Hyaluronic acid-based temperature-sensitive hydrogel SV-coated HP as well as preparation method and application of hyaluronic acid-based temperature-sensitive hydrogel SV-coated HP

Abstract

The invention belongs to the field of biological pharmacy, and relates to a temperature-sensitive hydrogel, in particular to a temperature-sensitive hydrogel SV (at) HP based on hyaluronic acid as well as a preparation method and application of the temperature-sensitive hydrogel SV (at) HP. The temperature-sensitive hydrogel SV (at) HP entraps CS-siRNA nanoparticles and VEGF (at) PLGA microspheres, and is used for realizing microenvironment responsive drug delivery to treat intrauterine adhesion. Chlorogenic acid and TGF beta1 siRNA are self-assembled through disulfide bonds to form CS-siRNA nano-particles, the CS-siRNA nano-particles are used for treating intrauterine adhesion through gene and chemical therapy, and cell and animal experiments prove that the CS-siRNA nano-particles have a good effect of improving intrauterine adhesion. Through evaluation of a real-time fluorescence quantification method, a western blot method, a cell scratch experiment, laser confocal microscope observation, inverted fluorescence microscope observation, an in-vivo experiment and the like, the SV-coated HP gel has the effects of resisting inflammation, improving endometrial fibrosis, promoting IUA rat endometrial angiogenesis and repair, inhibiting intrauterine adhesion and improving the intrauterine environment.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals and relates to a temperature-sensitive hydrogel and a preparation method and application thereof. Background Art

[0002] The uterus is formed by a mixture of endometrium, muscle tissue and connective tissue. A good endometrium is the premise and guarantee of reproductive and maternal and infant health. Damage to the basal layer of the endometrium caused by intrauterine manipulation or infection leads to menstruation, infertility, repeated pregnancy loss, infertility and other gynecological diseases, which seriously affect women's psychological and physical health. Intrauterine adhesions (IUAs), also known as Asherman's disease, are caused by the damage of the endometrium to the lower basal layer, resulting in the formation of scar tissue in the upper functional layer, which leads to partial or complete adhesion of the uterine cavity. It is the result of severe damage to the basal layer of the endometrium, accompanied by abdominal pain, oligomenorrhea, cyclical pelvic pain and amenorrhea, and has become the second most common cause of female infertility. Intrauterine adhesions can be divided into primary adhesions after pregnancy-related operations or hysteroscopic surgery, and secondary adhesions that occur again after adhesion lysis. There are many causes of IUA. Intrauterine adhesions are considered to be an iatrogenic disease caused by uterine trauma. With the increase of intrauterine surgeries such as dilation, gestational curettage, hysteroscopy and endometrial polypectomy, its incidence is also on the rise, which has a serious impact on women's fertility and mental health. In addition, the susceptibility of intrauterine adhesions may be related to non-specific factors such as age, race, nutritional status and infection process. It has become an urgent task to explore effective treatment methods for intrauterine adhesions.

[0003] At present, the treatment methods for intrauterine adhesions in clinical practice include: (1) Hysteroscopy is the gold standard for the diagnosis and treatment of intrauterine adhesions, because it provides a good perspective of the cavity and relatively accurately describes the location and degree of adhesions, which greatly facilitates treatment; (2) Methods for repairing damaged uterus also include the introduction of physical barriers such as stents, balloons, catheters, etc. into the uterine cavity; (3) Drug treatment of endometrial damage is also widely used. Studies have shown that whether estrogen is used alone or as an adjuvant treatment for intrauterine adhesions, there will be an increase in menstrual volume. None of these current methods can completely restore the structure and function of the endometrium. Therefore, new and effective solutions are urgently needed to improve the structure and function of the damaged uterus.

[0004] Studies have confirmed that transplantation of bone marrow-derived stem cells, hematopoietic stem cells, endometrial mesenchymal stem cells, embryonic stem cells, amniotic mesenchymal stem cells, and hematopoietic stem cells can effectively promote endometrial regeneration and menstrual recovery in IUA animal models or patients. They have the potential to replace damaged endometrial cells and are specific to the endometrium. Vascular endothelial growth factor (VEGF) can promote the growth of vascular endothelial cells, accelerate the migration of endothelial cells to the injured site, and promote the rapid refilling of the eroded area by new blood vessels to complete tissue reconstruction. Fibrosis markers highly expressed in endometrial tissue of IUA patients or animal models, such as the TGF-β1 / SMAD pathway, play a leading role in the study of the pathogenesis of IUA. Therefore, the occurrence of IUA can be inhibited by knocking down the expression of TGF-β1. The common disadvantage of drug therapy and stem cell therapy is that they are applied to systemic treatment, with low local retention rate in the uterus, resulting in limited therapeutic effect. The combination of conventional therapy and biomaterials can perfectly solve this limitation. Some common biomaterials, such as hydrogels and nanoparticles, can improve drug delivery efficiency and improve therapeutic effects. Among these biomaterials, hydrogels have been explored for endometrial wound repair and have good therapeutic effects, showing strong application prospects. Patent 202410017456.2 discloses a method for preparing a hydrogel for preventing intrauterine adhesions, by introducing dopamine groups into methacrylated hyaluronic acid to synthesize dopamine-modified methacrylated hyaluronic acid; the introduction of dopamine groups increases the sustained adhesion ability of the hydrogel to the endometrium and prolongs the retention time of stem cells; and the grafting of dopamine groups provides the hydrogel with integrin sites that interact with cells, which is beneficial to enhance the vitality and secretory activity of stem cells in the hydrogel, thereby further promoting endometrial regeneration and improving fertility.

