Preparation method and application of targeted lipid temperature-sensitive gel for vagina

Abstract

The invention provides a preparation method and application of targeted lipid temperature-sensitive gel for vagina. The preparation method comprises the following steps: mixing egg congelation fat, cholesterol, DSPE-MPEG2000, DSPE-PEG2000-NHS and mifepristone or olaparil, dissolving in dichloromethane, volatilizing an organic phase by adopting a rotary evaporation method to obtain a thin film material, hydrating a thin film by using a phosphate buffer solution under ultrasonic to obtain mifepristone or olaparil-loaded liposome, homogenizing the particle size by utilizing an emulsification ultrasonic technology, and preparing the mifepristone or olaparil-loaded liposome. Adding homing peptide for reaction, and then uniformly mixing poloxamer 188, poloxamer 407, glycerol, Tween 80 and the drug-loaded targeted liposome, so as to obtain the targeted liposome temperature-sensitive gel. The targeted lipid temperature-sensitive gel disclosed by the invention can be converted into a solid state in a vaginal cavity within 30 seconds, and by adopting a local administration mode, the dosage is reduced, the side effects of the whole body are relieved, and the adenomyosis can be effectively treated. The invention provides a choice for medicines for treating adenomyosis.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a preparation method and application of a vaginal targeted liposome temperature-sensitive gel. Background Art

[0002] Adenomyosis (AM) is a localized or diffuse lesion in which the endometrial glands and stroma invade the myometrium. It mainly occurs in multiparous women aged 30-50 years old, with persistent and worsening dysmenorrhea, excessive menstrual flow, and prolonged menstruation as the main manifestations. It usually occurs in combination with endometriosis and / or leiomyoma. It is a common gynecological disease that seriously affects women's physical and mental health and quality of life. The benign nature of the disease and the potential for malignant biological behaviors such as invasion and recurrence make it one of the reproductive system diseases that is difficult to cure and has great harm. The infertility caused by adenomyosis will significantly reduce the birth rate and profoundly affect the country's population dynamics, social cohesion and economic development.

[0003] The incidence of adenomyosis is on the rise, but the etiology and pathogenesis of the disease are still unclear. At present, traditional hysterectomy is generally not accepted by patients with adenomyosis. Residual lesions often exist when conservative surgery is performed to remove the lesions, and the recurrence rate after surgery is high. Drug therapy is still needed to control clinical symptoms, and pregnancy rupture is prone to occur. Although uterine artery embolization (UAE) and high-intensity focused ultrasound (HIFU) can also treat adenomyosis, the efficacy and safety of UAE and HIFU are still controversial. Therefore, in terms of the long-term management plan of adenomyosis, drug therapy is the mainstream treatment method.

[0004] Mifepristone (RU486) is a selective progesterone receptor modulator. It is the first clinically available antiprogestin and has attracted great interest from scientists since its discovery in 1982. RU486 was originally used to terminate early pregnancy. Recently, a large number of preclinical and clinical studies have confirmed that RU486 has significant efficacy against various types of tumors, including gastric cancer, breast cancer, prostate cancer, endometrial cancer and ovarian adenocarcinoma. RU486 mainly exerts its anti-tumor effect by inhibiting the proliferation and invasion of cancer cells and promoting apoptosis of cancer cells. In a series of short-term clinical studies using different doses, it was found that RU486 has been shown to be effective in treating uterine leiomyoma and endometriosis. In vivo and in vitro studies have shown that RU486 can also inhibit endometrial cell proliferation, reduce angiogenesis and oxidative stress and promote cell apoptosis. In experimentally induced animal studies, RU486 has shown the effect of inhibiting the growth of adenomyosis lesions and preventing the occurrence of adenomyosis. As an inhibitor of cell cycle-dependent protein kinases CDK1 and CDK2, RU486 can arrest the cell cycle and induce cell apoptosis. In recent years, especially in China, different doses of RU486 have been used clinically to treat adenomyosis and achieved significant therapeutic effects. However, the current clinical administration of mifepristone is mainly oral, which often produces some side effects, such as gastrointestinal irritation, allergic reactions, etc., and the response rate is not high.

