Drug carrier for targeting ovary as well as preparation method and application of drug carrier

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an ovary-targeting drug carrier as well as a preparation method and application thereof. Specifically, the invention provides a targeted ovarian drug carrier ZIF-8-GH-ZP3, so as to realize targeted treatment of POI and targeted ovarian release of growth hormone to improve the treatment effect and reduce the adverse reaction of the growth hormone. According to the design of the drug carrier, a zeolite imidazole skeleton (ZIF-8) and a zona pellucida antibody (ZP3Ab) are adopted, growth hormones can be released in a targeted mode according to follicles developed at different levels in an ovary, targeted therapy of primary ovarian insufficiency is finally achieved based on the dual functions of targeting and promoting development of dominant follicles of the growth hormones, and the drug carrier has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an ovary-targeted drug carrier and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Primary ovarian insufficiency (POI), also known as premature ovarian failure (POF), refers to the loss of ovarian function in women under 40 years of age for unknown reasons. The prevalence of POI is about 3.7%. POI is characterized by ovarian hypoplasia, insufficient follicular development, and reduced hormone secretion, which can lead to health problems such as infertility and menstrual disorders. At the same time, POI is also associated with other sequelae of estrogen deficiency such as osteoporosis, cardiovascular disease, fractures, and depression.

[0004] At present, human recombinant growth hormone (rhGH) is widely used to improve low ovarian reserve and enhance ovarian responsiveness in patients with low ovarian response. However, traditional rhGH preparations are not selective and cannot be specifically distributed in the body. For extraovarian tissues, high growth hormone can cause side effects such as glucose metabolism disorders, abnormal thyroid function, and the occurrence or recurrence of certain tumors. At the same time, while promoting follicular growth, it can cause primordial follicle activation, thereby affecting the ovarian reserve function of patients with premature ovarian failure and leading to follicular pool depletion. Therefore, various existing growth hormone drug preparations have certain safety issues, which greatly limits the application of growth hormone in the treatment of POI. Summary of the invention

[0005] In order to overcome the above technical problems, the present invention provides an ovarian-targeted drug carrier and its preparation method and application. Specifically, the present invention provides a novel ovarian-targeted drug carrier ZIF-8-GH@ZP3, which is designed with a zeolite imidazole framework (ZIF-8) and a zona pellucida antibody (ZP3Ab), and can release growth hormone according to the different levels of ovarian follicles. Based on its dual functions of targeting and promoting the development of dominant follicles, it can achieve targeted treatment of primary ovarian insufficiency. Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides an ovarian-targeted drug carrier, which is named ZIF-8-GH@ZP3. The drug carrier comprises at least a zeolitic imidazole framework (ZIF-8), and the ZIF-8 is loaded with growth hormone and zona pellucida antibody.

[0008] The second aspect of the present invention provides a method for preparing the above-mentioned ovarian-targeted drug carrier, the preparation method comprising:

[0009] S1. Synthesizing ZIF-8 loaded with growth hormone; specifically, mixing 2-methylimidazole with growth hormone and then adding zinc nitrate hexahydrate;

[0010] S2, adding zona pellucida antibody to the ZIF-8-GH prepared in step S1 to obtain the product.

[0011] The third aspect of the present invention provides a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the above-mentioned ovary-targeted drug carrier.

[0012] The fourth aspect of the present invention provides the use of the above-mentioned ovarian-targeted drug carrier or drug composition in any one or more of the following:

[0013] (a) preparing a product for inhibiting apoptosis of granulosa cells;

[0014] (b) preparing a product for inhibiting inflammation and senescence of KGN cells;

[0015] (c) preparing products for restoring ovarian endocrine function;

[0016] (d) preparing products for rescuing reduced fertility;

[0017] (e) preparing a product for preventing and / or treating primary ovarian insufficiency.

[0018] A fifth aspect of the present invention provides a method for preventing and / or treating primary ovarian insufficiency, the method comprising administering the above-mentioned ovary-targeted drug carrier or drug composition to a subject.

[0019] Beneficial technical effects of one or more of the above technical solutions:

[0020] Synthesis of drug carrier: The above technical scheme prepares the zeolitic imidazole framework ZIF-8-GH through a specific chemical synthesis process, and ZP3Ab is added after the preparation is completed.

[0021] Intelligent responsiveness: ZIF-8-GH@ZP3 can intelligently regulate the release of drugs by targeting the release of growth hormone according to the different levels of developing follicles in the ovaries.

[0022] Biocompatibility and safety: The biocompatibility evaluation of the materials used in the present invention proves that ZIF-8-GH@ZP3 has good cell compatibility and low toxicity, ensuring its safety and applicability.

[0023] Drug release kinetics: In vitro release experiments verified that the drug can be released continuously for 24 hours.

[0024] In vivo verification of therapeutic effects: Through application in animal models, it was confirmed that ZIF-8-GH@ZP3 can effectively alleviate primary ovarian insufficiency, increase the level of sex hormones in the body, reduce follicular atresia, and thus slow down the progression of the disease.

[0025] In summary, the ovarian targeted drug carrier ZIF-8-GH@ZP3 prepared by the above technical scheme, based on its dual functions of targeting and promoting the development of dominant follicles, achieves targeted treatment of primary ovarian insufficiency and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 This is a diagram of the action mechanism of the ovarian-targeted drug carrier ZIF-8-GH@ZP3 of the present invention.

