Temperature-sensitive degradable gel for treating intrauterine adhesion and method for preparing same

Abstract

Provided are a temperature-sensitive degradable gel for treating intrauterine adhesion and a method for preparing same. The present invention relates to the technical field of gels. The method for preparing same comprises the following steps: preparing an amygdalin-loaded polymer; preparing hydroxyproline-modified chitosan; and mixing the amygdalin-loaded polymer and the hydroxyproline-modified chitosan to prepare the temperature-sensitive degradable gel. First, a solution of soy protein isolate and a solution of carboxymethyl konjac glucomannan are prepared. Then, amygdalin is added to the solution of soy protein isolate, and the resulting mixture is mixed with the solution of carboxymethyl konjac glucomannan. After electrostatic self-assembly polymerization, a carrier having a three-dimensional macromolecular structure is formed. After amygdalin is loaded, an amygdalin-loaded polymer is obtained. After being added to a gel, the amygdalin-loaded polymer can enhance the resistance of the gel to deformation, thereby improving the supporting effect of the gel and reducing the recurrence rate of intrauterine adhesion.

Description

A thermosensitive biodegradable gel for treating intrauterine adhesions and its preparation method 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 Technology

[0002] Intrauterine adhesions are a common gynecological condition, often caused by surgical procedures (such as abortion, curettage, etc.) or inflammatory infections that damage the uterine cavity mucosa, leading to narrowing or closure of the uterine cavity. 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 adhesions, such as hysteroscopic adhesiolysis. While this method can directly remove the adhesions, the risk of re-adhesion in the uterine cavity post-surgery is extremely high. This is because, during the healing process, the surgical wound lacks effective physical barriers and tissue support, making it easy for the uterine mucosa to re-adhere before complete repair, leading to recurrence. This often requires repeated surgeries, causing significant physical pain and psychological burden 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 applying estrogen to promote endometrial growth. However, drug therapy alone has very limited effect on improving the physical structure of the uterine cavity and providing support. Without effectively maintaining the uterine cavity shape, drugs cannot form an effective concentration gradient and action environment locally, significantly reducing their effectiveness in promoting endometrial repair and failing to fundamentally solve the key problem 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. Furthermore, compared to surgical treatment, thermosensitive gel provides a non-invasive physical barrier, forming a physical barrier within the uterine cavity to effectively prevent re-contact of separated uterine tissues and reduce the risk of adhesion recurrence. However, existing gels are prone to insufficient support. In the physiological environment of the uterine cavity, due to factors such as human activity, tissue peristalsis, and gravity, gels with weak support cannot stably maintain the open shape of the uterine cavity. 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 uterine 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:

[0008] S1: Preparation of amygdalin-loaded polymer

[0009] 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.

[0010] S2: Preparation of hydroxyproline-modified chitosan

[0011] 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.

[0012] S3: Preparation of thermosensitive biodegradable gel by mixing

[0013] 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.

[0014] Furthermore, S1 specifically includes the following steps:

[0015] 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.

[0016] 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.

[0017] 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.

[0018] Furthermore, S2 specifically includes the following steps:

[0019] 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;

[0020] 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;

[0021] 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.

[0022] 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.

[0023] Furthermore, S3 specifically includes the following steps:

[0024] 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.

[0025] 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.

[0026] 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;

[0027] 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.

[0028] Furthermore, the volume ratio of the amygdalin mixture to the carboxymethyl konjac glucomannan solution is (2-3):1.

[0029] 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).

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects:

[0034] 1. This invention involves first preparing solutions of soy protein isolate and carboxymethyl konjac glucomannan, then adding amygdalin to the soy protein isolate solution, and finally mixing it with the carboxymethyl konjac glucomannan solution for electrostatic self-assembly polymerization to form a carrier with a three-dimensional macromolecular structure. Amygdalin is then loaded onto the carrier to obtain an amygdalin-loaded polymer. When this polymer is added to a gel, it can intertwine with the polymer molecular chains in the gel, acting as a "reinforcing phase" embedded in the gel's network structure, making the gel's network structure more compact and robust. Furthermore, its rigid structure can act as a "blocking" and "fixing" mechanism between the molecular chains, making it more difficult for the gel molecular chains to withstand external forces, thus enhancing the gel's resistance to deformation and ultimately improving the gel's support strength.

[0035] 2. This invention first dissolves hydroxyproline and chitosan separately, then activates hydroxyproline with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The activated hydroxyproline solution is then mixed with the chitosan solution to modify the chitosan. When hydroxyproline-modified chitosan is added to the gel, its abundant hydrogen bond-forming sites can rapidly form hydrogen bonds with other components in the gel system. During temperature increases, these additional hydrogen bonds accelerate the cross-linking between molecular chains, allowing the gel to form a three-dimensional network structure in a shorter time, thereby improving the gel's rapid gelation ability.

[0036] 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 uterine cavity and promote the synthesis of extracellular matrix, it is beneficial for filling and repairing damaged tissues. When used in combination, the anti-inflammatory effect of amygdalin and the tissue repair 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 uterine cavity tissue cells, promoting their proliferation and differentiation, accelerating the repair of damaged tissues, and preventing the 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 new blood vessels to better perform their nutrient transport function, providing sufficient nutrients to the tissues under repair, and further promoting the regeneration of intrauterine tissues 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

[0037] 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.

