Preparation method for curcumin-loaded recombinant collagen hydrogel and use of curcumin-loaded recombinant collagen hydrogel in treatment of intrauterine adhesion

Abstract

A preparation method for a curcumin-loaded recombinant collagen hydrogel and a use of the curcumin-loaded recombinant collagen hydrogel in treatment of intrauterine adhesion. Homogenized curcumin is introduced, so that the curcumin changes from fat-soluble to water-soluble and can be better absorbed by a human body, thereby enhancing the bioavailability. Meanwhile, by combining with a crosslinking agent at a specific concentration, crosslinking between collagen is promoted, so that the recombinant collagen can act on a lesion site for a long time, the loss of the recombinant collagen in delivery is reduced, and the therapeutic effect of the recombinant collagen is improved. The prepared hydrogel effectively solves the technical problems of severe loss of recombinant collagen and unsatisfactory treatment effect when recombinant collagen is directly and locally applied to an intrauterine adhesion site, significantly improves the treatment effect, and has a high clinical value in preventing tissue adhesion.

Description

Preparation method of curcumin-loaded recombinant collagen hydrogel and its application in the treatment of intrauterine adhesions Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for preparing a curcumin-loaded recombinant collagen hydrogel and application of the hydrogel in treating intrauterine adhesions. Background Art

[0002] The female uterus plays a vital role in physiological and reproductive functions. The normal endometrium is capable of self-repair and regeneration. However, conditions such as induced abortion, induced labor, curettage, severe endocrine disorders, and intrauterine infection can damage the endometrium, disrupting its normal structure and causing increased tissue fibrosis, decreased glandular volume, and partial or complete occlusion of the uterine cavity and / or cervical canal, leading to intrauterine adhesions (IUA). Intrauterine adhesions (IUA), also known as Asherman syndrome, can clinically present as decreased menstrual flow, amenorrhea, spermatogenesis impairment, premature menopause, cyclical lower abdominal pain, recurrent miscarriage, infertility, and obstetric complications such as placenta accreta and placenta accreta. This condition is a serious gynecological condition, severely impacting fertility and menstruation in women of childbearing age, often leading to reproductive impairment and menstrual abnormalities. Improper care after treatment can lead to recurrence, resulting in further adhesions. It is a serious public health issue that seriously endangers women's reproductive health.

[0003] The treatment of intrauterine adhesions (IUA) is a clinical challenge, particularly for moderate to severe cases, where existing treatments are ineffective. For example, hysteroscopic adhesiolysis and postoperative adhesion prevention, including intraoperative placement of physical barriers such as intrauterine devices, Cook balloons, and regenerated cellulose membranes, combined with postoperative high-dose estrogen to promote endometrial growth, are the mainstays of treatment for IUA. However, these treatments are ineffective for patients with moderate to severe IUA. Vaginal suppositories, douches, and oral tablets have been reported as endometrial repair agents, but their efficacy remains limited. This is primarily due to the inability of these existing agents to ensure close contact with the damaged endometrium and maintain effective concentrations over a prolonged period. Furthermore, the rapid secretion and turnover of endometrial mucus leads to rapid drug loss within the damaged uterine cavity, preventing the achievement of effective therapeutic concentrations. These factors hinder the application of current agents in endometrial repair. Therefore, drug loading into a gel allows for long-term drug delivery to the affected area, minimizing drug loss during delivery and enhancing efficacy.

[0004] Curcumin is a yellow pigment extracted from the rhizomes of turmeric, a plant of the ginger family. Turmeric and curcumin are natural plant components with medicinal value and no significant toxic side effects. However, despite its wide range of uses and high medicinal value, curcumin's poor solubility in water, poor absorption by the human body, and rapid metabolism significantly limit its application.

[0005] Recombinant collagen has demonstrated promising therapeutic effects in a variety of trauma repair diseases. Studies have found that recombinant collagen can modulate the disease microenvironment at the lesion site, promoting the repair and regeneration of damaged tissue, demonstrating significant potential in the treatment of traumatic diseases. However, direct topical application of recombinant collagen to intrauterine adhesions results in significant loss of recombinant collagen, a short duration of action at the lesion site, and unsatisfactory therapeutic effects. Improving the effectiveness of recombinant collagen in the treatment of intrauterine adhesions is a technical challenge that urgently needs to be addressed, and currently, no research has examined the combined use of curcumin and recombinant collagen for the treatment of intrauterine adhesions.

[0006] Summary of the Invention

[0007] In response to the deficiencies of the prior art, the present invention provides a method for preparing a curcumin-loaded recombinant collagen hydrogel and its application in the treatment of intrauterine adhesions. The present invention creatively uses homogenized curcumin as a raw material, combined with a cross-linking agent and recombinant collagen to prepare a hydrogel, which can be used to treat intrauterine adhesions and achieves significant technical effects.

