Temperature-sensitive hydrogel sv@hp based on hyaluronic acid and preparation method and application thereof

Abstract

The application belongs to the field of biological pharmacy, and relates to a temperature-sensitive hydrogel, in particular to a temperature-sensitive hydrogel SV@HP based on hyaluronic acid and a preparation method and application thereof; the temperature-sensitive hydrogel SV@HP is used for loading CS-siRNA nanoparticles and VEGF@PLGA microspheres, and is used for realizing microenvironment-responsive drug delivery to treat intrauterine adhesion. The application uses CS-siRNA nanoparticles of chlorogenic acid and TGFbeta1 siRNA self-assembled through a disulfide bond, is used for gene and chemotherapy to treat intrauterine adhesion, and proves to have a good effect of improving intrauterine adhesion through cell and animal experiments. Through evaluation by real-time fluorescent quantification, Western blotting, a cell scratch test, laser confocal microscope observation, inverted fluorescence microscope observation and in vivo experiments, it is found that the SV@HP gel has the effects of anti-inflammation, improvement of endometrial fibrosis, promotion of endometrial angiogenesis and repair of IUA rats, inhibition of intrauterine adhesion and improvement of intrauterine environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biopharmaceuticals, and relates to a temperature-sensitive hydrogel and a preparation method and application thereof. BACKGROUND

[0002] The uterus is formed by the mixture of endometrium, muscle tissue and connective tissue. Good endometrium is the premise and guarantee of reproduction and maternal and infant health. Damage to the basal layer of the endometrium caused by factors such as uterine cavity operation or infection leads to menstruation, infertility, repeated pregnancy loss, infertility and other gynecological diseases, which seriously affects the psychological and physiological health of women. Intrauterine adhesion (IUAs), also known as Asherman's syndrome, is caused by damage to the lower basal layer of the endometrium, leading to the formation of scar tissue in the upper functional layer, resulting in partial or complete adhesion of the uterine cavity. It is a result of severe damage to the basal layer of the endometrium, and is accompanied by abdominal pain, oligomenorrhea, cyclic pelvic pain and amenorrhea, and has become the second most common cause of female infertility. Intrauterine adhesion can be divided into primary adhesion after pregnancy-related operation or hysteroscopy, and secondary adhesion after adhesion lysis. There are many causes of IUA, and intrauterine adhesion is considered to be a iatrogenic disease caused by uterine trauma, and with the increase in intrauterine surgery such as dilation, pregnancy curettage, hysteroscopy and endometrial polyp resection, the incidence rate also shows an upward trend, which has a serious impact on the reproductive ability and psychological health of women. In addition, the susceptibility of intrauterine adhesion may be related to non-specific factors such as age, race, nutritional status and infection process, and exploring effective treatment methods for intrauterine adhesion has become a top priority.

[0003] At present, the treatment methods for intrauterine adhesion in clinical practice include: (1) hysteroscopy is the gold standard for diagnosing and treating intrauterine adhesion, as it provides a good perspective of the uterine cavity, relatively accurately describes the location and extent of adhesion, which greatly facilitates treatment; (2) methods for repairing damaged uteruses also include introducing physical barriers such as stents, balloons, catheters, etc. into the uterine cavity; (3) there is also a wide range of drug treatments for endometrial damage. Studies have shown that the use of estrogen alone or as an adjunctive therapy for intrauterine adhesion will result in increased menstrual flow. None of these current methods can completely restore the structure and function of the endometrium. Therefore, there is an urgent need for new and effective solutions to improve the structure and function of the damaged uterus.

[0004] Studies have confirmed that bone marrow-derived stem cells, hematopoietic stem cells, endometrial mesenchymal stem cells, embryonic stem cells, amniotic mesenchymal stem cells, and hematopoietic stem cell transplantation can effectively promote the endometrial regeneration and menstrual recovery of IUA animal models or patients, and 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, promote the rapid refilling of newly formed blood vessels to the eroded area, and complete tissue reconstruction. The fibrosis markers highly expressed in the 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, so the occurrence of IUA can be inhibited by knocking down the expression of TGF-β1. The common shortcomings of drug therapy and stem cell therapy are applied to systemic treatment, and the local retention rate in the uterus is low, leading to limited treatment effect, while 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 treatment effect. Among these biomaterials, hydrogels have been explored for endometrial wound repair and have shown good treatment effect, showing strong application prospects. Patent 202410017456.2 discloses a preparation method of a hyaluronic acid hydrogel for preventing intrauterine adhesion, which synthesizes dopamine-modified methacrylated hyaluronic acid by introducing dopamine groups into methacrylated hyaluronic acid; the introduction of dopamine groups increases the sustained adhesion capacity of the hydrogel to the endometrium, prolongs the retention time of stem cells; and the grafting of dopamine groups provides the hydrogel with integrin sites for cell interaction, which is beneficial to enhancing the viability and secretory activity of stem cells in the hydrogel, thereby further promoting endometrial regeneration and improving fertility.