[0028] Figure 2 Characterization of the material of the present invention. Wherein, A is the electron microscope image of ZIF-8. B is the electron microscope image of ZIF-8-GH. C is the zeta potential of ZIF-8, ZIF-8-GH, and ZIF-8-GH@ZP3. D is the hydrodynamic size distribution of ZIF-8, ZIF-8-GH, and ZIF-8-GH@ZP3. E is a line graph of growth hormone release rate. F is a silver staining image after polyacrylamide gel electrophoresis. G is the imaging after subcutaneous injection of targeted material. H is the imaging after subcutaneous injection of non-targeted material. I is the ovarian imaging after injection of targeted or non-targeted material. J is the expression of ZP3 on the surface of follicles of different levels.

[0029] Figure 3 The biocompatibility test of the material of the present invention is shown in Figure 1. A is HE staining of main organs in different groups. B is live and dead staining of cells in different groups. C is CCK-8-24h. D is CCK-8-48h.

[0030] Figure 4 The ovaries under the stereomicroscope of the present invention are shown in Figure 1. A is the ovary of the normal group of mice. B is the ovary of the CIS group of mice. C is the ovary of the ZIF-8 group of mice. D is the ovary of the GH group of mice. E is the ovary of the ZIF-8-GH group of mice. F is the ovary of the ZIF-8-GH@ZP3 group of mice.

[0031] Figure 5 The effect of the material of the present invention is shown in the figure. A is the weight of the mouse. B is the general image of the mouse ovary. C is the weight of the mouse ovary. D is the mouse ovary index.

[0032] Figure 6 The effect of the material of the present invention is shown in the figure. A is the FSH content in serum. B is the LH content in serum. C is the P content in serum. D is the E2 content in serum.

[0033] Figure 7 The following are HE slice images of mouse ovaries after the material of the present invention is applied. A is the HE staining image of the ovaries in the normal group. B is the HE staining image of the ovaries in the CIS group. C is the HE staining image of the ovaries in the ZIF-8 group. D is the HE staining image of the ovaries in the GH group. E is the HE staining image of the ovaries in the ZIF-8-GH group. F is the HE staining image of the ovaries in the ZIF-8-GH@ZP3 group. G is a bar chart showing the number of follicles at different levels in different groups.

[0034] Figure 8 The figure is a graph of mouse fertility after the material of the present invention acts on the mice. A is the pregnancy rate of mice. B is the number of suckling mice. C is suckling mice in the NC group. D is suckling mice in the CIS group. E is suckling mice in the ZIF-8 group. F is suckling mice in the GH group. G is suckling mice in the ZIF-8-GH group. H is suckling mice in the ZIF-8-GH@ZP3 group.

[0035] Fig. 9 The following are histochemical staining pictures of ovarian sections in each group after being treated with the material of the present invention.

[0036] Fig.10 The β-glycosidase staining diagram after the material of the present invention is acted upon. A is the glycosidase staining diagram. B is the senescent cell ratio diagram.

[0037] Fig.11 The fluorescence images of cells after the material of the present invention acts on them. A is Bax fluorescence. B is Bax fluorescence quantification. C is Bcl-2 fluorescence. D is Bcl-2 fluorescence quantification. E is Bax / Bcl-2 value.

[0038] Fig.12 The fluorescence images of cells after the material of the present invention acts on them. A is the fluorescence of P21. B is the quantification of P21 fluorescence. C is the fluorescence of IL-1-β. D is the quantification of IL-1-β fluorescence. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0041] As mentioned above, although existing technologies provide a variety of means for the treatment of premature ovarian failure, there are still some obvious limitations:

[0042] 1. For extra-ovarian tissues, high growth hormone can cause side effects such as glucose metabolism disorders, abnormal thyroid function, and the occurrence or recurrence of certain tumors.

[0043] 2. Follicular pool depletion: The current regimen promotes follicle growth while leading to follicular pool depletion, thus affecting the ovarian reserve function of patients with premature ovarian failure.

[0044] In view of this, the main purpose of the present invention is a new type of ovarian targeted drug carrier (ZIF-8-GH@ZP3) to achieve the following goals:

[0045] 1. Reduce side effects and complications: By targeting the ovaries, the overall drug load is reduced, thereby alleviating potential side effects and complications.

[0046] 2. Screen dominant follicles and promote their maturation.

[0047] 3. Achieve lasting therapeutic effects: Provide a sustained-release drug delivery system to prolong the therapeutic effect and slow down the progression of POI.

[0048] 4. Adopt a multi-mechanism treatment strategy: Combine multiple mechanisms of action such as anti-inflammatory, antioxidant and anti-apoptotic to provide a comprehensive treatment plan to address the complex pathological process of POI.

[0049] In summary, the present invention aims to overcome the deficiencies of the prior art and provide a new method for treating primary ovarian insufficiency, which is safer and more effective, through its unique design and multiple mechanisms of action.

[0050] Specifically, in a typical embodiment of the present invention, an ovarian-targeted drug carrier is provided, which is named ZIF-8-GH@ZP3. The drug carrier comprises at least a zeolite imidazole framework (ZIF-8), and the ZIF-8 is loaded with growth hormone and zona pellucida antibody.

[0051] Furthermore, the growth hormone is specifically human recombinant growth hormone, which can be obtained through commercial means.

[0052] The zona pellucida antibody is specifically zona pellucida 3 antibody (ZP3Ab), which can also be obtained commercially.

[0053] Furthermore, the drug carrier is a nanogel preparation, and the hydrodynamic diameter of the drug carrier is about 377 nm.

