Temperature-sensitive degradable gel for treating intrauterine adhesion and preparation method thereof

Abstract

The invention provides temperature-sensitive degradable gel for treating intrauterine adhesion and a preparation method thereof, and belongs to the technical field of gel. The preparation method comprises the following steps: preparing an amygdalin-loaded polymer; preparing hydroxyproline modified chitosan; and mixing to prepare the temperature-sensitive degradable gel. Soybean protein isolate and carboxymethyl konjac glucomannan are prepared into solutions, then amygdalin is added into the soybean protein isolate solution, then the mixture is mixed with the carboxymethyl konjac glucomannan solution for electrostatic self-assembly polymerization to form a carrier with a three-dimensional macromolecular structure, the amygdalin is loaded, and the amygdalin / carboxymethyl konjac glucomannan composite material is obtained. After the amygdalin-loaded polymer is added into the gel, the deformation resistance of the gel can be enhanced, so that the effect of improving the supporting force of the gel is achieved, and the recurrence rate of intrauterine adhesion is reduced.

Description

Technical Field

[0001] This invention relates to the field of gel technology, specifically to a thermosensitive biodegradable gel for treating intrauterine adhesions and its preparation method. Background Art

[0002] Intrauterine adhesions are a common gynecological condition, often caused by surgical procedures on the cervix (such as induced abortion, curettage, etc.), inflammation, or infection, leading to damage to the cervical canal mucosa and subsequently stenosis or closure of the cervical canal. This pathological condition seriously affects women's reproductive health, causing menstrual abnormalities such as decreased menstrual flow or even amenorrhea, as well as fertility problems including infertility and recurrent miscarriages. Statistics show that the incidence of intrauterine adhesions is increasing year by year among women who have undergone multiple intrauterine procedures, making it one of the most pressing issues in gynecological clinical practice.

[0003] Traditional treatments for intrauterine adhesions primarily rely on surgical separation of the adhered tissue, such as hysteroscopic adhesiolysis. While this method can directly remove the adhesions, the risk of re-adhesion in the cervical canal post-surgery is extremely high. This is because, during the healing process, the surgical wound lacks effective physical barriers and tissue support, making it prone to re-adhesion before the cervical canal mucosa is fully repaired. This often necessitates repeated surgeries, causing significant physical and psychological distress for patients, while also increasing medical costs and resource consumption. Drug therapy mainly focuses on preventing infection and promoting endometrial repair, such as using antibiotics to prevent infection and estrogen to promote endometrial growth. However, drug therapy alone has limited effectiveness in improving the physical structure of the cervical canal and providing support. Without effectively maintaining the cervical canal's morphology, drugs struggle to create an effective concentration gradient and action environment locally, significantly reducing their effectiveness in promoting endometrial repair and failing to fundamentally address the key issue of intrauterine adhesion recurrence.

[0004] Thermosensitive gel, as a novel local drug delivery and tissue repair material, can act more precisely on the adhesion site compared to drug treatment. It gels under body temperature, allowing the drug or therapeutic components to remain concentrated at the adhesion site for a longer period, improving the treatment's effectiveness. Compared to surgical treatment, thermosensitive gel provides a non-invasive physical barrier, forming a physical barrier within the cervical canal to effectively prevent re-contact of separated cervical tissues and reduce the risk of adhesion recurrence. However, existing gels are prone to insufficient support. Under the physiological environment of the cervix, due to factors such as human activity, tissue peristalsis, and gravity, gels with weak support cannot stably maintain the open shape of the cervical canal. They are prone to deformation and collapse under even small external forces, failing to provide sufficient space and stable physical support for the repair of the cervical canal mucosa. This limits their widespread application in the treatment of intrauterine adhesions.

[0005] Therefore, it is necessary to propose a thermosensitive biodegradable gel with good support for the treatment of intrauterine adhesions and its preparation method, so as to improve the efficacy and reduce the recurrence rate. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a thermosensitive biodegradable gel for treating intrauterine adhesions and its preparation method.