[0008] In order to achieve the above technical objectives, the technical solutions provided by the present invention are as follows:

[0009] The present invention first provides a curcumin-loaded recombinant collagen hydrogel, which is composed of the following raw materials:

[0010] Curcumin, a cross-linking agent, recombinant collagen, and water; the mass fraction of curcumin in the raw materials is 0.15-1.0%, and the mass fraction of recombinant collagen is 5-10%; the cross-linking agents are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (NHS) and N-hydroxysuccinimide (EDC), and the amount of each is 0.3-0.4% of the mass of the recombinant collagen; the balance is water; the curcumin is first dissolved in water when used, and then added to the homogenized liquid after homogenization.

[0011] Preferably, the water includes deionized water, water for injection, and physiological saline; the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide is 1:1; and the recombinant collagen is recombinant human type III collagen, preferably in an amount of 8%.

[0012] The present invention also provides a method for preparing a curcumin-loaded recombinant collagen hydrogel, comprising the following steps:

[0013] (1) First, a certain amount of curcumin powder is weighed and added to water, and then subjected to magnetic stirring and homogenization cycle treatment to obtain a curcumin homogenous solution; wherein the magnetic stirring time is 1.5-2 hours, and the number of homogenization cycles is 20-30 times;

[0014] (2) dissolving the recombinant collagen raw material in the curcumin homogenate and stirring evenly to form a curcumin-loaded collagen solution; then adding cross-linking agents N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, stirring evenly, reacting for a period of time to obtain a curcumin-loaded recombinant collagen absorbable hydrogel, cutting the curcumin-loaded recombinant collagen absorbable hydrogel into a size suitable for dialysis to obtain a curcumin-loaded recombinant collagen absorbable hydrogel block, placing it in a phosphate buffer for dialysis to exchange the cross-linker, setting the PBS osmotic pressure and dialysis time for dialysis, and after dialysis, pulverizing the block curcumin-loaded recombinant collagen absorbable hydrogel to obtain granules, and then filling and sterilizing to obtain the curcumin-loaded recombinant collagen hydrogel.

[0015] In the above step (2), the reaction time is 2-6 hours; the osmotic pressure of the phosphate buffer is set to 200-500 mOsm / kg, and the dialysis time is 4-6 days; the pH of the phosphate buffer is 7.5-8; the curcumin-loaded recombinant collagen absorbable hydrogel is cut into a size of 1-2 cm suitable for dialysis 3 .

[0016] The invention discloses an application of the curcumin-loaded recombinant collagen hydrogel in the preparation of products for preventing and treating intrauterine adhesions, resisting uterine fibrosis, and promoting endometrial recovery.

[0017] A composite preparation comprises the curcumin-loaded recombinant collagen hydrogel.

[0018] The composite preparation is used for preparing products for repairing various endometrial injuries, wherein the repairing of various endometrial injuries includes treating intrauterine adhesions, resisting uterine fibrosis, and promoting endometrial recovery.

[0019] The beneficial effects of the present invention are:

[0020] (1) The curcumin-loaded recombinant collagen hydrogel prepared by the present invention has good biocompatibility, does not cause pollution to the patient's affected area and clothing, and is highly comfortable; it also has an anti-adhesion effect and has high clinical value in preventing postoperative tissue adhesion.

[0021] (2) The curcumin used in the present invention is homogenized and added in the form of a homogenous liquid; its purpose is, on the one hand, to make curcumin from fat-soluble to water-soluble, so that it can be better absorbed by the human body and enhance bioavailability; on the other hand, it realizes the application of the hydrogel prepared based on curcumin in the treatment of intrauterine adhesions and achieves significant technical effects.

[0022] (3) The present invention further limits the amount of curcumin in the curcumin-loaded recombinant collagen hydrogel. If the amount of curcumin is too low, the viscosity of the curcumin-loaded recombinant collagen hydrogel will be too low, and the effect of treating intrauterine adhesions will be insignificant. If the amount of curcumin is too high, the viscosity will increase, making the hydrogel difficult to absorb and excrete from the body, and the effect of treating intrauterine adhesions will be poor. Therefore, the amount of curcumin is crucial. Too high or too low is not conducive to application and cannot effectively achieve the therapeutic effect of intrauterine adhesions.

[0023] (4) The present invention creatively introduces the synergistic effect of curcumin and cross-linking agent, which promotes cross-linking between collagens, allowing recombinant collagen to act on the lesion site for a long time, reducing the loss of recombinant collagen during delivery and improving the therapeutic effect of recombinant collagen; the combination of the two effectively solves the technical problems of direct local application of recombinant collagen to the uterine adhesion site, resulting in severe loss of recombinant collagen, short action time on the lesion, and unsatisfactory therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a diagram of the dynamic viscosity of recombinant collagen hydrogels in groups A and B in preliminary experiment 1.

[0025] Figure 2 shows the dynamic viscosity diagram of recombinant collagen hydrogels in groups C, D, E, and F in preliminary experiment 1.

[0026] FIG3 is a diagram of the uterine tissue morphology of rats in the model group in Example 1.

[0027] FIG4 is a diagram showing the uterine tissue morphology of rats in the treatment group in Example 1.

[0028] FIG5 is a white light scan of the left uterine scar (MASSON) of rats in the model group in Example 1.

