A thermosensitive degradable gel for treating intrauterine adhesions and a preparation method thereof

Abstract

The present invention provides a thermosensitive degradable gel for treating intrauterine adhesions and a preparation method thereof, belonging to the technical field of gels; the preparation method thereof comprises the following steps: preparing amygdalin-loaded polymer; preparing hydroxyproline-modified chitosan; and mixing to prepare a thermosensitive degradable gel. The present invention prepares a solution of soy protein isolate and carboxymethyl konjac glucomannan respectively, then adds amygdalin to the soy protein isolate solution, and then mixes with the carboxymethyl konjac glucomannan solution to perform electrostatic self-assembly polymerization to form a carrier with a three-dimensional macromolecular structure, loads amygdalin, obtains amygdalin-loaded polymer, and after adding the amygdalin-loaded polymer to the gel, the anti-deformation ability of the gel can be enhanced, thereby achieving the effect of improving the supporting force of the gel and reducing the recurrence rate of intrauterine adhesions.

Description

Technical Field

[0001] The invention relates to the technical field of gel, and in particular to a temperature-sensitive degradable gel for treating intrauterine adhesions and a preparation method thereof. Background Art

[0002] Intrauterine adhesions are a common gynecological disease, which is mostly caused by local surgical operations in the uterine cavity (such as artificial abortion, curettage, etc.), inflammatory infections and other factors that lead to damage to the uterine cavity mucosa, which in turn causes uterine cavity stenosis or atresia. This pathological state seriously affects women's reproductive health and can lead to menstrual abnormalities, such as decreased menstrual volume or even amenorrhea, as well as fertility disorders, including infertility and recurrent miscarriage. According to statistics, the incidence of intrauterine adhesions has been increasing year by year among women who have undergone multiple intrauterine operations, and has become one of the important problems that need to be solved in gynecological clinics.

[0003] Traditional treatment of intrauterine adhesions mainly relies on surgical separation of adhesion tissues, such as hysteroscopic adhesion separation. Although this method can directly remove the adhesion site, the risk of re-adhesion of the uterine cavity after surgery is extremely high. This is because the surgical wound lacks effective physical barriers and tissue support during the healing process. The intrauterine mucosa is very easy to adhere again when it is not completely repaired, resulting in recurrence of adhesions. Repeated surgeries are often required, which brings great physical pain and psychological burden to patients, and also increases medical costs and consumption of medical resources. Drug treatment mainly focuses on preventing infection and promoting endometrial repair, such as using antibiotics to prevent infection and using estrogen to promote endometrial growth. However, simple drug treatment has very limited effect on improving the physical structure of the uterine cavity and providing support. When the uterine cavity morphology is not effectively maintained, it is difficult for the drug to form an effective concentration gradient and action environment locally, and its effect in promoting endometrial repair is greatly reduced, and it cannot fundamentally solve the key problem of recurrence of intrauterine adhesions.

[0004] As a new type of local drug delivery and tissue repair material, thermosensitive gel can act more accurately on the adhesion site than drug therapy. It gels under the action of body temperature, so that the drug or therapeutic ingredients can be concentrated in the adhesion site for a long time, thereby improving the targeted treatment. Compared with surgical treatment, thermosensitive gel provides a non-invasive physical barrier method. It can form a physical barrier in the uterine cavity, effectively preventing the separated uterine cavity tissues from contacting again and reducing the risk of adhesion recurrence. However, existing gels are prone to insufficient support. In the physiological environment of the uterine cavity, due to the influence of factors such as human activities, 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 when subjected to small external forces, and cannot provide sufficient space and stable physical support for the repair of the uterine cavity mucosa, thereby failing to effectively reduce the recurrence rate of adhesions, limiting its wide application in the treatment of uterine cavity adhesions.

[0005] Therefore, it is necessary to propose a thermosensitive degradable gel with good supporting force for treating intrauterine adhesions and a preparation method thereof, so as to improve the efficacy and reduce the recurrence rate. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a thermosensitive degradable gel for treating intrauterine adhesions and a preparation method thereof.

[0007] A method for preparing a thermosensitive degradable gel for treating intrauterine adhesions comprises the following steps:

[0008] S1: Preparation of amygdalin-loaded polymers

[0009] 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;

[0010] S2: Preparation of hydroxyproline-modified chitosan

[0011] Dissolving and activating hydroxyproline, adding it to an acetic acid solution of chitosan, adding triethylamine and stirring to react, thereby obtaining hydroxyproline-modified chitosan;

[0012] S3: Mixing to prepare thermosensitive degradable gel

[0013] 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.