[0005] Therefore, it is of great significance to develop a new type of efficient microenvironment-responsive drug delivery system for improving the treatment effect of intrauterine adhesion. SUMMARY

[0006] To solve the above problems, the present application provides a temperature-sensitive hydrogel SV@HP based on hyaluronic acid and a preparation method and application thereof.

[0007] The technical scheme of the present application is as follows:

[0008] On the one hand, the present application provides a preparation method of a temperature-sensitive hydrogel SV@HP based on hyaluronic acid, and the steps are as follows:

[0009] (1) After the HA is activated with an activator, it is reacted with ADH, and after the reaction is completed, the pH is adjusted to neutral, dialyzed, centrifuged, and the supernatant is freeze-dried to obtain HA-ADH.

[0010] (2) P407 is dissolved in organic solvent I, triethylamine solution is added, after mixing, methacryloyl chloride solution is added, after stirring and reaction, reaction solution I is dialyzed, and the dialysis solution is freeze-dried to obtain P407-MC; after mixing reaction of P407-MC solution and HA-ADH solution, reaction solution II is mixed with P407 solution to obtain the temperature-sensitive hydrogel HP;

[0011] (3) CA-SS-COOH solution is obtained by adding chlorogenic acid to the DMF aqueous solution of 3,3'-dithiodipropionic acid under the condition that EDC and NHS are present; after dropwise addition of the DOPE aqueous solution of 50 OD TGFβ1 siRNA modified with amino to the aqueous solution of CA-SS-COOH, reaction, dialysis and freeze-drying, the CS-siRNA nanoparticles are obtained;

[0012] (4) After PLGA is dissolved in organic solvent II, the deionized aqueous solution of VEGF protein is ultrasonically mixed to form an oil-in-water white emulsion, the white emulsion is added to the PVA solution in batches and stirred to obtain VEGF@PLGA microspheres;

[0013] (5) After the CS-siRNA nanoparticles and VEGF@PLGA microspheres are simultaneously added to the temperature-sensitive hydrogel HP solution and stirred uniformly, the temperature-sensitive hydrogel SV@HP is formed.

[0014] Preferably, in the above step (1), the molar ratio of HA to ADH is 1:500-5000; the activator is EDC and NHS, and the molar ratio of EDC to NHS is 1:1; the reaction temperature is 10-40℃, and the reaction time is 20-55 min.

[0015] 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 reaction solution II to P407 solution is 1:2-30, the mass percentage of P407 solution is 15% wt, the mass percentage of HA-modified P407 in reaction solution II is 15% wt; and the mixing reaction condition is 25-55℃ water bath for 1-5 min.

[0016] 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 50 OD TGFβ1 siRNA modified with amino to CA-SS-COOH is 1:0.1-5; and the reaction condition is 20-55℃ for 6-48 h.

[0017] Preferably, the organic solvent II in step (4) 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 stirring at 500-5000 rpm for 1-24 h.

[0018] Preferably, the molar ratio of the temperature-sensitive hydrogel HP, the CS-siRNA nanoparticles and the VEGF@PLGA microspheres in step (5) is 500-50000:1:1-300; and the stirring temperature is 15-60℃.

[0019] Preferably, the dialysis bag used in the dialysis has a size of 1000-12000 Da, the dialysis liquid is deionized water, and the dialysis time is 12-50 h.

[0020] In the second aspect, the temperature-sensitive hydrogel SV@HP is prepared by the preparation method of the temperature-sensitive hydrogel SV@HP.

[0021] In the third aspect, the temperature-sensitive hydrogel SV@HP is used in anti-inflammatory, anti-fibrosis and pro-angiogenesis.

[0022] In the fourth aspect, the temperature-sensitive hydrogel SV@HP is used in treating intrauterine adhesion and improving intrauterine environment.

[0023] In the fifth aspect, the temperature-sensitive hydrogel SV@HP is used in preparing a drug for treating intrauterine adhesion.

[0024] The present application has the following beneficial effects:

[0025] 1. The present application prepares a temperature-sensitive hydrogel SV@HP based on hyaluronic acid, which is used for realizing microenvironment-responsive drug delivery to treat intrauterine adhesion. The SV@HP hydrogel has good in-vitro biological safety 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 the 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 is helpful for angiogenesis.

[0026] 2、The application observes the number of junctions, the number of meshes and the total length of CS@HP hydrogel to be lower than V@HP hydrogel group through inverted fluorescence microscope, and the SV@HP hydrogel group shows the best tube formation capacity, which confirms that VEGF@PLGA microspheres can improve the tube formation capacity, promote angiogenesis and wound healing; the SV@HP hydrogel is injected into the uterus for treatment, can form a physical barrier, prevent liquid leakage and adhesion after drug release, and at the same time provide a matrix environment to inhibit inflammation and fibrosis of the wound in the early stage and promote neovascularization in the later stage. The H&E staining results show that the endometrial thickness, blood vessels and gland number of the IUA rat uterus in the simple hydrogel group and the drug-loaded hydrogel group are significantly increased.