[0029] FIG6 is a white light scan of the right uterine scar (MASSON) of rats in the model group in Example 1.

[0030] FIG7 is a white light scan of the left uterine scar (MASSON) of rats in the treatment group in Example 1.

[0031] FIG8 is a white light scan of the right uterine scar (MASSON) of rats in the treatment group in Example 1.

[0032] FIG9 is a white light scanning image of endometrial cells (HE) on the left side of the rats in the model group in Example 1.

[0033] FIG10 is a white light scanning image of endometrial cells (HE) on the right side of the rats in the model group in Example 1.

[0034] FIG11 is a white light scanning image of endometrial cells (HE) on the left side of the rats in the treatment group in Example 1.

[0035] FIG12 is a white light scanning image of endometrial cells (HE) on the right side of the rats in the treatment group in Example 1.

[0036] FIG13 is a diagram of the uterine tissue morphology of rats in the model group in Example 2.

[0037] FIG14 is a diagram showing the uterine tissue morphology of rats in the treatment group in Example 2.

[0038] FIG15 is a white light scan of the left uterine scar (MASSON) of rats in the model group in Example 2.

[0039] FIG16 is a white light scan of the right uterine scar (MASSON) of rats in the model group in Example 2.

[0040] FIG17 is a white light scan of the left uterine scar (MASSON) of rats in the treatment group in Example 2.

[0041] FIG18 is a white light scan of the right uterine scar (MASSON) of rats in the treatment group in Example 2.

[0042] FIG19 is a white light scanning image of endometrial cells (HE) on the left side of the rats in the model group in Example 2.

[0043] FIG20 is a white light scanning image of endometrial cells (HE) on the right side of the rats in the model group in Example 2.

[0044] FIG21 is a white light scanning image of endometrial cells (HE) on the left side of the rats in the treatment group in Example 2.

[0045] FIG22 is a white light scanning image of endometrial cells (HE) on the right side of the rats in the treatment group in Example 2.

[0046] FIG23 shows the endometrial thickness of AD rats in the model group and the treatment group. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] (1) Raw material selection:

[0049] a. Solute: curcumin, recombinant type III collagen, 50% (w / w) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (C8H 17 N3HCl, EDC), 10% (w / w) N-hydroxysuccinimide (C4H5NO3, NHS);

[0050] b. Solvent water: deionized water;

[0051] (2) Dosage instructions: Curcumin is first dissolved in water and added to the homogenized solution at a mass fraction of 0.15-1.0%; the mass fraction of recombinant collagen is 5-10%; the cross-linking agents are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (NHS) and N-hydroxysuccinimide (EDC), and their usage amounts are 0.3-0.4% of the mass of recombinant collagen; the balance is deionized water.

[0052] Raw Materials: The reagents and raw materials used in this invention are all existing products. Curcumin was purchased from Adamas. The recombinant human type III collagen used in this invention is a genetically engineered collagen protein. The prepared recombinant human type III collagen protein is disclosed in patent application number: 201310033299.6, patent title: Genetically Recombinant Human Collagen. N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Tokyo Chemical Industry Co., Ltd. (TCI). Phosphate buffer was purchased from Solarbio.

[0053] A method for preparing a curcumin-loaded recombinant collagen hydrogel comprises the following steps:

[0054] (1) First, a certain amount of curcumin powder was weighed and added to water, magnetically stirred for 1.5-2 h, and homogenized 20-30 times to obtain a curcumin homogenate solution;

[0055] (2) Dissolving the recombinant collagen raw material in the curcumin homogenate and stirring evenly to form a curcumin-loaded collagen solution; then adding cross-linking agents N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, stirring evenly, and reacting for 2-6 hours to obtain a curcumin-loaded recombinant collagen absorbable hydrogel, cutting the curcumin-loaded recombinant collagen absorbable hydrogel into a size suitable for dialysis to obtain a curcumin-loaded recombinant collagen absorbable hydrogel block, placing it in a phosphate buffer for dialysis to exchange the cross-linker, the PBS osmotic pressure is 200-500mOsm / kg, the dialysis time is 4-6 days, after dialysis, the block curcumin-loaded recombinant collagen absorbable hydrogel is crushed and granulated, and then filled and sterilized to obtain the curcumin-loaded recombinant collagen hydrogel.

[0056] Preliminary experiment 1:

[0057] The preparation method of curcumin-loaded recombinant collagen hydrogel was the same as above; 6 groups of components with different proportions were set and the corresponding dynamic viscosities were tested. The specific raw material components and test results are shown in Table 1.

[0058] Table 1 Ratio of each component and corresponding dynamic viscosity

[0059] By comparing the data of groups A, B, C, D, E, and F, it can be seen that the use of cross-linking agents has a promoting effect on the improvement of dynamic viscosity; at the same time, the change in the dosage of curcumin has a more significant effect on the dynamic viscosity; from the data of group A, it can be seen that if the dosage of curcumin is too low, the viscosity of the recombinant collagen hydrogel loaded with curcumin will be too low, which is not conducive to application; if the dosage of curcumin in group B is too high, it will also lead to increased viscosity, making the hydrogel not conducive to absorption and excretion from the body; therefore, controlling the dosage of curcumin is crucial, and too high or too low is not conducive to application and cannot achieve effective treatment of intrauterine adhesions.