[0005] Therefore, developing a novel and efficient microenvironment-responsive drug delivery system is of great significance for improving the therapeutic effect of intrauterine adhesions. Summary of the invention

[0006] In order to solve the above problems, the present invention proposes a thermosensitive hydrogel SV@HP based on hyaluronic acid and a preparation method and application thereof.

[0007] The technical solution of the present invention is achieved in this way: On the one hand, the present invention provides a method for preparing a thermosensitive hydrogel SV@HP based on hyaluronic acid, the steps of which are as follows: (1) HA is activated with an activator and then reacted with ADH. After the reaction, the pH is adjusted to neutral, dialyzed, centrifuged, and the lyophilized supernatant is HA-ADH.

[0008] (2) P407 was dissolved in organic solvent I, triethylamine solution was added, methacryloyl chloride solution was added after mixing, and reaction solution I was dialyzed after stirring. The dialyzate was freeze-dried to obtain P407-MC; reaction solution II obtained by mixing the P407-MC solution with the HA-ADH solution was mixed with the P407 solution and soaked to obtain the thermosensitive hydrogel HP; (3) Adding chlorogenic acid to a DMF aqueous solution of 3,3'-dithiodipropionic acid to react in the presence of EDC and NHS to obtain a CA-SS-COOH solution; dropping an amino-modified 50 OD TGFβ1 siRNA in a DOPE aqueous solution into a CGA-SS-COOH aqueous solution to react, dialyze, and freeze-dry to obtain CS-siRNA nanoparticles; (4) Dissolving PLGA in organic solvent II and ultrasonically mixing it with a deionized water solution of VEGF protein to form a white oil-in-water emulsion, and then adding the white emulsion into the PVA solution in batches and stirring to obtain VEGF@PLGA microspheres; (5) CS-siRNA nanoparticles and VEGF@PLGA microspheres were added to the thermosensitive hydrogel HP solution at the same time and stirred evenly to form a thermosensitive hydrogel SV@HP.

[0009] Preferably, in the above step (1), the molar ratio of HA to ADH is 1:500-5000; the activators are EDC and NHS, and the molar ratio of EDC to NHS is 1:1; the reaction temperature is 10-40°C, and the reaction time is 20-55 min.

[0010] Preferably, in the above step (2), the organic solvent I is a dioxane solution; the molar ratio of P407, triethylamine and methacryloyl chloride is 1:0.5-8:0.5-8; the molar ratio of P407-MC to HA-ADH is 1:0.1-3; the volume ratio of the reaction solution II to the P407 solution is 1:2-30, the mass percentage of the P407 solution is 15% wt, and the mass percentage of HA-modified P407 in the reaction solution II is 15% wt; and the mixed reaction conditions are 25-55°C water bath for 1-5 min.

[0011] Preferably, in the above step (3), the molar ratio of 3,3'-dithiodipropionic acid, EDC, NHS and chlorogenic acid is 1:0.1-6:0.1-6:0.1-6; the molar ratio of amino-modified 50 OD TGFβ1 siRNA to CGA-SS-COOH is 1:0.1-5; and the reaction conditions are 20-55°C for 6-48 h.

[0012] Preferably, in step (4), the organic solvent II is dichloromethane; the mass ratio of VEGF to PLGA is 1:5000-50000; the mass concentration of the PVA solution is 1-10%, and the stirring condition is 500-5000 rpm for 1-24 h.

[0013] Preferably, in the above step (5), the molar ratio of the thermosensitive hydrogel HP, CS-siRNA nanoparticles and VEGF@PLGA microspheres is 500-50000:1:1-300; and the stirring temperature is 15-60°C.

[0014] Preferably, the dialysis bag model of the above dialysis is 1000-12000 Da, the dialysis fluid is deionized water, and the dialysis time is 12-50 h.

[0015] In a second aspect, the thermosensitive hydrogel SV@HP is prepared by the above-mentioned preparation method of the thermosensitive hydrogel SV@HP.

[0016] Thirdly, the application of the above-mentioned thermosensitive hydrogel SV@HP in anti-inflammation, anti-fibrosis and promoting angiogenesis.

[0017] Fourthly, the application of the above-mentioned thermosensitive hydrogel SV@HP in treating intrauterine adhesions and improving the intrauterine environment.

[0018] A fifth aspect is the use of the above-mentioned thermosensitive hydrogel SV@HP in the preparation of drugs for treating intrauterine adhesions.

[0019] The present invention has the following beneficial effects: 1. This application prepares a thermosensitive hydrogel SV@HP based on hyaluronic acid for the treatment of intrauterine adhesions by achieving microenvironment-responsive drug delivery. The SV@HP hydrogel has good in vitro biosafety and biocompatibility, and has the effect of promoting cell proliferation; the cell scratch experiment shows that the cell migration distance of the V@HP hydrogel and SV@HP hydrogel groups increases significantly, and the SV@HP hydrogel group is almost completely closed, which confirms that the SV@HP hydrogel has good cell migration ability and contributes to angiogenesis; 2. The present application observed through an inverted fluorescence microscope that the number of knots, mesh number and total length of CS@HP hydrogel were lower than those of V@HP hydrogel group, and SV@HP hydrogel group showed the best tube-forming ability, confirming that VEGF@PLGA microspheres can improve tube-forming ability, promote angiogenesis and wound healing; intrauterine injection of SV@HP hydrogel for treatment can form a physical barrier, prevent liquid leakage and adhesion after drug release, and provide a matrix environment to inhibit wound inflammation and fibrosis in the early stage, and promote angiogenesis in the later stage. H&E staining results showed that the endometrial thickness, blood vessels and gland numbers of IUA rats in the simple hydrogel group and the drug-loaded hydrogel group increased significantly.