[0005] Olaparib is a PARP inhibitor. Studies have shown that PARP plays a key role in repairing single-strand breaks in DNA, is overexpressed in a variety of cancers, and affects the body's immune response and inflammatory signaling pathways. PARP inhibitors can increase replication stress and genomic instability, leading to the accumulation of single-strand breaks in DNA and subsequent double-strand breaks in DNA, leading to cell death. In 2014, PARP inhibitors first entered the clinical field. The U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) approved Olaparib for the treatment of advanced ovarian cancer. Today, PARP inhibitors have been introduced as a tool to prevent inflammatory diseases. These selective inhibitors are being evaluated as nanotherapeutics in clinical trials as a targeted treatment strategy for solid tumors of ovarian, prostate, breast, colorectal and uterine tissues. Generally, endometriosis and adenomyosis are regarded as chronic benign diseases, but the biological behaviors of ectopic endometrial cells, such as invasion, migration, and reduced susceptibility to apoptosis, are similar to those of tumor cells. Studies have detected the immunohistochemical expression of PARP-1 in cancer and endometriosis, and found that benign ovarian lesions associated with endometriosis and ovarian cancer associated with endometriosis have similar PARP-1 expression levels. These findings demonstrate the great potential of PARP inhibitors in the treatment of adenomyosis, and the combination of the two drugs is expected to further enhance the effect of drug treatment for adenomyosis.

[0006] Studies have shown that vaginal administration is a more direct and effective route of administration for the treatment of adenomyosis. By utilizing the first-pass effect of the uterus, the drug can be locked in the lesion site, has high permeability and sustained drug release activity, and can avoid the disadvantages of oral administration such as the first-pass effect of the liver and gastrointestinal irritation and other adverse reactions. In addition, vaginal administration is a non-invasive route of administration. Vaginal gels can quickly coagulate at body temperature, allowing the drug to be retained in the vaginal cavity for a long time, thereby prolonging the release of the drug, reducing the frequency of drug administration and achieving local administration. However, vaginal mucus contains 95% water, 1-2% mucin glycoprotein fibers and some other trace components such as lactic acid, salt, protein, lipid and enzyme. Its viscosity is 1000-10000 times that of water. Free lipophilic mifepristone and olaparib cannot penetrate the reproductive tract mucus barrier to reach the adenomyosis lesion tissue. Therefore, the unique biomembrane-like structure of liposomes is used to encapsulate the drugs, thereby promoting drug delivery. In addition, EphB4 receptors are highly expressed in adenomyosis lesion tissues, and its ligand homing peptide TNYL is grafted onto the liposome surface, which can further enhance the targeting of drug delivery.

[0007] Therefore, constructing a pharmaceutical preparation to help mifepristone and olaparib penetrate mucus and target the lesion tissue of adenomyosis becomes a technical problem to be solved by the present invention. Summary of the invention

[0008] In order to achieve the specific delivery of lipophilic drugs mifepristone and olaparib to adenomyosis lesion tissue, the present invention aims to provide a method for preparing vaginal targeted liposome thermosensitive gel, (1) egg yolk egg gel, cholesterol, DSPE-MPEG 2000 , DSPE-PEG 2000 -NHS is mixed with mifepristone or olaparib and dissolved in dichloromethane; (2) the organic phase is volatilized by a rotary evaporator to obtain a thin film material; (3) the thin film is hydrated with a phosphate buffer under ultrasound to obtain liposomes; (4) the particle size is uniformed by high shear emulsification ultrasound technology, TNYL peptide is added and reacted at room temperature for 2 days to obtain TNYL peptide-modified targeted liposomes; (5) poloxamer 188, poloxamer 407, glycerol, Tween 80 and the drug-loaded targeted liposomes are evenly mixed and refrigerated to obtain mifepristone or olaparib targeted liposome thermosensitive gel.