[0054] In another specific embodiment of the present invention, a method for preparing the above-mentioned ovarian-targeted drug carrier is provided, and the preparation method comprises:

[0055] S1. Synthesizing ZIF-8 loaded with growth hormone; specifically, mixing 2-methylimidazole with growth hormone and then adding zinc nitrate hexahydrate;

[0056] S2, adding zona pellucida antibody to the ZIF-8-GH prepared in step S1 to obtain the product.

[0057] Wherein, in step S1, the concentration ratio of 2-methylimidazole, growth hormone and zinc nitrate hexahydrate is 200-300:1-5:50-150, further 216 mg / ml:3.33 mg / ml:100 mg / ml; the volume ratio is 10-20:5-10:1-5, preferably 18:9:2;

[0058] To ensure uniform mixing, stirring is performed at a speed of 500-1000 rpm (preferably 800 rpm); zinc nitrate hexahydrate can be added in small amounts and multiple times (such as dropwise addition), which is more conducive to the synthesis of ZIF-8.

[0059] Furthermore, growth hormone-loaded ZIF-8 was synthesized and diluted with buffer (PBS) for later use;

[0060] In the step S2, stirring is performed during the process of adding the zona pellucida antibody, and the stirring speed is 500-1000 rpm.

[0061] The volume-to-mass ratio of the zona pellucida antibody to growth hormone is 2-6:3 (μl:μg).

[0062] In another specific embodiment of the present invention, a pharmaceutical composition is provided, wherein the active ingredient of the pharmaceutical composition comprises the above-mentioned ovary-targeted drug carrier.

[0063] In another specific embodiment of the present invention, the pharmaceutical composition may further contain other pharmaceutically acceptable carriers, the dosage of which should be harmless to the subject, including but not limited to buffers, antioxidants, preservatives, bactericides, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, tension regulators, sugars, surfactants, salt-forming counterions, metal complexes and / or non-ionic surfactants, etc., which are not specifically limited here.

[0064] In another specific embodiment of the present invention, the drug administration subject can be human and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, and gorillas, with humans being preferred.

[0065] In another specific embodiment of the present invention, the use of the above-mentioned ovarian-targeted drug carrier or drug composition in any one or more of the following is provided:

[0066] (a) preparing a product for inhibiting apoptosis of granulosa cells;

[0067] (b) preparing a product for inhibiting inflammation and senescence of KGN cells;

[0068] (c) preparing products for restoring ovarian endocrine function;

[0069] (d) preparing products for rescuing reduced fertility;

[0070] (e) preparing a product for preventing and / or treating primary ovarian insufficiency.

[0071] In another embodiment of the present invention, the product can be a drug or a test reagent for non-pharmaceutical use. The test reagent is used for basic research.

[0072] In another specific embodiment of the present invention, a method for preventing and / or treating primary ovarian insufficiency is provided, the method comprising administering the above-mentioned ovarian-targeted drug carrier or drug composition to a subject.

[0073] The subject of the present invention refers to an animal that has been the object of treatment, observation or experiment, preferably a mammal, and most preferably a human.

[0074] The "effective amount" of the present invention refers to the amount of active compounds or agents, including the compounds of the present invention, which can induce the biological or medical response of the tissue system, animal or human sought by researchers, veterinarians, doctors or other medical personnel, including alleviating or partially alleviating the symptoms of the disease, syndrome, condition or disorder being treated.

[0075] In addition, it should be noted that technical improvements made by those skilled in the art to the technical solution of the present invention based on the inventive concept of the present invention without creative work also fall within the protection scope of the present invention. The inventor lists some of the improvements as follows:

[0076] Different drug carrier materials: Although the present invention uses ZIF-8 as a drug carrier and ZP3 as a target, other similar biocompatible and biodegradable materials can be considered as alternatives, such as the target can be replaced by ZP1, ZP2, and the carrier can be replaced by ZIF-1, ZIF-14.

[0077] Adjustment of drug combination: The drug growth hormone used in the present invention can be introduced or replaced by other therapeutic drugs to adapt to the needs and conditions of different patients.

[0078] Changes in drug administration methods: Currently designed for subcutaneous injection of metal nanoparticles, other forms such as intraperitoneal injection, microneedles, etc. can be used to adapt to different application requirements and injection methods.

[0079] Universal platform for drug delivery systems: Although the present invention is mainly aimed at the treatment of primary ovarian insufficiency, the targeting and anti-inflammatory, antioxidant and anti-apoptosis properties of ZIF-8@ZP3 may also be applicable to the treatment of other diseases, such as ovarian tumors, fallopian tube-related diseases, etc.

[0080] The present invention is further explained by the following examples, but it is not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In the following examples, the materials, reagents, etc. used, if not otherwise specified, are obtained from commercial sources.

[0081] Example

[0082] 1. Experimental Methods

[0083] 1.1 Synthesis of ZIF-8-GH@ZP3

[0084] The raw materials are 2-methylimidazole (C5H8N2 Aladdin M104839-100g) and zinc nitrate hexahydrate (Zn(NO3)2·6H2O Aladdin Z111706-500g). To synthesize ZIF-8, 1.8 ml of C5H8N2 (216 mg / ml) and 0.2 ml of Zn(NO3)2·6H2O (100 mg / ml) solution were added dropwise to 2-methylimidazole on a magnetic stirrer at a stirring speed of 800 rpm. To synthesize ZIF-8-GH, 1.8 ml of C5H8N2 (216 mg / ml) and growth hormone (rDNA source) injection (rhGH 3.33 mg / ml) were mixed in a volume ratio of 2:1. 0.2 ml Zn(NO3)2·6H2O solution (100 mg / ml) was added dropwise to the mixed system at 800 rpm on a magnetic stirrer. PBS was diluted to 40 ml before use. To synthesize ZIF-8-GH@ZP3, 1 ml of diluted ZIF-8-GH was taken and 100 μl ZP3 (Proteintech 21279-1-AP) antibody was added at 800 rpm on a magnetic stirrer and stirred for 24 h. All the above operations were completed on a clean bench.

