Application of secreting type curl-associated protein 2 gene as new target spot for treating uterine scars

Abstract

The invention discloses an application of a secretory curl-associated protein 2 gene as a new target for treating uterine scars, the secretory curl-associated protein 2 gene (sFRP2) promotes the progress of the uterine scars, interferes, silences and inhibits the uterine scars, and the sFRP2 is knocked out, so that the occurrence and development of the scar uterus can be relieved, the pregnancy rate of women can be increased, and the adverse pregnancy outcome can be reduced. In order to further improve the curative effect that sFRP2 gene expression is inhibited by sFRP2-targeted small interfering RNA (issFRP2) so as to inhibit uterine scar formation, ionizable lipid, distearoyl phosphatidylcholine (DSPC), cholesterol and 1, 2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol (DMG-PEG) are mixed and dissolved in ethanol according to a certain molar ratio, issFRP2 is dissolved in a citric acid buffer solution, and the issFRP2-targeted small interfering RNA (issFRP2) is added into the citric acid buffer solution so as to obtain a mixed solution; an ethanol phase and a water phase are mixed through a microfluid device, and the mixed solution is dialyzed in a PBS solution for two hours to prepare lipid nanoparticles. And then mixing the polycarbophil, the poloxamer and the lipid nanoparticles to prepare the composite hydrogel. The hydrogel can locally release sisFRP2 in the uterus, inhibit fibrosis, reduce collagen deposition and repair uterine scars. The hydrogel is good in biocompatibility and noninvasive, can realize slow release of the medicine, avoids repeated administration, has no obvious side effect on a maternal body and a fetus, and can also be used for other fibrosis-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of gene treatment of gynecological diseases, and specifically relates to the application of interference, silencing, inhibition and knockout of secreted frizzled-related protein 2 gene (sFRP2) in the treatment of uterine scars and verifies the effect of the gene on pregnancy outcomes. Background Art

[0002] Uterine scarring is characterized by the formation of scar tissue on the uterus and can be caused by a variety of factors, such as uterine surgery, infection, and pelvic radiotherapy. Among them, cesarean section is a common cause of uterine scarring, and the increase in the global cesarean section rate has led to an increasing prevalence of uterine scarring in women undergoing this surgery. Other surgical interventions that can cause uterine scarring include myomectomy, curettage, hysteroscopic resection, and uterine malformation correction. It is worth noting that myomectomy is often used to treat uterine fibroids, a benign gynecological tumor that affects 3-10% of women of childbearing age. Depending on the extent and location of uterine scarring, pelvic pain and irregular menstruation can result. In addition, both preclinical and clinical studies have shown that uterine scarring is an increased risk factor for many adverse pregnancy outcomes, such as infertility, placenta previa, placenta accreta, placental abruption, ectopic pregnancy, uterine rupture, intrauterine growth restriction, recurrent miscarriage, premature birth, postpartum hemorrhage, and even fetal death. Of particular concern is the impact of cesarean section-induced uterine scarring on adverse pregnancy outcomes, which may persist for years after the initial surgery.

[0003] In order to reduce the incidence of cesarean section-related uterine scarring, various prevention strategies have been implemented in clinical practice, including improving the surgical technique of uterine incision, improving the suturing method, and avoiding incision infection. For women with previous uterine scarring, surgical treatments such as curettage, hysteroscopy, laparotomy, and laparoscopic scar resection are usually used to eliminate scar tissue and restore normal uterine function. Unfortunately, existing results show that surgical treatments to remove and repair cesarean scars do not reduce the occurrence of cesarean scar pregnancy. Nonsurgical methods are also used to promote healing and inhibit the formation of uterine scars, including hormone therapy, methotrexate therapy, needle aspiration, high-intensity focused ultrasound, and uterine artery embolization. Despite these efforts, nonsurgical interventions have limited effectiveness in preventing uterine scarring. At present, no pharmacological strategies have been reported to effectively address the problem of collagen deposition in uterine scars.