[0060] According to the ratio of each component listed in Table 1, a recombinant collagen hydrogel loaded with curcumin was prepared and subjected to rheological testing. Figures 1 and 2 are dynamic viscosity diagrams based on rheology. At a fixed temperature of 25°C and a fixed shear rate of 0.25 (1 / s), the viscosity increases with the increase of the cross-linker ratio and the curcumin concentration. At the same shear rate, the viscosity at 25°C is close to that at 37°C. From this phenomenon, it can be seen that the viscosity of the recombinant collagen hydrogel loaded with curcumin does not change when the temperature rises to close to human body temperature (37°C), and it has a certain stability.

[0061] As shown in Figure 1, a low curcumin dosage results in a low viscosity of the curcumin-loaded recombinant collagen hydrogel, making it ineffective in treating intrauterine adhesions. A high curcumin dosage also increases the viscosity, making it difficult for the hydrogel to be absorbed and excreted from the body. Consequently, a high curcumin dosage is not effective in treating intrauterine adhesions. The viscosity shown in the data for groups C, D, E, and F in Figure 2 indicates that the hydrogel is suitable for intrauterine adhesions, supporting the application of curcumin-loaded recombinant collagen hydrogel in preventing uterine adhesions.

[0062] Preliminary experiment 2:

[0063] The proportion of each component in the heterogeneous group and the corresponding dynamic viscosity;

[0064] The preparation method is the same as that of Preliminary Experiment 1, except that in step (1), curcumin powder is added to water without homogenization. Two groups G and H are set up, and the specific results are shown in Table 2.

[0065] According to the results of G and H dynamic viscosity measurements, the dynamic viscosity of the curcumin solution treated with inhomogeneous treatment decreased compared with the mean group, but was still within the applicable range; however, combined with Figure 23, under the same conditions, there was no significant difference in the endometrial thickness between the inhomogeneous group and the model group (Figure 23), and the inhomogeneous curcumin hydrogel had no significant effect on the recovery of the rat endometrium, which further illustrates the unexpected substantial effect of the homogenized curcumin.

[0066] Animal experiments, method introduction:

[0067] (1) Selection of experimental subjects:

[0068] Eight-week-old, sexually mature female rats weighing approximately 250 g were selected and subjected to adaptive culture for one week. Surgery was performed during the estrus period (estrus cycle is 4-5 days).

[0069] (2) Required materials:

[0070] Reagents: physiological saline, PBS buffer, 95% ethanol, 75% ethanol (or iodine disinfection), 0.1% crystal violet solution, 10% chloral hydrate, 4% paraformaldehyde;

[0071] 4% paraformaldehyde: Weigh 40g of paraformaldehyde powder and dissolve it in 1000ml of phosphate buffered saline (PBS). Heat to 60°C to fully dissolve it. Cool it to room temperature and store it in a sealed container in a cool place for no more than 1 month.

[0072] Cleaning solution: Soak in 30% hydrochloric acid or 5% phenol solution for 24 hours, then rinse thoroughly with water. This method can be used to clean coverslips or slides.

[0073] (3) Group establishment and processing methods:

[0074] Twelve rats were randomly divided into four groups, namely, model group, low-concentration homogeneous curcumin hydrogel group (treatment group A), high-concentration homogeneous curcumin hydrogel group (treatment group B), low-concentration heterogeneous curcumin hydrogel group (treatment group C), and high-concentration heterogeneous curcumin hydrogel group (treatment group D).

[0075] Model group: Bilateral curettage was performed to establish the model of intrauterine adhesions; 100 μL of deionized water was injected into both uteri.

[0076] Treatment groups (treatment groups A-D): Based on the established intrauterine adhesion model, 100 μL of recombinant collagen hydrogel loaded with curcumin was injected into both uteri.

[0077] (4) Determination of estrus:

[0078] Vaginal cytology:

[0079] Note: Vaginal smear collection and estrous cycle assessment should be performed between 8:30 and 9:30 am every day to prevent cells from transitioning to the next stage and the assessment should be conducted for at least one week.

[0080] Open the base of your left hand and quickly insert your thumb and index finger under the base of the rat's tail. With the base of your left hand facing backward, press the remaining three fingers and palm of your hand on the back half of the rat's body. Be careful not to use too much force. Lift the tail, let the front paws rest on the cage, and the hind legs hang in the air. You can see the vaginal opening open. Use a pipette and a pipette to draw in (50μL) of normal saline, place the pipette tip 3-5mm from the female rat's vaginal opening, slowly release the liquid into the vagina and then draw it back out. Repeat 4-5 times, then drop the liquid onto a glass slide, dry, fix with 95% alcohol, and then stain with 0.1% crystal violet solution for 3 minutes. Rinse and observe under a microscope.