[0085] 1.2 Material characterization of ZIF-8-GH@ZP3

[0086] The morphology and structure were evaluated using scanning electron microscopy (SEM). The surface charge characteristics of the material were analyzed using a zeta potential instrument. The growth hormone release mode was confirmed by in vitro release experiments, and the concentration of released growth hormone was monitored by radioimmunoassay. Rapid silver staining was used to verify whether ZIF-8-GH@ZP3 was successfully loaded with GH and ZP3.

[0087] 1.3 Establishment of POI mouse model

[0088] Eight-week-old female C57BL / 6 mice were housed in an SPF environment for 7 days to avoid health problems. All mice were in accordance with the experimental protocol approved by the Ethics Committee of the Model Animal Research Institute of the Provincial Hospital Affiliated to Shandong First Medical University. Throughout the experiment, mice were maintained in an SPF environment with 12 h of light and 12 h of darkness per day, and had free access to water and pelleted feed. We chose POI induced by cisplatin chemotherapy. Mice with a body weight of 19.5-20.5 g and regular estrous cycle were selected. Mice in the blank control group were intraperitoneally injected with 0.9% Nacl solution, and mice in the POI group were intraperitoneally injected with cisplatin (CDDP MCE HY-17394) 2 mg / Kg for 7 consecutive days. The estrous cycle was monitored at the same time. After intraperitoneal injection of cisplatin, the treatment group was given subcutaneous injection of PBS, ZIF-8 (100 mg / kg), GH (1.6 mg / kg), ZIF-8-GH (100 mg / kg), and ZIF-8-GH@ZP3 (100 mg / kg) for 7 consecutive days. The mice were weighed on the 7th and 14th days of the experiment. On the 14th day of the experiment, the eyeballs were taken for blood collection, and the ovaries were separated and weighed.

[0089] 1.4 HE staining and follicle counting

[0090] The ovaries, hearts, livers, spleens, lungs, and kidneys of mice were fixed with 4% paraformaldehyde for 24 hours, dehydrated, and embedded in paraffin. The hearts, livers, spleens, and one side of the ovaries, lungs, and kidneys of rats in each group were cut into 5 μm thick continuous sections. The sections were used to count follicles and confirm whether the intervention caused damage to the mouse organs. The sections were stained with hematoxylin-eosin solution (ServicebioG1076). The number of various follicles was counted under a light microscope, and every 10 sections were analyzed, and the total number of follicles in each group was calculated from the total number of sections.

[0091] 1.5 TUNEL staining

[0092] TUNEL staining was performed using a TUNEL kit. The ovaries were embedded in 5 μm thick paraffin as described above, and the sections were immersed in dewaxing solution (ervicebio, G1128), anhydrous ethanol and ddH2O in turn for dewaxing. Proteinase K was incubated at 37°C for 25 min. Reagent 1, reagent 2 and buffer were then mixed at a ratio of 1:5:50, incubated in a 37°C humidifier for 1 h, and then incubated in 3% H2O2 in the dark for 15 min. After washing three times with PBS solution, reagent 3 and TBST were fixed at a ratio of 1:200 and incubated at 37°C for 30 min. Finally, DAB was used for color development and hematoxylin was used for nuclei staining.

[0093] 1.6 Immunohistochemical staining

[0094] Immunohistochemistry was used to detect the expression of IGF-1 (1:200HUABIO ER1911-10), VEGF (1:250), Bax (1:800), Bcl-2 (1;250), and cd31 (1:500). Paraffin-embedded sections were dewaxed in xylene and dehydrated in different concentrations of ethanol. The tissue sections were placed in a box containing EDTA antigen retrieval solution and placed in a pressure cooker for antigen retrieval. The sections were blocked with 3% bovine serum albumin for 30 minutes at room temperature. The sections were then incubated with the primary antibody in a 4°C wet box overnight. The secondary antibody was incubated at room temperature for 1 hour, and the DAB color reaction was performed under a microscope by controlling the color development time. Finally, the sections were counterstained with hematoxylin. Images were collected using an OLYMPUSBX63 microscope.

[0095] 1.7 Construction and intervention of granulosa cell in vitro model

[0096] KGN cell line was cultured at 37°C and 5% CO2. The experiment was divided into 4 groups: blank control group, KGN cells were cultured in DMEM medium containing 1% PBS for 48h; cisplatin group: KGN cells were cultured in DMEM medium containing cisplatin (1.25mg / ml) for 48h; CIS+ZIF-8 group: on the basis of the cisplatin group, ZIF-8 (4mg / ml) was added to treat KGN cells for 24h; CIS+growth hormone group, on the basis of the cisplatin group, KGN cells were treated with growth hormone injection (0.8IU / ml) for 24h;

[0097] 1.8 β-Galactosidase staining

[0098] KGN cells were detected using a senescent β-galactosidase staining kit. KGN cells were seeded on 6-well plates. After 4 groups of treatments, KGN cells were fixed in 4% paraformaldehyde, washed three times with PBS, and then different staining solutions were added in sequence according to the instructions of the reagent manufacturer. After overnight at 37°C, the cells were observed under a microscope and photographed.