[0038] Figure 1 is a flowchart of the preparation method of the thermosensitive biodegradable gel for treating intrauterine adhesions used in the embodiments of the present invention. Detailed Implementation

[0039] 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.

[0040] Example 1

[0041] A method for preparing a thermosensitive biodegradable gel for treating intrauterine adhesions, as shown in Figure 1, includes the following steps:

[0042] S1: Preparation of amygdalin-loaded polymer

[0043] 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;

[0044] 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.

[0045] 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.

[0046] S2: Preparation of hydroxyproline-modified chitosan

[0047] 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;

[0048] S2.2: Add hydroxyproline to deionized water at a solid-liquid ratio of 1g:10mL, stir thoroughly to dissolve, and obtain a hydroxyproline solution;

[0049] 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;

[0050] 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.

[0051] S3: Preparation of thermosensitive biodegradable gel by mixing

[0052] 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.

[0053] 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.

[0054] 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;

[0055] 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.

[0056] Example 2

[0057] A method for preparing a thermosensitive biodegradable gel for treating intrauterine adhesions, as shown in Figure 1, includes the following steps:

[0058] S1: Preparation of amygdalin-loaded polymer

[0059] 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;

[0060] 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.

[0061] 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.

[0062] S2: Preparation of hydroxyproline-modified chitosan

[0063] 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;

[0064] S2.2: Add hydroxyproline to deionized water at a solid-liquid ratio of 1g:15mL, stir thoroughly to dissolve, and obtain a hydroxyproline solution;

[0065] 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;

[0066] 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.

[0067] S3: Preparation of thermosensitive biodegradable gel by mixing

[0068] 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.

[0069] 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.

[0070] 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;

[0071] 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.

[0072] Example 3

[0073] A method for preparing a thermosensitive biodegradable gel for treating intrauterine adhesions, as shown in Figure 1, includes the following steps:

[0074] S1: Preparation of amygdalin-loaded polymer

[0075] 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;

[0076] 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.

[0077] 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.

[0078] S2: Preparation of hydroxyproline-modified chitosan

[0079] 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;

[0080] S2.2: Add hydroxyproline to deionized water at a solid-liquid ratio of 1g:20mL, stir thoroughly to dissolve, and obtain a hydroxyproline solution;

[0081] 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;

[0082] 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.

[0083] S3: Preparation of thermosensitive biodegradable gel by mixing

[0084] 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.

[0085] 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.

[0086] 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;

[0087] 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.

[0088] Comparative Example 1

[0089] The difference between Comparative Example 1 and Example 1 is that the amygdalin-loaded polymer in step S3.4 is removed.

[0090] Comparative Example 2

[0091] 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.

[0092] Comparative Example 3

[0093] 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.

[0094] Comparative Example 4

[0095] 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.

[0096] Test case

[0097] 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.

[0098] Table 1: Comparison of Compression Strength Test Results

[0099] 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.

[0100] 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 is taken.

[0101] Table 2: Comparison of gel time test results

[0102] 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.

[0103] 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.

[0104] Table 3: Comparison of uterine fibrosis area analysis results in mice

[0105] 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.

[0106] 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 temperature-sensitive degradable gel for the treatment of intrauterine adhesions, characterized in that, 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. Then, autologous platelet-rich plasma and the above-mentioned amygdalin-loaded polymer were added and homogenized to obtain a thermosensitive biodegradable gel. 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 them a second time. After discarding the upper layer, add thrombin and 8-10 wt% 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 8-12%, and the thrombin content is 15-25 U / mL.

2. A process for the preparation of a temperature sensitive degradable gel for the treatment of intrauterine adhesions according to claim 1, characterized in that, S1 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.

3. A process for the preparation of a temperature sensitive degradable gel for the treatment of 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 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.

4. A process for the preparation of a temperature sensitive degradable gel for the treatment of intrauterine adhesions according to claim 3, characterized in that, S4 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.

5. The method of claim 2, wherein the temperature-sensitive degradable gel for the treatment of intrauterine adhesion is prepared by the steps of: The volume ratio of the amygdalin mixture to the carboxymethyl konjac glucomannan solution was (2-3):

1.

6. The method of claim 3, wherein the temperature-sensitive degradable gel for the treatment of intrauterine adhesion is prepared by the steps of: 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 activated hydroxyproline solution to chitosan solution is 1:(8-9).

7. The method of claim 4, wherein the temperature-sensitive degradable gel for the treatment of intrauterine adhesion is prepared by the steps of, The volume ratio of autologous platelet-rich plasma to mixed sol is 1:(8-10), and the mass ratio of amygdalin-loaded polymer to autologous platelet-rich plasma is (3-5):

1.

8. A temperature-sensitive degradable gel for the treatment of intrauterine adhesions, characterized in that, It is prepared by the method for preparing a thermosensitive biodegradable gel for treating intrauterine adhesions as described in any one of claims 1-7.

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