Smad3-siRNA loaded injectable hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medical treatment, in particular to Smad3-siRNA loaded injectable hydrogel and a preparation method and application thereof.The preparation method comprises the following steps that hyaluronic acid is dissolved in deionized water, then sodium periodate and an ethylene glycol solution are added, dialysis and freeze drying are conducted after the reaction is completed, an oxidized hyaluronic acid freeze-dried sample is obtained, and the Smad3-siRNA loaded injectable hydrogel is obtained; dissolving the QOHA in deionized water, adding a Gelaltt reagent-T, adjusting the pH value, dialyzing after the reaction is finished, and freeze-drying to obtain a QOHA freeze-dried sample; dissolving the QOHA freeze-dried sample in a phosphoric acid buffer salt solution, adding the dendrimer polyamidoamine solution, and obtaining QOHP hydrogel after the reaction is finished; and adding the QOHP hydrogel into the Smad3-siRNA solution, and after the reaction is finished, obtaining the hydrogel carrying the Smad3-siRNA, and recording the hydrogel as QOHP (at) siRNA. The injectable hydrogel loaded with Smad3-siRNA disclosed by the invention has the characteristic of inhibiting tissue fibrosis and is capable of promoting endometrial regeneration and fertility recovery.

Description

Technical Field

[0001] The invention relates to the field of medical technology, in particular to an injectable hydrogel loaded with Smad3-siRNA and a preparation method and application thereof. Background Art

[0002] The endometrium undergoes periodic proliferation and shedding during the female reproductive cycle, and is essential for fertility and reproductive health. However, some congenital abnormalities or acquired severe uterine damage, such as improper curettage and infection-induced intrauterine adhesions (IUA), may lead to uterine infertility, recurrent miscarriage, abnormal uterine bleeding and other obstetric complications.

[0003] For intrauterine adhesions, the current common treatments include hysteroscopic adhesion lysis, artificial hormone therapy, and placement of intrauterine contraceptive devices. Among them, hysteroscopic adhesion lysis is regarded as the gold standard for the treatment of intrauterine adhesions. Although a variety of treatments are used after surgery, such as balloon dilatation and hormone therapy, the therapeutic effect on moderate to severe intrauterine adhesions is still limited. In addition, the recurrence rate of severe intrauterine adhesions is as high as over 62%. Even if the initial treatment achieves good results, the high recurrence rate leads to a poor prognosis. What is particularly tricky is that the formation of severe fibrosis and scar tissue is often irreversible, and the TGF / β1-SMAD3 signaling pathway is considered to play a key role in this process. When the endometrium is damaged, this signaling pathway will be activated, thereby promoting the process of fibrosis. Fan Cunyi's research team from the Sixth People's Hospital of Shanghai Jiao Tong University found that Smad3-siRNA nanoparticles have a silencing effect on the Smad3 gene of about 75%, which can significantly improve local tissue fibrosis, reduce inflammation and inhibit adhesion. The relevant research results have been published in Advanced Materials [Adv Mater. 2022Feb; 34(5): e2106564].

[0004] RNA interference (RNAi), as a key gene silencing technology, has been widely used in the field of gene silencing and drug development due to its high specificity, mild side effects and easy synthesis. Among them, siRNA, as a powerful tool for RNAi therapy, shows higher specificity, individualization and safety compared to traditional therapeutic drugs. However, how to efficiently deliver biologically active siRNA to the target tissue is still a major challenge in the implementation of siRNA therapy.

[0005] As an important natural polymer for biomedical hydrogels, hyaluronic acid is widely distributed in joints, skin and connective tissues in the human body. Its excellent biocompatibility, degradability and low immunogenicity make it shine in the biomedical field and widely used in orthopedics, ophthalmology, rhinology, cosmetic medicine and cancer treatment. However, traditional hyaluronic acid hydrogels are usually weak in strength, simple in structure, prone to permanent fracture, lack of special functions, and difficult to adapt to the complex and changeable intrauterine environment, which greatly limits its scope of application.

