Ritodrine-loaded decellularized amniotic membrane matrix temperature-sensitive hydrogel and pharmaceutical application thereof

Abstract

The invention discloses a ritodrine-loaded decellularized amnion matrix temperature-sensitive hydrogel and pharmaceutical application thereof, i.e., an amnion is subjected to decellularization treatment and then is digested by pepsin to obtain blank gel, and then drug loading is performed to obtain drug-loaded gel for treating premature delivery. The ritodrine-loaded vagina temperature-sensitive gel with high biocompatibility is prepared by taking a premature delivery treatment drug ritodrine commonly used in clinic as an active compound and taking an acellular amniotic membrane as a carrier, so that the enrichment of the drug in the uterus is improved, and the systematic exposure of the drug and the side effects caused by the systematic exposure are reduced. The invention provides a medicine delivery and treatment strategy based on the uterus first-pass effect for premature delivery treatment, and provides a simple and effective technical scheme for developing clinically effective premature delivery treatment medicines and reducing side effects of clinical medicines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a ritodrine-loaded decellularized amniotic membrane matrix thermosensitive hydrogel and its pharmaceutical use. Background Art

[0002] Premature birth is defined as childbirth before 37 weeks of gestation and is a major global health problem that affects approximately 15 million pregnancies each year and results in a significant increase in neonatal morbidity and mortality. The causes of premature birth are multifaceted, and there is currently a large amount of evidence from mouse models and human studies supporting that premature birth is caused by the disruption of fetal-maternal interface immune tolerance and excessive and premature inflammation. Currently, although some drugs have shown efficacy in preventing premature birth, truly safe and effective products are still limited. Hydroxyprogesterone (such as Makena) is widely used in women with symptoms of threatened premature birth. However, the latest evaluations have shown that its clinical effectiveness has not met expectations. Magnesium sulfate is also used in the treatment of acute premature birth and has a neuroprotective effect, but potential side effects need to be closely monitored. In summary, exploring drug preparations with definite efficacy, safety, convenience, and low toxicity and side effects is an urgent need for clinical prevention of premature birth.

[0003] Ritodrine hydrochloride is a β2-adrenergic agonist that is used to treat by relaxing uterine muscles and inhibiting contractions. Its use is associated with various side effects such as pulmonary edema and was withdrawn from the market by the FDA in 1995. However, in fact, due to its good clinical efficacy and low cost, it is still widely used in many developing countries including China. Studies have demonstrated that vaginal preparations can achieve a first-pass effect in the uterus, thus preferentially concentrating the drug in the uterine site and reducing systemic distribution. This strategy is expected to solve the clinical application dilemma of ritodrine and reduce maternal adverse reactions while ensuring the drug efficacy.

[0004] The latest progress in biomaterials has paved the way for the development of innovative drug delivery systems. The application of natural materials in biomedicine is a promising research field, such as decellularized tissues that retain the beneficial properties of the extracellular matrix (ECM). Decellularized amniotic membrane is an ECM. Due to its easy access and ethical basis, decellularized amniotic membrane (dAM) has shown extensive application potential in fields such as wound healing, regenerative medicine, and gynecology. In addition, decellularized amniotic membrane has advantages due to its excellent biocompatibility, low immunogenicity, anti-inflammatory properties, and ability to promote cell regeneration. Summary of the Invention

[0005] The object of the present invention is to provide a preparation method of a thermosensitive hydrogel based on decellularized amniotic membrane, which forms a ritodrine-loaded thermosensitive hydrogel by subjecting amniotic membrane to decellularization, freeze-drying, digestion, freeze-drying, and swelling for drug loading, and is achieved through the following steps: (1) Preparation of decellularized amniotic membrane: The fetal membranes were obtained after cesarean section (approved by the Medical Ethics Committee of the affiliated institution). After blunt dissection to remove the chorionic tissue, the amniotic membrane was cut into pieces about 10 cm × 10 cm. The fresh amniotic membrane was repeatedly rinsed with normal saline and then soaked in 10% TritonX-100 solution, incubated at 37 °C for 24 h. After taking out, it was rinsed clean with normal saline, fully oscillated in 25 g / L trypsin and 0.2 g / L EDTA, and incubated at 37 °C for 4 h. After rinsing clean with normal saline, it was freeze-dried and stored for later use; (2) Examination of decellularization: The natural and decellularized amniotic membrane tissues were fixed with paraformaldehyde, embedded in paraffin, sectioned, stained with Sirius red, and observed under a microscope to quantitatively analyze the collagen content; (3) Preparation of drug-loaded gel: The freeze-dried decellularized amniotic membrane was cut into pieces and digested with 1 mg / ml pepsin (dissolved in 0.1 M HCl) at 25 °C for 48 h to obtain a light blue solution (amniotic membrane concentration 20 mg / mL), and the pH was adjusted to 4.8 with 1 M NaOH. At this time, the extracellular matrix solution derived from the amniotic membrane could gel at 37 °C to obtain a thermosensitive blank gel based on the decellularized amniotic membrane, which was freeze-dried and stored for later use.