[0004] Therefore, it is very necessary to develop a preparation that has high patient compliance, is safe and reliable, and can inhibit fibrosis and collagen deposition and thus prevent and treat uterine scars. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide a target gene related to uterine scar - secretory frizzled-related protein 2 gene (sFRP2). The present invention uses human specimens, cell experiments and animal models to discover and verify for the first time that sFRP2 can cause uterine scar progression. Interference, silencing, inhibition, and knockout of the target gene sFRP2 can alleviate the occurrence and development of scarred uterus. Interference, silencing, inhibition, and knockout of target genes or proteins can be achieved through gene editing and protein synthesis technologies such as inhibitors, viruses, interfering RNA, CRISPR / Cas9. In order to further improve the efficiency of gene therapy, the present invention uses lipid nanoparticles to encapsulate small interfering RNA that interferes with the secretory frizzled-related protein 2 gene, and then forms an adhesive composite hydrogel by mixing poloxamer with polycarbophil and the lipid nanoparticles. The hydrogel is instilled locally in the uterine scar, avoiding the first-pass elimination effect, and has good biocompatibility and is non-invasive. It can achieve sustained release of drugs, avoid multiple administrations, and reduce secondary damage.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides an application of a secretory frizzled-related protein 2 gene as a new target for preventing and treating uterine scars. Interference and knockout of the target gene sFRP2 can be used to prevent and treat uterine scars, and the application means include small interfering RNA (sisFRP2) that interferes with the secretory frizzled-related protein 2 gene, lentivirus containing short hairpin RNA to silence the secretory frizzled-related protein 2 gene, inhibitors and CRISPR / Cas9 gene technology. The therapeutic concentration of the small interfering RNA (sisFRP2) that interferes with the secretory frizzled-related protein 2 gene is 10-200nM. The small interfering RNA that interferes with the secretory frizzled-related protein 2 gene includes: SEQ ID NO.1: 5′-CAUCAACCGAGAUACCAAA-3′ (sisFRP2-h64231, human), SEQ ID NO.2: 5′-GACAACGACAUCAUGGAAATT-3′ (sisFRP2-763, mouse), SEQ ID NO.3: 5′-GCAAGACCAUUUACAAGCUTT-3′ (sisFRP2-872, mouse). A lentivirus containing short hairpin RNA silencing secreted frizzled-related protein 2 gene (shsFRP2) was constructed. The sequence of shsFRP2 is SEQ ID NO.4: 5′-GCAAGACCAUUUACAAGCUTT-3′.

[0008] On the other hand, the present invention also provides a drug for preventing and treating uterine scars, comprising one or more of the following substances: small interfering RNA that interferes with secretory frizzled-related protein 2 gene, a lentivirus containing short hairpin RNA to silence secretory frizzled-related protein 2 gene, a sFRP2 inhibitor, a lipid nanoparticle containing small interfering RNA that interferes with secretory frizzled-related protein 2 gene, or a composite hydrogel thereof.

[0009] Specifically, the small interfering RNA that interferes with the secretory frizzled-related protein 2 gene includes sisFRP2-h64231, sisFRP2-763 and sisFRP2-872, the nucleotide sequence of sisFRP2-h64231 is shown in SEQ ID NO.1, the nucleotide sequence of sisFRP2-763 is shown in SEQ ID NO.2, and the nucleotide sequence of sisFRP2-872 is shown in SEQ ID NO.3.

[0010] Specifically, the nucleotide sequence of the lentivirus containing short hairpin RNA for silencing secretory frizzled-related protein 2 gene is shown in SEQ ID NO.4.

[0011] On the other hand, the present invention also provides a lipid nanoparticle for preventing and treating uterine scars, comprising small interfering RNA interfering with secretory frizzled-related protein 2 gene, ionizable lipid, distearoylphosphatidylcholine (DSPC), cholesterol, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), the molar ratio of ionizable lipid, DSPC, cholesterol, and DMG-PEG being 40-60:10-20:15-40:1-20, and the mass ratio of ionizable lipid to small interfering RNA interfering with secretory frizzled-related protein 2 gene being 5:1-30:1. The ionizable lipid is selected from DLin-MC3-DMA or ALC-0315.

[0012] The present invention also provides a method for preparing the above-mentioned lipid nanoparticles for preventing and treating uterine scars, comprising: (1) mixing and dissolving ionizable lipids, DSPC, cholesterol and DMG-PEG in ethanol according to the above-mentioned molar ratio, (2) dissolving small interfering RNA that interferes with the secretory frizzled-related protein 2 gene in a citric acid buffer, (3) mixing the ethanol phase and the aqueous phase through a microfluidic device, dialyzing the mixed solution in a PBS solution for two hours to prepare lipid nanoparticles, and storing the lipid nanoparticles at 4°C.