[0009] The preparation method of the present invention is specifically achieved by the following steps:

[0010] (1) First weigh egg yolk lecithin, cholesterol, DSPE-MPEG 2000 , DSPE-PEG 2000 -NHS and mifepristone or olaparib dissolved in 24 ml of dichloromethane; Wherein 105 mg, 10 mg, 12 mg, 3 mg, and 10 mg of mifepristone were dissolved in 24 ml of dichloromethane; Olaparib was dissolved in 24 ml of dichloromethane with ultrasound assistance, with the dosage of 95 mg, 5 mg, 12 mg, 3 mg and 10 mg respectively.

[0011] (2) The speed of the rotary evaporator was adjusted to 35 rpm, the temperature of the water bath was adjusted to 28°C, and the pressure was adjusted to 320 mbar. Then, 24 ml of the dissolved organic phase (dichloromethane) was injected into a clean, anhydrous 500 ml round-bottom flask and placed under the vacuum adsorption port to ensure that the flask could be effectively adsorbed by the vacuum adsorption system. The height of the flask was adjusted so that the water surface of the water bath was above the liquid surface in the container. The vacuum rotary evaporation was timed for 20 minutes to allow the organic phase to fully volatilize to obtain a thin film material.

[0012] (3) Add 4 ml of phosphate buffer to the resulting film-forming flask, shake it manually and slowly for 5 minutes to make the film fall off, then shake it under ultrasound for 3 minutes to fully hydrate the film, and collect the liquid in a 5 ml centrifuge tube;

[0013] (4) Place the centrifuge tube containing liposomes under the ultrasonic probe, with the height of the ultrasonic probe to the liquid level being 1 cm. Adjust the power of the ultrasonic probe device to 200 watts, adjust the ultrasonic mode to interval ultrasonic, set the time to ultrasonic for 2 seconds, and the interval for 5 seconds. Start the ultrasonic device, ultrasonic for a total of 10 seconds, collect the ultrasonic liposomes and inject 5 ml into it, remove the needle and connect it to a 0.8 micron water system membrane (filter the drug through the 0.8 micron water system membrane after ultrasonication), slowly push the syringe, collect the liposomes that have passed the membrane into a new centrifuge tube, and the free fat-soluble drug can be removed. Weigh 19 mg of TNYL peptide and dissolve it in 1 ml of DMSO, add 40 μl of TNYL peptide to the liposomes that have passed the membrane (the mass of the added TNYL peptide is 0.75 mg), dissolve it with 40 μl of dimethyl sulfoxide, mix well, and react at room temperature for 2 days.

[0014] (5) Weigh 2100 mg, 100 mg, 200 mg, and 500 mg of poloxamer 188, poloxamer 407, glycerol, and Tween 80, respectively, and place them in a 50 ml centrifuge tube. Add 3.55 ml of each of the prepared two targeted liposomes (mifepristone or olaparib), stir with a 1 ml pipette tip, leave the pipette tip in the centrifuge tube after stirring to prevent the gel material from being taken out, and place in a refrigerator to form a thermosensitive gel. The refrigeration temperature is 2-6° C., and the refrigeration time is more than 2 hours.

[0015] Another object of the present invention is to provide the use of the thermosensitive gel as a carrier in the preparation of a vaginal targeted treatment drug for adenomyosis. The thermosensitive gel encapsulates the drug, completes phase transition when entering the vaginal cavity, becomes a semisolid and adheres to the mucosa, increases the retention time of the drug, and achieves the purpose of slowly releasing the drug. The drug is mifepristone or olaparib.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The thermosensitive gel of the present invention encapsulates drug-loaded targeted liposomes, reduces the frequency of drug administration, avoids the disadvantages of low oral administration response rate and many side effects, increases the drug concentration in female pelvic reproductive organs, and reduces the adverse reactions of systemic administration. Since free lipophilic drugs mifepristone and olaparib are difficult to penetrate the reproductive tract mucus barrier, the present invention constructs liposomes to encapsulate drugs therein, realizes drug delivery, combines the therapeutic effects of mifepristone and olaparib on adenomyosis, and simultaneously grafts the homing peptide TNYL on the surface of the liposomes, and completes the targeted convergence of drugs to the lesions through the "ligand-receptor" binding principle. The present invention solves the problem that free lipophilic mifepristone and olaparib cannot penetrate the reproductive tract mucus barrier to reach the lesion tissue. The targeted liposome thermosensitive gel provided by the present invention can effectively treat adenomyosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 2 is the particle size distribution and potential diagram of mifepristone and olaparib targeted liposomes; A is the particle size diagram of TNYL-modified mifepristone-loaded liposomes, and B is its potential diagram; C is the particle size diagram of TNYL-modified olaparib-loaded liposomes, and D is its potential diagram.