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

[0015] 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;

[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 1 g: (320-330) mL, and perform ultrasonic treatment at 160-180 W for 20-30 min to obtain amygdalin mixed solution;

[0017] 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.

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

[0019] 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;

[0020] 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;

[0021] 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;

[0022] 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.

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

[0024] 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;

[0025] 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;

[0026] 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;

[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, and then place in a homogenizer and homogenize for 20-30 minutes to obtain a temperature-sensitive degradable 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 the 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.

[0031] Furthermore, the preparation steps of autologous platelet-rich plasma are as follows: first collect the patient's peripheral blood 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.

[0032] Furthermore, a thermosensitive degradable gel for treating intrauterine adhesions is prepared by any of the above methods for preparing a thermosensitive degradable gel for treating intrauterine adhesions.

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

[0034] 1, the present invention is by first respectively being mixed with solution by soy protein isolate and carboxymethyl konjac glucomannan, then in soy protein isolate solution, add amygdalin, then mix with carboxymethyl konjac glucomannan solution to carry out electrostatic self-assembly polymerization to form a carrier with three-dimensional macromolecular structure, amygdalin is loaded, obtain carrying amygdalin polymer, after being added in gel, on the one hand, it can be intertwined with the polymer molecular chain in the gel, as a kind of "enhanced phase", be embedded in the network structure of the gel, make the network structure of the gel more tight and firm, on the other hand, its rigid structure can play the effect of "blocking" and "fixing" between molecular chains, make the gel molecular chain more difficult when being subjected to external force, strengthen the anti-deformation ability of gel, thereby reach the effect of improving gel supporting power;

[0035] 2. The present 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 for reaction to modify the chitosan. After the hydroxyproline-modified chitosan is added to the gel, its abundant hydrogen bond forming sites can quickly form hydrogen bonds with other components in the gel system. During the temperature increase process, these additional hydrogen bonds can accelerate the mutual cross-linking between molecular chains, so that the gel forms a three-dimensional network structure in a shorter time, thereby improving the rapid gelling ability of the gel;

[0036] 3. When the thermosensitive degradable gel of the present 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 to the filling and repair of damaged tissues. When the two are used in combination, the anti-inflammatory effect of amygdalin and the tissue repair effect of autologous platelet-rich plasma can cooperate with each other. In the early stage of inflammation, amygdalin reduces the inflammatory response. When the oxidative stress and cell damage caused by inflammation are relieved, the growth factors in the autologous platelet-rich plasma can be more effective. It effectively acts on the cells of the uterine cavity tissue, promotes their proliferation and differentiation, accelerates the repair of damaged tissues, and prevents the further development of adhesions. In addition, the growth factors in autologous platelet-rich plasma promote the formation of new blood vessels at the site of uterine cavity adhesions. Amygdalin may improve the nutritional intake and metabolic state of cells, enable the new blood vessels to better play the nutrient transport function, provide sufficient nutrients for the repairing tissues, and further promote the regeneration of uterine cavity tissues and the separation of adhesions. Therefore, autologous platelet-rich plasma and amygdalin can synergistically treat intrauterine adhesions and further improve the efficacy of the treatment of intrauterine adhesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable those skilled in the relevant art to make and use the present disclosure.

[0038] Figure 1 The present invention is a flow chart of a method for preparing a thermosensitive degradable gel for treating intrauterine adhesions used in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] The following describes in detail a thermosensitive degradable gel for treating intrauterine adhesions and a preparation method thereof provided by the present invention in conjunction with the accompanying drawings and specific embodiments.

[0040] Embodiment 1:

[0041] A method for preparing a thermosensitive degradable gel for treating intrauterine adhesions, such as Figure 1 As shown, the following steps are included:

[0042] S1: Preparation of amygdalin-loaded polymers

[0043] S1.1: Soybean protein isolate and carboxymethyl konjac glucomannan are added to distilled water at a material-liquid ratio of 1 g:90 mL, respectively, and heated to 35° C. with stirring to dissolve and keep warm to obtain a soy protein isolate solution and a 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 1 g:320 mL, and perform ultrasonic treatment at 160 W for 20 min to obtain amygdalin mixed solution;