[0027] 3、The application finds that the expression and co-localization of Ki67 and CD31 increase after treatment of the drug-loaded hydrogel SV@HP through in vivo experiments, which indicates stronger neovascularization, so that the vascular structure of the intrauterine adhesion site develops faster, the angiogenic response is rapid, stimulates endothelial cell proliferation and tissue formation, promotes the up-regulation of angiogenic factors, accelerates wound healing, epithelialization faster, improves granulation tissue maturation and enhances vascular formation. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0029] Figure 1 is a synthesis route of HA-ADH, and (b) is a synthesis route of P407-MC.

[0030] Figure 2 is a synthesis route of HA and HA-ADH. 1 HNMR (500 MHz, D2O) spectrum.

[0031] Figure 3 is a CCK-8 method for determining the influence of hydrogel on HUVECs cytotoxicity.

[0032] Figure 4 is the proliferation of HUVECs cells in different hydrogel groups; (a) is the proliferation of HUVECs cells after co-culturing with hydrogel for 24 h, and (b) is a statistical diagram of cell proliferation rate.

[0033] Figure 5Effects of different hydrogels on HUVECs cell migration, wherein (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, (b) is a cell migration rate histogram.

[0034] Figure 6 Effects of different hydrogels on HUVECs cell migration, wherein (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, (b) is a cell migration rate histogram.

[0035] Figure 7 Effects of different hydrogels on HUVECs cell migration, wherein (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, (b) is a cell migration rate histogram.

[0036] Figure 8 Effects of different hydrogels on HUVECs cell migration, wherein (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, (b) is a cell migration rate histogram.

[0037] Figure 9 Effects of different hydrogels on HUVECs cell migration, wherein (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, (b) is a cell migration rate histogram.

[0038] Figure 10 Effects of different hydrogels on HUVECs cell migration, wherein (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, (b) is a cell migration rate histogram. DETAILED DESCRIPTION

[0039] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0040] The test methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained through commercial channels unless otherwise specified.

[0041] The main experimental reagents and instruments and equipment used in the application are briefly introduced as follows: hyaluronic acid (HA, 98000 Da), poloxamer (P407), adipic acid dihydrazide (ADH), methacryloyl chloride, 1-ethyl-(3-dimethylaminopropyl) carbon dihydride.

[0042] amine hydrochloride (EDCI·HCl), N-hydroxy succinimide (NHS), dialysis bag (MW8000); fully digital superconducting nuclear magnetic resonance hydrogen spectrum, freeze dryer, etc.

[0043] Example 1

[0044] The preparation method of the synthesis of hyaluronic acid temperature-sensitive gel (HP) of the embodiment is as follows:

[0045] 1. Preparation of HP hydrogel:

[0046] 1.1 The synthesis route of HA-ADH is as shown in Figure 1 (a), and the specific steps are as follows:

[0047] (1) Precisely weigh HA (300.0 mg, 0.79 mmol) into a flask, add distilled water as a 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 group on HA.

[0048] (2) Add ADH (413.3 mg, 2.37 mmol) to the above flask, and after completely dissolving, adjust the pH to 4.7 with 1N hydrochloric acid, and protect it with argon for 2 h.

[0049] (3) After the reaction is completed, adjust the pH to neutral with 1 mol / L NaOH solution, dialyze with distilled water for 24 h, collect the liquid in the dialysis bag, centrifuge, and freeze-dry the supernatant to obtain HA-ADH.

[0050] 1.2 The synthesis route of P407-MC is as shown in Figure 1 (b), and the specific steps are as follows:

[0051] (1) In order to obtain methacrylate-modified poloxamer P407, first dissolve 5 g of P407 in 20 mL of dioxane, and add 333 μL of triethylamine solution at a ratio of 1:4 M. Stir the reaction mixture at 37°C using a magnetic stir bar for 1 h.

[0052] (2) Then add 220 μL of methacryloyl chloride solution at a ratio of 1:4 M, and react by stirring at 37°C for 12 h under argon protection.

[0053] (3) Then, the reaction solution was poured into a 8000 Da dialysis bag and dialyzed for 48 h, during which the dialysis solution was replaced with purified water every 6 h, and the dialysis solution was freeze-dried in a vacuum freeze dryer to obtain double-bond P407 (P407-MC).

[0054] 1.3 Preparation of H-P hydrogel

[0055] A 15% (w / w) double-bond P407 solution was mixed with 1% (w / v) amino-modified hyaluronic acid at a ratio of 1:2 M, and the mixture was reacted for 12 h to obtain HA-modified P407 by Michael addition reaction, and then the reaction product was mixed with a 15% (w / w) P407 solution at a volume ratio of 1:10, and immersed in a 37°C water bath for 60 s to obtain a temperature-sensitive hydrogel HP.

[0056] 2. Synthesis of chemogene particles

[0057] 2.1 Preparation of CA-SS-COOH

[0058] (1) First, 3,3'-dithiodipropionic acid (1.052 g, 5 mmol) was dissolved in 35 mL of a DMF aqueous solution (DMF:water = 4:3), and 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.