[0006] In view of this, we propose an injectable hydrogel loaded with Smad3-siRNA and its preparation method and application. Summary of the invention

[0007] The purpose of the present invention is to provide an injectable hydrogel loaded with Smad3-siRNA and a preparation method and application thereof, so as to solve the problems raised in the above background technology.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A method for preparing a Smad3-siRNA loaded injectable hydrogel comprises the following steps:

[0010] S1. Dissolve hyaluronic acid in deionized water and then add sodium periodate and ethylene glycol solution. After the reaction is completed, dialyze and freeze-dry to obtain oxidized hyaluronic acid, which is recorded as OHA freeze-dried sample;

[0011] S2, dissolving the OHA freeze-dried sample in deionized water, adding Girardt's reagent-T, adjusting the pH, and performing a quaternization reaction. After the reaction is completed, dialyzing is performed, and freeze-drying is performed to obtain a quaternary ammonium salt-modified oxidized hyaluronic acid, which is recorded as a QOHA freeze-dried sample;

[0012] S3, dissolving the QOHA freeze-dried sample in a phosphate buffered saline solution to obtain a QOHA solution; diluting the dendrimer polyamide amine solution with a phosphate buffered saline solution to obtain a mixed solution; and then mixing the two solutions to obtain a QOHP hydrogel;

[0013] S4. Mix the Smad3-siRNA liquid with the transfection reagent, and add them to the QOHP hydrogel. The resulting mixture is recorded as QOHP@siRNA hydrogel.

[0014] As a further solution of the present invention: in the step S1, the concentration of hyaluronic acid after dissolution is 2% to 3%, and the molar ratio of hyaluronic acid: sodium periodate: ethylene glycol is 1:1:(0.5-1).

[0015] As a further solution of the present invention: in the step S1, the hyaluronic acid is dissolved in deionized water and stirred at room temperature; the sodium periodate and ethylene glycol solution are added and stirred at room temperature in the dark.

[0016] As a further solution of the present invention: in the step S2, the concentration of the OHA freeze-dried sample after dissolution is 2% to 3%, and the molar ratio of the OHA freeze-dried sample to Girardt's reagent-T is 2:(0.5-1).

[0017] As a further solution of the present invention: in the step S2, the pH is adjusted to stabilize the pH of the solution to 4.4-4.6; the quaternization reaction is carried out under stirring at room temperature in the dark.

[0018] As a further solution of the present invention: in the step S1 and step S2, the dialysis is to place the reaction solution into a 3500Da dialysis bag, and dialyze it in deionized water at room temperature and in the dark for 4 days; the freeze-drying is to transfer the solution in the dialysis bag to a centrifuge tube, and perform the dialysis in a vacuum freeze dryer at -80°C, and the obtained freeze-dried sample is sealed and stored at -20°C in the dark.

[0019] As a further solution of the present invention: in the step S3, the concentration of the QOHA freeze-dried sample after dissolution is 30% to 40%, and the concentration of the dendritic polymer polyamide amine solution after dilution is 20% to 30%, and phosphate buffer is used as the solvent; the molar ratio of the QOHA solution and the mixed solution is 1:(1-2).

[0020] As a further solution of the present invention: in step S4, the concentration of Smad3-siRNA is 50 nM, the molar concentration ratio of Smad3-siRNA solution to transfection reagent is 1:(1-2), and the reaction is carried out under sufficient stirring at room temperature.

[0021] The present invention provides a Smad3-siRNA loaded injectable hydrogel, which is prepared by the above-mentioned method for preparing the Smad3-siRNA loaded injectable hydrogel.

[0022] The present invention provides an application of a Smad3-siRNA loaded injectable hydrogel prepared by the above-mentioned preparation method in treating intrauterine adhesions.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention adds sodium periodate and ethylene glycol solution to hyaluronic acid (HA) to oxidize it into oxidized hyaluronic acid (OHA), and then adds Girardt's reagent-T to react with the oxidized hyaluronic acid to obtain quaternary ammonium salt-modified oxidized hyaluronic acid, which is denoted as QOHA. Since the quaternary ammonium salt has antibacterial properties, it can inhibit inflammation caused by local operation after surgical operation.