[0020] 3. Through in vivo experiments, the present application found that the expression and co-localization of Ki67 and CD31 increased after treatment with the drug-loaded hydrogel SV@HP, indicating stronger angiogenesis, which accelerated the development of the vascular structure at the site of intrauterine adhesion, and had a rapid angiogenic response, stimulating endothelial cell proliferation and tissue formation, promoting the upregulation of angiogenic factors, accelerating wound healing, faster epithelialization, improving granulation tissue maturation and enhancing vascularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 Figure 2 is a synthetic route map; (a) is the synthetic route of HA-ADH, and (b) is the synthetic route of P407-MC.

[0023] Figure 2 For HA and HA-ADH 1 H NMR (500 MHz, D2O) spectrum.

[0024] Figure 3 The CCK-8 method was used to determine the effect of hydrogel on the cytotoxicity of HUVECs.

[0025] Figure 4 The proliferation effects of different hydrogel groups on HUVECs cells; (a) shows the proliferation of HUVECs cells after co-culture of hydrogel and HUVECs cells for 24 h, and (b) shows the statistical chart of cell proliferation rate.

[0026] Figure 5 The effects of different hydrogel groups on HUVECs cell migration, where (a) is the scratch healing experiment to detect the effects of each group of hydrogels on HUVECs cell migration after 0 h and 24 h of treatment, and (b) is the statistical graph of cell migration rate.

[0027] Figure 6 The effects of different hydrogel treatments on the in vitro tube-forming ability of HUVECs cells; (a) is a representative picture of the in vitro tube-forming ability of HUVECs cells after 6 h of hydrogel treatment, (b) is the number of grids, (c) is the number of knots, and (d) is a statistical chart of the total tube length.

[0028] Figure 7The effects of different hydrogel treatments on fibroblast migration; (a) is a wound healing experiment, (b) is a transwell migration experiment to detect the effect of hydrogel treatment on NIH-3T3 cell migration, (c) is the cell migration area, and (d) is a statistical chart of the migration number.

[0029] Figure 8 The following are pictures of the uterine morphology 14 and 28 days after administration.

[0030] Fig. 9 The effects of drug treatment on the morphology of the endometrium and the number of blood vessels and glands in rats; (a) is the H&E staining picture, (b) is the endometrial thickness, (c) is the number of glands, and (d) is the statistical chart of the number of blood vessels.

[0031] Fig.10 The effects of drug treatment on endometrial neovascularization; (a) is the immunofluorescence co-localization staining image of CD31 and Ki67 in uterine sections; (b) is Ki67, and (c) is the average absorbance quantification of CD31. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with 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.

[0033] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0034] The main experimental reagents and instruments used in the present invention are briefly introduced as follows: hyaluronic acid (HA, 98000Da), poloxamer (P407), adipic acid dihydrazide (ADH), methacryloyl chloride, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide.

[0035] Amine hydrochloride (EDCI·HCl), N-hydroxysuccinimide (NHS), dialysis bag (MW8000); fully digital superconducting nuclear magnetic resonance hydrogen spectrometer, freeze dryer, etc.

[0036] Example 1 The preparation method of the synthesis of the hyaluronic acid thermosensitive gel (HP) of this embodiment comprises the following specific steps: 1. Preparation of HP hydrogel: 1.1 Synthesis route of HA-ADH Figure 1 As shown in (a), the specific steps are as follows: (1) Accurately weigh HA (300.0 mg, 0.79 mmol) into a flask, add distilled water as solvent, and stir until the solution is clear. Add activator EDCI (181.7 mg, 0.95 mmol) and NHS (109.1 mg, 0.95 mmol) at 0°C and react at room temperature for 30 min to activate the carboxyl groups on HA.

[0037] (2) Add ADH (413.3 mg, 2.37 mmol) to the above flask. After it is completely dissolved, adjust the pH to 4.7 with 1N hydrochloric acid and protect with argon for 2 h.

[0038] (3) After the reaction is completed, the pH is adjusted to neutral with 1 mol / L NaOH solution, and dialyzed with distilled water for 24 h. The liquid in the dialysis bag is collected, centrifuged, and the supernatant is freeze-dried to obtain HA-ADH.

[0039] 1.2 Synthesis route of P407-MC Figure 1 (b), the specific steps are as follows: (1) To obtain methacrylate-modified poloxamer P407, 5 g of P407 was first dissolved in 20 mL of dioxane, and 333 μL of triethylamine solution was added at a ratio of 1:4 M. The reaction mixture was stirred at 37 °C using a magnetic stirring bar for 1 h.

[0040] (2) Then, 220 μL of methacryloyl chloride solution was added at a ratio of 1:4 M, and the reaction was stirred at 37 °C for 12 h under argon protection.