[0081] (5) Surgical procedures:

[0082] Curettage: The abdomen is shaved and disinfected, and then anesthetized and fixed.

[0083] · Make a longitudinal incision of about 3 cm in the abdominal wall skin 2-3 cm above the pubic symphysis. Cut the tissue layer by layer, enter the abdominal cavity, and slowly pick out the Y-shaped uterus of the rat.

[0084] ·Simultaneous surgery on both uterine corners

[0085] Make a longitudinal incision about 5 mm in length with ophthalmic straight scissors approximately 5 mm above the cervix. Use a homemade rat uterine curette to perform curettage through the incision above the cervix. Stop curettage when the uterine cavity walls feel rough.

[0086] Use sterile gauze to stop bleeding thoroughly, suture the uterus with interrupted 7-0 sutures, return the uterus to the abdominal cavity, flush the abdominal cavity with normal saline, and close the abdomen with 3-0 sutures. Administer 200 mg / kg of antibiotics intramuscularly every 48 hours after surgery.

[0087] The model group only made a longitudinal incision of the same length in the uterus

[0088] After the second estrous cycle, the rats were sacrificed, fixed with 4% paramethylol for more than 24 hours, dehydrated with gradient ethanol, then embedded in paraffin, and sectioned for staining and observation.

[0089] (6) Result processing (testing): Masson staining and hematoxylin-eosin (HE) staining were used to evaluate the healing effect of the uterus in each group. The tissue morphology, Masson staining, HE staining, endometrial cells and endometrial thickness were tested.

[0090] Example 1:

[0091] 1. Raw material selection:

[0092] a. Solute: curcumin, recombinant type III collagen, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (C8H 17 N3HCl, EDC), N-hydroxysuccinimide (C4H5NO3, NHS);

[0093] b.Solvent: deionized water

[0094] (2) Preparation of curcumin-loaded recombinant collagen hydrogel:

[0095] a. Weigh a certain amount of curcumin powder and add it to deionized water to prepare a curcumin aqueous solution. Stir magnetically for 2 hours and homogenize 30 times. The mass fraction of curcumin is 0.15%.

[0096] b. The recombinant type III collagen raw material was dissolved in the above-mentioned curcumin aqueous solution and stirred to form a curcumin-loaded collagen solution, and then a cross-linking agent was added, the cross-linking agent being 10% (w / w) of N-hydroxysuccinimide (NHS) and 50% (w / w) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in combination; stirred and reacted for 2 h to obtain a curcumin-loaded recombinant type III collagen absorbable hydrogel; wherein the mass fraction of recombinant type III collagen was 8%; according to the mass fraction of recombinant type III collagen: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was 0.37% and N-hydroxysuccinimide was 0.37%;

[0097] Cut the block of recombinant type III collagen absorbable hydrogel into 2 cm 3 Dialysis size: The recombinant collagen absorbable hydrogel block was placed in phosphate buffered saline (PBS) for dialyzation to exchange the crosslinker. The PBS osmotic pressure was 200mOsm / kg and the dialysis time was 4 days. The material was crushed by a homogenizer and poured into a centrifuge bottle for centrifugation (9000rpm / 20min); the gel was loaded into a pre-filled needle and sterilized in a sterilizer at 121°C for 15min to finally obtain a recombinant collagen hydrogel loaded with curcumin.

[0098] 2. Animal Experiment Results

[0099] ①Observation of rat uterine tissue morphology:

[0100] First, the abdomens of the two groups of rats were disinfected, anesthetized and fixed. A longitudinal incision of about 3 cm was made on the abdominal wall skin 2-3 cm above the pubic symphysis, and the tissue was cut layer by layer to enter the abdominal cavity, and the Y-shaped uterus of the rat was slowly picked out. Figure 3 is a diagram of the uterine tissue morphology of the rats in the model group; Figure 4 is a diagram of the uterine tissue morphology of the rats in treatment group A. As shown in Figures 3 and 4, there was obvious abnormal tissue hyperplasia on the left side of the uterus of the rats in the model group, while the left side of the uterus of the rats in the treatment group was relatively flat and had no obvious hyperplasia. Similarly, the right side of the uterus of the rats in the model group was also significantly thickened relative to the treatment group. Compared with the uterine tissue morphology of the modeled rats, the uterine tissue morphology of the rats after treatment was significantly improved, indicating that the recombinant collagen hydrogel loaded with low concentration of curcumin (0.15%) has a significant effect on the restoration of rat uterine tissue morphology.