[0099] 1.9 Immunofluorescence staining

[0100] Immunofluorescence staining was used to detect the expression of Bax, Bcl-2, P21, and IL-1β in KGN cells. After culture and washing, the cells were fixed with 4% paraformaldehyde for 30 min, treated with 0.5% Triton X-100 for 10 min, and blocked with 2% BSA at 37°C for 1 h. After washing with PBS, the cells were stained with Bax (1:500) or Bcl-2 (1:200) or P21 (1:50) or IL-1-β (1:50) antibodies at 4°C overnight. The cells were incubated with goat anti-rabbit IgG-H&L polyclonal secondary antibody (1:500) at room temperature in the dark for 1.5 h, and the cell nuclei were stained with DAPI. The fluorescence changes of Bax, Bcl-2, P21, and IL-1-β were observed under a fluorescence microscope.

[0101] 1.10 Cell counting kit (CCK-8) detection

[0102] The cytotoxicity of ZIF-8 was detected using a cell counting kit. KGN cells were inoculated in a 96-well culture plate at a cell density of 3000 cells / well. KGN cells were inoculated in DMEM culture medium and cultured for about 12 hours. Different concentrations of ZIF-8 (0, 2, 4, 6, 10, 20, 40, 80 mg / ml) were added in sequence. KGN cells were cultured with the above mixed culture medium for 24 and 48 hours. Then, according to the manufacturer's instructions, KGN cells were inoculated with 10% CCK-8 culture medium for 2 hours, and the absorbance value of the 96-well plate was measured at 450nm using an ELISA reader.

[0103] 2. Experimental results

[0104] 2.1 Characterization of ZIF-8, ZIF-8-GH, and ZIF-8-GH@ZP3 Nanogels

[0105] Synthesize ZIF-8, TEM studies show that ZIF-8-GH has an octahedral structure, and the ZIF-8-GH material with in-situ addition of GH is rougher than ZIF ( Figure 2 B). After the synthesis of ZIF-8-GH, the ZP3 target was added, and the hydrodynamic diameter of ZIF-8-GH@ZP3 was about 377 nm ( Figure 2 D). Analyze the surface charge characteristics of the material using a Zeta potential instrument, see ( Figure 2 C), growth hormone is negatively charged in the physiological environment, so the Zeta potential of ZIF8-GH is lower than that of ZIF-8. ZP3 antibody also carries a small amount of negative charge in the physiological environment, so after modifying ZP3, the Zeta potential of the material is further reduced. In vitro release experiments have confirmed that growth hormone can be released continuously for 24 hours ( Figure 2 E). Silver staining showed that ZIF-8-GH successfully loaded GH and ZIF-8-GH@ZP3 successfully loaded ZP3 and GH ( Figure 2 F).

[0106] 2.2 In vitro cytocompatibility assessment

[0107] First, the cytocompatibility of ZIF-8 on ovarian granulosa cells was evaluated. The cells were co-cultured with different concentrations of ZIF-8 (0, 2, 4, 6, 10 mg / ml), and the cell viability was quantified after 24 h and 48 h using the CCK-8 assay ( Figure 3 C, D). At 24h, the cell activity of 6mg / mL concentration reached 98.76%±2.47% ( Figure 3 C), indicating that this concentration has good cell compatibility. Live / dead cell staining experiments further confirmed the activity of ovarian granulosa cells at a concentration of 6 mg / mL ( Figure 3 B). This indicates that ZIF-8 has excellent cell compatibility. Our study shows that within a concentration of 6 mg / mL, ZIF-8 exhibits good cell compatibility.

[0108] 2.3 Effect of in-vitro treatment of primary ovarian insufficiency

[0109] In this study, cisplatin was selected to induce POI. Briefly, cisplatin (2 mg / kg) was injected intraperitoneally for 7 consecutive days. In cisplatin-induced POI mice, PBS, ZIF-8 (100 mg / kg), GH (1.6 mg / kg), ZIF-8-GH (100 mg / kg), and ZIF-8-GH@ZP3 (100 mg / kg) were injected subcutaneously every day for 7 consecutive days. The body weight of mice was measured at 0, 4, 7, 11, and 14 days after cisplatin injection; the body weight of mice in each group decreased significantly on the 4th and 8th days after cisplatin injection. The body weight of mice rebounded from the 11th day. The GH group had the fastest weight gain. The ZIF-8 group (19.67±1.20) g, the ZIF-8-gh group (19.7±1.31) g, the ZIF-8-GH@ZP3 group (19.37±0.35) g, and the CIS group recovered the slowest. We speculate that the effect of GH on weight gain is delayed due to the sustained release and targeting of ZIF8@ZP3 ( Figure 5 A). After 7 consecutive days of intraperitoneal injection of cisplatin, the ovarian morphology was observed under a microscope. Compared with the NC group, the ovaries in the cisplatin group were significantly atrophied. The ovaries in the CIS group were white in color and smooth in surface ( Figure 4 B). The ovaries of the NC group mice were pink in color, and the surface of the normal ovaries was rough. This is because the ovaries of the normal group mice contained normal-looking follicles ( Figure 4 A). The ovarian morphology of different intervention groups also improved. ZIF-8-GH@ZP3 showed significant improvement ( Figure 4 C, D, E, F). The study also showed that ZIF-8 had a clear benefit in improving ovarian morphology. Next, the ovaries of the mice were surgically removed and placed on a medium sheet ( Figure 5B). The ovaries of the treatment group were significantly larger than those of the CIS group. Compared with the CIS group (3.27±0.32) mg, the ovarian mass of the mice in the ZIF-8 group (4.03±0.71) mg, GH group (5.53±0.35) mg, ZIF-8-GH group (6.27±0.15) mg, and ZIF-8-GH@ZP3 group (7.03±0.15) mg were significantly increased (p<0.05) ( Figure 4 C). We speculate that the addition of ZIF-8 and ZP3 allows the drugs to act locally on the ovaries, and the ovarian weight is significantly increased compared with the GH group. Compared with the GH group, the body weight of mice in the ZIF-8-GH@ZP3 group (7.03±0.15 mg) was significantly increased (p<0.01). The ovarian / body weight index was calculated by dividing the ovarian mass by the body weight. The ovarian / body weight index of the ZIF-8-GH@ZP3 group (0.036334±0.0014511) was significantly higher than that of the GH group (0.025881±0.0020551) (P<0.01)( Figure 4 D). ZP3-mediated targeting of GH to the ovary better restored ovarian weight and reduced the effects of GH on other parts of the mice.