[0041] (3) Next, the reaction solution was poured into an 8000 Da dialysis bag and dialyzed for 48 h. During this period, purified water was replaced every 6 hours. The dialyzed solution was lyophilized in a vacuum freeze dryer to obtain double bond P407 (P407-MC).

[0042] 1.3 Preparation of HP hydrogel 15% (w / w) double-bond P407 solution and 1% (w / v) amino-modified hyaluronic acid were mixed in a ratio of 1:2 M, and the mixture was reacted for 12 h to obtain HA-modified P407 through Michael addition reaction. The reaction product was then mixed with 15% (w / w) P407 solution in a volume ratio of 1:10 and immersed in a 37°C water bath for 60 s to obtain the thermosensitive hydrogel HP.

[0043] 2. Synthesis of Chemogene Particles 2.1 Preparation of CA-SS-COOH (1) First, 3,3'-dithiodipropionic acid (1.052 g, 5 mmol) was dissolved in 35 mL of DMF aqueous solution (DMF: water = 4:3), EDC (0.958 g, 5 mmol) and NHS (0.575 g, 5 mmol) were added, and the reaction was stirred at 40 °C for 30 min.

[0044] (2) Chlorogenic acid (1.771 g, 5 mmol) was slowly added to the above solution under continuous stirring and the reaction was continued at 40 °C for 12 h to obtain CA-SS-COOH.

[0045] 2.2 Preparation of CA-SS-TGFβ1 siRNA (1) Dissolve 0.25 g of CGA-SS-COOH in 25 mL of deionized water, add EDC (0.048 g, 0.25 mmol) and NHS (0.029 g, 0.25 mmol), and stir at 40 °C for 30 min.

[0046] (2) Amino-modified 50 OD TGFβ1 siRNA (purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.) (250 nmol) was dissolved in DOPE water and added dropwise to the CGA-SS-COOH solution. The mixture was stirred at 40 °C for 12 h. The solution was dialyzed with distilled water in a dialysis bag (MW8000) for 24 h and freeze-dried to obtain the product CS-siRNA nanoparticles.

[0047] 3. Preparation of VEGF@PLGA: PLGA microspheres were prepared by water-in-oil-in-water (W / O / W) emulsification technology to encapsulate VEGF protein.

[0048] (1) Dissolve 150 mg of PLGA in 3 mL of dichloromethane and 0.01 mg of VEGF protein in 0.8 mL of deionized water containing 75 mg of VEGF.

[0049] (2) The two solutions were emulsified by ultrasonic mixing (60 W, 90 s) to form a white oil-in-water (W / O) emulsion.

[0050] (3) The white oil-in-water (W / O) emulsion was added to 20 mL of 4% (w / v) PVA solution and stirred at 1000 rpm for 5 min to form a water-in-oil-in-water (W / O / W) emulsion.

[0051] (4) The water-in-oil-in-water (W / O / W) emulsion was added to 400 mL of 0.4% (w / v) PVA solution and stirred at 500 rpm for 6 h to evaporate dichloromethane to obtain VEGF@PLGA microspheres.

[0052] (5) The VEGF@PLGA microspheres prepared above were washed three times with distilled water, centrifuged at 4000 rpm for 10 min and freeze-dried.

[0053] 4. Preparation of drug-containing hydrogel: Thermosensitive hydrogel HP was added with CS-siRNA nanoparticles and VEGF@PLGA microspheres separately and together in the solution state, and stirred to make them evenly dispersed. CS@HP gel, V@HP gel and SV@HP gel were formed at 37°C.

[0054] Weigh an appropriate amount (5-10 mg) of the sample HA-ADH powder to be tested, use D2O as solvent, and determine the 1 H-NMR. The results are as follows Figure 2 As shown by Figure 2 It can be seen that HA is successfully synthesized by connecting to ADH through the newly formed amide bond between its own carboxyl group and the amine group of ADH.

[0055] Application Example 1 Evaluation of the toxic effect of SV@HP gel on HUVECs cells: (1) Digest and collect HUVECs cells in the logarithmic growth phase and add 5×10 3 The cells were inoculated into 96-well plates and placed in a constant temperature incubator for 12 h.

[0056] (2) After the cells adhered to the wall, HUVECs were co-cultured with five groups of 500 μg / mL sample suspensions (blank control group, HPhydrogel group, CS@HP hydrogel group, V@HP hydrogel group, and SV@HP hydrogel group).

[0057] (3) After 24 h, 48 h, and 72 h of co-culture, add 100 μL of 10% CCK-8 solution to each well and incubate for 1 h.

[0058] (4) Record the absorbance of the suspension at 450 nm using an ELISA reader The results are as follows Figure 3As shown in the figure, CCK-8 is an enzyme-linked immunosorbent assay that measures its light absorption value at a wavelength of 450 nm, which can indirectly reflect the number of living cells to achieve the purpose of reflecting cell proliferation activity. We used CCK8 experiments to analyze the effects of different groups of hydrogel treatment on HUVECs cell proliferation, such as Figure 3 As shown. The results showed that after the hydrogel treatment in each group, the cell viability did not decrease significantly compared with the control group, indicating that the hydrogel had good in vitro biosafety for HUVECs cells. After the hydrogel groups were co-incubated with HUVECs cells for 3 days, the cell viability of SV@HP hydrogel increased significantly; after co-incubation for 5 days, both the V@HP hydrogel and SV@HP hydrogel groups increased significantly, indicating that the hydrogel loaded with VEGF microspheres can promote cell proliferation and improve cell viability. The other groups did not significantly decrease, proving that the hydrogel material has good biocompatibility.