[0007] A method for preparing a thermosensitive biodegradable gel for treating intrauterine adhesions includes the following steps: S1: Preparation of amygdalin-loaded polymer Soy protein isolate and carboxymethyl konjac glucomannan were prepared into soy protein isolate solution and carboxymethyl konjac glucomannan solution, respectively. Amygdalin was added to the soy protein isolate solution and then mixed with the carboxymethyl konjac glucomannan solution for polymerization to obtain amygdalin-loaded polymer. S2: Preparation of hydroxyproline-modified chitosan Hydroxyproline was dissolved and activated, then added to an acetic acid solution of chitosan, triethylamine was added and the mixture was stirred to obtain hydroxyproline-modified chitosan. S3: Preparation of thermosensitive biodegradable gel by mixing Polygalacturonic acid, sodium β-glycerophosphate, and the above-mentioned hydroxyproline-modified chitosan were dissolved separately, then stirred and mixed in an ice-water bath. Autologous platelet-rich plasma and the above-mentioned amygdalin-loaded polymer were then added and homogenized to obtain a thermosensitive biodegradable gel.

[0008] Furthermore, S1 specifically includes the following steps: S1.1: Add soy protein isolate and carboxymethyl konjac glucomannan to distilled water at a ratio of 1g:(90-100)mL, heat and stir at 35-45℃ to dissolve and keep warm to obtain soy protein isolate solution and carboxymethyl konjac glucomannan solution; S1.2: Add hydrochloric acid to the above soy protein isolate solution to adjust the pH to 3-4, then add amygdalin at a solid-liquid ratio of 1g:(320-330)mL, and sonicate at 160-180W for 20-30min to obtain amygdalin mixture. S1.3: Add the above carboxymethyl konjac glucomannan solution to the above amygdalin mixture, stir at a rate of 700-800 r / min for 1-2 h to carry out polymerization, and obtain amygdalin-loaded polymer.

[0009] Furthermore, S2 specifically includes the following steps: S2.1: Add chitosan to 1% acetic acid solution at a solid-liquid ratio of 1g:(80-90)mL, stir thoroughly to dissolve, and obtain chitosan solution; S2.2: Add hydroxyproline to deionized water at a solid-liquid ratio of 1g:(10-20)mL, stir thoroughly to dissolve, and obtain a hydroxyproline solution; S2.3: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the above hydroxyproline solution, and stir the reaction in an ice-water bath at 1-3℃ for 1-2 hours to obtain an activated hydroxyproline solution. S2.4: While stirring, add the activated hydroxyproline solution to the chitosan solution, then add triethylamine, adjust the pH to 7-8, and continue stirring for 16-20 hours. After dialysis with deionized water and freeze-drying, hydroxyproline-modified chitosan is obtained.

[0010] Furthermore, S3 specifically includes the following steps: S3.1: Add the hydroxyproline-modified chitosan obtained in step S2.4 to a 1% acetic acid solution at a solid-liquid ratio of 1g:(50-60)mL, stir thoroughly to dissolve, and obtain a hydroxyproline-modified chitosan solution. S3.2: Add polygalacturonic acid and sodium β-glycerophosphate to deionized water and stir until completely dissolved to prepare a 1 wt% polygalacturonic acid solution and a 1 wt% sodium β-glycerophosphate solution. S3.3: Under ice-water bath conditions of 2-4℃, stir equal volumes of the above hydroxyproline-modified chitosan solution, the above polygalacturonic acid solution and the above β-glycerophosphate sodium solution for 20-30 min to obtain a mixed sol solution; S3.4: Add autologous platelet-rich plasma and the amygdalin-loaded polymer obtained in step S1.3 to the above mixed sol solution, stir and mix thoroughly, then place in a homogenizer and homogenize for 20-30 minutes to obtain a thermosensitive biodegradable gel.

[0011] Furthermore, the volume ratio of the amygdalin mixture to the carboxymethyl konjac glucomannan solution is (2-3):1.

[0012] Furthermore, the mass ratios of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to hydroxyproline are 1:(2-3) and 1:(3-5), respectively, and the volume ratio of the activated hydroxyproline solution to the chitosan solution is 1:(8-9).

[0013] Furthermore, the volume ratio of autologous platelet-rich plasma to the mixed sol solution is 1:(8-10), and the mass ratio of amygdalin-loaded polymer to autologous platelet-rich plasma is (3-5):1.

