A drug carrier targeting ovary and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a drug carrier targeting ovaries and a preparation method and application thereof. Specifically, the application provides a drug carrier ZIF-8-GH@ZP3 targeting ovaries, so that targeted treatment for POI is realized, growth hormone is released in a targeted manner to improve the treatment effect and reduce the adverse reactions of growth hormone. The drug carrier is designed by using a zeolite imidazole framework (ZIF-8) and a zona pellucida antibody (ZP3Ab), can release growth hormone in a targeted manner according to different levels of developing follicles in the ovaries, based on the dual functions of targeting and promoting the development of dominant follicles, finally realizes targeted treatment for primary ovarian insufficiency, and has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a drug carrier targeting ovaries and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or any kind of suggestion that this information forms the general prior art already known to a person of ordinary skill in the art.

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

[0004] At present, human recombinant growth hormone (rhGH) is widely used to improve low ovarian reserve and improve the ovarian response of patients with low ovarian response. However, the traditional rhGH preparation lacks selectivity and cannot be specifically distributed in the body. For tissues outside the ovary, high growth hormone can cause glucose metabolism disorders, thyroid function abnormalities, and the occurrence or recurrence of certain tumors and other side effects. At the same time, promoting follicle growth can cause primordial follicle activation, thereby affecting the ovarian reserve function of patients with premature ovarian failure and leading to follicular pool depletion. Therefore, the existing various growth hormone drug preparations all have certain safety problems, which greatly limits the application of growth hormone in the treatment of POI. SUMMARY

[0005] In order to overcome the above technical problems, the present application provides a drug carrier targeting ovaries and a preparation method and application thereof. Specifically, the present application provides a novel drug carrier targeting ovaries ZIF-8-GH@ZP3, which is designed using zeolitic imidazolate framework (ZIF-8) and zona pellucida antibody (ZP3Ab), and can target release growth hormone according to different levels of developing follicles in the ovary. Based on its targeting and dual functions of promoting the development of dominant follicles, the targeted treatment of primary ovarian insufficiency is achieved. Based on the above research results, the present application is completed.

[0006] To achieve the above technical purpose, the technical scheme adopted by the present application is as follows:

[0007] In a first aspect of the present application, a drug carrier targeting ovaries is provided, which is named ZIF-8-GH@ZP3. The drug carrier comprises at least a zeolitic imidazolate framework (ZIF-8), and the ZIF-8 is loaded with growth hormone and zona pellucida antibody.

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

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

[0010] S2. Add 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-targeting drug carrier.

[0012] A fourth aspect of the present invention provides use of the above-mentioned ovarian-targeted drug carrier or pharmaceutical 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 that inhibits 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, comprising administering the above-mentioned ovary-targeted drug carrier or pharmaceutical 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 solution prepared the zeolitic imidazole framework ZIF-8-GH through a specific chemical synthesis process, and ZP3Ab was added after the preparation was 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 ovary.

[0022] Biocompatibility and safety: The biocompatibility evaluation of the materials used in the present invention demonstrated 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, 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 mechanism of action of the ovarian-targeted drug carrier ZIF-8-GH@ZP3 of the present invention.

[0028] Figure 2 Characterization of the materials of the present invention. Wherein, A is an electron microscope image of ZIF-8. B is an 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 the growth hormone release rate. F is a silver staining image after polyacrylamide gel electrophoresis. G is an image after subcutaneous injection of a targeted material. H is an image after subcutaneous injection of a non-targeted material. I is an image of the ovary after injection of a targeted or non-targeted material. J is the expression level of ZP3 on the surface of follicles of different levels.

[0029] Figure 3 This is a biocompatibility test for the materials of the present invention. A shows HE staining of major organs in different groups. B shows live / dead cell staining in different groups. C shows CCK-8 staining for 24 hours. D shows CCK-8 staining for 48 hours.

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

[0031] Figure 5 This figure demonstrates the effects of the material of the present invention. A represents mouse weight. B represents a gross image of the mouse ovary. C represents mouse ovary weight. D represents the mouse ovary index.

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

[0033] Figure 7 The HE section image of mouse ovary after the material of the present application is shown. Wherein, A is the HE staining image of normal group ovary. B is the HE staining image of CIS group ovary. C is the HE staining image of ZIF-8 group ovary. D is the HE staining image of GH group ovary. E is the HE staining image of ZIF-8-GH group ovary. F is the HE staining image of ZIF-8-GH@ZP3 group ovary. G is the column chart of the number of follicles at different levels in different groups.