[0006] Another object of the present invention is to provide the application of the gel in the preparation of a drug for inhibiting uterine smooth muscle contraction. The drug is a thermosensitive hydrogel of decellularized amniotic membrane matrix loaded with ritodrine, which can prevent premature birth. The drug is a uterine contraction inhibitor drug.

[0007] The drug is achieved through the following steps: PBS was added to the blank gel lyophilized powder and vortexed for dispersion, swollen at 4 °C, and a high-concentration ritodrine solution was added and vortexed again to obtain a thermosensitive hydrogel of decellularized amniotic membrane matrix loaded with ritodrine (dAM@Rit, final concentration of ritodrine 15 mg / mL), stored at 4 °C and used within 48 h.

[0008] In the present invention, a commonly used drug for the treatment of premature birth in clinical practice is used as an active compound, and decellularized amniotic membrane is used as a carrier to prepare a vaginal thermosensitive gel with high biocompatibility to improve the enrichment of the drug in the uterus, reduce the systemic exposure of the drug and the resulting side effects. The present invention provides a drug delivery and treatment strategy based on the uterine first-pass effect for the treatment of premature birth, and provides a simple and effective technical solution for developing clinically effective drugs for the treatment of premature birth and reducing clinical drug side effects. The present invention uses the drug-loaded special gel for vaginal delivery of ritodrine. This innovative method helps ritodrine to directly enter the uterus after local release, effectively inhibiting uterine smooth muscle contraction. At the same time, the decellularized amniotic membrane component has a significant anti-inflammatory effect, thus enhancing the curative effect. This hydrogel provides a promising strategy for preventing premature birth and protecting the health of pregnant women and fetuses. Description of the Drawings

[0009] Figure 1 This is the comparison diagram of Sirius red staining before and after decellularization of amniotic membrane in Example 1 of the present invention.

[0010] Figure 2 This is the comparison diagram of the content of collagen before and after decellularization in Example 1 of the present invention.

[0011] Figure 3 This is the diagram of the gel formation of the ritodrine-loaded decellularized amniotic membrane thermosensitive gel at 37 °C in Example 1 of the present invention.

[0012] Figure 4 This is the scanning electron microscope diagram of the blank decellularized amniotic membrane hydrogel and the ritodrine-loaded decellularized amniotic membrane thermosensitive gel in Test Example 1 of the present invention.

[0013] Figure 5 This is the in vitro release diagram of the ritodrine-loaded decellularized amniotic membrane thermosensitive gel in Test Example 1 of the present invention.

[0014] Figure 6 This is the cytotoxicity diagram of the ritodrine-loaded decellularized amniotic membrane thermosensitive gel in Test Example 2 of the present invention.

[0015] Figure 7 This is the diagram of the vaginal injury after multiple vaginal administrations of the ritodrine-loaded decellularized amniotic membrane thermosensitive gel in Test Example 3 of the present invention.

[0016] Figure 8 This is the biocompatibility diagram of the ritodrine-loaded decellularized amniotic membrane thermosensitive gel in Test Example 3 of the present invention.

[0017] Figure 9 This is the lung and uterus distribution diagram of the Sulfo-Cy5-loaded decellularized amniotic membrane thermosensitive gel in Test Example 4 of the present invention.

[0018] Figure 10 This is the blood drug concentration curve after vaginal administration of the ritodrine-loaded decellularized amniotic membrane thermosensitive gel in Test Example 5 of the present invention.

[0019] Figure 11 This is the effect diagram of the ritodrine-loaded decellularized amniotic membrane thermosensitive gel in preventing premature birth in Test Example 6 of the present invention.