[0101] ②Masson scar:

[0102] Rats in both groups were sacrificed after the second estrous cycle, fixed with 4% paramethanol for more than 24 hours, dehydrated with gradient ethanol, and then embedded in paraffin. Sections were dewaxed in distilled water and stained with Weigert iron hematoxylin for 3 minutes. The sections were differentiated with hydrochloric acid and alcohol for 15 seconds (if the iron hematoxylin staining was too deep, the differentiation time could be appropriately increased), washed with water, blued with Masson bluing solution, washed with water, and washed with distilled water for 1 minute. The sections were stained with Ponceau fuchsin for 7-8 seconds (the staining time was controlled according to tissue density), washed with 0.2% weak acid for 1 minute, washed with phosphomolybdic acid solution for 2 minutes, washed with 0.2% weak acid for 1 minute, stained with aniline blue for 1 minute 30 seconds (the staining time was controlled according to tissue density), washed with 0.2% weak acid for 1 minute, and dehydrated with 95% ethanol for 2-3 seconds and anhydrous ethanol for 3 times, each for 5-10 seconds. The sections were cleared with xylene for 3 times, each for 1-2 minutes, and sealed. The rat uterine scars were observed by MASSON staining. As shown in Figures 5 and 6, which are the white light scans of the left and right uterine scars (MASSON) of the rats in the model group, it can be clearly seen that the left and right uteri of the rats in the model group had obvious adhesions, respectively; as shown in Figures 7 and 8, which are the white light scans of the left and right uterine scars (MASSON) of the rats in the treatment group A, the left and right uteri of the rats in the treatment group had normal morphology and no adhesions, indicating that the recombinant collagen hydrogel loaded with curcumin has a significant effect on the treatment of uterine adhesions.

[0103] On the other hand, it can be seen from the picture that the optical density and area of ​​the collagen fibers (blue) on the left and right sides of the uterus of the rats in the treatment group are significantly smaller than those in the uterus of the rats in the model group, indicating that the recombinant collagen hydrogel loaded with low concentration of curcumin also has a significant promoting effect on the anti-fibrosis of the rat uterus.

[0104] ③ Endometrial cells (HE):

[0105] The two groups of rats were killed after the second estrous cycle, fixed with 4% polymethanol for more than 24 hours, dehydrated with ethanol gradient, and then embedded in paraffin. The sections were dewaxed to distilled water. Hematoxylin staining of cell nuclei: the sections were stained with Harris hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, turned blue with 0.6% ammonia water, and rinsed with running water. Eosin staining of cytoplasm: the sections were stained with eosin staining solution for 1-3 minutes. Dehydration and sealing: the sections were dehydrated with ethanol gradient to make them transparent, the sections were taken out of xylene and slightly dried, and then sealed with neutral gum. Rat endometrial cells were observed by HE staining (chromatin in the cell nucleus and nucleic acids in the cytoplasm were purple-blue; components in the cytoplasm and extracellular matrix were red);

[0106] As shown in Figures 9-12, the endometrial cells on the left side (Figure 9) and right side (Figure 10) of the uterus of the rats in the model group were significantly reduced compared with the endometrial cells on the left side (Figure 11) and right side (Figure 12) of the uterus of the rats in the treatment group; and it can be seen from the pictures that the left and right uterine areas of the rats in the model group are larger, and the uterine cavity area of ​​the treatment group A is smaller than that of the model group, indicating that the endometrium recovered better after treatment, maintained the basic shape of the uterine cavity, and had no adhesions.

[0107] Example 2:

[0108] 1. Raw material selection:

[0109] a. Solute: curcumin, recombinant type III collagen, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (C8H 17 N3HCl, EDC), N-hydroxysuccinimide (C4H5NO3, NHS);

[0110] b.Solvent: deionized water;

[0111] (2) The preparation method of curcumin-loaded recombinant collagen hydrogel is as follows:

[0112] a. Weigh a certain amount of curcumin powder and add it to deionized water to prepare a curcumin aqueous solution. Stir magnetically for 2 hours and homogenize 30 times. The mass fraction of curcumin is 1%.

[0113] b. The recombinant type III collagen raw material was dissolved in the above-mentioned curcumin aqueous solution and stirred to form a curcumin-loaded collagen solution, and then a cross-linking agent was added, the cross-linking agent being 10% (w / w) of N-hydroxysuccinimide (NHS) and 50% (w / w) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in combination; stirred and reacted for 2 h to obtain a curcumin-loaded recombinant type III collagen absorbable hydrogel; wherein the mass fraction of recombinant type III collagen was 8%; according to the mass fraction of recombinant type III collagen: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was 0.35%, and N-hydroxysuccinimide was 0.35%;

[0114] Cut the block of recombinant type III collagen absorbable hydrogel into 2 cm 3 Dialysis size: Recombinant collagen absorbable hydrogel blocks were dialyzed against phosphate-buffered saline (PBS) at an osmotic pressure of 200 mOsm / kg for four days to exchange the crosslinker. The block was pulverized in a homogenizer and centrifuged in a centrifuge bottle (9000 rpm / 20 min). The gel was then filled into pre-filled syringes and sterilized in a sterilizer at 121°C for 15 min to obtain a curcumin-loaded recombinant collagen hydrogel.