[0013] The molar ratio of the above ionizable lipid, DSPC, cholesterol, and DMG-PEG is 40-60:10-20:15-40:1-20, and the mass ratio of the ionizable lipid to the small interfering RNA interfering with the secretory frizzled-related protein 2 gene is 5:1-30:1. The ionizable lipid includes DLin-MC3-DMA or ALC-0315.

[0014] Finally, the present invention also provides a composite hydrogel, comprising the above-mentioned lipid nanoparticles, polycarbophil and poloxamer, wherein the final concentration of polycarbophil is 0.1%-0.5%, and the final concentration of poloxamer is 16%-23%.

[0015] On the basis of the above-mentioned preparation of lipid nanoparticles, a composite hydrogel was prepared, including dispersing and swelling polycarbophil (PCP) in ultrapure water, adding different concentrations of poloxamer 407 (PX) thereto, and incubating at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of lipid nanoparticles was added to the gel solution to prepare a composite hydrogel. The final concentration of the lipid nanoparticles was 0.5-10 mg / mL. The final concentration of polycarbophil was 0.1%-0.5%, and the final concentration of poloxamer was 16%-23%.

[0016] Application of the lipid nanoparticles or composite hydrogel in the preparation of medicines for treating uterine scar diseases.

[0017] The lipid nanoparticles and composite hydrogel are used in the preparation of medicines for improving the pregnancy rate of women with a history of uterine scar and reducing adverse pregnancy outcomes.

[0018] The lipid nanoparticles and composite hydrogels are used in the preparation of medicines for preventing and treating fibrosis-related diseases.

[0019] The target gene secretory frizzled-related protein 2 gene (sFRP2) can promote fibrosis and collagen accumulation, thereby leading to the development of uterine scars. The present invention uses small interfering RNA and virus that interfere with the secretory frizzled-related protein 2 gene to effectively reduce local uterine fibrosis and inhibit the occurrence of uterine scars. This is the first study on the prevention and treatment of uterine scars by the target gene sFRP2 at home and abroad. At the same time, other fibrosis-related scar diseases can also be prevented and treated.

[0020] The present invention relates to reducing, inhibiting, silencing and interfering with the expression of the target gene, which can improve the pregnancy rate of pregnant women and reduce adverse pregnancy outcomes.

[0021] The beneficial effects of the present invention include at least:

[0022] (1) The present invention is the first to discover that the target gene (sFRP2) can lead to fibrosis and collagen deposition and promote the occurrence and development of uterine scars, laying a foundation for subsequent research on the prevention and treatment of uterine scars or other fibrotic diseases.

[0023] (2) The interfering RNA and virus provided by the present invention are simple to synthesize and can reduce, interfere with, and silence the target gene (sFRP2) to achieve the purpose of preventing and treating uterine scars, thus filling the gap in drug treatment of uterine scars.

[0024] (3) The lipid nanoparticles provided by the present invention are formed by using ionizable lipids to encapsulate small interfering RNA (sisFRP2) that interferes with the secretory frizzled-related protein 2 gene, which can increase the circulation time of sisFRP2 in the body. The lipid nanoparticles are neutral in the body and have been proven to be reliable in safety by clinical studies.

[0025] (4) The composite hydrogel provided by the present invention is drip-injected into the local uterine wound, avoiding the first-pass elimination effect, and has good biocompatibility and is non-invasive. It can also achieve sustained release of drugs. The drug is administered once after uterine surgery and before abdominal closure, avoiding multiple administrations and improving patient compliance.

[0026] (5) The composite hydrogel provided by the present invention contains polycarbophil adhesive, which can expand on the surface of the uterus and adhere tightly to the uterus. It has strong viscosity and can be retained in the uterus for more than 28 days, thereby prolonging the drug action time, reducing the number of dosing times, and achieving good therapeutic effects.