[0059] Application Example 2 Evaluation of the effect of SV@HP gel on HUVECs cell proliferation: (1) Digest and collect HUVECs cells in the logarithmic growth phase and add 5×10 3 The cells were inoculated into 96-well plates and placed in a constant temperature incubator for 12 h.

[0060] (2) After the cells adhered to the wall, HUVECs were co-cultured with five groups of 500 μg / mL sample suspensions (blank control group, HPhydrogel group, CS@HP hydrogel group, V@HP hydrogel group, and SV@HP hydrogel group).

[0061] (3) After drug treatment, the drug-containing culture medium was discarded, and 50 μM EDU culture medium was added to each group and incubated with PC-3 cells for 2 h. After incubation, the cells were washed with PBS.

[0062] (4) After fixing the cells, wait for 30 min and discard the fixative. Then add 50 μL of glycine (concentration 2 mg / mL), incubate for 5 min and discard the glycine solution. Finally, add 100 μL of permeabilization agent (0.5% TritonX-100 in PBS), incubate for 10 min and wash with PBS.

[0063] (5) Add Apollo staining reaction solution in a dark place and incubate for 30 min. Then discard the Apollo staining reaction solution, add permeabilization agent (0.5% TritonX-100 in PBS), and wash 2-3 times.

[0064] (6) Add DAPI staining working solution and stain for 30 min. Then wash the cells with PBS and store in a dark place.

[0065] (7) Observe the cells using an inverted fluorescence microscope and calculate the cell proliferation rate using Image J.

[0066] The results are as follows Figure 4 As shown in the figure, the proliferation activity of HUVECs cells after being treated with different hydrogel groups was evaluated by EdU experiment. After being treated with HUVECs, each hydrogel group showed a proliferation trend of increasing cell number, which was significantly increased compared with the control group. This may be because hyaluronic acid coordinates cell communication by interacting with various cell receptors, affecting cell migration, proliferation, and differentiation, and has the effect of promoting cell proliferation. Among them, the proliferation effect of V@HP hydrogel group and SV@HP hydrogel was particularly significant, which was related to the effect of VEGF on promoting cell migration of HUVECs.

[0067] Application Example 3 Scratch test of SV@HP gel on HUVECs cells: (1) Digest and collect HUVECs cells in the logarithmic growth phase, and plate HUVECs at a rate of 1×10 6 The cells were inoculated into 6-well plates and placed in a constant temperature incubator for 12 h.

[0068] (2) Replace the medium with serum-free Gibco basal medium and starve the cells for 12 h.

[0069] (3) The monolayer cells were scraped with a 10 μL pipette tip, and after washing away the cell debris with PBS, the cells were incubated with five different cell suspensions (blank control group, HP hydrogel group, CS@HP hydrogel group, V@HP hydrogel group, and SV@HPhydrogel group).

[0070] (4) Take photos using a microscope at 0 h and 24 h.

[0071] (5) Calculate the scratch area and cell migration rate (%) using Image J software. ×100, where A0 and At are the initial scratch area and the scratch area after different culture times, respectively.

[0072] The results are as follows Figure 5As shown in the figure, impaired vascular function is a key factor affecting the recovery of intrauterine adhesions, and cell migration is the basis of angiogenesis. Therefore, the effect of hydrogel on HUVECs migration was evaluated by cell scratch test. After HUVECs were incubated with hydrogel solutions of each group for 24 h, the migration distance of each group increased differently compared with the control group. The migration distance of V@HP hydrogel and SV@HP hydrogel groups increased significantly, and the SV@HP hydrogel group was almost completely closed. The results show that VEGF plays a key role in the mid-term of cell migration. It shows that HP hydrogel also has good cell migration ability, and the reason may be that hyaluronic acid can also improve cell migration to the wound and contribute to angiogenesis.

[0073] Application Example 4 Effect of SV@HP gel on in vitro tube formation: (1) Add 40 μL of pre-cooled matrix gel to a 96-well plate and wait for 30 min at 37°C until gel formation. Then, plate HUVECs at a rate of 5×10 4 100 / well were seeded onto the surface of the Matrigel pre-treated well in a 96-well plate.

[0074] (2) After the cells adhered to the wall, HUVECs were co-cultured with five groups of 500 μg / mL sample suspensions (blank control group, HPhydrogel group, CS@HP hydrogel group, V@HP hydrogel group, and SV@HP hydrogel group). 50 μL of each hydrogel suspension was added to each well.

[0075] (3) After 6 h of observation, observe the tube formation under an inverted fluorescence microscope.

[0076] (4) Image J software was used to analyze the number of connections, number of meshes, and total length.

[0077] The results are as follows Figure 6 As shown in the figure, tube formation in endothelial cells is essential for angiogenesis in the repair of intrauterine adhesions. In vitro tube formation assays were used to evaluate the angiogenesis of the hydrogels. Compared with the control group, the V@HP hydrogel and SV@HP hydrogel groups presented a well-developed tubular network. Quantitative analysis further validated this observation. As shown in the figure, by comparing the number of knots, meshes, and total length of the HP hydrogel, CS@HP hydrogel, and SV@HP hydrogel groups, it was found that the number of knots, meshes, and total length of the CS@HP hydrogel were lower than those of the V@HP hydrogel group, and the SV@HP hydrogel group showed the best tube-forming ability. The results indicate that the addition of VEGF@PLGA will improve the tube-forming ability and promote angiogenesis and wound healing.