[0025] 2. The present invention dissolves the QOHA freeze-dried sample in phosphate buffered saline (PBS), and then dissolves siRNA in a dendrimer polyamide amine solution (PAMAM DENDRIMERGENERATION, PAMAM) to obtain an injectable hydrogel carrying Smad3-siRNA, which is recorded as QOHP@siRNA. When QOHP@siRNA is injected into the uterus, due to the mutual attraction and cross-linking forces between the molecular chains of the polymer complex, QOHP@siRNA has adhesion and can adhere to the surface of the uterine cavity, playing a role of physical support and preventing uterine cavity adhesion; at the same time, the carried Smad3-siRNA can be absorbed by endometrial cells after stable release, inhibiting the expression of fibrosis-related genes, preventing the occurrence and development of uterine cavity adhesion, and playing a role in repairing the endometrium. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The structures of HA, OHA and QOHA are 1 HNMR characterization result diagram;

[0027] Figure 2 The results of FT-IR characterization of the structures of HA, OHA and QOHA;

[0028] Figure 3 Schematic diagram of QOHP@siRNA gel formation and injectability test diagram;

[0029] Figure 4 G' and G" change amplitude diagram of QOHP hydrogel and QOHP@siRNA hydrogel;

[0030] Figure 5 The microstructure diagram of QOHP hydrogel and QOHP@siRNA hydrogel;

[0031] Figure 6 Schematic diagram of QOHP@siRNA hydrogel sustained release of siRNA;

[0032] Figure 7 HE staining and Masson staining evaluation diagram;

[0033] Figure 8 Diagram for evaluating the effect of endometrial function recovery for pregnancy experiment. DETAILED DESCRIPTION

[0034] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Example 1

[0036] The present invention provides an injectable hydrogel loaded with Smad3-siRNA, and the preparation method thereof comprises the following steps:

[0037] S1. (a) Weigh HA and dissolve it in deionized water. Stir with a magnetic stirrer at room temperature for 2 hours until it is completely dissolved; (b) Dissolve sodium periodate and slowly add it to the above solution. Stir thoroughly for 4 hours at room temperature in the dark; (c) Slowly add ethylene glycol solution to the above solution. Stir thoroughly for 1 hour at room temperature in the dark; (d) Transfer the resulting solution into a dialysis bag with a molecular weight cutoff of 3500Da, dialyze at room temperature in the dark for 4 days, and replace the deionized water dialysate 4 times a day; (e) Transfer the solution in the dialysis bag to a centrifuge tube, freeze-dry it in a vacuum freeze dryer at -80°C to obtain an OHA freeze-dried sample, and seal it and store it in the dark at -20°C. Among them, the concentration of HA after dissolution is 2% to 3% (w / v), and the molar ratio of hyaluronic acid: sodium periodate: ethylene glycol is 1:1:(0.5~1);

[0038] S2, (a) weigh the OHA freeze-dried sample and add it to deionized water, stir it on a magnetic stirrer at room temperature for 2 hours until it is completely dissolved; (b) slowly add Giraert's reagent-T to the above solution, adjust the pH to 4.4-4.6, and stir it for 4 hours at room temperature in the dark; (c) transfer the resulting solution into a dialysis bag with a molecular weight cutoff of 3500Da, dialyze it at room temperature in the dark for 4 days, and replace the deionized water dialysate 4 times a day; (d) transfer the solution in the dialysis bag to a centrifuge tube, freeze-dry it in a vacuum freeze dryer at -80°C to obtain a QOHA freeze-dried sample, and seal it and store it in the dark at -20°C. Among them, the concentration of the OHA freeze-dried sample after dissolution is 2%-3% (w / v), and the molar ratio of OHA freeze-dried sample: Giraert's reagent-T is 2: (0.5-1).

[0039] S3, (a) weigh the QOHA freeze-dried sample and add it to PBS to fully dissolve; (b) add PAMAM to PBS for dilution; (c) mix the above solutions for 20 minutes to obtain QOHP hydrogel. The concentration of the QOHA freeze-dried sample after dissolution is 30% to 40% (w / v); the concentration of PAMAM after dilution is 20% to 30%, and the molar ratio of QOHA solution:PAMAM solution is 1:(1-2);

[0040] S4, (a) fully dissolving Smad3-siRNA in a transfection reagent; (b) adding the dissolved Smad3-siRNA to the above QOHP hydrogel and mixing thoroughly for 20 minutes to obtain QOHP@siRNA hydrogel. The concentration of Smad3-siRNA is 50 nM, the molar concentration ratio of Smad3-siRNA solution to transfection reagent is 1:(1-2), and the reaction is fully stirred at room temperature.