[0027] (6) The composite hydrogel provided by the present invention is a thermosensitive in situ hydrogel, which is convenient and quick to apply locally, has low cost, improves the pregnancy rate of the mother while treating uterine scars, reduces the adverse pregnancy outcomes caused by scars, and has no obvious side effects on the mother and fetus. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The transcriptome sequencing results (heat map, volcano map) of the myometrium tissue of the normal uterus (Normal) and uterine scar (US) of pregnant women, as well as the results of Western blot and qPCR quantitative statistics of sFRP2 expression;

[0029] Figure 2 Results of Western blot and qPCR quantitative analysis of sFRP2 in uterine tissues of normal mice (Control) and mice with uterine scars (Model);

[0030] Figure 3 To quantify the expression of fibrinogen, type I collagen and type III collagen in uterine tissue of sFRP2 knockout mice by Western blot and qPCR;

[0031] Figure 4 Transwell experiment was used to observe the migration of primary uterine fibroblasts from uterine scar mice (Model) and sFRP2 knockout uterine scar mice (sFRP2KO+Model);

[0032] Figure 5 Transmission electron microscopy images and particle size distribution of small interfering RNA lipid nanoparticles encapsulating secretory frizzled-related protein 2 gene interference;

[0033] Figure 6 This is an experiment on the inhibition of collagen contraction by blank lipid nanoparticles and lipid nanoparticles with different concentrations encapsulating small interfering RNA that interferes with the secretory frizzled-related protein 2 gene;

[0034] Figure 7 is the scanning electron microscopy image of the composite hydrogel;

[0035] Figure 8 The composite hydrogel was instilled into the uterus of mice with uterine scars, and the retention of small interfering RNA that interferes with the secreted frizzled-related protein 2 gene was observed at different times;

[0036] Fig. 9 After different treatments were performed on mice with uterine scar, Masson staining and H&E staining were used to observe collagen deposition, myometrial defects and glandular distribution in tissue sections. DETAILED DESCRIPTION

[0037] The embodiments are provided to better illustrate the present invention, but the present invention is not limited to the embodiments. Various substitutions and changes made according to common technical knowledge and conventional means in the art without departing from the technical concept of the present invention should be included in the scope of the present invention.

[0038] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, materials or combinations thereof may exist or may be added.

[0039] The embodiment of the present invention provides a product for preventing and treating uterine scars based on the secretory frizzled-related protein 2 gene as a new target, including small interfering RNA (sisFRP2) interfering with the secretory frizzled-related protein 2 gene, lentivirus containing short hairpin RNA silencing the secretory frizzled-related protein 2 gene (shsFRP2), sFRP2 inhibitor, sFRP2 gene knockout mice, small interfering RNA lipid nanoparticles containing the secretory frizzled-related protein 2 gene interfering, and composite hydrogel.

[0040] It should be noted that the small interfering RNA (sisFRP2) that interferes with the secretory frizzled-related protein 2 gene, the lentivirus containing shsFRP2, and the sFRP2 inhibitor have low biocompatibility and short circulation time in the body. The lipid nanoparticles in the present invention can not only prolong the circulation time of sisFRP2 in the body by combining sisFRP2 with ionizable lipids, but also increase the effect of preventing and treating uterine scars; and, since the lipid nanoparticles are not charged in the body and are neutral, the lipid nanoparticles have reliable safety. Some ionizable lipid nanoparticles have been marketed and applied in clinical practice, so it is feasible to use ionizable lipid nanoparticles as carriers of sisFRP2.

[0041] It should be noted that the lipid nanoparticles have low adhesion in the local application of the uterus, so the retention time in the uterus is short. We use gel matrix poloxamer mixed with the above-mentioned lipid nanoparticles to extend the retention time of sisFRP2 in the uterus. In order to further improve the adhesion of the hydrogel, the present invention uses polycarbophil mixed with poloxamer and lipid nanoparticles to prepare a mixed hydrogel, which is instilled in the local uterine wound to avoid the first-pass elimination effect, and has good biocompatibility and non-traumatic properties. It can also achieve the sustained release of drugs, and is administered after surgery to avoid multiple administrations, thereby improving the patient's compliance. The hydrogel can be retained in the vagina for more than 28 days, reducing the number of administrations and improving the therapeutic effect. The composite hydrogel provided by the present invention is a thermosensitive in situ hydrogel, which is convenient and fast for local application, has low cost, can reduce adverse pregnancy outcomes on the basis of preventing and treating uterine scars, and has no obvious side effects on the mother and fetus.

[0042] It should also be noted that the secreted frizzled-related protein 2 gene involved in the present invention is used as a new target not only for the prevention and treatment of uterine scars, but can also be further applied to diseases related to fibrosis and collagen deposition.