[0020] Figure 12 This is the effect diagram of the ritodrine-loaded decellularized amniotic membrane thermosensitive gel in reducing adverse reaction pulmonary edema in Test Example 7 of the present invention.

[0021] Figure 13 This is the effect diagram of the ritodrine-loaded decellularized amniotic membrane thermosensitive gel in inhibiting inflammatory reaction in Test Example 8 of the present invention. Detailed implementation manners

[0022] The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] Reagents and drugs: Triton-100 and pepsin were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Trypsin-EDTA (0.25%) was provided by Beyotime Biotechnology Co., Ltd. (Shanghai, China), and ritodrine hydrochloride was provided by Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Hydrochloric acid solution (0.1 M) for volumetric analysis was provided by Guangzhou Howe Pharmaceutical Technology Co., Ltd. (Guangzhou, China), and sodium hydroxide was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS, derived from Escherichia coli 0111:B4) was obtained from Sigma Aldrich (MO, USA). Sulfo-Cy5 was purchased from MedChemExpress (New Jersey, USA), and paraformaldehyde (4% v / v) was purchased from Sevier Biotechnology Co., Ltd. (Wuhan, China). PBS was obtained from Gibco BRL (MD, USA), and ToxinSensor TM Chromogenic LAL endotoxin detection kit was obtained from GenScript Biotech Corporation (New Jersey, USA). Normal saline and vaginal simulation fluid were prepared in the laboratory. ELISA kits for interleukin-6 (IL-6), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX-2) were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China).

[0024] Cells and animals: Human normal endometrial mesenchymal cells (hESCs) were derived from the Obstetrics and Gynecology Hospital Affiliated to Zhejiang University School of Medicine and cultured in DMEM medium containing 10% FBS. ICR mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All experiments were carried out in accordance with the guiding principles and approvals of the Scientific Research Committee of Zhejiang Chinese Medical University. The ethical approval number was IACUC-20240415-20.

[0025] Example 1: Preparation of ritodrine-loaded decellularized amniotic thermosensitive gel

[0026] (1) Take the fetal membranes after full-term cesarean section without pregnancy complications (approved by the Medical Ethics Committee of the affiliated institution), and obtain the informed consent of the parturient before taking. The parturient is negative for hepatitis B, hepatitis C, AIDS, and syphilis. After bluntly separating and removing the chorionic tissue, cut the amniotic membrane into pieces about 10 cm × 10 cm in size. Rinse the fresh amniotic membrane repeatedly with normal saline, then soak it in 10% TritonX-100 solution and store it at 37°C for 24 h. After rinsing it clean with normal saline, freeze-dry it for future use.

[0027] (2) Rinse TritonX-100 clean with normal saline, add 25 g / L trypsin and 0.2 g / L EDTA, soak, shake well, and store at 37°C for 4 h.

[0028] (3) Rinse off the residual trypsin with normal saline, drain the water, freeze at -80°C, and lyophilize at -50°C under 0.02 mbar for 48 h for future use.

[0029] (4) Detection of decellularization effect: Fix the natural and decellularized amniotic membrane tissues with paraformaldehyde, embed them in paraffin, prepare sections and stain them with Sirius red. Observe the changes in cell structure and collagen fiber content under a microscope ( Figure 1-2 ).

[0030] (5) Pepsin digestion: Digest the freeze-dried decellularized amniotic membrane with 1 mg / mL pepsin (dissolved in 0.1 M HCl) by stirring for 48 h to form a light blue solution (amniotic membrane concentration 20 mg / mL), and adjust the pH to 4.8 with 1 M NaOH. The obtained amniotic membrane-derived extracellular matrix solution is incubated at 37°C until it gels to obtain a blank hydrogel, and freeze-dry it again under the same conditions for future use.

[0031] (6) Drug loading: Disperse the freeze-dried blank hydrogel powder with PBS by vortexing and swell it at 4°C. Mix the high-concentration ritodrine hydrochloride PBS solution (150 mg / mL) with the blank gel at a ratio of 1:9 (v / v), and vortex well to obtain the temperature-sensitive hydrogel of decellularized amniotic membrane matrix loaded with ritodrine (dAM@Rit), store it at 4°C, and use it within 72 h (final concentration of ritodrine 15 mg / mL).

[0032] (7) Verification of temperature-sensitive properties: Take 1 mL of dAM@Rit and incubate it at 37°C for 2 min, and observe the change in fluidity by the inversion method ( Figure 3 ).