[0014] Further, the preparation steps of autologous platelet-rich plasma are as follows: First, peripheral blood from the patient is collected and anticoagulated, then centrifuged to separate the peripheral blood into upper, middle and lower layers. Then, the upper and middle layers are aspirated and centrifuged a second time. After discarding the upper layer, thrombin and 8-10 wt% calcium chloride solution are added to the remaining portion to obtain autologous platelet-rich plasma. The volume percentage of calcium chloride solution in the autologous platelet-rich plasma is 8-12%, and the thrombin content is 15-25 U / mL.

[0015] Furthermore, a thermosensitive biodegradable gel for treating intrauterine adhesions is prepared by the method described in any one of the above-mentioned methods for preparing a thermosensitive biodegradable gel for treating intrauterine adhesions.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: 1. This invention first prepares solutions of soy protein isolate and carboxymethyl konjac glucomannan separately, then adds amygdalin to the soy protein isolate solution, and then mixes it with the carboxymethyl konjac glucomannan solution for electrostatic self-assembly polymerization to form a carrier with a three-dimensional macromolecular structure. Amygdalin is loaded onto the carrier to obtain an amygdalin-loaded polymer. When this polymer is added to a gel, on the one hand, it can intertwine with the polymer molecular chains in the gel and be embedded in the gel network structure as a "reinforcing phase", making the gel network structure more compact and robust. On the other hand, its rigid structure can play a "blocking" and "fixing" role between the molecular chains, making it more difficult for the gel molecular chains to be subjected to external forces, thus enhancing the gel's resistance to deformation and thereby improving the gel's support strength. 2. This invention first dissolves hydroxyproline and chitosan separately, then activates hydroxyproline with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then mixes the activated hydroxyproline solution with the chitosan solution to modify the chitosan. After adding the hydroxyproline-modified chitosan to the gel, its abundant hydrogen bond forming sites can quickly form hydrogen bonds with other components in the gel system. During the temperature rise process, these additional hydrogen bonds can accelerate the cross-linking between molecular chains, so that the gel can form a three-dimensional network structure in a shorter time, thereby improving the rapid gelation ability of the gel. 3. When the thermosensitive biodegradable gel of this invention is used to treat intrauterine adhesions, the main drug components are autologous platelet-rich plasma and amygdalin. Since autologous platelet-rich plasma can stimulate the proliferation and migration of fibroblasts in the cervix and promote the synthesis of extracellular matrix, it is beneficial for filling and repairing damaged tissue. When used in combination, the anti-inflammatory effect of amygdalin and the tissue-repairing effect of autologous platelet-rich plasma can work synergistically. In the early stages of inflammation, amygdalin reduces the inflammatory response. Once the oxidative stress and cell damage caused by inflammation are relieved, the growth factors in autologous platelet-rich plasma can further enhance their effects. It effectively acts on cervical tissue cells, promoting their proliferation and differentiation, accelerating the repair of damaged tissues, and preventing further development of adhesions. Furthermore, the growth factors in autologous platelet-rich plasma promote the formation of new blood vessels at the site of intrauterine adhesions. Amygdalin may improve the nutrient uptake and metabolic state of cells, enabling newly formed blood vessels to better perform their nutrient transport function, providing sufficient nutrients to the repairing tissue, and further promoting the regeneration of cervical tissue and the separation of adhesions. Therefore, autologous platelet-rich plasma and amygdalin can synergistically treat intrauterine adhesions, further improving the efficacy of intrauterine adhesion treatment. Attached Figure Description

[0017] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate embodiments of the present disclosure and, together with the specification, further serve to explain the principles of the present disclosure and enable those skilled in the art to implement and use the present disclosure.

[0018] Figure 1 This is a flowchart illustrating the preparation method of a thermosensitive biodegradable gel for treating intrauterine adhesions, as used in an embodiment of the present invention. Detailed Implementation

[0019] The following describes in detail, with reference to the accompanying drawings and specific embodiments, a thermosensitive biodegradable gel for treating intrauterine adhesions and its preparation method, provided by the present invention.