[0043] It should be noted that a lentivirus containing short hairpin RNA (shsFRP2) was constructed to knock out the target gene sFRP2. The sequence of the constructed shsFRP2 was 5′-GCAAGACCAUUUACAAGCUTT-3′.

[0044] It should be noted that in order to reduce the expression of the target gene sFRP2, the present invention constructed small interfering RNA (sisFRP2) for humans and mice respectively, and the constructed sisFRP2 sequences are: 5′-CAUCAACCGAGAUACCAAA-3′ (sisFRP2-h64231, human), 5′-GACAACGACAUCAUGGAAATT-3′ (sisFRP2-763, mouse), 5′-GCAAGACCAUUUACAAGCUTT-3′ (sisFRP2-872, mouse).

[0045] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0046] Example 1

[0047] Ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 50:10:38.5:1.5; small interfering RNA (sisFRP2-h64231) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of DLin-MC3-DMA to sisFRP2-h64231 was 15:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0048] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 1 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0049] Example 2

[0050] Ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 50:10:38.5:1.5; small interfering RNA (sisFRP2-763) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of DLin-MC3-DMA to sisFRP2-763 was 15:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0051] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 1 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0052] Example 3

[0053] Ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 50:10:38.5:1.5; small interfering RNA (sisFRP2-872) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of DLin-MC3-DMA to sisFRP2-872 was 15:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0054] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 1 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0055] Example 4

[0056] Ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 50:10:38.5:1.5; small interfering RNA (sisFRP2-h64231) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of DLin-MC3-DMA to sisFRP2-h64231 was 15:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0057] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 2 mg / mL. The final concentrations of PCP and PX were 0.5% and 16%, respectively.

[0058] Example 5

[0059] Ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 50:10:38.5:1.5; small interfering RNA (sisFRP2-763) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of DLin-MC3-DMA to sisFRP2-763 was 15:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0060] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 2 mg / mL. The final concentrations of PCP and PX were 0.5% and 16%, respectively.

[0061] Example 6

[0062] Ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 50:10:38.5:1.5; small interfering RNA (sisFRP2-872) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of DLin-MC3-DMA to sisFRP2-872 was 15:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0063] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 2 mg / mL. The final concentrations of PCP and PX were 0.5% and 16%, respectively.

[0064] Example 7

[0065] Ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 60:20:15:5; small interfering RNA (sisFRP2-h64231) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of DLin-MC3-DMA to sisFRP2-h64231 was 15:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0066] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 1 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0067] Example 8

[0068] Ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 60:20:15:5; small interfering RNA (sisFRP2-763) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of DLin-MC3-DMA to sisFRP2-763 was 15:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0069] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 1 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0070] Example 9

[0071] Ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 60:20:15:5; small interfering RNA (sisFRP2-872) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of DLin-MC3-DMA to sisFRP2-872 was 15:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0072] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 1 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0073] Example 10

[0074] Ionizable lipid (ALC-0315), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 40:10:30:20; small interfering RNA (sisFRP2-h64231) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of ALC-0315 to sisFRP2-h64231 was 5:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0075] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 0.5 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0076] Embodiment 11

[0077] Ionizable lipid (ALC-0315), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 40:10:30:20; small interfering RNA (sisFRP2-763) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of ALC-0315 to sisFRP2-763 was 10:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0078] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 0.5 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0079] Example 12

[0080] Ionizable lipid (ALC-0315), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 40:10:30:20; small interfering RNA (sisFRP2-872) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of ALC-0315 to sisFRP2-872 was 30:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0081] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 0.5 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0082] Embodiment 13

[0083] Ionizable lipid (ALC-0315), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 40:10:30:20; small interfering RNA (sisFRP2-h64231) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of ALC-0315 to sisFRP2-h64231 was 5:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0084] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 5 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0085] Embodiment 14

[0086] Ionizable lipid (ALC-0315), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 40:10:30:20; small interfering RNA (sisFRP2-763) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of ALC-0315 to sisFRP2-763 was 10:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0087] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 5 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0088] Embodiment 15

[0089] Ionizable lipid (ALC-0315), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 40:10:30:20; small interfering RNA (sisFRP2-872) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of ALC-0315 to sisFRP2-872 was 30:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0090] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 5 mg / mL. The final concentrations of PCP and PX were 0.3% and 18%, respectively.