[0033] Example 2: Preparation of temperature-sensitive gel of decellularized amniotic membrane loaded with ritodrine

[0034] The freeze-dried acellular amniotic membrane was digested with 1 mg / mL pepsin (dissolved in 0.1 M HCl) by stirring for 48 h to form a light blue solution (amniotic membrane concentration 15 - 25 mg / mL), and the pH was adjusted to 4.8 with 1 M NaOH. The amniotic membrane-derived extracellular matrix solution obtained at this time was incubated at 37 °C until it gelated to obtain a blank hydrogel. The remaining steps were the same as those in Example 1 for preparing the ritodrine-loaded acellular amniotic membrane thermosensitive gel.

[0035] Example 3: Preparation of ritodrine-loaded acellular amniotic membrane thermosensitive gel

[0036] A high-concentration ritodrine hydrochloride solution (125 - 175 mg / mL) was added to the blank hydrogel in Example 1, and dAM@Rit was prepared by vortexing and stored at 4 °C for use within 72 h (final ritodrine concentration 12.5 - 17.5 mg / mL). The remaining steps were the same as those in Example 1 for preparing the ritodrine-loaded acellular amniotic membrane thermosensitive gel.

[0037] Test Example 1: In vitro characterization of ritodrine-loaded acellular amniotic membrane thermosensitive gel:

[0038] Morphology: Take 1 mL of dAM@Rit and incubate it at 37 °C for 2 min. After solidification, it was quickly frozen in liquid nitrogen and freeze-dried at -50 °C and 0.02 mbar for 48 h. The microscopic three-dimensional structure of the gel was observed using a scanning electron microscope ( Figure 4 ).

[0039] Thermosensitivity: The tube inversion method was used to observe the phase transition process of the hydrogel. Place dAM@Rit (1 mL) in an incubator at a constant temperature of 37 °C, invert it every 1 min, and observe its fluidity. The gelation time was within 2 min, indicating that the preparation could quickly form a gel after administration.

[0040] Rheological properties: A rheometer was used to perform rheological analysis on the thermosensitive hydrogel. The effect of shear rate on the viscosity of the hydrogel was tested at 37 °C, and the shear rate range was 0.1 - 100 s -1 . The storage modulus G' and loss modulus G" were measured at 0 - 40 °C.

[0041] Drug loading: Take an appropriate amount of dAM@Rit and freeze-dry it. Collect the dried hydrogel, weigh it, and then dissolve it in PBS. The ritodrine content was determined using an ultraviolet spectrophotometer, and the drug loading was calculated according to the following formula. Five drug loadings were tested in parallel and averaged. Drug loading (%) = (Content of ritodrine in dAM@Rit) / (Total weight of dAM@Rit after freeze-drying) × 100%

[0042] In vitro drug release: Using simulated vaginal fluid (SVF) as the release medium, the dialysis method was adopted to study the in vitro release characteristics of ritodrine in dAM@Rit. Free ritodrine and dAM@Rit were separately added into dialysis bags. Then the dialysis bags were placed in SVF and incubated in a shaker at 37 °C and 100 rpm. At specified time points (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 11 h, 24 h), 2 mL of release solution was collected from the release solution outside the dialysis bag, and an equal volume of blank SVF was supplemented. The ritodrine content of these samples was evaluated using an ultraviolet spectrophotometer. The cumulative release rate of the drug was calculated according to the following formula. The release curve of dAM@Rit showed its sustained-release effect ( Figure 5 ). Cumulative release rate (%) = (Cumulative amount of ritodrine released) / (Total amount of ritodrine in the dialysis bag) × 100%

[0043] Test Example 2: Detection of the cytotoxicity of ritodrine-loaded acellular amniotic thermosensitive gel on human endometrial stromal cells by CCK-8 method

[0044] Detection of the cytotoxicity of ritodrine solution, blank dAM or dAM@Rit on hESCs by CCK-8 method: hESCs (4×10 3 cells / well) were seeded in 96-well plates. After culturing for 24 h, media containing free ritodrine or dAM@Rit (drug concentration gradients were 5 - 200 μg / mL) were added respectively and incubated for 24 h. Subsequently, 10 μL of CCK-8 reagent was added to each well, and after a total incubation of 2 h, the absorbance value was measured at 450 nm. In addition, hESCs (4×10 3 cells / well) were seeded in 96-well plates. The blank gel (0.5 mL / well) was evenly spread on the bottom of 12-well plates and incubated at 37 °C for 5 min. 2 mL of complete medium was added to each well and leached for 24 h. The leachate was diluted into concentration gradients of 0, 25, 50, 100% with complete medium. The medium in 96-well plates was replaced with leachate at different concentrations and incubated for 24 h. After adding CCK-8, the absorbance was measured in the same way. As Figure 6 shown, when the concentration of ritodrine hydrochloride was in the range of 5 - 200 μg / mL, dAM@Rit had no toxicity to hESCs, and the leachate of the blank acellular amniotic thermosensitive gel promoted the proliferation of hESCs ( Figure 6 ).