[0078] Application Example 5 Effect of SV@HP hydrogel on fibroblast migration: 1. Fibroblast scratch assay (1) Take NIH-3T3 cells in logarithmic phase and add 5×10 5 The cells were seeded at a density of 100 μg / well in 6-well plates and cultured in DMEM complete medium for 24 h.

[0079] (2) The culture medium was replaced with DMEM containing 2% serum and starved for 12 h.

[0080] (3) Use the tip of a 10 μL pipette to draw a line perpendicular to the bottom of the well plate and wash three times with PBS to remove cell debris.

[0081] (4) Culture medium containing different hydrogel suspensions (blank control group, HP hydrogel group, CS@HP hydrogel group, V@HP hydrogel group, and SV@HP hydrogel group) was added for culture.

[0082] (5) Take photos of the cells in the same field of view at 0 h and 24 h after scratching.

[0083] (6) Use Image J to calculate the scratch area and cell migration rate (%) = ×100, where A0 and At are the initial scratch area and the scratch area after different culture times, respectively.

[0084] 2. Transwell migration assay (1) Cell starvation: 6 h before the experiment, the complete culture medium was switched to low serum culture medium for cell starvation. NIH3T3 cells were resuspended in DMEM medium containing 2% FBS and counted. 4 × 10 4 The cells were added at a density of 1 / well into the upper chamber of a Transwell chamber in a 24-well plate.

[0085] (2) Drug treatment: A blank control group, HP hydrogel group, CS@HP hydrogel group, V@HPhydrogel group, and SV@HP hydrogel group were set up. The grouped solutions of different drugs were added to the lower chamber, which was placed in an incubator and waited for migration for 6 h before taking out the chamber.

[0086] (3) Fixation: Fix the cells with 4% paraformaldehyde for 10 min and wash three times with PBS.

[0087] (4) Staining: Stain with crystal violet for 5 min and wash twice with PBS.

[0088] (5) Cleaning: Use a cotton swab to gently wipe off the cells in the upper chamber.

[0089] (6) Photography: Use an inverted fluorescence microscope to observe the cells that migrated to the bottom of the chamber and take photos. Select three fields of view for each group for observation and statistics.

[0090] The results are as follows Figure 7 As shown in the figure, after each group of hydrogels were treated with NIH-3T3 cells, the scratch test was used to detect their migration ability. The results showed that SV@HP hydrogel could significantly inhibit the migration area compared with the control group. After various hydrogels were treated with NIH-3T3 cells, the Transwell test results detected their migration ability. After staining the upper chamber cells with crystal violet, it was found that SV@HP hydrogel could significantly reduce the number of migration compared with the control group.

[0091] Application Example 6 H&E staining was used to observe the histopathological changes of rats in each group 1. Establishment of IUA model in SD rats In this study, the IUA rat model was established by unilateral mechanical injury. After the rats were anesthetized with isoflurane (2-3%, inhalation), the surgery was performed after the corneal reflex and righting reflex disappeared. The rats were placed in the supine position, and the limbs were properly fixed on the surgical board. The abdominal surgical area was prepared and disinfected with 75% ethanol, and the aseptic operation was strictly followed. A longitudinal wound of about 1.0 cm long was made at the midpoint between the second nipple and the midline of the abdomen on the right side, and the skin, fat and muscle layers were cut in sequence. The right uterus of the rat was exposed, and a transverse incision of about 2 mm was made at the junction of the cervical opening and the long axis of the uterus. The rats were subjected to curettage through the incision above the cavity with a T10 scalpel, and the uterus was repeatedly scraped from top to bottom. During this period, 0.9% sodium chloride solution was used to keep the exposed uterus moist. When the walls of the uterine cavity became rough, the curettage was stopped, and the hydrogel solution was injected in situ into the injury site and irradiated under a warm lamp for 1 min until solidified. No treatment was performed on the left side as a natural control. The abdominal cavity was flushed with 0.9% sodium chloride solution, and the abdominal wall of the rat was sutured layer by layer. Animals receiving the same treatment were placed in the same cage. The survival rate of rats after surgery was improved by maintaining body temperature through infrared irradiation, supplementing glucose and saline to provide energy, and changing bedding in time to prevent wound infection.

[0092] The results are as follows Figure 8As shown in the figure, the results of uterine anatomical morphology showed that the uterus in the sham operation group had a smooth surface, uniform thickness, regular shape, normal color, elastic tissue, no congestion and necrosis, and no adhesion to the surrounding tissues; the uterus in the model group was congested and swollen, with irregular shape, pale surface, uneven thickness of uterine wall, irregular edges, narrowed uterine cavity, adhesion and wrapping with surrounding tissues, and decreased tissue elasticity; the uterus of the rats in the drug-loaded hydrogel group had a morphology similar to that of the sham operation group, with a relatively uniform thickness and elastic tissue, slightly thickened uterus, and partially congested. Only partial adhesions were seen in the simple hydrogel group, and the uterine morphology was better than that of the model group. This is related to the fact that after we injected HP solution, a physical barrier of hydrogel was formed at 37°C, which prevented liquid leakage and adhesion after drug release, and provided a matrix environment to inhibit wound inflammation and fibrosis in the early stage and promote angiogenesis in the later stage.