[0091] Example 16

[0092] Ionizable lipid (ALC-0315), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 45:15:38:2; small interfering RNA (sisFRP2-h64231) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of ALC-0315 to sisFRP2-h64231 was 5:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0093] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 10 mg / mL. The final concentrations of PCP and PX were 0.1% and 23%, respectively.

[0094] Embodiment 17

[0095] Ionizable lipid (ALC-0315), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 45:15:38:2; small interfering RNA (sisFRP2-763) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of ALC-0315 to sisFRP2-763 was 10:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0096] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 10 mg / mL. The final concentrations of PCP and PX were 0.1% and 23%, respectively.

[0097] Embodiment 18

[0098] Ionizable lipid (ALC-0315), distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) were mixed and dissolved in 112.5 μL ethanol at a molar ratio of 45:15:38:2; small interfering RNA (sisFRP2-872) that interferes with the secretory frizzled-related protein 2 gene was dissolved in 337.5 μL 10 mM citric acid buffer, and the mass ratio of ALC-0315 to sisFRP2-872 was 30:1; then the ethanol phase and the aqueous phase were mixed through a microfluidic device, and the mixed solution was dialyzed in PBS solution for two hours to obtain lipid nanoparticles, which were stored at 4°C.

[0099] Polycarbophil (PCP) was dispersed and swollen in ultrapure water, to which poloxamer 407 (PX) was added and incubated at 4°C to obtain a gel solution. After 24 hours, an appropriate amount of the above lipid nanoparticles was added to the gel solution, and the final concentration of the lipid nanoparticles was 10 mg / mL. The final concentrations of PCP and PX were 0.1% and 23%, respectively.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

[0101] Figure 1 Heatmaps, volcano plots, and Western blot and qPCR quantitative statistical results of transcriptome analysis for three myometrial samples from human uterus in the uterine scar group (US) and normal group (Normal). Written informed consent was obtained from all participants before tissue collection. Female patients aged 25-35 years, with singleton pregnancy, at least one history of cesarean section, and diagnosed with uterine scar were included in our study as the US group. Primiparas aged 25-35 years with full-term pregnancy who were scheduled for elective cesarean section were included in the Normal group. Participants were excluded if they had pregnancy complications such as diabetes, cardiovascular disease, infectious diseases, autoimmune diseases, chronic kidney disease, chronic hypertension, and metabolic diseases. The collected samples were immediately frozen in liquid nitrogen and stored at -80°C for subsequent transcriptome analysis, Western blot, and qPCR experiments. According to Figure 1 The results showed that sFRP2 was highly expressed in human uterine scar tissue.

[0102] Figure 2 The Western blot and qPCR quantitative statistical results of sFRP2 in the uterine tissue of mice in the control group and the model group. Figure 2 It can be concluded that compared with the control group, sFRP2 was highly expressed in the model group mice, which is consistent with the conclusion in humans.

[0103] Figure 2 The construction methods of the control and model mice involved are as follows:

[0104] Female C57BL / 6J mice (5-6 weeks, 15-20 g) were anesthetized with isoflurane, and the skin was incised in the lower abdomen to expose the uterine horns. A longitudinal incision (1 cm) was made in the middle of the uterine horn using ophthalmic scissors. The incision penetrated the myometrium and endometrium on the surgical side. It was closed with 8-0 absorbable sutures, and the ends of the incision edges were marked with 8-0 non-absorbable sutures. The surgery was performed on the left uterine horn. The uterus was placed back into the abdominal cavity, and the abdominal skin was sutured using 3-0 non-absorbable sutures. Control animals underwent a lower abdominal incision but no uterine incision. 30 days after surgery, the mice were killed, and uterine tissue (1 cm) from the scar formation site (model) or the control group (control) was collected for experiments.

[0105] The Western blot and qPCR experiments in the present invention all adopt conventional experimental operation steps in the art.

[0106] Figure 3Western blot and qPCR quantitative statistics of the expression of fibrinogen, type I collagen and type III collagen in the uterine tissue of model mice (Model) and sFRP2 knockout model mice (sFRP2KO+Model). Figure 3 It can be seen that knocking out sFRP2 using lentivirus can reduce the expression of fibrinogen, type I collagen and type III collagen in uterine tissue.