[0020] Example 1: A method for preparing a thermosensitive biodegradable gel for treating intrauterine adhesions, such as... Figure 1 As shown, it includes the following steps: S1: Preparation of amygdalin-loaded polymer: S1.1: Add soy protein isolate and carboxymethyl konjac glucomannan to distilled water at a ratio of 1g:90mL, heat and stir at 35℃ to dissolve and keep warm to obtain soy protein isolate solution and carboxymethyl konjac glucomannan solution; S1.2: Add hydrochloric acid to the above soy protein isolate solution to adjust the pH to 3, then add amygdalin at a solid-liquid ratio of 1g:320mL, and sonicate at 160W for 20min to obtain amygdalin mixture. S1.3: The above carboxymethyl konjac glucomannan solution was added to the above amygdalin mixture and stirred at a rate of 700 r / min for 1 h to carry out polymerization, thereby obtaining amygdalin-loaded polymer, wherein the volume ratio of amygdalin mixture to carboxymethyl konjac glucomannan solution was 2:1. S2: Preparation of hydroxyproline-modified chitosan S2.1: Add chitosan to 1% acetic acid solution at a solid-liquid ratio of 1g:80mL, stir thoroughly to dissolve, and obtain chitosan solution; S2.2: Add hydroxyproline to deionized water at a solid-liquid ratio of 1g:10mL, stir thoroughly to dissolve, and obtain a hydroxyproline solution; S2.3: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the above hydroxyproline solution, and stir the reaction in an ice-water bath at 1°C for 1 h to obtain an activated hydroxyproline solution, wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to hydroxyproline is 1:2 and 1:3, respectively; S2.4: While stirring, add the above activated hydroxyproline solution to the above chitosan solution, then add triethylamine, adjust the pH to 7, and continue stirring for 16 hours. After dialysis with deionized water and freeze-drying, hydroxyproline-modified chitosan is obtained. The volume ratio of activated hydroxyproline solution to chitosan solution is 1:8. S3: Preparation of thermosensitive biodegradable gel by mixing S3.1: Add the hydroxyproline-modified chitosan obtained in step S2.4 to a 1% acetic acid solution at a solid-liquid ratio of 1g:50mL, stir thoroughly to dissolve, and obtain a hydroxyproline-modified chitosan solution. S3.2: Add polygalacturonic acid and sodium β-glycerophosphate to deionized water and stir until completely dissolved to prepare a 1 wt% polygalacturonic acid solution and a 1 wt% sodium β-glycerophosphate solution. S3.3: Under the condition of ice-water bath at 2℃, equal volumes of the above hydroxyproline-modified chitosan solution, the above polygalacturonic acid solution and the above β-glycerophosphate sodium solution were stirred for 20 min to obtain a mixed sol solution; S3.4: Add autologous platelet-rich plasma and the amygdalin-loaded polymer obtained in step S1.3 to the above mixed sol solution, stir thoroughly, and then place in a homogenizer for homogenization for 20 min to obtain a thermosensitive biodegradable gel. The volume ratio of autologous platelet-rich plasma to mixed sol solution is 1:8, and the mass ratio of amygdalin-loaded polymer to autologous platelet-rich plasma is 3:1. The preparation steps of autologous platelet-rich plasma are as follows: first, collect peripheral blood from the patient and perform anticoagulation treatment, then centrifuge to separate the peripheral blood into upper, middle and lower layers. Then, aspirate the upper and middle layers and centrifuge again. After discarding the upper layer, add thrombin and 8wt% calcium chloride solution to the remaining part to obtain autologous platelet-rich plasma. The volume percentage of calcium chloride solution in the autologous platelet-rich plasma is 8%, and the thrombin content is 15 U / mL.