[0018] Figure 2 The flow diagram of the gel at low temperature and the solidification diagram at 37°C, A is the flow diagram and B is the solidification diagram.

[0019] Figure 3 These are transmission electron micrographs of mifepristone- and olaparib-targeted liposomes; A and B are transmission electron micrographs of mifepristone-targeted liposomes, and C and D are transmission electron micrographs of olaparib-targeted liposomes.

[0020] Figure 4 Figure 3 is the scanning electron micrograph of blank gel, mifepristone targeted liposome gel and olaparib targeted liposome gel, A is the scanning electron micrograph of blank gel, B is the scanning electron micrograph of mifepristone targeted liposome gel, and C is the scanning electron micrograph of olaparib targeted liposome gel.

[0021] Figure 5These are the modulus test graphs of blank gel, mifepristone targeted liposome gel and olaparib targeted liposome gel; A is the modulus test graph of blank gel, B is the modulus test graph of mifepristone targeted liposome gel, and C is the modulus test graph of olaparib targeted liposome gel.

[0022] Figure 6 These are the images of lesions collected after drug treatment of adenomyosis mice, and the weight, volume and number analysis graphs; A is the image of the solid lesions, B is the image of the total weight of the uterus and lesions, C is the image of the number of lesions, and D is the image of the volume of lesions.

[0023] Figure 7 The statistical graphs of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) in mice with adenomyosis after drug treatment; A is the ALT distribution graph, B is the AST distribution graph, and C is the BUN distribution graph. DETAILED DESCRIPTION

[0024] The technical scheme of the present invention is further described in detail below in conjunction with the accompanying drawings and through specific embodiments. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the raw materials, reagents, methods, etc. used in the embodiments are all conventional raw materials, reagents, methods in the art.

[0025] Example 1 Preparation of mifepristone and olaparib targeted liposomes First, weigh egg yolk lecithin, cholesterol, DSPE-MPEG 2000 , DSPE-PEG 2000 -NHS and mifepristone 105 mg, 10 mg, 12 mg, 3 mg, 10 mg respectively, dissolved in 24 ml of dichloromethane; weigh egg yolk condensate, cholesterol, DSPE-MPEG 2000 , D SPE-PEG 2000-NHS and Olaparib were dissolved in 24 ml of dichloromethane at 95 mg: 5 mg: 12 mg: 3 mg: 10 mg, respectively, and ultrasonic dissolution was performed. Then, the speed of the rotary evaporator was adjusted to 35 rpm, the temperature of the water bath was adjusted to 28°C, and the pressure was 320 mbar. Subsequently, 24 ml of the dissolved organic phase was injected into a clean, anhydrous 500 ml round-bottom flask, and then the flask was placed under the vacuum adsorption port to ensure that the flask could be effectively adsorbed by the vacuum system. The height of the bottle was adjusted so that the water surface of the water bath was above the liquid surface in the container. After decompression, the organic phase was fully volatilized by rotary evaporation for 20 minutes; after 20 minutes, 4 ml of phosphate buffer was added to the obtained film-forming flask, and the film was fully hydrated under ultrasound. The liquid was collected in a 5 ml centrifuge tube, and the power of the ultrasonic probe device was adjusted to 200 watts, and the ultrasonic mode was adjusted to interval ultrasound. The time was set to ultrasound for 2 seconds and intervals of 5 seconds. The centrifuge tube containing the liposomes was placed under an ultrasonic probe with the height of the ultrasonic probe to the liquid level being 1 cm. The ultrasonic device was started for a total of 10 seconds. The sonicated liposomes were passed through a 0.8-micron aqueous membrane to remove free drugs. 0.75 mg of TNYL peptide (dissolved in 40 μl of dimethyl sulfoxide) was added to the liposomes that passed through the membrane, and the mixture was reacted at room temperature for 2 days to obtain mifepristone / olaparib targeted liposomes.