[0107] Figure 3 The experiments involving mice are as follows:

[0108] The method for constructing the sFRP2 knockout model mouse (sFRP2KO+Model) was to apply a lentivirus containing a short hairpin RNA (shsFRP2, 5′-GCAAGACCAUUUACAAGCUTT-3′) to the mouse uterus for 7 days, and then Figure 2 The construction method of mice described in some contents was to undergo uterine scar surgery, and uterine tissue was collected 1 month later for Western blot and qPCR experiments. The model mice (Model) were constructed by replacing the lentivirus containing short hairpin RNA (shsFRP2) with a lentivirus containing scrambled short hairpin RNA, and the rest of the steps were the same.

[0109] Figure 4 Transwell experiment was used to observe the migration of primary uterine fibroblasts from mice with uterine scars (Model) and mice with uterine scars with target gene sFRP2 knocked out (sFRP2KO+Model). Figure 4 It can be seen that knocking out sFRP2 can inhibit the migration of fibroblasts.

[0110] Figure 4 Experiments with mice Figure 3 Consistent with the experiment shown, the collected uterine tissue was mixed with type II collagenase (0.1%) to digest the tissue and extract primary fibroblasts. Primary fibroblasts (1×10 5 Cells / well) were added to the upper chamber of each Transwell (8 μm), and fresh culture medium was added to the bottom chamber. After culturing at 37°C for 3 days, the cells that had passed through the chamber membrane were fixed and observed under a microscope after staining with 0.5% crystal violet.

[0111] The transmission electron microscopy image and particle size distribution of lipid nanoparticles prepared according to the method of Example 1 above are as follows Figure 5 shown.

[0112] Figure 6 This is an experiment on the inhibition of collagen contraction by blank lipid nanoparticles and lipid nanoparticles with different concentrations encapsulating small interfering RNA that interferes with the secretory frizzled-related protein 2 gene. Figure 6 It can be seen that small interfering RNA lipid nanoparticles encapsulating the secretory frizzled-related protein 2 gene can inhibit fibroblast contraction in a dose-dependent manner.

[0113] Figure 6 The experiments involved are as follows:

[0114] 4×10 4 Cells (400 μL) were mixed with 200 μL rat tail type I collagen matrix and then added to the wells of a 24-well plate. Cells were treated with empty lipid nanoparticles (LNPs), lipid nanoparticles encapsulating small interfering RNA that interferes with the secretory frizzled-related protein 2 gene (LNP / sisFRP2-h) at 50 and 150 nM sisFRP2-h64231 concentrations for 3 days, and then activated with 10 ng / mL TGF-β for 3 days. Cells stimulated with TGF-β for 3 days without LNP or LNP / sisFRP2-h treatment were used as the model group. The control group treated cells with PBS instead of TGF-β. The diameter changes of the collagen gel were captured using a Canoscan 9000F at 0 and 24 hours. The lipid nanoparticles were prepared according to Example 1, and the empty lipid nanoparticles were prepared according to Example 1 without adding sis FRP2-h64231.

[0115] The scanning electron microscopy image of the composite hydrogel prepared according to the method of Example 3 above is as follows Figure 7 shown.

[0116] Figure 8 The composite hydrogel was dripped into the uterus of mice with uterine scars, and the retention of small interfering RNA (Cy3-sisFRP2-872) that interferes with secretory frizzled-related protein 2 gene was observed at different times. Figure 8 It can be seen that the composite hydrogel can keep Cy3-sisFRP2-872 in the uterus for at least 28 days.

[0117] Figure 8 The experiments involved are as follows:

[0118] After inducing uterine scars in mice, a composite hydrogel (Cy3-PLNP / sisFRP2, 0.5 mg / kg of sisFRP2) containing Cy3-sisFRP2-872 was immediately applied to the surgical site. The abdominal skin was then sutured using 3-0 non-absorbable sutures. On days 0, 1, 7, 14, 21, 28, and 35 after Cy3-PLNP / sisFRP2 treatment, the uterus was isolated for ex vivo fluorescence imaging. The composite hydrogel (Cy3-PLNP / sisFRP2) was prepared according to Example 3, replacing sisFRP2-872 with Cy3-sisFRP2-872.