[0115] 2. Animal Experiment Results

[0116] ①Observation of rat uterine tissue morphology:

[0117] First, the abdomens of the two groups of rats were disinfected, anesthetized and fixed. A longitudinal incision of about 3 cm was made on the abdominal wall skin 2-3 cm above the pubic symphysis, the tissue was cut layer by layer, the abdominal cavity was entered, and the Y-shaped uterus of the rat was slowly picked out. As shown in Figures 13 and 14, the model group and treatment group B showed obvious abnormal tissue hyperplasia on the left side of the uterus of the rats in the model group, while the left side of the uterus of the rats in treatment group B was relatively flat and had no obvious hyperplasia. Similarly, the right side of the uterus of the rats in the model group was also significantly thickened relative to that of the treatment group B. Compared with the uterine tissue morphology of the modeled rats, the uterine tissue morphology of the rats treated with treatment group B was significantly improved, indicating that high-concentration curcumin hydrogel has a significant effect on the restoration of rat uterine tissue morphology.

[0118] ②Masson scar:

[0119] Rats in both groups were sacrificed after the second estrous cycle, fixed with 4% paramethanol for more than 24 hours, dehydrated with gradient ethanol, and then embedded in paraffin. Sections were dewaxed in distilled water and stained with Weigert iron hematoxylin for 3 minutes. The sections were differentiated with hydrochloric acid and alcohol for 15 seconds (if the iron hematoxylin staining was too deep, the differentiation time could be appropriately increased), washed with water, blued with Masson bluing solution, washed with water, and washed with distilled water for 1 minute. The sections were stained with Ponceau fuchsin for 7-8 seconds (the staining time was controlled according to tissue density), washed with 0.2% weak acid for 1 minute, washed with phosphomolybdic acid solution for 2 minutes, washed with 0.2% weak acid for 1 minute, stained with aniline blue for 1 minute 30 seconds (the staining time was controlled according to tissue density), washed with 0.2% weak acid for 1 minute, and dehydrated with 95% ethanol for 2-3 seconds and anhydrous ethanol for 3 times, each for 5-10 seconds. The sections were cleared with xylene for 3 times, each for 1-2 minutes, and sealed.

[0120] Uterine scars in rats were observed using MASSON staining. As shown in Figures 15-16, the uteri of the model group rats showed significant adhesions. As shown in Figures 17 and 18, the left and right uteri of the rats in treatment group B were normal in morphology and showed no adhesions, demonstrating that the high-concentration curcumin hydrogel can effectively prevent adhesions. Furthermore, the images show that the optical density and area of ​​the collagen fibers (blue) on the left and right sides of the uteri of the rats in treatment group B, which were treated with the high-concentration curcumin hydrogel, were significantly smaller than those in the model group rats, indicating that the high-concentration curcumin hydrogel also significantly promotes anti-fibrosis in the rat uterus.

[0121] ③ Endometrial cells (HE):

[0122] Rats from both groups were sacrificed after their second estrous cycle. The sections were fixed with 4% paramethanol for at least 24 hours, dehydrated with gradient ethanol, and then embedded in paraffin. The sections were dewaxed in distilled water. For hematoxylin staining of cell nuclei, the sections were stained with Harris hematoxylin for 3-8 minutes, washed with tap water, and then incubated with 1% hydrochloric acid alcohol for a few seconds. The sections were rinsed with tap water, then blued with 0.6% ammonia solution, and rinsed with running water. For eosin staining of cytoplasm, the sections were stained with eosin for 1-3 minutes. Dehydration and mounting were performed: the sections were dehydrated with gradient ethanol to make them transparent. The sections were removed from the xylene and air-dried, then mounted with neutral gum. Rat endometrial cells were visualized using HE staining (chromatin in the nucleus and nucleic acids in the cytoplasm appear purple-blue; components of the cytoplasm and extracellular matrix appear red).

[0123] As shown in Figures 19-22, the number of endometrial cells on the left side (Figure 19) and right side (Figure 20) of the uterus of the rats in the model group was significantly reduced compared with the endometrial cells on the left side (Figure 21) and right side (Figure 22) of the uterus of the rats in the treatment group B. At the same time, it can be seen from the pictures that the left and right uterine areas of the rats in the model group are larger, while the uterine cavity area of ​​the treatment group B is smaller than that of the model group, indicating that the endometrium recovered well after treatment, and the basic shape of the uterine cavity was maintained without adhesion.

[0124] ④ Endometrial thickness:

[0125] The endometrial thickness of rats was observed by HE staining (average endometrial thickness = area / circumference, and the endometrial area and circumference were calculated using image analysis software (Image Pro Plus)). As shown in FIG23 , the endometrial thickness of rats was observed by HE staining (average endometrial thickness = area / circumference, and the endometrial area and circumference were calculated using image analysis software (Image Pro-Plus). As shown in FIG23 , the endometrial thickness of the left endometrium of rats in treatment group A (571 μm) was significantly thicker than that of the left endometrium of rats in model group A (360.5 μm); the endometrium thickness of the right endometrium of rats in treatment group A (675 μm) was significantly thicker than that of the right endometrium of rats in model group A (389.25 μm), indicating that the low-concentration curcumin hydrogel has a significant effect on the recovery of the rat endometrium.