[0093] 2. H&E staining to observe the morphology of rat endometrium and the changes in the number of blood vessels and glands On the 14th and 28th day after drug treatment, the animals were killed, the uterine tissue was removed, the excess fat tissue was cut off, and the uterine anatomical morphology was observed and recorded after rinsing the uterine tissue with 0.9% sodium chloride solution. The uterine tissue was fixed in 4% paraformaldehyde. The uterine tissue fixed in 4% paraformaldehyde was removed, embedded in paraffin, and the slice thickness was 6 μm. Two slices were randomly selected from each wax block for dewaxing and rehydration, hematoxylin staining for 3 min, washing with water for 2 s, 1% hydrochloric acid ethanol differentiation for 1 s, washing with water for 10 min, eosin staining for 3 min, ethanol dehydration, xylene transparent, and neutral gum sealing. The overall uterine morphology was observed under an optical microscope, and three fields of view were randomly selected from each slice for photography, and the endometrial thickness was measured, and the number of blood vessels and glands was counted. The average value was taken for statistical analysis.

[0094] The results are as follows Fig. 9As shown in the figure, the results of H&E staining showed that the uterine structure of the rats in the sham operation group was intact, and the uterine cavity had a regular morphology, with the endometrium, myometrium, and exometrium from the inside to the outside. The endometrium was thicker and had clear boundaries. It was composed of neatly arranged single-layer columnar epithelial cells and stroma. Glands, blood vessels, and fibrous connective tissue were visible in the stroma. The tissue structure of the uterine cavity wall was clear, and the uterine cavity was smooth and unobstructed. On the 14th and 28th days after the modeling surgery, the endometrial layer of the rats in the model group was destroyed, the wrinkles were reduced, the epithelial cells were disordered, the fibrous connective tissue was loose, the uterine cavity was obviously narrowed and adhesions occurred, the interstitial congestion and edema occurred. Compared with the endometrium of the sham-operated rats, the number of endometrial wrinkles was reduced, the uterine cavity was narrow and almost completely closed, there were fibrotic adhesion bands in the uterine cavity, some epithelial peeling, and morphological changes were observed on the surface of some uterine cavities. The single-layer columnar epithelial cells disappeared and were covered by stratified flat epithelial cells, cuboidal epithelial cells or low columnar epithelial cells. Very few glands were visible in the basal layer, the integrity of the glands was destroyed, the interstitial inflammatory infiltration was obvious, the capillaries ruptured and bled, more inflammatory cells infiltrated, and the glands were sparse. In the drug-loaded hydrogel group, the endometrial epithelium was mostly intact 14 days after administration, with only a few epithelial cells completely falling off, some single-layer columnar epithelial cells disappeared, and were covered by flat epithelial cells, cuboidal epithelial cells or low columnar epithelial cells. The number of submucosal glands decreased, local bleeding and congestion were observed, and some angiogenesis was observed. After 28 days of administration, the uterine tissue structure was basically intact, close to the normal uterus, with more new glands and a small amount of inflammatory cell infiltration. Compared with the model group, the endometrial thickness, blood vessels and gland numbers of the simple hydrogel group and the drug-loaded hydrogel group increased significantly.

[0095] Application Example 7 Immunohistofluorescence staining to evaluate endometrial neovascularization: On the 14th and 28th days after drug treatment, the animals were killed, the uterine tissue was removed, the excess fat tissue was cut off, the uterine tissue was rinsed with 0.9% sodium chloride solution, the uterine anatomical morphology was observed and recorded, and the uterine tissue was fixed in 4% paraformaldehyde. The uterine tissue fixed in 4% paraformaldehyde was taken out and wrapped with wax, and then the uterine tissue was sliced ​​into 6 μm sections using a hand-wheeled slicer for immunohistofluorescence staining, and the steps were as follows: (1) Antigen repair: Take a certain amount of pH 6 citrate buffer solution, microwave it until boiling, add the solution, put it in the slide rack, heat it on medium-high heat for 8 minutes, stop heating for 5 minutes, add the solution, repeat the above steps, and repair it 3 times in total. After the repair is completed, dry the slide and wash it 3 times with PBS buffer, 5 minutes each time.

[0096] (2) Reduce nonspecific staining: After the slide is dried, draw a circle with a tissue pen. Then add tissue autofluorescence quencher A, leave it at room temperature for 30 minutes, and wash it with pure water for 5 minutes; then add tissue autofluorescence quencher B, leave it at room temperature for 5 minutes, and rinse it with running water for 3 minutes.

[0097] (3) Punching: After the slides are dried, add 50 μL of 0.2% TritonX-100 solution, leave at room temperature for 10 minutes, and wash with PBS for 5 minutes.

[0098] (4) Blocking: After the slides are dried, add 50 μL of 5% BSA solution and block at room temperature for 30 minutes.

[0099] (5) Primary antibody incubation: Remove the blocking solution and add 50 μL of primary antibody diluted in 0.5% BSA (1:150). Place the sections flat in a humidified chamber and incubate overnight at 4°C.