[0026] Example 2 Preparation of mifepristone and olaparib targeted liposomes First, weigh egg yolk lecithin, cholesterol, DSPE-MPEG 2000 , DSPE-PEG 2000 -NHS and mifepristone were dissolved in 20 ml of dichloromethane at 88 mg, 5 mg, 10 mg, 3 mg, and 5 mg, respectively; egg yolk condensate, cholesterol, DSPE-MPEG were weighed 2000 , DSPE-PEG 2000-NHS and Olaparib were dissolved in 20 ml of dichloromethane at 88 mg: 5 mg: 10 mg: 3 mg: 5 mg, respectively, and ultrasonic dissolution was performed. Then, the speed of the rotary evaporator was adjusted to 60 rpm, the temperature of the water bath was adjusted to 28°C, and the pressure was 320 mbar. Subsequently, 20 ml of the dissolved organic phase was injected into a clean, anhydrous 500 ml round-bottom flask, and then the flask was placed under the vacuum adsorption port to ensure that the flask could be effectively adsorbed by the vacuum system. The height of the bottle was adjusted so that the water surface of the water bath was above the liquid surface in the container bottle, and the organic phase was fully volatilized by vacuum rotary evaporation for 20 minutes; after 20 minutes, 5 ml of phosphate buffer was added to the obtained film-forming flask, and the film was fully hydrated under ultrasound. The liquid was collected in a 10 ml centrifuge tube, and the power of the ultrasonic probe device was adjusted to 200 watts, and the ultrasonic mode was adjusted to interval ultrasound. The time was set to ultrasound for 2 seconds and intervals of 5 seconds. The centrifuge tube containing the liposomes was placed under an ultrasonic probe with the height of the ultrasonic probe to the liquid level being 1 cm. The ultrasonic device was started for a total of 10 seconds. The sonicated liposomes were passed through a 0.8-micron aqueous membrane to remove free drugs. 0.75 mg of TNYL peptide (dissolved in 20 μl of dimethyl sulfoxide) was added to the liposomes that passed through the membrane, and the mixture was reacted at room temperature for 2 days to obtain mifepristone / olaparib targeted liposomes.

[0027] Example 3 Preparation of mifepristone and olaparib targeted liposomes First, weigh egg yolk lecithin, cholesterol, DSPE-MPEG 2000 , DSPE-PEG 2000 -NHS and mifepristone 105 mg, 10 mg, 15 mg, 3 mg, 10 mg respectively, dissolved in 36 ml of dichloromethane; weigh egg yolk condensate, cholesterol, DSPE-MPEG 2000 , DSPE-PEG 2000-NHS and Olaparib were dissolved in 36 ml of dichloromethane at 95 mg: 10 mg: 15 mg: 3 mg: 10 mg, respectively, and ultrasonic dissolution was performed. Then, the speed of the rotary evaporator was adjusted to 60 rpm, the temperature of the water bath was adjusted to 28°C, and the pressure was 320 mbar. Subsequently, 36 ml of the dissolved organic phase was injected into a clean, anhydrous 500 ml round-bottom flask, and then the flask was placed under the vacuum adsorption port to ensure that the flask could be effectively adsorbed by the vacuum system. The height of the bottle was adjusted so that the water surface of the water bath was above the liquid surface in the container bottle, and the organic phase was fully volatilized by vacuum rotary evaporation for 25 minutes; after 25 minutes, 5 ml of phosphate buffer was added to the obtained film-forming flask, and the film was fully hydrated under ultrasound. The liquid was collected in a 10 ml centrifuge tube, and the power of the ultrasonic probe device was adjusted to 200 watts, and the ultrasonic mode was adjusted to interval ultrasound. The time was set to ultrasound for 2 seconds and intervals of 5 seconds. The centrifuge tube containing the liposomes was placed under an ultrasonic probe with the height of the ultrasonic probe to the liquid level being 1 cm. The ultrasonic device was started for a total of 10 seconds. The sonicated liposomes were passed through a 0.8-micron aqueous membrane to remove free drugs. 0.75 mg of TNYL peptide (dissolved in 20 μl of dimethyl sulfoxide) was added to the liposomes that passed through the membrane, and the mixture was reacted at room temperature for 2 days to obtain mifepristone / olaparib targeted liposomes.