[0045] S1.3: adding the carboxymethyl konjac glucomannan solution to the amygdalin mixture, stirring at a rate of 700 r / min for 1 h to polymerize to obtain amygdalin-loaded polymer, wherein the volume ratio of the amygdalin mixture to the carboxymethyl konjac glucomannan solution is 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 a chitosan solution;

[0048] S2.2: Add hydroxyproline into deionized water at a solid-liquid ratio of 1 g:10 mL, 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 in an ice-water bath at 1°C for 1 hour to obtain an activated hydroxyproline solution, wherein the mass ratios of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to hydroxyproline are 1:2 and 1:3, respectively;

[0050] S2.4: adding the activated hydroxyproline solution to the chitosan solution while stirring, and then adding triethylamine to adjust the pH to 7, and continuing to stir and react for 16 hours, and then dialyzing against deionized water and freeze-drying to obtain hydroxyproline-modified chitosan, wherein the volume ratio of the activated hydroxyproline solution to the chitosan solution is 1:8;

[0051] S3: Mixing to prepare thermosensitive degradable gel

[0052] S3.1: Add the hydroxyproline-modified chitosan prepared in step S2.4 into 1% acetic acid solution at a solid-liquid ratio of 1 g:50 mL, stir and dissolve thoroughly to obtain a hydroxyproline-modified chitosan solution;

[0053] 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;

[0054] 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°C for 20 minutes 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-mentioned mixed sol liquid, stir and mix thoroughly, and then place in a homogenizer and homogenize for 20 minutes to obtain a temperature-sensitive degradable gel, wherein the volume ratio of autologous platelet-rich plasma to the mixed sol liquid is 1:8, and the mass ratio of the amygdalin-loaded polymer to the autologous platelet-rich plasma is 3:1; the preparation steps of autologous platelet-rich plasma are as follows: first collect the patient's peripheral blood for anticoagulation treatment, and then centrifuge to separate the peripheral blood into an upper layer, a middle layer and a lower layer, then absorb the upper layer and the middle layer for secondary centrifugation, discard the upper layer liquid, and add thrombin and 8wt% calcium chloride solution to the remaining part to obtain autologous platelet-rich plasma; wherein the volume percentage of calcium chloride solution in the autologous platelet-rich plasma is 8%, and the thrombin content is 15U / mL.

[0056] Embodiment 2:

[0057] A method for preparing a thermosensitive degradable gel for treating intrauterine adhesions, such as Figure 1 As shown, the following steps are included:

[0058] S1: Preparation of amygdalin-loaded polymers

[0059] S1.1: Soybean protein isolate and carboxymethyl konjac glucomannan are added to distilled water at a material-liquid ratio of 1 g:95 mL, respectively, and heated to 40° C. with stirring to dissolve and keep warm to obtain a soy protein isolate solution and a carboxymethyl konjac glucomannan solution;

[0060] S1.2: hydrochloric acid was added to the above soy protein isolate solution to adjust the pH to 3.5, and amygdalin was added at a solid-liquid ratio of 1 g:325 mL, and ultrasonic treatment was performed at 170 W for 25 min to obtain amygdalin mixed solution;

[0061] S1.3: adding the carboxymethyl konjac glucomannan solution to the amygdalin mixture, stirring at a rate of 750 r / min for 1.5 h to polymerize to obtain amygdalin-loaded polymer, wherein the volume ratio of the amygdalin mixture to the carboxymethyl konjac glucomannan solution is 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 a chitosan solution;

[0064] S2.2: Add hydroxyproline into deionized water at a solid-liquid ratio of 1 g:15 mL, 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 mixture in an ice-water bath at 2°C for 1.5 hours to obtain an activated hydroxyproline solution, wherein the mass ratios of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to hydroxyproline are 1:2.5 and 1:4, respectively;

[0066] S2.4: adding the activated hydroxyproline solution to the chitosan solution while stirring, and then adding triethylamine to adjust the pH to 7.5, and continuing to stir and react for 18 hours, dialyzing with deionized water and freeze-drying to obtain hydroxyproline-modified chitosan, wherein the volume ratio of the activated hydroxyproline solution to the chitosan solution is 1:8.5;

[0067] S3: Mixing to prepare thermosensitive degradable gel

[0068] S3.1: Add the hydroxyproline-modified chitosan prepared in step S2.4 into 1% acetic acid solution at a solid-liquid ratio of 1 g:55 mL, stir and dissolve thoroughly to obtain a hydroxyproline-modified chitosan solution;