[0110] Ovarian sections were stained with hematoxylin-eosin (HE) Figure 7). The ovaries in the NC group were larger and had the largest number of follicles. The ovaries in the CIS group were significantly atrophied, with disordered cortical and medullary structures, reduced volume, disappearance of mature follicles, and an increase in atretic follicles. The number of mature follicles in each treatment group increased significantly, while the number of atretic follicles decreased. The follicles were classified and counted. The true ovarian reserve is reflected by the number of follicles. The follicles were classified using the following criteria. Primordial follicles: There is a layer of flat granulosa cells around the oocyte; primary follicles: There are 1 to 6 layers of cuboidal granulosa cells around the oocyte; secondary follicles: There are at least 2 layers of cuboidal granulosa cells around the oocyte, and there is an antral cavity between the follicle cells; mature follicles: The diameter of ovarian follicles is significantly enlarged and protrudes above the ovarian surface; atretic follicles: The zona pellucida and oocytes are deformed. The number of primordial follicles, primary follicles, secondary follicles, and mature follicles were counted in consecutive sections of the ovaries. HE staining pathological examination showed that the number of follicles in rats in the CIS group was significantly reduced. Mature follicles disappeared. The number of primordial follicles, primary ovarian follicles, and mature follicles increased significantly in the GH group, and the number of mature follicles in the ZIF-8-GH@ZP3 group increased significantly compared with the GH group. Subcutaneous injection of ZIF-8-GH@ZP3 significantly restored the development of ovarian follicles compared with the GH group. The number of mature follicles in the ZIF-8-GH@ZP3 group was significantly higher than that in the ZIF-8-GH group, and treatment with ZIF8-GH@ZP3 has been shown to be superior to treatment with ZIF-8-GH in restoring the development of mature follicles. Interestingly, the number of secondary follicles in the ZIF8-GH@ZP3 group decreased in each treatment group, so we speculate that ZIF8-GH@ZP3 accelerates the transition from secondary follicles to mature follicles. In addition, the number of follicles of different grades showed that the ZIF-8 group was better than the CIS group in restoring follicle development. This suggests that ZIF-8 is also effective against POI. In summary, ZIF-8-GH@ZP3 can protect the ovaries from premature ovarian failure caused by CIS ( Figure 7 G). Serum follicle stimulating hormone (FSH), luteinizing hormone (LH), estrogen (E2), and progesterone (P) levels were measured to evaluate the protective effect of ovarian endocrine function. The secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) is controlled by gonadotropin-releasing hormone (GnRH). FSH and LH are continuously secreted from the pituitary gland and then reach the ovaries in the serum to stimulate the production of E2 and P. LH and FSH are in turn subject to feedback control by E2 and P. This leads to a decrease in the secretion of E2 and P. Due to the loss of ovarian negative feedback, serum LH and FSH concentrations increased. Compared with the control group, the levels of estradiol (4.76±1.47pg / ml) and progesterone (4.00±1.51ng / ml) in the CIS group were significantly decreased, and the levels of follicle stimulating hormone (2.24±0.29mIU / ml) and luteinizing hormone (7.54±0.09mIU / ml) were significantly increased. ( Figure 6) after treatment, the changes in serum sex hormone levels of mice in different treatment groups showed a significant recovery effect. The ZIF-8-GH@ZP3 group was the most significant, especially the estrogen level was equivalent to the NC group (p<0.05). It is worth noting that the use of ZIF-8 alone also has a certain restorative effect on ovarian hormone secretion. Compared with the CIS group, the serum E2 level increased (p<0.05) and the serum FSH level decreased (p<0.05). The serum FSH level of the treatment group was significantly higher than that of the CIS group, and the differences were statistically significant, with the ZIF-8-GH@ZP3 group (0.74±0.06pg / ml) being the most obvious. Compared with the CIS group, the serum E2 levels in the ZIF-8 (8.60±0.26pg / ml), GH (9.36±0.28pg / ml), ZIF-8-GH (10.48±0.51pg / ml), and ZIF-8-GH@ZP3 (12.28±1.08pg / ml) groups were significantly increased, with the ZIF-8-GH@ZP3 group increasing most significantly (p<0.01). Compared with the CIS group, the serum LH level in the ZIF-8-GH@ZP3 group (4.34±0.21mIU / ml) was decreased, and the difference was statistically significant (p<0.001). Compared with the GH group, the serum FSH level in the ZIF-8-GH@ZP3 group was decreased, and the difference was statistically significant (p<0.0001). The P level was significantly higher in the ZIF-8-GH@ZP3 group (9.49±0.26) than in the CIS group (4.00±1.51ng / ml) (P<0.001), as shown below. ZIF-8-GH@ZP3 treatment significantly restored the serum P level compared with the GH group (7.12±0.63ng / ml) (p<0.01). In addition to the increased P level, the serum E2 level was significantly different between the two groups. In summary, subcutaneous injection of ZIF-8-GH@ZP3 can significantly restore ovarian function compared with the GH group. ZIF-8 treatment significantly restored serum estrogen levels (p<0.05) and reduced serum FSH levels (1.53±0.08mIU / ml) compared with the CIS group, and the differences were statistically significant (p<0.05). These data indicate that both ZIF8-GH@ZP3 and ZIF-8 can restore ovarian endocrine function in POI mice.