[0059] (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.

[0060] 2.2 Preparation of CA-SS-TGFβ1 siRNA

[0061] (1) 0.25 g of CA-SS-COOH was dissolved in 25 mL of deionized water, and EDC (0.048 g, 0.25 mmol) and NHS (0.029 g, 0.25 mmol) were added, and the reaction was stirred at 40°C for 30 min.

[0062] (2) Amino-modified 50 OD TGFβ1 siRNA (purchased from Shanghai Jimabio Pharmaceutical Technology Co., Ltd.) (250 nmol) was dissolved in DOPE water and added dropwise to the CA-SS-COOH solution, and the reaction was stirred at 40°C for 12 h, and the solution was dialyzed against distilled water in a dialysis bag (MW8000) for 24 h, and freeze-dried to obtain the product CS-siRNA nanoparticles.

[0063] 3. Preparation of VEGF@PLGA:

[0064] PLGA microspheres were prepared using a water-in-oil-in-water (W / O / W) emulsification technique to encapsulate VEGF protein.

[0065] (1) Dissolve 150 mg PLGA in 3 mL of dichloromethane and dissolve 0.01 mg VEGF protein in 0.8 mL of deionized water containing 75 mg.

[0066] (2) The two solutions were emulsified by ultrasonic mixing (60 W, 90 s) to form a white (W / O) emulsion in water.

[0067] (3) Add the above-mentioned water-in-oil white (W / O) emulsion to 20 mL of 4% (w / v) PVA solution and stir at 1000 rpm for 5 min to form a water-in-oil-in-water (W / O / W) emulsion.

[0068] (4) Add the water-in-oil-in-water (W / O / W) emulsion to 400 mL of 0.4% (w / v) PVA solution and stir at 500 rpm for 6 h to evaporate dichloromethane to obtain VEGF@PLGA microspheres.

[0069] (5) The VEGF@PLGA microspheres prepared above were washed three times with distilled water, centrifuged at 4000 rpm for 10 min and then freeze-dried.

[0070] 4. Preparation of drug-containing hydrogels:

[0071] Thermosensitive hydrogels (HP) were prepared by adding CS-siRNA nanoparticles and VEGF@PLGA microspheres separately and together in solution, followed by stirring to ensure uniform dispersion. CS@HP gel, V@HP gel, and SV@HP gel were formed at 37°C.

[0072] Weigh an appropriate amount (5-10 mg) of the HA-ADH powder sample to be tested, and use D2O as a solvent to determine the HA and HA-NH content. 1 H-NMR. Results are as follows: Figure 2 As shown, by Figure 2 It can be seen that HA-ADH was successfully synthesized by connecting HA to ADH through a newly formed amide bond between its own carboxyl group and the amino group of ADH.

[0073] Application Example 1

[0074] Evaluation of the toxic effects of SV@HP gel on HUVECs cells:

[0075] (1) Digest and collect HUVECs cells in the logarithmic growth phase, according to 5×10 3The 5x10 3 cells / well were inoculated in 96-well plates and placed in a constant temperature incubator for continuous culture for 12 h.

[0076] (2) After the cells adhered, the HUVECs were co-cultured with five groups of 500 μg / mL sample suspensions (blank control group, HP hydrogel group, CS@HP hydrogel group, V@HP hydrogel group and SV@HP hydrogel group) respectively.

[0077] (3) After co-culturing for 24 h, 48 h and 72 h, 100 μL of 10% CCK-8 solution was added to each well, and incubated for 1 h.

[0078] (4) The absorbance of the suspension at 450 nm was recorded by an enzyme labeler

[0079] The results are shown in Figure 3 CCK-8 was measured by an enzyme-linked immunoassay instrument at a wavelength of 450 nm, which can indirectly reflect the number of living cells to achieve the purpose of reacting cell proliferation activity. We used CCK8 experiment to analyze the effect of different groups of hydrogel on HUVECs cell proliferation after treatment, as shown in Figure 3 The results showed that after treatment of hydrogel in each group, the cell viability did not decrease significantly compared with the control group, indicating that the hydrogel had good in vitro biological safety for HUVECs cells. After co-incubation of hydrogel groups and HUVECs cells for 3 d, the cell viability of SV@HP hydrogel group increased significantly; after co-incubation for 5 d, the V@HP hydrogel and SV@HP hydrogel groups both increased significantly, indicating that the hydrogel loaded with VEGF microspheres can promote cell proliferation and improve cell viability, and the other groups did not decrease significantly, proving that the hydrogel material has good biocompatibility.

[0080] Application Example 2

[0081] Evaluation of the effect of SV@HP gel on HUVECs cell proliferation:

[0082] (1) The HUVECs cells in logarithmic growth phase were digested and collected, and inoculated in 96-well plates at 5x10 3 cells / well, and placed in a constant temperature incubator for continuous culture for 12 h.