[0028] Example 4 Preparation of mifepristone and olaparib targeted lipid thermosensitive gel Weigh 2100 mg, 100 mg, 200 mg and 500 mg of poloxamer 188, poloxamer 407, glycerol and Tween 80 respectively, put them into a 50 ml centrifuge tube, add 3.55 ml of each of the two prepared targeted liposomes, stir to mix, keep the pipette tip in the centrifuge tube, seal the centrifuge tube with a lid and place it in a 4°C refrigerator to form a thermosensitive gel in about 2 hours.

[0029] Example 5 Preparation of mifepristone and olaparib targeted lipid thermosensitive gel Weigh 2400 mg, 100 mg, 200 mg and 500 mg of poloxamer 188, poloxamer 407, glycerol and Tween 80 respectively, put them into a 50 ml centrifuge tube, add 3.4 ml of each of the two prepared targeted liposomes, stir to mix, keep the gun tip in the centrifuge tube, seal the centrifuge tube with a lid and place it in a 4°C refrigerator to form a thermosensitive gel in about 2 hours.

[0030] Example 6 Preparation of mifepristone and olaparib targeted lipid thermosensitive gel Weigh 2200 mg, 200 mg, 200 mg, and 500 mg of poloxamer 188, poloxamer 407, glycerol, and Tween 80 respectively, put them into a 50-ml centrifuge tube, add 3.45 ml of each of the two prepared targeted liposomes, stir to mix, keep the gun tip in the centrifuge tube, seal the centrifuge tube with a lid, and place it in a 4°C refrigerator to form a thermosensitive gel in about 2 hours.

[0031] Example 7 Characterization of mifepristone and olaparib targeted liposomes The particle size and surface potential of mifepristone / olaparib targeted liposomes were measured using a particle size and surface potential instrument. Figure 1 The results showed that the particle size of TNYL-mifepristone was 119.2±2.02 nanometers, and the polydispersity index (PDI) was 0.22±0.05; the particle size of TNYL-olaparib was 167±2.91 nanometers, and the PDI was 0.25±0.03, indicating that the particle size distribution of the two liposomes was narrow and relatively uniform. The potential of TNYL-mifepristone was: -16.04±1.44 millivolts, and the potential of TNYL-olaparib was: -19.72±1.18 millivolts, indicating that both had good negative charge, indicating good stability. In addition, the drug content of mifepristone and olaparib liposomes was determined by ultraviolet spectrophotometry. The mifepristone liposomes were demulsified with dimethyl sulfoxide at 304 nanometers, and the olaparib liposomes were demulsified with acetonitrile at 254 nanometers. The standard curve is shown as follows: Figure 2 As shown, the results show that the encapsulation efficiency and drug loading of mifepristone are: 70.73% ± 0.0464 and 5.05% ± 0.0033; the encapsulation efficiency and drug loading of olaparib are 56.57% ± 0.0329 and 4.525% ± 0.0026, indicating that fat-soluble drugs can be well encapsulated in liposomes. The transmission electron microscopy results of the two liposomes are shown in Figure 3 As shown, the spatial structure of the nanoparticles is demonstrated.