[0021] Example 2: A method for preparing a thermosensitive biodegradable gel for treating intrauterine adhesions, such as... Figure 1 As shown, it includes the following steps: S1: Preparation of amygdalin-loaded polymer S1.1: Add soy protein isolate and carboxymethyl konjac glucomannan to distilled water at a ratio of 1g:95mL, heat and stir at 40℃ to dissolve and keep warm to obtain soy protein isolate solution and carboxymethyl konjac glucomannan solution; S1.2: Add hydrochloric acid to the above soy protein isolate solution to adjust the pH to 3.5, then add amygdalin at a solid-liquid ratio of 1g:325mL, and sonicate at 170W for 25min to obtain amygdalin mixture. S1.3: The above carboxymethyl konjac glucomannan solution was added to the above amygdalin mixture and stirred at a rate of 750 r / min for 1.5 h to carry out polymerization, thereby obtaining amygdalin-loaded polymer, wherein the volume ratio of amygdalin mixture to carboxymethyl konjac glucomannan solution was 2.5:1. S2: Preparation of hydroxyproline-modified chitosan S2.1: Add chitosan to 1% acetic acid solution at a solid-liquid ratio of 1g:85mL, stir thoroughly to dissolve, and obtain chitosan solution; S2.2: Add hydroxyproline to deionized water at a solid-liquid ratio of 1g:15mL, stir thoroughly to dissolve, and obtain a hydroxyproline solution; S2.3: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the above hydroxyproline solution, and stir the reaction in an ice-water bath at 2°C for 1.5 h to obtain an activated hydroxyproline solution, wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to hydroxyproline is 1:2.5 and 1:4, respectively; S2.4: While stirring, add the above activated hydroxyproline solution to the above chitosan solution, then add triethylamine, adjust the pH to 7.5, and continue stirring for 18 hours. After dialysis with deionized water and freeze-drying, hydroxyproline-modified chitosan is obtained. The volume ratio of activated hydroxyproline solution to chitosan solution is 1:8.5. S3: Preparation of thermosensitive biodegradable gel by mixing S3.1: Add the hydroxyproline-modified chitosan obtained in step S2.4 to a 1% acetic acid solution at a solid-liquid ratio of 1g:55mL, stir thoroughly to dissolve, and obtain a hydroxyproline-modified chitosan solution. S3.2: Add polygalacturonic acid and sodium β-glycerophosphate to deionized water and stir until completely dissolved to prepare a 1 wt% polygalacturonic acid solution and a 1 wt% sodium β-glycerophosphate solution. S3.3: Under the condition of ice-water bath at 3℃, equal volumes of the above hydroxyproline-modified chitosan solution, the above polygalacturonic acid solution and the above β-glycerophosphate sodium solution were stirred for 25 min to obtain a mixed sol solution; S3.4: Add autologous platelet-rich plasma and the amygdalin-loaded polymer obtained in step S1.3 to the above mixed sol solution, stir thoroughly, and then place in a homogenizer for homogenization for 25 min to obtain a thermosensitive biodegradable gel. The volume ratio of autologous platelet-rich plasma to mixed sol solution is 1:9, and the mass ratio of amygdalin-loaded polymer to autologous platelet-rich plasma is 4:1. The preparation steps of autologous platelet-rich plasma are as follows: first, collect peripheral blood from the patient and perform anticoagulation treatment, then centrifuge to separate the peripheral blood into upper, middle and lower layers. Then, aspirate the upper and middle layers and centrifuge again. After discarding the upper layer, add thrombin and 9wt% calcium chloride solution to the remaining part to obtain autologous platelet-rich plasma. The volume percentage of calcium chloride solution in the autologous platelet-rich plasma is 10%, and the thrombin content is 20 U / mL.