[0069] 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;

[0070] 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 3°C ​​for 25 minutes 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-mentioned mixed sol liquid, stir and mix thoroughly, and then place in a homogenizer and homogenize for 25 minutes to obtain a temperature-sensitive degradable gel, wherein the volume ratio of autologous platelet-rich plasma to the mixed sol liquid is 1:9, and the mass ratio of the amygdalin-loaded polymer to the autologous platelet-rich plasma is 4:1; the preparation steps of autologous platelet-rich plasma are as follows: first collect the patient's peripheral blood for anticoagulation treatment, and then centrifuge to separate the peripheral blood into an upper layer, a middle layer and a lower layer, then absorb the upper layer and the middle layer for secondary centrifugation, discard the upper layer liquid, and add thrombin and 9wt% calcium chloride solution to the remaining part to obtain autologous platelet-rich plasma; wherein the volume percentage of calcium chloride solution in the autologous platelet-rich plasma is 10%, and the thrombin content is 20U / mL.

[0072] Embodiment 3:

[0073] A method for preparing a thermosensitive degradable gel for treating intrauterine adhesions, such as Figure 1 As shown, the following steps are included:

[0074] S1: Preparation of amygdalin-loaded polymers

[0075] S1.1: Soybean protein isolate and carboxymethyl konjac glucomannan are added to distilled water at a material-liquid ratio of 1 g:100 mL, respectively, and heated to 45° C. with stirring to dissolve and keep warm to obtain a soy protein isolate solution and a 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 1 g:330 mL, and perform ultrasonic treatment at 180 W for 30 min to obtain amygdalin mixed solution;

[0077] S1.3: adding the carboxymethyl konjac glucomannan solution to the amygdalin mixture, stirring at a rate of 800 r / min for 2 h to polymerize to obtain amygdalin-loaded polymer, wherein the volume ratio of the amygdalin mixture to the carboxymethyl konjac glucomannan solution is 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 a chitosan solution;

[0080] S2.2: Add hydroxyproline into deionized water at a solid-liquid ratio of 1 g:20 mL, 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 in an ice-water bath at 3°C ​​for 2 hours to obtain an activated hydroxyproline solution, wherein the mass ratios of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to hydroxyproline are 1:3 and 1:5, respectively;

[0082] S2.4: adding the activated hydroxyproline solution to the chitosan solution while stirring, and then adding triethylamine to adjust the pH to 8, and continuing to stir and react for 20 hours, dialyzing with deionized water and freeze-drying to obtain hydroxyproline-modified chitosan, wherein the volume ratio of the activated hydroxyproline solution to the chitosan solution is 1:9;

[0083] S3: Mixing to prepare thermosensitive degradable gel

[0084] S3.1: Add the hydroxyproline-modified chitosan prepared in step S2.4 into 1% acetic acid solution at a solid-liquid ratio of 1 g:60 mL, stir and dissolve thoroughly to obtain a hydroxyproline-modified chitosan solution;

[0085] 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;

[0086] 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 4°C for 30 minutes 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-mentioned mixed sol liquid, stir and mix thoroughly, and then place in a homogenizer and homogenize for 30 minutes to obtain a temperature-sensitive degradable gel, wherein the volume ratio of autologous platelet-rich plasma to the mixed sol liquid is 1:10, and the mass ratio of the amygdalin-loaded polymer to the autologous platelet-rich plasma is 5:1; the preparation steps of autologous platelet-rich plasma are as follows: first collect the patient's peripheral blood for anticoagulation treatment, and then centrifuge to separate the peripheral blood into an upper layer, a middle layer and a lower layer, then absorb the upper layer and the middle layer for secondary centrifugation, discard the upper layer liquid, and add thrombin and 10wt% calcium chloride solution to the remaining part to obtain autologous platelet-rich plasma; wherein the volume percentage of calcium chloride solution in the autologous platelet-rich plasma is 12%, and the thrombin content is 25U / 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 by an equal amount of chitosan.

[0092] Comparative Example 3:

[0093] The difference between Comparative Example 3 and Example 1 is that the amygdalin in step S1.2 is replaced by 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 example:

[0097] Test 1: The temperature-sensitive degradable gels prepared in Examples 1-3 and Comparative Example 1 were heated to 37°C to form a gel, which was then cut into cubes of 10 mm×10 mm×10 mm. The compressive strength was then tested using a universal material testing machine. The results are shown in Table 1.