[0032] Example 8 Characterization of Mifepristone and Olaparib Targeted Lipid Thermosensitive Gel After the thermosensitive hydrogel was dehydrated and freeze-dried, the internal morphology of mifepristone and olaparib targeted lipid thermosensitive gel was detected by scanning electron microscopy. Figure 4 As shown in the results, the scanning electron micrographs of the blank gel and the two liposome thermosensitive gels all showed a spatial network structure with structural gaps. Rheological tests were then performed to evaluate the storage modulus, loss modulus, and viscosity. Figure 5As shown, the critical temperatures of blank gel, mifepristone targeted thermosensitive gel, and olaparib targeted thermosensitive gel are approximately 17.5°C, 14°C, and 15°C, respectively. These results indicate that the blank gel and the two drug-loaded liposomes are flowable at low temperatures (14°C) and can become solid gels at body temperature. Therefore, after the gel is applied to the vagina, it can be effectively retained in the vagina-cervix, allowing the drug to enter the target tissue. At the same time, the gelation time of the blank gel and the drug-loaded gel at 37°C was tested, and the results showed that the above gels can be transformed into a solid state within 30 seconds.

[0033] Example 9 Construction of ICR Mouse Animal Model of Adenopathy An ICR mouse experimental model of adenomyosis was established by pituitary transplantation: ICR male mice were killed, the pituitary gland was removed by craniotomy, and placed in sterile saline for later use; atropine (40 μL / mouse) and Zota (8 μL / g) were used to anesthetize the model mice by intraperitoneal injection respectively, and the abdomen of the anesthetized female mice was shaved and disinfected with iodine solution, a 2 cm longitudinal incision was made in the middle of the lower abdomen, the bifurcation of the uterus was found under the bladder, and a small incision was made to separate the right uterus; the pituitary gland in the saline was sucked out with a homemade set, as little liquid as possible was sucked out, and it was gently pushed to the end of the uterus, the uterus was sutured with a needle, and then an appropriate amount of penicillin powder was sprinkled in the abdominal cavity, the abdominal endometrium was sutured with a needle, and the outer skin was clamped with a mouse-specific wound clip.

[0034] Example 10 Efficacy of mifepristone and olaparib targeted lipid thermosensitive gel in the treatment of adenomyosis model animals Three months after modeling, the animals were given drug treatment. The experiment was divided into five groups: oral saline, oral mifepristone, mifepristone liposome gel, mifepristone + olaparib liposome gel, and TNYL-targeted mifepristone + olaparib liposome gel. Mifepristone and olaparib were administered at a dose of 2.5 mg / kg body weight and 1.8 mg / kg body weight, respectively. The drug was given every three days for 15 consecutive times, and the weight changes of the mice were recorded. At the end of the experiment, the volume and weight of the mouse lesions were collected and counted, and the changes in ALT, AST and BUN in the serum were analyzed to evaluate the effects on liver and kidney function. Figure 6 As shown in the figure, after the adenomyosis ICR mice were treated with drugs, there was no significant difference in the total volume of the uterus and lesions in the vaginal administration group compared with other groups, but the number of lesions was significantly reduced after vaginal administration, especially after the combination of the two drugs, the volume of the lesions was significantly reduced, and the effect was more prominent. The serological examination analyzed ALT, AST, BUN, such as Figure 7 , showing no significant differences among the groups, the existing therapeutic effects demonstrate that nanocarriers can treat adenomyosis to a certain extent without obvious liver and kidney side effects.

[0035] In summary, the preparation method of the mifepristone and olaparib targeted liposome thermosensitive gel provided by the present invention is simple, which solves the problem that free lipophilic mifepristone and olaparib cannot penetrate the mucus barrier of the reproductive tract. The vaginal administration method can increase the drug concentration in the female pelvic reproductive organs, thereby reducing the adverse reactions of systemic administration. The hydrogel of the present invention encapsulates the drug-loaded targeted liposomes, completes the phase transition when entering the vaginal cavity, becomes a semi-solid and adheres to the mucosa, increases the retention time of the drug, and the sustained release of the liposome further enhances the sustained release effect of the drug. At the same time, the application of homing peptides completes the precise delivery from the reproductive tract to the target target cells through the "ligand-receptor" binding principle.