[0022] Example 3: A method for preparing a thermosensitive biodegradable gel for treating intrauterine adhesions, such as... Figure 1 As shown, it includes the following steps: S1: Preparation of amygdalin-loaded polymer S1.1: Add soy protein isolate and carboxymethyl konjac glucomannan to distilled water at a ratio of 1g:100mL, heat and stir at 45℃ to dissolve and keep warm to obtain soy protein isolate solution and carboxymethyl konjac glucomannan solution; S1.2: Add hydrochloric acid to the above soy protein isolate solution to adjust the pH to 4, then add amygdalin at a solid-liquid ratio of 1g:330mL, and sonicate at 180W for 30min to obtain amygdalin mixture. S1.3: The above carboxymethyl konjac glucomannan solution was added to the above amygdalin mixture and stirred at a rate of 800 r / min for 2 h to carry out polymerization, thereby obtaining amygdalin-loaded polymer, wherein the volume ratio of amygdalin mixture to carboxymethyl konjac glucomannan solution was 3:1. S2: Preparation of hydroxyproline-modified chitosan S2.1: Add chitosan to 1% acetic acid solution at a solid-liquid ratio of 1g:90mL, stir thoroughly to dissolve, and obtain chitosan solution; S2.2: Add hydroxyproline to deionized water at a solid-liquid ratio of 1g:20mL, stir thoroughly to dissolve, and obtain a hydroxyproline solution; S2.3: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the above hydroxyproline solution, and stir the reaction in an ice-water bath at 3°C ​​for 2 hours to obtain an activated hydroxyproline solution, wherein the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to hydroxyproline is 1:3 and 1:5, respectively; S2.4: While stirring, add the above activated hydroxyproline solution to the above chitosan solution, then add triethylamine, adjust the pH to 8, and continue stirring for 20 hours. After dialysis with deionized water and freeze-drying, hydroxyproline-modified chitosan is obtained. The volume ratio of activated hydroxyproline solution to chitosan solution is 1:9. S3: Preparation of thermosensitive biodegradable gel by mixing S3.1: Add the hydroxyproline-modified chitosan obtained in step S2.4 to a 1% acetic acid solution at a solid-liquid ratio of 1g:60mL, stir thoroughly to dissolve, and obtain a hydroxyproline-modified chitosan solution. S3.2: Add polygalacturonic acid and sodium β-glycerophosphate to deionized water and stir until completely dissolved to prepare a 1 wt% polygalacturonic acid solution and a 1 wt% sodium β-glycerophosphate solution. S3.3: Under the condition of ice-water bath at 4℃, equal volumes of the above hydroxyproline-modified chitosan solution, the above polygalacturonic acid solution and the above β-glycerophosphate sodium solution were stirred for 30 min to obtain a mixed sol solution; S3.4: Add autologous platelet-rich plasma and the amygdalin-loaded polymer obtained in step S1.3 to the above mixed sol solution, stir thoroughly, and then place in a homogenizer for homogenization for 30 min to obtain a thermosensitive biodegradable gel. The volume ratio of autologous platelet-rich plasma to mixed sol solution is 1:10, and the mass ratio of amygdalin-loaded polymer to autologous platelet-rich plasma is 5:1. The preparation steps of autologous platelet-rich plasma are as follows: first, collect peripheral blood from the patient and perform anticoagulation treatment, then centrifuge to separate the peripheral blood into upper, middle and lower layers. Then, aspirate the upper and middle layers and centrifuge again. After discarding the upper layer, add thrombin and 10wt% calcium chloride solution to the remaining part to obtain autologous platelet-rich plasma. The volume percentage of calcium chloride solution in the autologous platelet-rich plasma is 12%, and the thrombin content is 25 U / mL.

[0023] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that the amygdalin-loaded polymer in step S3.4 is removed.

[0024] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that the hydroxyproline-modified chitosan in step S3.1 is replaced with an equal amount of chitosan.

[0025] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that amygdalin in step S1.2 is replaced with an equal amount of autologous platelet-rich plasma.

[0026] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that the autologous platelet-rich plasma in step S3.4 is replaced with an equal amount of amygdalin.

[0027] Test example: Test 1: The thermosensitive biodegradable gels prepared in Examples 1-3 and Comparative Example 1 were heated to 37°C to form a gel body, which was then cut into cubes with dimensions of 10mm×10mm×10mm. The compressive strength of the cubes was then tested using a universal testing machine. The results are shown in Table 1. Table 1: Comparison of Compression Strength Test Results;

[0028] As shown in Table 1, the compressive strength of the thermosensitive biodegradable gel prepared in Comparative Example 1 without the addition of the amygdalin-loaded polymer is approximately 0.46 MPa, which is much lower than that in Example 1. This indicates that by first preparing solutions of soy protein isolate and carboxymethyl konjac glucomannan, then adding amygdalin to the soy protein isolate solution, and then mixing it with the carboxymethyl konjac glucomannan solution for electrostatic self-assembly polymerization to form a carrier with a three-dimensional macromolecular structure, and loading amygdalin onto it, the amygdalin-loaded polymer can be obtained. Adding this polymer to the gel can effectively improve the compressive strength of the gel, thereby improving the gel's support strength.