[0083] (2) After the cells adhered, the HUVECs were co-cultured with five groups of 500 μg / mL sample suspensions (blank control group, HP hydrogel group, CS@HP hydrogel group, V@HP hydrogel group and SV@HP hydrogel group) respectively.

[0084] (3) After drug treatment, discard the drug-containing medium, and add 50 μM EDU medium to each group, and incubate with PC-3 cells for 2 h. After incubation, wash with PBS.

[0085] (4) After fixing the cells, wait for 30 min, and then discard the fixing solution. Then add 50 μL glycine (concentration of 2 mg / mL), incubate for 5 min, and then discard the glycine solution. Finally, add 100 μL permeabilization reagent (0.5% TritonX-100 in PBS), incubate for 10 min, and then wash with PBS.

[0086] (5) Add Apollo staining reagent in the dark, incubate for 30 min, then discard the Apollo staining reagent, add permeabilization reagent (0.5% TritonX-100 in PBS), and wash 2-3 times.

[0087] (6) Add DAPI staining working solution, stain for 30 min, then wash the cells with PBS, and store in the dark.

[0088] (7) Observe the cells with an inverted fluorescence microscope, and count and calculate the cell proliferation rate by Image J.

[0089] The results are shown in Figure 4 Fig. 6. The proliferation activity of HUVECs treated with different hydrogels was evaluated by EdU experiment. After treatment of the hydrogels with HUVECs, the number of cells in each group showed a proliferation trend, and the number of cells in each group was significantly increased compared with the control group. This may be because hyaluronic acid promotes cell proliferation by interacting with various cell receptors to coordinate cell communication, affecting cell migration, proliferation, and differentiation. Among them, the proliferation effect of V@HP hydrogel group and SV@HP hydrogel group is particularly significant, which is related to the promotion of VEGF on HUVECs cell migration.

[0090] Application Example 3

[0091] Scratch test of SV@HP gel on HUVECs cells:

[0092] (1) Digest and collect HUVECs cells in logarithmic growth phase, and inoculate HUVECs in a 6-well plate at a concentration of 1×10 6 cells / well, and place in a constant temperature incubator for continuous culture for 12 h.

[0093] (2) Replace the serum-free Gibco basal medium and starve for 12 h.

[0094] (3) Scrape the monolayer cells with a 10 μL pipette tip, and after washing the cell debris with PBS, incubate the cells with five different cell suspensions (blank control group, HP hydrogel group, CS@HP hydrogel group, V@HP hydrogel group and SV@HP hydrogel group).

[0095] (4) Take photos by microscope at 0 h and 24 h.

[0096] (5) Calculate the scratch area by Image J software, and calculate the cell migration rate (%) = (At-A0) / A0 ×100, where A0 and At are the initial scratch area and the scratch area after different culture times, respectively.

[0097] The results are shown in Figure 5 , impaired vascular function is a key factor affecting the recovery of intrauterine adhesion, and cell migration is the basis of angiogenesis. Therefore, the effect of hydrogel on the migration of HUVECs was evaluated by cell scratch experiment. After incubation of each group of hydrogel solution with HUVECs for 24 h, the migration distance of each group was increased compared with the control group, and the migration distance of V@HP hydrogel and SV@HP hydrogel groups was significantly increased, and the SV@HP hydrogel group was almost completely closed. The results show that VEGF plays a key role in the middle stage 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 the migration of cells to the wound, which is helpful for angiogenesis.

[0098] Application Example 4

[0099] Effect of SV@HP gel on in vitro tube formation:

[0100] (1) Add 40 μL pre-cooled Matrigel to the 96-well plate, and wait for 30 min at 37°C to form a gel. Then inoculate HUVECs cells at 5×10 4 cells / well into the 96-well Matrigel pretreated surface.

[0101] (2) After the cells adhere, co-culture HUVECs with five groups of 500 μg / mL sample suspensions (blank control group, HP hydrogel group, CS@HP hydrogel group, V@HP hydrogel group and SV@HP hydrogel group). Add 50 μL of each hydrogel suspension to each well.

[0102] (3) After 6 h of observation, observe the test tube formation under an inverted fluorescence microscope.

[0103] (4) Analyze the number of connections, the number of meshes and the total length by Image J software.

[0104] Results as shown in Figure 6 Figure 6, tube formation in endothelial cells is essential for angiogenesis in the repair of intrauterine adhesion. The angiogenesis of hydrogels was evaluated by in vitro tube formation experiment. Compared with the control group, V@HP hydrogel and SV@HP hydrogel groups showed a developed tubular network. Quantitative analysis further verified this observation. As shown in the figure, by comparing the node number, mesh number and total length of HP hydrogel, CS@HP hydrogel and SV@HP hydrogel groups, it was found that the node number, 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 formation capacity. The results showed that the addition of VEGF@PLGA would improve the tube formation capacity, promote angiogenesis and wound healing.

[0105] Example 5

[0106] Effect of SV@HP hydrogel on fibroblast migration:

[0107] 1. Fibroblast scratch experiment

[0108] (1) Take the logarithmic phase growth of NIH-3T3 cells, inoculate into 6-well plates at a density of 5×10 5 cells / well, and use DMEM complete culture medium to culture for 24 h.