[0100] (6) Secondary antibody incubation: Wash with PBS three times, 5 minutes each time. After drying slightly, add 50 μL of the secondary antibody corresponding to the primary antibody to cover the tissue and incubate at room temperature for 50 minutes in the dark.

[0101] (7) Sealing: Wash with PBS three times, 5 minutes each time. After drying, seal the slides with antifade mounting medium.

[0102] (8) Microscopic examination: Images were captured using a Leica inverted fluorescence microscope.

[0103] The experimental results are as follows Fig.10 As shown in the figure, immunofluorescence staining was used to detect endometrial angiogenesis. We used Ki67 and CD31 immunofluorescence colocalization to verify the proliferation of new blood vessels. CD31 is a platelet-endothelial cell adhesion factor that participates in the composition of the vascular wall. It can prove the existence of vascular endothelial tissue and reflect the density of microvessels. Therefore, it is often used as a marker to evaluate microangiogenesis. VEGF is a key stimulatory factor that directly acts on vascular endothelial cells. It can promote angiogenesis, increase microvessel density, and thus promote endometrial regeneration. Compared with the model group, the expression and colocalization of Ki67 and CD31 in the drug-loaded hydrogel increased after 14 and 28 days of treatment, indicating stronger angiogenesis, which accelerated the development of vascular structure at the site of intrauterine adhesion, the angiogenesis response was rapid, stimulated endothelial cell proliferation and tissue formation, promoted the upregulation of angiogenic factors, and successfully improved the healing of the wound site, accelerated healing, faster epithelialization, improved granulation tissue maturation, and enhanced angiogenesis.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a thermosensitive hydrogel SV@HP based on hyaluronic acid, characterized in that: Here are the steps: (1) P407 is dissolved in an organic solvent I, a triethylamine solution is added, the mixture is mixed, and then a methacryloyl chloride solution is added. After stirring for reaction, the reaction solution I is dialyzed, and the dialyzate is freeze-dried to obtain P407-MC; the reaction solution II obtained by mixing the P407-MC solution with the HA-ADH solution is mixed with the P407 solution, and the mixture is soaked to obtain a thermosensitive hydrogel HP; (2) Adding chlorogenic acid to a DMF aqueous solution of 3,3'-dithiodipropionic acid to react in the presence of EDC and NHS to obtain a CA-SS-COOH solution; dropping a DOPE aqueous solution of 50 OD TGFβ1 siRNA modified with amino groups into a CGA-SS-COOH aqueous solution to react, dialyze, and freeze-dry to obtain CS-siRNA nanoparticles; (3) PLGA was dissolved in organic solvent II and then ultrasonically mixed with a deionized water solution of VEGF protein to form a white oil-in-water emulsion. The white emulsion was added to the PVA solution in batches and stirred to obtain VEGF@PLGA microspheres; (4) Add CS-siRNA nanoparticles and VEGF@PLGA microspheres to the thermosensitive hydrogel HP solution at the same time and stir evenly to form a thermosensitive hydrogel SV@HP.

2. The method for preparing the thermosensitive hydrogel SV@HP according to claim 1, characterized in that: In the step (1), the organic solvent I is a dioxane solution; the molar ratio of P407, triethylamine and methacryloyl chloride is 1:0.5-8:0.5-8; the molar ratio of P407-MC to HA-ADH is 1:0.1-3; the volume ratio of the reaction solution II to the P407 solution is 1:2-30, the mass percentage of the P407 solution is 15% wt, and the mass percentage of HA-modified P407 in the reaction solution II is 15% wt; and the mixed reaction conditions are 25-55°C water bath for 1-5 min.

3. The method for preparing the thermosensitive hydrogel SV@HP according to claim 2, characterized in that: In the step (2), the molar ratio of 3,3'-dithiodipropionic acid, EDC, NHS and chlorogenic acid is 1:0.1-6:0.1-6:0.1-6; the molar ratio of amino-modified 50OD TGFβ1 siRNA to CGA-SS-COOH is 1:0.1-5; and the reaction conditions are 20-55°C for 6-48 hours.

4. The method for preparing the thermosensitive hydrogel SV@HP according to claim 3, characterized in that: In the step (3), the organic solvent II is dichloromethane; the mass ratio of VEGF to PLGA is 1:5000-50000; and the mass concentration of the PVA solution is 1-10%.

5. The method for preparing the thermosensitive hydrogel SV@HP according to claim 4, characterized in that: In the step (4), the molar ratio of the temperature-sensitive hydrogel HP, CS-siRNA nanoparticles and VEGF@PLGA microspheres is 500-50000:1:1-300; and the stirring temperature is 15-60°C.

6. The method for preparing the thermosensitive hydrogel SV@HP according to claim 5, characterized in that: The dialysis bag model of the dialysis is 1000-12000 Da, the dialysis fluid is deionized water, and the dialysis time is 12-50 h.

7. Thermosensitive hydrogel SV@HP prepared by the preparation method of the thermosensitive hydrogel SV@HP according to any one of claims 1 to 6.

8. Use of the thermosensitive hydrogel SV@HP according to claim 7 in anti-inflammation, anti-fibrosis and promoting angiogenesis.

9. Use of the thermosensitive hydrogel SV@HP according to claim 7 in treating intrauterine adhesions and improving the intrauterine environment.

10. Use of the thermosensitive hydrogel SV@HP according to claim 7 in preparing a drug for treating intrauterine adhesions.

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