[0045] Test Example 3: Biocompatibility of ritodrine-loaded acellular amniotic thermosensitive gel

[0046] Injury of long-term vaginal administration: The vaginal tissues of pregnant mice that underwent repeated vaginal administration of dAM@Rit were fixed with paraformaldehyde and stained with hematoxylin and eosin (H&E) to detect the local injury of vaginal tissues after repeated administration and exposure to high concentrations of dAM@Rit. The tissue injury was determined by histopathological analysis, and the results showed that repeated vaginal injection of dAM@Rit did not cause injury( Figure 7 ).

[0047] Biocompatibility of decellularized amniotic gel: PBS, blank dAM, and dAM@Rit (100 μL each on the left and right sides) were injected subcutaneously into the back of pregnant mice. The animals were sacrificed 15 days after the operation, and the hydrogels that were not completely absorbed and the adjacent skin tissues were collected. Tissue sections were prepared and stained with H&E to evaluate the tissue injury. The results showed that dAM@Rit had good biocompatibility( Figure 8 ).

[0048] Test Example 4: In vivo distribution of ritodrine-loaded decellularized amniotic thermosensitive gel

[0049] Before injecting the dAM@Sulfo-Cy5 solution, fluorescence images of ICR pregnant mice were collected by the IVIS imaging system as a blank control. After administering free Sulfo-Cy5 and dAM@Sulfo-Cy5 respectively, fluorescence images of pregnant mice were collected at specified time points (0.5 h, 2 h, and 4 h) using the IVIS imaging system and the fluorescence intensity was analyzed. Meanwhile, 3 pregnant mice in each group were sacrificed at 0.5 h, 2 h, and 4 h respectively, and the liver, placenta, and fetus were taken out. Fluorescence distribution images of tissues were collected using the IVIS imaging system and analyzed using LivingImage software. Semi-quantitative data confirmed that vaginal administration of the decellularized amniotic thermosensitive gel could target the drug distribution in the uterus, reduce systemic distribution without increasing fetal exposure, and had good maternal-fetal safety( Figure 9 ).

[0050] Test Example 5: Kinetic study of ritodrine-loaded decellularized amniotic thermosensitive gel

[0051] Female SD rats (200 - 220 g) were intravenously administered free ritodrine or vaginally administered dAM@Rit (9 mg / kg). 0.5 mL of blood samples were collected from the central retinal vein at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration. The drug content in the samples was determined by liquid chromatography-tandem mass spectrometry LC-MS / MS (Bruker, Germany). The blood concentration curve showed that the ritodrine-loaded decellularized amniotic thermosensitive gel greatly reduced the amount of ritodrine entering the systemic circulation after vaginal administration, which helped to reduce maternal adverse reactions of ritodrine such as pulmonary edema and hypokalemia( Figure 10 ).

[0052] Test Example 6: Treatment of Chorioamnionitis-Induced Preterm Birth in Mice with Ritodrine-Loaded Decellularized Amniotic Membrane Thermosensitive Gel

[0053] At 13.5 d.p.c., a preterm birth model induced by chorioamnionitis was constructed. Oral or intravenous injection of free ritodrine was continuously carried out, dAM@Rit (7.5 mg / kg) was administered vaginally, and maintenance treatment with dAM@Rit was performed after a single intravenous injection of free ritodrine. The preterm birth situation was observed and recorded. Pregnant mice with a weight loss of more than 3 g on the first day after model establishment or a weight gain of less than 1 g for two consecutive days before 18.5 d.p.c. were defined as preterm birth. Ritodrine-loaded decellularized amniotic membrane thermosensitive gel effectively reduced the preterm birth rate induced by chorioamnionitis. At the same time, the maintenance treatment plan of ritodrine-loaded decellularized amniotic membrane thermosensitive gel after a single intravenous injection of free ritodrine showed excellent treatment effects on preterm birth( Figure 11 ).