[0109] (2) Replace the 2% serum DMEM medium and starve for 12 h.

[0110] (3) Use the 10 μL pipette gun head to draw a line vertically on the bottom of the plate, and use PBS to clean 3 times to remove cell debris.

[0111] (4) Add 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) for culture.

[0112] (5) Photograph and record the cells in the same field of view at 0 h and 24 h after scratching.

[0113] (6) Calculate the scratch area using Image J, and calculate the cell migration rate (%) = (At-A0) / A0x100, where A0and Atare the initial scratch area and the scratch area after different culture times, respectively.

[0114] 2. Transwell migration experiment

[0115] ​(1) Cell starvation treatment: 6 h before the experiment, the complete culture medium was converted to low serum culture medium to starve the cells. NIH3T3 cells were resuspended in DMEM medium containing 2% FBS and counted at 4×10⁻⁶ cells / year. 4 The density of the pores is added to the upper chamber of the Transwell cell in the 24-well plate.

[0116] (2) Drug treatment: A blank control group, HP hydrogel group, CS@HP hydrogel group, V@HP hydrogel group and SV@HP hydrogel group were set up. The grouped solutions of different drugs were added to the lower chamber and placed in an incubator for 6 h of migration before the chamber was removed.

[0117] (3) Fixation: Fix cells with 4% paraformaldehyde for 10 min and wash with PBS 3 times.

[0118] (4) Staining: Stain with crystal violet for 5 min, and wash twice with PBS.

[0119] (5) Cleaning: Gently wipe away the cells in the upper chamber with a cotton swab.

[0120] (6) Photographs: Use an inverted fluorescence microscope to observe and photograph cells that have migrated to the bottom of the chamber. Three fields of view are selected for observation and statistical analysis for each group.

[0121] The results are as follows Figure 7 As shown, after treating NIH-3T3 cells with various hydrogels, their migration ability was detected by scratch assay. The results showed that SV@HP hydrogel significantly inhibited the migration area compared with the control group. After treating NIH-3T3 cells with various hydrogels, the migration ability was detected by Transwell assay. After staining the upper chamber cells with crystal violet, it was found that SV@HP hydrogel significantly reduced the number of migrating cells compared with the control group.

[0122] Application Example 6

[0123] H&E staining was used to observe the histopathological changes in rats in each group.

[0124] 1. Establishment of the IUA model in SD rats

[0125] The IUA rat model was established by unilateral mechanical injury. After the rats were anesthetized with isoflurane (2-3%, inhalation), the corneal reflex and righting reflex disappeared, the rats were placed in a supine position, the limbs were fixed on the operation board, the abdominal surgical area was prepared, 75% ethanol was used for disinfection, and strict aseptic operation was performed. A longitudinal incision about 1.0 cm was made at the right second nipple and the midpoint of the abdominal midline. The skin, fat and muscle layers were cut in turn. The right uterus of the rat was exposed, a transverse incision about 2 mm was made at the junction of the cervix and the long axis of the uterus. The uterus of the rat was scraped from top to bottom repeatedly using a T10 scalpel through the upper cavity incision. During the operation, 0.9% sodium chloride solution was used to keep the exposed uterus moist. When the rough feeling appeared on the four walls of the uterine cavity, the curettage was stopped, and the hydrogel solution was injected in situ to the injury site. The hydrogel was irradiated under a warm lamp for 1 min to solidify. The left side was not treated 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. The same treatment animals were placed in the same cage. The body temperature was maintained by postoperative infrared radiation, glucose and normal saline were supplemented to provide energy, and the bedding was changed in time to prevent wound infection, etc., to improve the survival rate of the rats after operation.

[0126] The results are shown in Figure 8 The results of uterine anatomical morphology showed that the uterine surface of the sham operation group was smooth, uniform in thickness, regular in shape, normal in color, and the tissue was elastic, without congestion and necrosis, and no adhesion with the surrounding tissue; the uterus of the model group was congestive and swollen, irregular in shape, with a pale surface, uneven uterine wall thickness, irregular edge, narrow uterine cavity, adhesion and encapsulation with the surrounding tissue, and decreased tissue elasticity; the uterus of the drug-loaded hydrogel group was similar to the sham operation group, with uniform uterine thickness and elastic tissue, slightly thickened uterine shape, and partial congestion. The simple hydrogel group only showed partial adhesion, and the uterine morphology was better than that of the model group. This is related to the physical barrier of the hydrogel formed at 37°C after injection of the HP solution, which prevents liquid leakage and adhesion after drug release, and provides a matrix environment to inhibit inflammation and fibrosis of the wound in the early stage and promote angiogenesis in the later stage.