[0041] Test Example 1

[0042] 10 mg of HA, 10 mg of the OHA freeze-dried sample obtained in step S1 of Example 1, and 10 mg of the QOHA freeze-dried sample obtained in step S2 of Example 1 were weighed and dissolved in 600 μL of deuterium oxide to prepare sample A, sample B, and sample C, respectively. Sample A, sample B, and sample C were transferred to nuclear magnetic resonance tubes, and sample A, sample B, and sample C were respectively subjected to nuclear magnetic resonance spectroscopy using a Bruker Avance NEO 400 MHz nuclear magnetic resonance instrument. 1 HNMR scan, as shown in Figure 1 HA, HASH, DAHASH shown 1 HNMR characterization diagram.

[0043] from Figure 1 It can be seen that: 1. The characteristic peak of deuterium oxide solvent is located at δ4.7ppm, and the peak of OHA 1 HNMR spectrum and HA 1 Compared with the HNMR spectrum, new characteristic peaks appeared near δ5.0ppm and δ5.1ppm, corresponding to the protons of the aldehyde group and the adjacent hydroxyl group, indicating that the aldehyde modification of HA was successful; 1 HNMR spectrum and OHA 1 Compared with the HNMR spectrum, new characteristic peaks appeared at δ7.3 ppm and δ7.5 ppm, corresponding to the formation of Schiff base between aldehyde group and hydrazine group, indicating that the quaternary ammonium salt group had been successfully grafted onto the OHA side chain.

[0044] Test Example 2

[0045] Take 10 mg of HA, OHA prepared in step S1 of Example 1, and QOHA prepared in step S2 of Example 1, and use Fourier transform infrared spectrometer Nicolet iS20 to detect the infrared absorption spectra of HA, OHA, and QOHA, and obtain the following: Figure 2 FT-IR characterization spectra of HA, OHA and QOHA are shown.

[0046] from Figure 2 The FT-IR characterization spectrum of HA shows that: 1. The main stretching vibration peak of the side chain carboxyl group of HA molecules appears at 1618 cm -1 and 1413cm -1 Compared with the FT-IR characterization spectrum of HA, the FT-IR characterization spectrum of OHA is -1 A new vibration absorption peak appeared around , which was attributed to C=O stretching, confirming the existence of aldehyde groups compared with HA; 2. The FT-IR characterization spectrum of QOHA was compared with the FT-IR characterization spectrum of OHA, at 1475cm -1 A new vibration absorption peak appeared around , which was attributed to the influence of the primary amine group (-NH2), and also confirmed that the quaternary ammonium group had been successfully grafted onto the OHA side chain.

[0047] Test Example 3

[0048] Take an appropriate amount of QOHA in Example 1 and dissolve it in PBS to a concentration of 30% (w / v); dilute PAMAM with PBS to a concentration of 20%; then mix the two solutions in a ratio of 1:1 to obtain Figure 3 The gelling ability diagram is shown.

[0049] Depend on Figure 3 A shows that: 1. QOHA solution and PAMAM solution are in liquid state at room temperature. When the two are mixed in a 1:1 ratio, they form a non-flowing state, indicating that QOHP hydrogel is successfully synthesized; the hydrogel is colorless, and for the convenience of display, it is marked with methylene blue staining; 2. Figure 3 B It can be seen that QOHP hydrogel can be injected into PBS through a syringe (26-gauge needle) and maintain the shape of the hydrogel. The word SMAD3 can be written in vitro through a 26-gauge needle. The hydrogel is colorless and is marked with eosin staining for easy display. At the same time, the above experiments prove that the hydrogel was successfully synthesized and has an injectable effect in vitro, providing an experimental basis for the non-invasive injection of the hydrogel.

[0050] Test Example 4

[0051] The QOHP hydrogel and QOHP@siRNA hydrogel prepared in Example 1 were used to prepare circular samples with a diameter of 8 mm. The two were prepared using the same mold and the rheometer was used to obtain the following at 37°C: Figure 4 The storage modulus (G') and loss modulus (G") of QOHP hydrogel and QOHP@siRNA hydrogel are shown.