[0126] The thickness of the left endometrium of the rats in the treatment group B (633 μm) was significantly thicker than that of the rats in the model group (360.5 μm); the thickness of the right endometrium of the rats in the treatment group (729 μm) was significantly thicker than that of the rats in the model group (389.25 μm), indicating that high-concentration curcumin hydrogel has a better effect on the recovery of the rat endometrium than low-concentration curcumin hydrogel.

[0127] The thickness of the left endometrium of the rats in treatment group C (379.7 μm) was compared with that of the rats in model group (360.5 μm), and the endometrium thickening of the rats in treatment group C was not obvious; the thickness of the right endometrium of the rats in treatment group C (407.3 μm) was compared with that of the rats in model group (389.25 μm), and the endometrium thickening of the rats in treatment group C was not obvious either, indicating that the low-concentration heterogeneous curcumin hydrogel had a poor effect on the recovery of the rat endometrium.

[0128] The thickness of the left endometrium of the rats in the treatment group D (405.5 μm) was compared with the thickness of the left endometrium of the rats in the model group (360.5 μm), and the endometrium of the rats in the treatment group D was not significantly thickened; the thickness of the right endometrium of the rats in the treatment group D (414.7 μm) was compared with the thickness of the right endometrium of the rats in the model group (389.25 μm), and the endometrium of the rats in the treatment group D was not significantly thickened either, indicating that the high-concentration heterogeneous curcumin hydrogel had a poor recovery effect on the rat endometrium.

[0129] From the above analysis, it can be seen that based on the uterine tissue morphology, uterine scars, endometrial cells and endometrial thickness, it can be seen that the heterogeneous curcumin hydrogel has no significant effect on the recovery of the rat endometrium in specific applications; the recombinant collagen hydrogel of curcumin prepared within a certain concentration range (0.15-1%) and after homogenization has a good repair effect on the damaged endometrium and has significant anti-fibrosis and anti-adhesion effects.

[0130] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although this specification has described the present invention in detail with reference to the above embodiments, it should be understood by those skilled in the art that the present invention may still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A recombinant collagen hydrogel loaded with curcumin, characterized in that: It is composed of the following ingredients: curcumin, cross-linking agent, reconstituted collagen and water; The mass fraction of curcumin is 0.15-1.0%; the mass fraction of recombinant collagen is 5-10%; the cross-linking agents are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and the amount of each is 0.3-0.4% of the mass of recombinant collagen; the balance is water; the curcumin is first dissolved in water when used, and is added in the homogenized liquid after homogenization; The method for preparing the recombinant collagen hydrogel loaded with curcumin comprises the following steps: (1) first weighing a certain amount of curcumin powder and adding it to water, and obtaining a curcumin homogenous solution after magnetic stirring and homogenization circulation treatment; wherein the magnetic stirring time is 1.5-2h, and the number of homogenization cycles is 20-30 times; (2) dissolving the recombinant collagen raw material in the curcumin homogenate and stirring evenly to form a curcumin-loaded collagen solution; then adding crosslinking agents N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, stirring evenly, reacting for a period of time to obtain a curcumin-loaded recombinant collagen absorbable hydrogel, cutting the curcumin-loaded recombinant collagen absorbable hydrogel into a size suitable for dialysis to obtain a curcumin-loaded recombinant collagen absorbable hydrogel block, placing the block in phosphate buffer for dialysis to exchange the crosslinker, setting the PBS osmotic pressure and dialysis time for dialysis, and after dialysis, pulverizing the block of curcumin-loaded recombinant collagen absorbable hydrogel to obtain granules, and then filling and sterilizing to obtain the curcumin-loaded recombinant collagen hydrogel.

2. A curcumin-loaded recombinant collagen hydrogel according to claim 1, characterized in that: The mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide is 1:1; the recombinant collagen is recombinant human type III collagen, and the mass fraction is 8%.

3. A curcumin-loaded recombinant collagen hydrogel according to claim 1, characterized in that: The water includes deionized water, water for injection, and physiological saline.

4. The recombinant collagen hydrogel loaded with curcumin according to claim 1, characterized in that: The reaction time in step (2) is 2-6 hours; the osmotic pressure of the phosphate buffer is set to 200-500 mOsm / kg, and the dialysis time is 4-6 days; the pH of the phosphate buffer is 7.5-8.

5. The curcumin-loaded recombinant collagen hydrogel according to claim 1, characterized in that: In step (2), the curcumin-loaded recombinant collagen absorbable hydrogel is cut into a size of 1-2 cm suitable for dialysis. 3 .

6. Use of the curcumin-loaded recombinant collagen hydrogel according to any one of claims 1 to 5 in the preparation of products for preventing and treating intrauterine adhesions, anti-uterine fibrosis, and promoting endometrial recovery.

7. A composite preparation, characterized in that: The invention relates to a recombinant collagen hydrogel loaded with curcumin as described in any one of claims 1 to 5.

8. Use of a composite preparation according to claim 7 in preparing a product for repairing various endometrial injuries, characterized in that the repair of various endometrial injuries includes treating intrauterine adhesions, anti-fibrosis of the uterus, and promoting endometrial recovery.

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