[0111] Improvement of POI mice's reproductive capacity. We counted the fertility results of 6 groups ( Figure 8), pregnancy rate and number of offspring. There were 20 mice in each group. Each group of mice was observed for 4 weeks after treatment, and the number of pregnancies was recorded. The number of pups 2 to 5 days after delivery was recorded. On the 28th day, the pregnancy rate in the CIS group was 15% (3 / 20), and that in the ZIF-8-GH@ZP3 group was 60% (12 / 20). The number of pups in the CIS group was (2.33±0.58), and that in the ZIF8-GH@ZP3 group was (6.67±0.58). This result indicates that the ZIF-8-GH@ZP3 group can significantly rescue the fertility of mice compared with the CIS group.

[0112] Cytokines such as VEGF, CD31, and IGF-1 play an important role in cell proliferation, survival, migration, and invasion, and play an important role in promoting ovarian repair. VEGF is a marker for new blood vessels and can promote angiogenesis. CD31 is a marker for angiogenesis. Growth hormone stimulates the liver to produce IGF-1, and IGF-1 in the circulation mediates the effects of growth hormone through the GH-IGF-1 axis. Immunohistochemical staining was used to detect cytokines. Immunohistochemical results showed that after CIS-induced ovarian damage, the expression of VEGF and CD31 was significantly reduced, and the ovarian repair capacity was insufficient ( Fig. 9 A, E). After ZIF-8-GH@ZP3 treatment, ovarian function was promoted, which may be related to the significant increase of VEGF and CD31 levels by ZIF8-GH@ZP3. IGF-1 expression was decreased in the ovaries of POI mice, and ZIF-8-GH@ZP3 intervention promoted IGF-1 expression ( Fig. 9 B). The anti-apoptotic protein Bcl-2 can inhibit the endogenous apoptotic pathway. The pro-apoptotic protein Bax can initiate cell death. Therefore, we used Bcl-2 and Bax immunohistochemistry to detect apoptosis of ovarian tissue cells. The results showed that CIS increased Bax-positive granulosa cells in mouse follicles and decreased Bcl-2-positive granulosa cells in mouse follicles ( Fig. 9 C, D). ZIF-8-GH@ZP3 intervention can reverse the expression of apoptosis-related factors. We believe that ZIF-8-GH@ZP3 intervention can restore damaged ovarian function by changing the expression ratio of anti-apoptotic and pro-apoptotic molecules. ZIF-8-GH@ZP3 intervention can affect the expression levels of multiple ovarian cytokines, providing a favorable microenvironment for angiogenesis, apoptosis inhibition and repair and regeneration of damaged ovarian tissue. TUNEL staining showed that granulosa cells in the CIS group showed obvious apoptosis, while ZIF-8-GH@ZP3 intervention could inhibit granulosa cell apoptosis ( Fig. 9 F).

[0113] 2.4 Ovarian targeting

[0114] In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP3 can form a biologically functional structure independent of other glycoproteins. In addition, we found through immunofluorescence analysis that the expression of ZP3 gradually increases with the maturation of follicles ( Figure 2 J), with the theoretical feasibility of targeting the dominant follicle. ZIF-8-GH is combined with the ZP3 antibody branch to enhance the interaction between the drug delivery system and the ovary; ZIF-8-GH actively targets the ovary, enhances the aggregation of GH on the ovary, and can improve the drug delivery efficiency. ZIF-8-GH is first labeled with Cy5 and then loaded with ZP3. Then ZIF-8-GH(Cy5)@ZP3 is injected intraperitoneally, and the mice are killed 12 hours later, and the organs are dissected. In order to make the fluorescence intensity more obvious, the ovaries, hearts, livers, spleens, lungs and kidneys of mice are separated from each group and placed on a culture dish. The in vivo targeting of the ZIF-8-GH@ZP3 group is monitored by an in vivo imaging system (IVIS). The fluorescence intensity generated by Cy5 can be used as an indicator of ovarian GH content. The ovarian fluorescence intensity of the ZIF-8-GH(Cy5)@ZP3 group is significantly enhanced compared with the non-targeted group, verifying that ZP3-mediated ovarian targeting and drug delivery will enhance the efficacy of GH treatment. Therefore, ZIF-8-GH(Cy5)@ZP3 can accumulate more GH in the ovaries, thus providing better treatment ( Figure 2 G, H, I).