[0052] from Figure 4 It can be seen that: 1. The time scanning results of QOHP hydrogel and QOHP@siRNA hydrogel show that the storage modulus (G') is always higher than the loss modulus (G"), which proves that both have the properties of viscoelastic solids and have formed a relatively stable network structure ( Figure 4 A); Second, the embodiment also detected the strain scan of QOHP hydrogel and QOHP@siRNA hydrogel, confirming that the addition of Smad3-siRNA does not affect the formation of hydrogel ( Figure 4 B); 3. Figure 4 C It can be seen that QOHP hydrogel and QOHP@siRNA hydrogel have a satisfactory "sol-gel" cyclic conversion process, switching to the "sol" state (G">G') at a high shear strain of 300%, and quickly recovering 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 ( Figure 4 C).

[0053] Test Example 5

[0054] The QOHP hydrogel and QOHP@siRNA hydrogel sample-80 prepared in Example 1 were freeze-dried and the microstructure of the hydrogel was observed using a scanning electron microscope (SEM).

[0055] from Figure 5 It can be seen that the QOHP hydrogel and QOHP@siRNA hydrogel prepared in Example 1 both have a highly interconnected reticular porous structure, which is conducive to the diffusion of Smad3-siRNA. Compared with the QOHP hydrogel, the loading of Smad3-siRNA in QOHP@siRNA does not affect the microstructure of the hydrogel, proving the stability of QOHP as a Smad3-siRNA carrier.

[0056] Test Example 6

[0057] The stable and continuous release of Smad3-siRNA by QOHP@siRNA hydrogel plays an important role in the cell uptake of Smad3-siRNA and the inhibition of fibrosis. The sustained release effect of QOHP@siRNA hydrogel was evaluated by using endometrial mesenchymal stem cells to uptake Smad3-siRNA. The specific steps are as follows:

[0058] ① Take the third generation endometrial stromal cells with good growth status and plant them in a 12-well plate. When they grow to 50% of the culture dish, select them for subsequent experiments;

[0059] ② 12 hours before the experiment, the cell culture medium was replaced with serum-free medium and divided into 4 groups:

[0060] 1) Fibrosis cell model: normal cells were added with 10ug / ml transforming growth factor-β (TGF-β), denoted as T group;

[0061] 2) QOHP hydrogel was added to the fibrosis cell model, and the concentration of QOHP hydrogel added to the culture medium was 10% (w / v), which was recorded as the T+QOHP group;

[0062] 3) Smad3-siRNA was added to the fibrotic cell model, and the final concentration of Smad3-siRNA after transfection was 50 nM, which was recorded as T+siRNA group;

[0063] 4) The fibrosis cell model was added with QOHP@siRNA hydrogel, denoted as T+QOHP@siRNA group;

[0064] Hoechest was used to label the cell nucleus (blue), Dio was used to label the cell membrane (green), and Cy-3 was used to label Smad3-siRNA (red). Immunofluorescence photography was performed on the cells on the 1st, 3rd, and 5th days to evaluate the cellular uptake of Smad3-siRNA.

[0065] from Figure 6 It can be seen that: 1. One day after transfection, there were red markers in the hydrogel cells of the T+siRNA group and the T+QOHP@siRNA group, indicating that Smad3-siRNA was successfully taken up by the cells, while there were no red markers in the hydrogel cells of the T group and the T+QOHP group, indicating that there was no Smad3-siRNA in the cells ( Figure 6 A); at the same time, 3 days and 5 days after transfection, there was still no red marker in the hydrogel group cells of the T group and T+QOHP group, while there was red marker in the hydrogel group cells of the T+siRNA group and T+QOHP@siRNA group ( Figure 6 B, Figure 6 C); 2. According to Figure 6D shows that there is no red marker in the cells of T and T+QOHP hydrogel groups at different time points, indicating that no Smad3-siRNA has entered the cells; the hydrogel groups of T+siRNA group and T+QOHP@siRNA group have red markers in the cells at three different time points, indicating that Smad3-siRNA has entered the cells; III. Figure 6 From the statistical results, it can be seen that the percentage of red markers in the cells of the hydrogel group of the T+QOHP@siRNA group remained at a high level at different time points, and that of the T+siRNA group was only maintained at a high level one day after transfection. As time went on, the percentage of red markers in the cells gradually decreased. The above results indicate that QOHP hydrogel can continuously and stably release Smad3-siRNA, ensuring that cells continue to take up Smad3-siRNA.

