution is 16-22%; and / or the mass volume concentration of the second gelling agent in the second solution is 2-4%.

7. Use of the hydrogel according to any one of claims 1 to 6 in the preparation of a composition for reducing the expression level of cell fibrosis genes.

8. The use according to claim 7, wherein the cells comprise human endometrial stromal cells.

9. The use according to claim 7, wherein the fibrosis gene comprises at least one of Col IA1, Col IA2, fibronectin, and α-SMA.

10. Use of the hydrogel according to any one of claims 1 to 6 in the preparation of a composition for medical or general surgical use for treating endometrial damage or intrauterine adhesions.

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