[0127] 2. H&E staining method was used to observe the changes of the number of blood vessels and glands in the endometrium of rats

[0128] On days 14 and 28 after drug treatment, animals were euthanized, uterine tissue was harvested, excess adipose tissue was removed, and the uterine tissue was rinsed with 0.9% sodium chloride solution. The anatomical morphology of the uterus was observed and recorded. The uterine tissue was then fixed in 4% paraformaldehyde. The uterine tissue fixed in 4% paraformaldehyde was removed, embedded in paraffin, and sectioned to a thickness of 6 μm. Two sections were randomly selected from each paraffin block for dewaxing and rehydration, hematoxylin staining for 3 min, washing with water for 2 s, differentiation with 1% hydrochloric acid ethanol for 1 s, washing with water for 10 min, eosin staining for 3 min, dehydration with ethanol, clearing with xylene, and mounting with neutral resin. The overall uterine morphology was observed under an optical microscope. Three fields of view were randomly selected from each section for photography, and the endometrial thickness was measured. The number of blood vessels and glands was counted, and the average values ​​were used for statistical analysis.

[0129] The results are as follows Figure 9 As shown, H&E staining results indicated that the uterine structure of the rats in the sham-operated group was intact, and the uterine cavity had a regular shape, consisting of the endometrium, myometrium, and adventitia from the inside out. The endometrium was relatively thick and had a clear boundary. It was composed of a single layer of columnar epithelial cells arranged in an orderly manner and stroma. Glands, blood vessels, and fibrous connective tissue were visible in the stroma. The uterine cavity wall tissue structure was clear, and the uterine cavity was smooth and unobstructed. On days 14 and 28 after the modeling surgery, the endometrial layer of the rats in the model group was destroyed, the number of folds was reduced, the epithelial cells were arranged in a disordered manner, the fibrous connective tissue was loose, the uterine cavity was significantly narrowed and adhesions appeared, and the interstitium was congested and edematous. Compared with the endometrium of the sham-operated rats, the number of endometrial folds was reduced, the uterine cavity was narrowed and almost completely closed, there were fibrotic adhesions in the uterine cavity, some epithelial peeling, and morphological changes were also visible on the surface of some uterine cavities. The single layer of columnar epithelial cells disappeared and was covered by stratified squamous epithelial cells, cuboidal epithelial cells or low columnar epithelial cells. A very small number of glands were visible in the basal layer, and the integrity of the glands was destroyed. There was obvious inflammatory infiltration in the interstitium, capillary rupture and hemorrhage, and a large number of inflammatory cell infiltrations were visible. The glands were sparse. In the drug-loaded hydrogel group, after 14 days of drug administration, the endometrial epithelium remained mostly intact, with only a small portion of epithelial cells completely sloughing off. Some single-layer columnar epithelial cells disappeared, replaced by squamous, cuboidal, or low columnar epithelial cells. The number of submucosal glands decreased, and local hemorrhage and congestion were observed, along with some angiogenesis. After 28 days of drug administration, the uterine tissue structure remained largely intact, approaching that of a normal uterus, with numerous newly formed glands and a small amount of inflammatory cell infiltration. Compared to the model group, both the simple hydrogel group and the drug-loaded hydrogel group showed significant increases in endometrial thickness, the number of blood vessels, and the number of glands.

[0130] Application Example 7

[0131] Immunohistofluorescence staining method for assessing endometrial neovascularization:

[0132] On the 14th day and the 28th day after the drug treatment, the animals were sacrificed, the uterine tissues were taken out, the excess adipose tissues were cut off, the uterine tissues were rinsed with 0.9% sodium chloride solution, and then the uterine anatomical morphology was observed and recorded after the uterine tissues were fixed in 4% paraformaldehyde. The uterine tissues fixed in 4% paraformaldehyde were taken out for paraffin embedding, and then the uterine tissues were sliced into 6 μm for immunofluorescence staining by using a handwheel type microtome, as follows:

[0133] (1) Antigen repair: a certain amount of citric acid buffer solution with pH 6 was taken, and was heated to boiling by microwave, and then was placed in a slide rack after the solution was supplemented, and was heated at medium-high fire for 8 minutes, and was stopped for 5 minutes, and then the solution was supplemented, and the above operation was repeated for 3 times. After the repair was completed, the slide was dried, and was washed with PBS buffer solution for 3 times, each for 5 minutes.

[0134] (2) Reduce non-specific staining: after the slide was dried, the slide was circled by using a histological pen. Then tissue autofluorescence quencher A was added dropwise, and was placed at room temperature for 30 minutes, and was washed with pure water for 5 minutes; and then tissue autofluorescence quencher B was added dropwise, and was placed at room temperature for 5 minutes, and was washed with flowing water for 3 minutes.

[0135] (3) Punching: after the slide was dried, 50 μL of 0.2% Triton X-100 solution was added dropwise, and was placed at room temperature for 10 minutes, and was washed with PBS for 5 minutes.

[0136] (4) Blocking: after the slide was dried, 50 μL of 5% BSA solution was added dropwise, and was blocked at room temperature for 30 minutes.