[0098] Table 1: Comparison of compression strength test results

[0099] ;

[0100] As can be seen from Table 1, the compressive strength of the temperature-sensitive degradable gel prepared without adding the amygdalin-loaded polymer in Comparative Example 1 is about 0.46 MPa, which is much smaller than that in Example 1. It can be seen that by first respectively preparing soy protein isolate and carboxymethyl konjac glucomannan into solutions, adding amygdalin to the soy protein isolate solution, and then mixing with the carboxymethyl konjac glucomannan solution to perform electrostatic self-assembly polymerization to form a carrier with a three-dimensional macromolecular structure, loading amygdalin to obtain the amygdalin-loaded polymer, and adding the amygdalin-loaded polymer to the gel, the compressive strength of the gel can be effectively improved, thereby achieving the effect of improving the supporting force of the gel.

[0101] Test 2: Take 2 mL of the thermosensitive degradable gel prepared in Examples 1-3 and Comparative Example 2, place it in a vial at room temperature for 30 minutes, then place it in a 37°C water bath, start timing, and observe the flow of the gel in the vial. When the thermosensitive degradable gel in the vial does not flow with the tilt of the vial, stop timing, and this time is the gelation time. Each sample is measured 3 times and the average is taken.

[0102] Table 2: Comparison of gel time test results:

[0103] Example 1 Example 2 Example 3 Comparative Example 2 Gel time (s) 77 77 78 183 ;

[0104] As can be seen from Table 2, when chitosan is not modified with hydroxyproline in Comparative Example 2, the obtained thermosensitive degradable gel has a gel time of about 183 s at 37°C, which is much longer than that in Example 1. It can be seen 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 for reaction, chitosan is modified, and after the hydroxyproline-modified chitosan is added to the gel, the rapid gelling ability of the gel can be improved.

[0105] Test 3: 7-week-old BALB / c adult female mice were selected, and the clinical mechanical curettage injury was simulated by the scraping method. The mice were anesthetized by inhalation of 5% isoflurane, and the abdominal cavity was entered from the back of the mouse to expose the uterine horn. A 24G needle was used to repeatedly rotate and scrape the endometrium. At the same time, the uterus was observed to be congested and thin under a stereomicroscope. The needle touched the surface of the uterus and felt rough, and the wound on the surface of the uterus caused by the needle was sutured with sterile absorbable surgical sutures. After the operation, the abdomen was closed layer by layer with absorbable sutures. After the operation, the mouse was placed on a 37°C heating pad and observed for 2 hours. If there was no special condition, it was returned to the animal room. After the mouse woke up from anesthesia, it was given water and mouse food to obtain a sample mouse with intrauterine adhesion. Then the sample mice were divided into 3 groups, the first group was Example 1 group, and 50 μL of the thermosensitive degradable gel prepared in Example 1 was injected into the mouse uterine cavity in situ; the second group was Comparative Example 3 group, and 50 μL of the thermosensitive degradable gel prepared in Comparative Example 3 was injected into the mouse uterine cavity in situ; the third group was Comparative Example 4 group, and 50 μL of the thermosensitive degradable hydrogel prepared in Comparative Example 4 was injected into the mouse uterine cavity in situ. 14 days after the injection, the three groups of mice were subjected to Masson chromosome analysis, and the area of ​​uterine fibrosis in the mice was analyzed, and the results are shown in Table 3.

[0106] Table 3: Comparison of mouse uterine fibrosis area analysis results

[0107] Example 1 Comparative Example 3 Comparative Example 4 Uterine fibrosis area (%) 26.8 49.3 75.6 ;

[0108] As can be seen from Table 3, when only autologous platelet-rich plasma was added in Comparative Example 3 and only amygdalin was added in Comparative Example 4, the area of ​​uterine fibrosis in the mice with intrauterine adhesion was greater than that in Example 1 14 days after the thermosensitive degradable gel was injected into the mice. This shows that autologous platelet-rich plasma and amygdalin can synergistically treat intrauterine adhesion and further improve the efficacy of the treatment of intrauterine adhesion.

[0109] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall 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 Dissolve polygalacturonic acid, sodium β-glycerophosphate and the hydroxyproline-modified chitosan separately, stir and mix in an ice water bath, then add autologous platelet-rich plasma and the amygdalin-loaded polymer, mix homogenously, and obtain a temperature-sensitive degradable gel; 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.

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. 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 7.

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