[0034] Figure 8 The fertility of mice after the material of the present application is shown. Wherein, A is the pregnancy rate of mice. B is the number of pups. C is the number of pups in NC group. D is the number of pups in CIS group. E is the number of pups in ZIF-8 group. F is the number of pups in GH group. G is the number of pups in ZIF-8-GH group. H is the number of pups in ZIF-8-GH@ZP3 group.

[0035] Figure 9 The HE section image of mouse ovary after the material of the present application is shown.

[0036] Figure 10 The glycosidase staining image after the material of the present application is shown. Wherein, A is the glycosidase staining image. B is the proportion of senescent cells.

[0037] Figure 11 The fluorescence image of cells after the material of the present application is shown. Wherein, A is the fluorescence of Bax. B is the quantification of Bax fluorescence. C is the fluorescence of Bcl-2. D is the quantification of Bcl-2 fluorescence. E is the value of Bax / Bcl-2.

[0038] Figure 12 The fluorescence image of cells after the material of the present application is shown. Wherein, 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 description is illustrative only and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0040] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. It will be understood that the scope of the application is not limited to the specific specific embodiments described below; it being understood that the terms used in the present application description are intended to describe the specific embodiments and not to limit the protective scope of the present application.

[0041] As previously mentioned, although the prior art provides 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 disorder, thyroid dysfunction, and occurrence or recurrence of certain tumors.

[0043] 2. Follicular pool depletion: the current scheme promotes follicular growth while causing follicular pool depletion, thereby affecting the ovarian reserve function of patients with premature ovarian failure.

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

[0045] 1. Reduce side effects and complications: reduce the overall drug load by targeting the ovary, thereby reducing potential side effects and complications.

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

[0047] 3. Achieve long-lasting therapeutic effect: provide a sustained-release drug delivery system to prolong the therapeutic effect and slow down the POI process.

[0048] 4. Adopt a multi-mechanism treatment strategy: combine various 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 application, through its unique design and multiple mechanisms of action, aims to overcome the shortcomings of the prior art and provide a new method for treating primary ovarian insufficiency that is safer and more effective.

[0050] Specifically, in one exemplary embodiment of the present application, a drug carrier targeting the ovary is provided, which is named ZIF-8-GH@ZP3, the drug carrier at least comprises a zeolitic imidazolate framework (ZIF-8), and the ZIF-8 is loaded with growth hormone and zona pellucida antibody.

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

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

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

[0054] In another specific embodiment of the present application, a preparation method of the above-mentioned drug carrier targeting ovaries is provided, and the preparation method comprises:

[0055] S1, synthesizing ZIF-8 loaded with growth hormone; specifically, 2-methylimidazole is mixed with growth hormone, and then zinc nitrate hexahydrate is added;

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

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

[0058] In order to mix uniformly, stirring treatment is performed, and the stirring speed is 500-1000 rpm (preferably 800 rpm); and the addition of zinc nitrate hexahydrate can be performed in the form of multiple small additions (such as dropwise addition), so as to be more conducive to the synthesis of ZIF-8.

[0059] Further, ZIF-8 loaded with growth hormone is synthesized, and a buffer (PBS) is added for dilution for standby;

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

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

[0062] In another specific embodiment of the present application, a pharmaceutical composition is provided, and the active ingredient of the pharmaceutical composition comprises the above-mentioned drug carrier targeting ovaries.

[0063] In still another embodiment of the present application, the pharmaceutical composition can further comprise other pharmaceutically acceptable carriers, the dosage of which should be harmless to the subject, specifically including but not limited to buffers, antioxidants, antiseptics, bactericides, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, tonicity adjusting agents, sugars, surfactants, salt-forming counterions, metal complexes and / or non-ionic surfactants, etc., which are not specifically limited herein.

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

[0065] In still another embodiment of the present application, the use of the above-mentioned drug carrier or pharmaceutical composition targeting the ovary is provided in any one or more of the following:

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

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

[0068] (c) preparing a product for restoring endocrine function in the ovary;

[0069] (d) preparing a product for saving reduced fertility;

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

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

[0072] In still another embodiment of the present application, a method for preventing and / or treating primary ovarian insufficiency is provided, which comprises administering the above-mentioned drug carrier or pharmaceutical composition targeting the ovary to a subject.