[0066] Test Example 7

[0067] The thickness, number of glands and degree of fibrosis of the endometrium are crucial for the evaluation of the degree of repair of the endometrium. In this test example, the effects of the QOHP@siRNA hydrogel prepared in Example 1, the QOHP hydrogel prepared in Example 1 and Smad3-siRNA on the thickness, number of glands and fibrosis of the endometrium of rats were evaluated by HE and MASSON staining. The specific steps are as follows:

[0068] ① Use isoflurane inhalation anesthetic to anesthetize SD female rats. After adequate anesthesia, make a 2-3 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;

[0069] ② Use sterile forceps to lift the uterus, cut a 2mm to 3mm incision parallel to the long axis of the uterus on the surface of the uterine wall, and use the end of the forceps to penetrate 0.5cm to 2cm deep into the uterus, repeatedly scrape the endometrium to produce endometrial damage, until the appearance of the uterus becomes significantly thinner, and finally use 6-0 absorbable sutures to suture the transverse wound of the uterine wall;

[0070] ③ Use a vascular clamp to clamp the upper end 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:

[0071] (A) Control group: the uterus was only incised and then sutured;

[0072] (B) IUA group: 200 μL PBS buffer solution was injected into the uterus after physical injury of the uterus;

[0073] (C) IUA+QOHP hydrogel group: 200 μL QOHP hydrogel therapeutic agent was injected into the uterine cavity after physical injury of the uterus;

[0074] (D) IUA+siRNA group: after physical injury of the uterus, 200 μL of PBS buffer solution containing Smad3-siRNA (50 nM) was injected into the uterine cavity.

[0075] (E) IUA+QOHP@siRNA hydrogel group: After physical injury of the uterus, 200 μL of QOHP@siRNA hydrogel therapeutic agent was injected into the uterine cavity.

[0076] HE histological staining was performed on the 14th day after treatment. Figure 7 Shown are HE staining and MASSON evaluation images of rats with treated intrauterine adhesions.

[0077] Figure 7 The results showed that compared with the Control group, the uterine cavity structure disappeared in the IUA group, accompanied by thinning of the endometrium, disappearance of glands and increased degree of fibrosis. Compared with the IUA group, the IUA+QOHP hydrogel group, IUA+siRNA group and IUA+QOHP@siRNA hydrogel group all showed a certain degree of increase in endometrial thickness, regeneration of gland number and reduction of fibrosis. Among them, the IUA+QOHP@siRNA hydrogel group had the best transplantation effect, which could restore the endometrial thickness and gland number closer to those of the Control group, and the percentage of fibrosis area was also closer to that of the Control group, indicating that QOHP@siRNA had a better repair effect.

[0078] Test Example 8

[0079] Pregnancy testing is considered the standard for assessing the recovery of endometrial function and is performed as follows:

[0080] ① On the 14th day after treatment, SD female rats were housed together with SD male rats in a 1:1 ratio;

[0081] ② Collect the pregnant uterus from the 14th to 18th day after cage combination, record the pregnancy rate and number of pregnancy fetuses in the uterus of the rats, and obtain the following: Figure 8 The figure shows the number of embryo implanted in the mouse uterus from the 14th to the 18th day of pregnancy. At the same time, the weight of the offspring rats in different groups was evaluated, and 10 offspring rats in each group were weighed.

[0082] Figure 8The results showed that the number of pregnant embryos in the IUA group was significantly reduced compared with the Control group, but the number of embryos in the rats was significantly increased after treatment with PBS buffer containing Smad3-siRNA (50nM), QOHP hydrogel and QOHP@siRNA hydrogel. At the same time, the number of embryos in the rats after QOHP@siRNA hydrogel treatment was closer to that of the Control group, indicating that QOHP@siRNA hydrogel can promote the recovery of endometrial function in rats; and there was no significant difference in body weight between the offspring, indicating that the treatment with PBS buffer containing Smad3-siRNA (50nM), QOHP hydrogel and QOHP@siRNA hydrogel would not affect the body weight of the offspring, proving its safety.