[0054] Test Example 7: Reduction of Maternal Adverse Reactions of Ritodrine during the Treatment of Chorioamnionitis-Induced Preterm Birth in Mice with Ritodrine-Loaded Decellularized Amniotic Membrane Thermosensitive Gel

[0055] After the lung tissues of pregnant mice in different treatment groups in Test Example 6 were fixed, embedded, sectioned, stained with H&E, and the tissue damage was evaluated( Figure 12 ).

[0056] Test Example 8: Inhibition of Inflammatory Response by Ritodrine-Loaded Decellularized Amniotic Membrane Thermosensitive Gel

[0057] At 13.5 d.p.c., a preterm birth model induced by chorioamnionitis was constructed. Free ritodrine was administered orally or intravenously once, and dAM or dAM@Rit (7.5 mg / kg) was administered vaginally. The pregnant mice were sacrificed 4 h later, and serum, amniotic fluid, and uterus were collected. The levels of inflammatory cytokines IL-6, IL-1β, and COX-2 in serum and uterine tissues were detected, and the endotoxin level in amniotic fluid was measured. The increase in the levels of IL-6 and IL-1β is closely related to the occurrence of preterm birth. As Figure 13 shown, compared with the LPS group, dAM@Rit significantly reduced the levels of IL-6 and IL-1β in the serum and uterine smooth muscle of pregnant mice. COX-2 plays a key role in inducing uterine contractions, and dAM@Rit effectively reduced the level of COX-2 in serum and uterine smooth muscle tissues. In addition, after evaluating the endotoxin level in amniotic fluid, it was found that both dAM and dAM@Rit could reverse the increase in endotoxin caused by modeling, thus providing a protective effect for the fetus. Overall, dAM@Rit has a significant anti-inflammatory effect, effectively reducing the levels of inflammatory factors in serum and uterine smooth muscle tissues, and at the same time also plays a role in protecting the fetus. Ritodrine-loaded decellularized amniotic membrane thermosensitive gel has superior effects on the treatment of inflammation-induced preterm birth, and decellularized amniotic membrane thermosensitive gel is a promising biomaterial for the treatment of pregnancy-related diseases(Figure 13 )。

Claims

1. A thermosensitive hydrogel containing ritodrine-loaded acellular amniotic membrane matrix, characterized in that: This is accomplished by following these steps: (1) Preparation of decellularized amniotic membrane: fetal membrane was obtained, and after separation and removal of chorionic tissue, the amniotic membrane was cut into pieces of approximately 10 cm × 10 cm. The fresh amniotic membrane was repeatedly rinsed with saline and then immersed in a 10% TritonX-100 solution and incubated at 37°C for 24 h. After being taken out, it was rinsed with saline, fully shaken in 25 g / L trypsin and 0.2 g / L EDTA, incubated at 37°C for 4 h, rinsed with saline and freeze-dried for later use. (2) Decellularization examination: The native and decellularized amniotic tissues were fixed with paraformaldehyde, processed and embedded in paraffin to prepare 10 μm sections, and stained with Sirius red. The collagen content was observed under a microscope and quantified. (3) Preparation of drug-loaded gel: The freeze-dried decellularized amniotic membrane was cut into pieces and digested with 1 mg / ml pepsin dissolved in 0.1 M HCl at 25°C for 48 h to obtain a light blue solution with an amniotic membrane concentration of 20 mg / mL. The pH was adjusted to 4.8 with 1 M NaOH. The amniotic membrane-derived extracellular matrix solution was gelled at 37°C to obtain a blank gel, which was freeze-dried and stored for later use.

2. Use of the hydrogel prepared by the method of claim 1 in preparing a drug for inhibiting uterine smooth muscle contraction, characterized in that: The drug is a decellularized amniotic membrane matrix thermosensitive hydrogel loaded with ritodrine.

3. The use according to claim 2, characterized in that: The drug is achieved by the following steps: Add PBS to the blank gel lyophilized powder and vortex disperse it, swell it at 4°C, add high concentration ritodrine hydrochloride solution and vortex disperse it again to obtain the decellularized amniotic membrane matrix thermosensitive hydrogel loaded with ritodrine. The final concentration of ritodrine is 15 mg / mL. Store it at 4°C and use it within 48 hours.

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