[0029] Test 2: Take 2 mL of the thermosensitive biodegradable gel prepared in Examples 1-3 and Comparative Example 2, place it in a vial and let it stand at room temperature for 30 min. Then place it on a 37°C water bath, start timing, and observe the flow of the gel in the vial. Stop timing when the thermosensitive biodegradable gel in the vial no longer flows with the inclination of the vial. This time is the gelation time. Each sample is measured 3 times, and the average value is taken. Table 2: Comparison of gel time test results;

[0030] As can be seen from Table 2, when chitosan was not modified with hydroxyproline in Comparative Example 2, the thermosensitive biodegradable gel had a gelation time of approximately 183 s at 37°C, which was much longer than that in Example 1. This shows that by first dissolving hydroxyproline and chitosan separately, then activating hydroxyproline with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and then mixing the activated hydroxyproline solution with the chitosan solution to react, the chitosan was modified. Adding hydroxyproline-modified chitosan to the gel can improve the rapid gelation ability of the gel.

[0031] Test 3: Seven-week-old adult female BALB / c mice were selected. The scraping method was used to simulate clinical mechanical curettage injury. Mice were anesthetized with 5% isoflurane by inhalation. The abdominal cavity was entered from the back of the mouse to expose the uterine horn. The endometrium was repeatedly scraped by rotating a 24G needle. At the same time, the uterine congestion and thinning were observed under a stereomicroscope. The procedure was stopped when the needle touched the surface of the uterus and felt rough. The uterine surface wound caused by the needle was sutured with sterile absorbable surgical sutures. The abdomen was closed layer by layer with absorbable sutures after the operation. The mice were placed on a 37°C heating pad and observed for 2 hours. If there were no abnormalities, they were returned to the animal room. After the mice recovered from anesthesia, they were given water and rat food to obtain sample mice with intrauterine adhesions. The sample mice were then divided into three groups: Group 1 (Example 1) received an in situ injection of 50 μL of the thermosensitive biodegradable gel prepared in Example 1 into the uterine cavity; Group 2 (Comparative Example 3) received an in situ injection of 50 μL of the thermosensitive biodegradable gel prepared in Comparative Example 3 into the uterine cavity; and Group 3 (Comparative Example 4) received an in situ injection of 50 μL of the thermosensitive biodegradable hydrogel prepared in Comparative Example 4 into the uterine cavity. Fourteen days after injection, Masson chromosome analysis was performed on the three groups of mice, and the area of ​​uterine fibrosis was analyzed. The results are shown in Table 3. Table 3: Comparison of results of uterine fibrosis area analysis in mice;

[0032] As shown in Table 3, when only autologous platelet-rich plasma was added in Comparative Example 3 and only amygdalin was added in Comparative Example 4, the uterine fibrosis area of ​​the thermosensitive biodegradable gel prepared was larger than that of mice with intrauterine adhesions after 14 days of injection. This shows that autologous platelet-rich plasma and amygdalin can synergistically treat intrauterine adhesions and further improve the efficacy of the treatment.

[0033] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a thermosensitive degradable gel for treating intrauterine adhesions, characterized in that: The steps include: S1: Preparation of amygdalin-loaded polymers The soy protein isolate solution and the carboxymethyl konjac glucomannan solution are prepared respectively, amygdalin is added to the soy protein isolate solution, and then the solution is mixed with the carboxymethyl konjac glucomannan solution for polymerization to obtain amygdalin-loaded polymer; S2: Preparation of hydroxyproline-modified chitosan Dissolving and activating hydroxyproline, and then adding it into an acetic acid solution of chitosan, adding triethylamine and stirring to react, thereby obtaining hydroxyproline-modified chitosan; S3: Mixing to prepare thermosensitive degradable gel Polygalacturonic acid, sodium β-glycerophosphate and the above hydroxyproline modified chitosan are dissolved separately, and then stirred and mixed in an ice water bath. Then, autologous platelet-rich plasma and the above amygdalin-loaded polymer are added and homogenized to obtain a temperature-sensitive degradable gel.

2. The method for preparing a thermosensitive degradable gel for treating intrauterine adhesions according to claim 1, characterized in that: S1 includes the following steps: S1.1: Add soy protein isolate and carboxymethyl konjac glucomannan into distilled water at a material-liquid ratio of 1 g: (90-100) mL, respectively, heat and stir to dissolve at 35-45°C and keep warm to obtain soy protein isolate solution and carboxymethyl konjac glucomannan solution; S1.2: Add hydrochloric acid to the above soy protein isolate solution to adjust the pH to 3-4, then add amygdalin at a solid-liquid ratio of 1 g: (320-330) mL, and perform ultrasonic treatment at 160-180 W for 20-30 min to obtain amygdalin mixed solution; S1.3: Add the carboxymethyl konjac glucomannan solution to the amygdalin mixture, stir at a rate of 700-800 r / min for 1-2 hours to polymerize and obtain amygdalin-loaded polymer.