[0083] Although this specification is described according to implementation modes, not every implementation mode includes only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

[0084] Therefore, the above description is only a preferred embodiment of the present application and is not intended to limit the scope of implementation of the present application; that is, all equivalent changes made according to the scope of the claims of the present application are within the protection scope of the claims of the present application.

Claims

1. A method for preparing an injectable hydrogel loaded with Smad3-siRNA, characterized in that: The steps include: S1. Dissolve hyaluronic acid in deionized water and then add sodium periodate and ethylene glycol solution. After the reaction is completed, dialyze and freeze-dry to obtain oxidized hyaluronic acid, which is recorded as OHA freeze-dried sample; S2, dissolving the OHA freeze-dried sample in deionized water, adding Girardt's reagent-T, adjusting the pH, and performing a quaternization reaction. After the reaction is completed, dialyzing is performed, and freeze-drying is performed to obtain a quaternary ammonium salt-modified oxidized hyaluronic acid, which is recorded as a QOHA freeze-dried sample; S3, dissolving the QOHA freeze-dried sample in a phosphate buffered saline solution to obtain a QOHA solution; diluting the dendrimer polymer polyamide amine solution with a phosphate buffered saline solution to obtain a mixed solution; and then mixing the two solutions to obtain a QOHP hydrogel; S4. Mix the Smad3-siRNA liquid with the transfection reagent, and add them to the QOHP hydrogel. The resulting mixture is recorded as QOHP@siRNA hydrogel.

2. The method for preparing the Smad3-siRNA loaded injectable hydrogel according to claim 1, characterized in that: In the step S1, the concentration of hyaluronic acid after dissolution is 2% to 3%, and the molar ratio of hyaluronic acid: sodium periodate: ethylene glycol is 1:1:(0.5-1).

3. The method for preparing the Smad3-siRNA loaded injectable hydrogel according to claim 1, characterized in that: In the step S1, the hyaluronic acid is dissolved in deionized water and stirred at room temperature; the sodium periodate and ethylene glycol solution are added and stirred at room temperature in the dark.

4. The method for preparing the Smad3-siRNA loaded injectable hydrogel according to claim 1, characterized in that: In the step S2, the concentration of the OHA freeze-dried sample after dissolution is 2% to 3%, and the molar ratio of the OHA freeze-dried sample to Girardt's reagent-T is 2:(0.5-1).

5. The method for preparing the Smad3-siRNA loaded injectable hydrogel according to claim 1, characterized in that: In the step S2, the pH is adjusted to stabilize the pH of the solution to 4.4-4.6; the quaternization reaction is carried out under stirring at room temperature in the dark.

6. The method for preparing the Smad3-siRNA loaded injectable hydrogel according to claim 1, characterized in that: In step S1 and step S2, the dialysis is performed by placing the reaction solution into a 3500Da dialysis bag and dialyzing it in deionized water at room temperature and in the dark for 4 days; the freeze-drying is performed by transferring the solution in the dialysis bag into a centrifuge tube and performing the dialysis in a vacuum freeze dryer at -80°C, and the obtained freeze-dried sample is sealed and stored in the dark at -20°C.

7. The method for preparing the Smad3-siRNA loaded injectable hydrogel according to claim 1, characterized in that: In step S3, the concentration of the QOHA freeze-dried sample after dissolution is 30% to 40%, and the concentration of the dendrimer polyamide amine solution after dilution is 20% to 30%, and phosphate buffer is used as the solvent; the molar ratio of the QOHA solution to the mixed solution is 1:(1-2).

8. The method for preparing the Smad3-siRNA loaded injectable hydrogel according to claim 1, characterized in that: In the step S4, the concentration of Smad3-siRNA is 50 nM, the molar concentration ratio of the Smad3-siRNA solution to the transfection reagent is 1:(1-2), and the reaction is carried out under sufficient stirring at room temperature.

9. A Smad3-siRNA loaded injectable hydrogel, characterized in that: The hydrogel is prepared by the method for preparing the Smad3-siRNA-loaded injectable hydrogel according to any one of claims 1 to 8.

10. Use of the Smad3-siRNA loaded injectable hydrogel prepared by the preparation method according to any one of claims 1 to 8 in treating intrauterine adhesions.

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