[0036] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for preparing a vaginal targeted liposome thermosensitive gel, characterized in that: The following steps are involved: (1) Egg yolk lecithin, cholesterol, DSPE-MPEG 2000 , DSPE-PEG 2000 -NHS is mixed with mifepristone or olaparib and dissolved in dichloromethane; (2) the organic phase is volatilized by a rotary evaporator to obtain a thin film material; (3) the thin film is hydrated with a phosphate buffer under ultrasound to obtain liposomes; (4) the particle size is uniformed by high shear emulsification ultrasound technology, TNYL peptide is added and reacted at room temperature for 2 days to obtain TNYL peptide-modified targeted liposomes; (5) poloxamer 188, poloxamer 407, glycerol, Tween 80 and the drug-loaded targeted liposomes are evenly mixed and refrigerated to obtain mifepristone or olaparib targeted liposome thermosensitive gel.

2. The preparation method according to claim 1, characterized in that: The specific preparation steps are as follows: (1) Egg yolk lecithin, cholesterol, DSPE-MPEG 2000 , DSPE-PEG 2000 -NHS and mifepristone or olaparib dissolved in 24 ml of dichloromethane; (2) The rotation speed of the rotary evaporator is 35 rpm, the temperature of the water bath is 28° C., the pressure is 320 mbar, the dissolved dichloromethane organic phase is injected, and the rotary evaporation time is 20 min to obtain a thin film material; (3) Add 4 ml of phosphate buffer, shake slowly for 5 minutes, and then shake under ultrasound to fully hydrate the film. The ultrasound power is 200 watts and the ultrasound time is 10 seconds in total. Collect the liquid in a centrifuge tube; (4) The centrifuge tube containing the liposomes was placed under an ultrasonic probe with an ultrasonic power of 200 watts for a total of 10 seconds. The liposomes that passed through the membrane were collected in a new centrifuge tube to remove free lipophilic drugs. TNYL peptide was dissolved in DMSO, and TNYL peptide was added to the liposomes that passed through the membrane. The mixture was mixed evenly and reacted at room temperature for 2 days to obtain targeted liposomes. (5) Poloxamer 188, poloxamer 407, glycerol, Tween 80 and targeted liposomes were mixed, the targeted liposomes were added, stirred, and placed in a refrigerator to form a thermosensitive gel.

3. The preparation method according to claim 2, characterized in that: In step (1), the amounts of mifepristone added are 105 mg, 10 mg, 12 mg, 3 mg, and 10 mg, respectively, which are dissolved in 24 ml of dichloromethane; the amounts of olaparib added are 95 mg, 5 mg, 12 mg, 3 mg, and 10 mg, respectively, which are dissolved in 24 ml of dichloromethane, and ultrasonic dissolution is performed.

4. The preparation method according to claim 2, characterized in that: The mass of the TNYL peptide added in step (4) was 0.75 mg, and it was dissolved in dimethyl sulfoxide.

5. The preparation method according to claim 2, characterized in that: The mass ratio of poloxamer 188, poloxamer 407, glycerol and Tween 80 in step (5) is 2100 mg: 100 mg: 200 mg: 500 mg.

6. The preparation method according to claim 2, characterized in that In step (5), the refrigeration temperature is 2-6°C and the refrigeration time is more than 2 hours.

7. Use of the thermosensitive gel prepared according to claim 1 as a carrier in the preparation of vaginal targeted treatment drugs for adenomyosis, characterized in that: The drugs contained are mifepristone or olaparib.

8. The use according to claim 7, characterized in that: The thermosensitive gel encapsulates the drug, completes phase transition after entering the vaginal cavity, becomes a semi-solid and adheres to the mucosa, increases the retention time of the drug, and achieves the purpose of slowly releasing the drug. The drug contained is mifepristone or olaparib.

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