[0119] Fig. 9 After different treatment measures were taken to intervene in uterine scar mice, the collagen deposition, myometrial defects and glandular distribution were observed by Masson staining and H&E staining of uterine tissue sections. Fig. 9 The results showed that lipid nanoparticles LNP / sisFRP2 and composite hydrogels PLNP / sisFRP2 could inhibit local collagen deposition in the uterus, improve myometrial defects, increase the number of glands, and thus prevent and treat uterine scars, and the composite hydrogel had a better effect. Blank lipid nanoparticles had no obvious effect.

[0120] Fig. 9 The experiments involved are as follows:

[0121] The mouse uterine scar model was constructed, and blank lipid nanoparticles LNP, lipid nanoparticles LNP / sisFRP2 (0.5 mg / kg of sisFRP2-872) and composite hydrogel PLNP / sisFRP2 (0.5 mg / kg of sisFRP2-872) were instilled locally in the wound, and the model group was treated with normal saline. The control group mice underwent lower abdominal incision without uterine incision and were treated with saline. After one month of treatment, uterine tissue was taken, sliced, and collagen deposition, myometrial defects and glandular distribution were observed by Masson staining and H&E staining. The preparation method of lipid nanoparticles LNP / sisFRP2 and composite hydrogel PLNP / sisFRP2 is referenced to Example 3, and the blank nanoparticles do not contain sisFRP2.

Claims

1. Application of secreted frizzled-related protein 2 gene as a new target for the prevention and treatment of uterine scars.

2. A drug for preventing and treating uterine scar, characterized in that: The invention comprises one or more of the following substances: small interfering RNA interfering with secretory frizzled-related protein 2 gene, lentivirus containing short hairpin RNA silencing secretory frizzled-related protein 2 gene, sFRP2 inhibitor, lipid nanoparticles containing small interfering RNA interfering with secretory frizzled-related protein 2 gene, or composite hydrogel thereof.

3. The drug for preventing and treating uterine scar according to claim 2, characterized in that: The small interfering RNA that interferes with the secretory frizzled-related protein 2 gene includes sisFRP2-h64231, sisFRP2-763 and sisFRP2-872, the nucleotide sequence of sisFRP2-h64231 is shown in SEQ ID NO.1, the nucleotide sequence of sisFRP2-763 is shown in SEQ ID NO.2, and the nucleotide sequence of sisFRP2-872 is shown in SEQ ID NO.

3.

4. The drug for preventing and treating uterine scar according to claim 2, characterized in that: The nucleotide sequence of the lentivirus containing short hairpin RNA for silencing secretory frizzled-related protein 2 gene is shown in SEQ ID NO.

4.

5. A lipid nanoparticle for preventing and treating uterine scar, characterized in that: The invention comprises small interfering RNA interfering with secretory frizzled-related protein 2 gene, ionizable lipid, distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG), wherein the molar ratio of the ionizable lipid, DSPC, cholesterol and DMG-PEG is 40-60:10-20:15-40:1-20, and the mass ratio of the ionizable lipid to the small interfering RNA interfering with secretory frizzled-related protein 2 gene is 5:1-30:

1.

6. The lipid nanoparticles for preventing and treating uterine scar according to claim 5, characterized in that: The ionizable lipid is selected from DLin-MC3-DMA or ALC-0315.

7. The lipid nanoparticles for preventing and treating uterine scar according to claim 5 or 6, characterized in that: The lipid nanoparticles are prepared by the following method: (1) ionizable lipids, DSPC, cholesterol, and DMG-PEG are mixed and dissolved in ethanol according to the molar ratio described in claim 5; (2) small interfering RNA that interferes with the secretory frizzled-related protein 2 gene is dissolved in citric acid buffer; (3) the ethanol phase and the aqueous phase are mixed through a microfluidic device, and the mixed solution is dialyzed in a PBS solution for two hours to prepare lipid nanoparticles.

8. A composite hydrogel, characterized in that: The lipid nanoparticles include those described in any one of claims 5 to 7.

9. A composite hydrogel according to claim 8, characterized in that: Polycarbophil and poloxamer are also included, with the final concentration of polycarbophil being 0.1%-0.5% and the final concentration of poloxamer being 16%-23%.

10. Use of the lipid nanoparticles according to any one of claims 5 to 7, or the composite hydrogel according to claim 8 or 9 in the preparation of a drug for treating uterine scar diseases.

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