[0115] 2.5ZIF-8 has a protective effect on KGN cells

[0116] The β-galactosidase activity of KGN cells after treatment in the above four groups was stained, and three independent experiments were performed in each group for counting. When the concentration of cisplatin was 1.5 mg / ml, the expression of senescence-related β-galactosidase in KGN cells increased significantly (37.02±3.66). After GH treatment, the senescence of KGN cells was significantly reduced (14.37±0.40) ( Fig.10 ); In order to further study the effects of ZIF-8 and GH on KGN cell apoptosis, we used immunofluorescence staining to detect the effects of Bax and Bcl-2 on KGN cell apoptosis. The Bcl-2 protein family includes Bax and Bcl-2. Bax promotes cell death by antagonizing the anti-apoptotic function of Bcl-2. The fluorescence intensity of Bax in the ZIF-8 group (8.93±0.46) decreased more significantly than that in the CIS group (18.21±1.77). P<0.001. The results of Bcl-2 staining showed that the fluorescence intensity of the CIS group (7.21±0.35) was significantly lower than that of the control group (27.05±0.92), and the ZIF-8 group (14.87±0.95) could increase the fluorescence intensity of Bcl-2 (P<0.001). ( Fig.11) In vitro experiments showed that ZIF-8 could directly inhibit apoptosis of KGN cells. Compared with the CIS group, the Bax / Bcl-2 ratio in the ZIF-8 group was significantly reduced (P<0.001). P21 is a marker of cell senescence. The P21 fluorescence intensity in the ZIF-8 group and the GH group was significantly lower than that in the CIS group ( Fig.12 A)(P<0.01). IL-1β is a potent pro-inflammatory cytokine and a key molecule in inflammation. Compared with the CIS group, the fluorescence intensity of the ZIF-8 group and the GH group was significantly reduced ( Fig.12 C), indicating that both ZIF-8 and GH treatment inhibited inflammation and senescence of KGN cells.

[0117] It can be seen from the above embodiments that the technical solution of the present invention has the following advantages:

[0118] Provide targeted drug release: Develop a drug release system that can be intelligently adjusted according to different levels of follicle development to achieve more precise therapeutic effects.

[0119] Achieving durable therapeutic effects: Designing a sustained-release drug delivery system to prolong the effectiveness of treatment and thereby slow the progression of premature ovarian insufficiency (POI).

[0120] Adopt a multi-mechanism treatment strategy: combining multiple mechanisms of action such as anti-inflammatory, antioxidant and anti-apoptotic to provide a comprehensive treatment plan to effectively deal with the complex pathological process of POI.

[0121] Reduced side effects and complications: By targeting drug delivery to the ovaries, the overall drug load is reduced, thereby reducing the risk of potential side effects and complications.

[0122] Personalized treatment plan: Design a personalized treatment plan based on the patient's specific situation and the stage of follicular development to improve the targeting and effectiveness of the treatment.

[0123] Simplified drug preparation process: Compared with other complex drug carrier systems, the preparation process of ZIF-8-GH@ZP3 is simpler, which is convenient for large-scale production and commercial application.

[0124] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A drug carrier targeting ovaries, characterized in that: The drug carrier at least comprises zeolite imidazole framework (ZIF-8), and the ZIF-8 is loaded with growth hormone and zona pellucida antibody.

2. The ovarian-targeted drug carrier according to claim 1, characterized in that: The growth hormone is specifically human recombinant growth hormone; The zona pellucida antibody is specifically the zona pellucida 3 antibody.

3. The ovarian-targeted drug carrier according to claim 1, characterized in that: The drug carrier is a nanogel preparation.

4. The method for preparing the ovarian-targeted drug carrier according to any one of claims 1 to 3, characterized in that: The preparation method comprises: S1. Synthesizing ZIF-8 loaded with growth hormone; specifically, mixing 2-methylimidazole with growth hormone and then adding zinc nitrate hexahydrate; S2, adding zona pellucida antibody to the ZIF-8-GH prepared in step S1 to obtain the product.

5. The preparation method according to claim 4, characterized in that: In the step S1, the concentration ratio of 2-methylimidazole, growth hormone and zinc nitrate hexahydrate is 200-300:1-5:50-150; the volume ratio is 10-20:5-10:1-5; The mixture was stirred at a speed of 500-1000 rpm.

6. The preparation method according to claim 4, characterized in that: Synthesize ZIF-8 loaded with growth hormone, add buffer (PBS) to dilute and set aside.

7. The preparation method according to claim 4, characterized in that: In the step S2, stirring is performed during the process of adding the zona pellucida antibody, and the stirring speed is 500-1000 rpm. The volume-to-mass ratio of the zona pellucida antibody to growth hormone is 2-6:3 (μl:μg).

8. A pharmaceutical composition, characterized in that The active ingredient of the pharmaceutical composition comprises the ovary-targeted drug carrier according to any one of claims 1 to 3.

9. Use of the ovarian-targeted drug carrier according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in any one or more of the following: (a) preparing a product for inhibiting apoptosis of granulosa cells; (b) preparing a product for inhibiting inflammation and senescence of KGN cells; (c) preparing products for restoring ovarian endocrine function; (d) preparing products for rescuing reduced fertility; (e) preparing a product for preventing and / or treating primary ovarian insufficiency.

10. A method for preventing and / or treating primary ovarian insufficiency, characterized in that: The method comprises administering the ovary-targeted drug carrier according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 to a subject.

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