3. The method for preparing a thermosensitive degradable gel for treating intrauterine adhesions according to claim 2, characterized in that: S2 includes the following steps: S2.1: Add chitosan to 1% acetic acid solution at a solid-liquid ratio of 1 g: (80-90) mL, stir thoroughly to dissolve, and obtain a chitosan solution; S2.2: Add hydroxyproline into deionized water at a solid-liquid ratio of 1 g: (10-20) mL, stir thoroughly to dissolve, and obtain a hydroxyproline solution; S2.3: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the above hydroxyproline solution, and stir in an ice-water bath at 1-3°C for 1-2h to obtain an activated hydroxyproline solution; S2.4: Add the activated hydroxyproline solution to the chitosan solution while stirring, then add triethylamine, adjust the pH to 7-8, and continue stirring the reaction for 16-20 hours, then dialyze against deionized water and freeze-dry to obtain hydroxyproline-modified chitosan.

4. The method for preparing a thermosensitive degradable gel for treating intrauterine adhesions according to claim 3, characterized in that: S4 includes the following steps: S3.1: Add the hydroxyproline-modified chitosan prepared in step S2.4 to 1% acetic acid solution at a solid-liquid ratio of 1 g: (50-60) mL, stir and dissolve thoroughly to obtain a hydroxyproline-modified chitosan solution; S3.2: adding polygalacturonic acid and sodium β-glycerophosphate into deionized water respectively, stirring until completely dissolved, to prepare a 1 wt % polygalacturonic acid solution and a 1 wt % sodium β-glycerophosphate solution; S3.3: Stir equal volumes of the hydroxyproline-modified chitosan solution, the polygalacturonic acid solution and the sodium β-glycerophosphate solution in an ice-water bath at 2-4°C for 20-30 minutes to obtain a mixed sol solution; S3.4: Add autologous platelet-rich plasma and the amygdalin-loaded polymer obtained in step S1.3 to the above mixed sol solution, stir and mix thoroughly, and then place in a homogenizer and homogenize for 20-30 minutes to obtain a temperature-sensitive degradable gel.

5. The method for preparing a thermosensitive degradable gel for treating intrauterine adhesions according to claim 2, characterized in that: The volume ratio of the amygdalin mixture to the carboxymethyl konjac glucomannan solution is (2-3):

1.

6. The method for preparing a thermosensitive degradable gel for treating intrauterine adhesions according to claim 3, characterized in that: The mass ratios of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to hydroxyproline are 1:(2-3) and 1:(3-5), respectively, and the volume ratio of the activated hydroxyproline solution to the chitosan solution is 1:(8-9).

7. The method for preparing a thermosensitive degradable gel for treating intrauterine adhesions according to claim 4, characterized in that: The volume ratio of autologous platelet-rich plasma to the mixed sol solution is 1:(8-10), and the mass ratio of the amygdalin-loaded polymer to the autologous platelet-rich plasma is (3-5):

1.

8. The method for preparing a thermosensitive degradable gel for treating intrauterine adhesions according to claim 4, characterized in that: The preparation steps of autologous platelet-rich plasma are as follows: first collect peripheral blood from the patient for anticoagulation treatment, then centrifuge to separate the peripheral blood into an upper layer, a middle layer and a lower layer, then draw the upper layer and the middle layer for secondary centrifugation, discard the upper layer liquid, and add thrombin and 8-10wt% calcium chloride solution to the remaining part to obtain autologous platelet-rich plasma; wherein the volume percentage of the calcium chloride solution in the autologous platelet-rich plasma is 8-12%, and the thrombin content is 15-25U / mL.

9. A thermosensitive degradable gel for treating intrauterine adhesions, characterized in that: The gel is prepared by the method for preparing a thermosensitive degradable gel for treating intrauterine adhesions as described in any one of claims 1 to 8.

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