[0137] (5) Incubation of primary antibody: the blocking solution was removed, 50 μL of primary antibody diluted with 0.5% BSA (1:150) was added dropwise, and the slice was placed in a wet box and was incubated at 4°C overnight.

[0138] (6) Incubation of secondary antibody: PBS was washed for 3 times, each for 5 minutes. After the slide was slightly shaken, 50 μL of secondary antibody corresponding to the primary antibody was added dropwise to cover the tissue, and was incubated at room temperature for 50 minutes in the dark.

[0139] (7) Mounting: PBS was washed for 3 times, each for 5 minutes. After the slide was dried, the slide was mounted by using an anti-quenching mounting agent.

[0140] (8) Microscopy: images were captured by using a Leica inverted fluorescence microscope.

[0141] The experimental results are as follows: Figure 10Immunofluorescence staining was used to detect the angiogenesis of endometrium. We used Ki67 and CD31 immunofluorescence co-localization to verify the proliferation of new blood vessels. CD31 is a platelet-endothelial cell adhesion factor involved in the composition of the blood vessel wall, which can prove the existence of vascular endothelial tissue and react to microvascular density, so it is often used as a marker to evaluate microvascular angiogenesis. VEGF is a key stimulator that directly acts on vascular endothelial cells, which can promote angiogenesis, increase microvascular density, and thus promote endometrial regeneration. Compared with the model group, the expression and co-localization of Ki67 and CD31 increased after 14 days and 28 days of treatment with drug-loaded hydrogel, suggesting stronger angiogenesis, which made the vascular structure of the intrauterine adhesion site develop faster, the angiogenic response was rapid, stimulated endothelial cell proliferation and tissue formation, promoted the up-regulation of angiogenic factors, successfully improved the healing of the wound site, accelerated healing, faster epithelialization, improved granulation tissue maturation and enhanced vascularization.

[0142] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a hyaluronic acid-based thermosensitive hydrogel SV@HP, characterized by, The steps are as follows: (1) Poloxamer 407 is dissolved in a dioxane solution, then triethylamine solution is added, and after mixing, methyl acryloyl chloride solution is added and stirred to react, and the solution after stirring and reaction is dialyzed, and the dialysate is freeze-dried to obtain P407-MC; P407-MC solution is mixed and reacted with hyaluronic acid-adipic acid dihydrazide to obtain HA-modified P407 solution, and the HA-modified P407 solution is mixed and soaked with P407 solution to obtain a temperature-sensitive hydrogel HP; (2) CA-SS-COOH solution is obtained by adding chlorogenic acid to a DMF aqueous solution of 3,3'-dithiodipropionic acid under the condition of EDC and NHS, and then adding the amino-modified 50 OD TGFβ1 siRNA DOPE aqueous solution to the CA-SS-COOH aqueous solution to react, dialyze, and freeze-dry to obtain CS-siRNA nanoparticles; (3) PLGA is dissolved in dichloromethane, and then mixed with a deionized water solution containing VEGF protein under ultrasonic mixing to form an oil-in-water white emulsion, and the oil-in-water white emulsion is added to a PVA solution to stir to form a water-in-oil-in-water emulsion, and the water-in-oil-in-water emulsion is again added to a PVA solution to stir, and the solvent is evaporated, and then freeze-dried to obtain VEGF@PLGA microspheres; (4) CS-siRNA nanoparticles and VEGF@PLGA microspheres are added to the temperature-sensitive hydrogel HP solution at the same time, and then stirred uniformly to form a temperature-sensitive hydrogel SV@HP.

2. The method for preparing the temperature-sensitive hydrogel SV@HP according to claim 1, characterized in that: In step (1), the molar ratio of poloxamer 407, triethylamine, and methyl acryloyl chloride is 1:0.5-8:0.5-8; the molar ratio of P407-MC and hyaluronic acid-adipic acid dihydrazide is 1:0.1-3; the volume ratio of HA-modified P407 solution to P407 solution is 1:2-30, the mass percentage of P407 solution is 15% wt, and the mass percentage of HA-modified P407 is 15% wt; the mixing reaction conditions are 25-55℃ water bath for 1-5 min.

3. The method for preparing temperature-sensitive hydrogel SV@HP according to claim 2, characterized in that: In 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 50 OD TGFβ1 siRNA to CA-SS-COOH is 1:0.1-5; and the reaction conditions are 20-55℃ for 6-48 h.

4. The method for preparing temperature-sensitive hydrogel SV@HP according to claim 3, characterized in that: In step (3), 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 temperature-sensitive hydrogel SV@HP according to claim 4, characterized in that: In step (4), the molar ratio of temperature-sensitive hydrogel HP, CS-siRNA nanoparticles, and VEGF@PLGA microspheres is 500-50000:1:1-300; and the stirring temperature is 15-60℃.

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

7. The temperature-sensitive hydrogel SV@HP prepared by the method of any one of claims 1-6.

8. The use of the thermosensitive hydrogel SV@HP of claim 7 in the preparation of a drug for anti-inflammatory, improving endometrial fibrosis or treating intrauterine adhesion.

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