[0073] The subject of the present application 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 application refers to the amount of active compounds or agents, including the compounds of the present application, which can cause a biological or medical response of a tissue system, animal or human that is being sought by a researcher, veterinarian, doctor or other medical personnel, which includes 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 expending creative effort also fall within the scope of protection of the present invention. The inventors list 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. For example, the target can be replaced by ZP1 or ZP2, and the carrier can be replaced by ZIF-1 or ZIF-14.

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

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

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

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

[0081] Example

[0082] 1. Experimental Methods

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

[0084] The raw materials were 2-methylimidazole (C5H8N2 Aladdin M104839-100g) and zinc nitrate hexahydrate (Zn(NO3)2·6H2O Aladdin Z111706-500g) for the synthesis of ZIF-8, 1.8 ml C5H8N2 (216 mg / ml) and 0.2 ml Zn(NO3)2·6H2O (100 mg / ml) solution were mixed, and the zinc nitrate hexahydrate was added dropwise into the 2-methylimidazole on a magnetic stirrer at a stirring speed of 800 rpm. For the synthesis of ZIF-8-GH, 1.8 ml 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 into the mixed system on a magnetic stirrer at a stirring speed of 800 rpm. PBS was diluted to 40 ml and then used. In order to synthesize ZIF-8-GH@ZP3, 1 ml of diluted ZIF-8-GH was taken, and 100 μl of ZP3 (Proteintech 21279-1-AP) antibody was added on a magnetic stirrer at a stirring speed of 800 rpm, and stirred for 24 h. The above operations were all completed on an ultra-clean bench.

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

[0086] The morphological structure was evaluated using a scanning electron microscope (SEM). The surface charge characteristics of the material were analyzed by a Zeta potential instrument, the release mode of growth hormone was verified by in vitro release experiment, and the concentration of released growth hormone was monitored by radioimmunoassay. By rapid silver staining, it was verified whether ZIF-8-GH@ZP3 successfully loaded GH and ZP3.

[0087] 1.3 Establishment of POI mouse model

[0088] Eight-week-old female C57BL / 6 mice were housed in an SPF-free environment for 7 days to avoid health problems. All mice were housed according to the experimental protocol approved by the Ethics Committee of the Model Animal Research Institute, Provincial Hospital Affiliated to Shandong First Medical University. Throughout the experiment, mice were maintained in an SPF environment with 12 hours of light and 12 hours of darkness per day and had free access to water and pelleted chow. We selected mice with a body weight of 19.5-20.5 g and regular estrous cycles for POI induced by cisplatin chemotherapy. Mice in the blank control group were intraperitoneally injected with 0.9% NaCl solution, while mice in the POI group were intraperitoneally injected with cisplatin (CDDP MCE HY-17394) at 2 mg / kg for 7 consecutive days. Estrous cycles were monitored. Following the intraperitoneal injection of cisplatin, the treatment groups received subcutaneous injections 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 day of the experiment. On the 14th day of the experiment, the eyeballs were removed for blood collection, and the ovaries were isolated and weighed.

[0089] 1.4 HE staining and follicle counting

[0090] The mouse ovaries, hearts, livers, spleens, lungs, and kidneys were fixed with 4% paraformaldehyde for 24 hours, dehydrated, and embedded in paraffin. The heart, liver, spleen, and one ovary, lung, and kidney of each group of rats were cut into 5-μm-thick serial sections. These sections were used to count follicles and determine whether the intervention had caused damage to the mouse organs. The sections were stained with hematoxylin-eosin solution (Servicebio G1076). The number of follicles of each type was counted under a light microscope, and every 10th section was analyzed. 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. Ovaries were embedded in 5 μm thick paraffin as described above, and sections were dewaxed by sequentially immersion in deparaffinization solution (servicebio, G1128), anhydrous ethanol, and ddH2O. Proteinase K was incubated at 37°C for 25 minutes. Reagent 1, Reagent 2, and buffer were then mixed at a ratio of 1:5:50 and incubated in a 37°C humidified chamber for 1 hour, followed by incubation in 3% H2O2 in the dark for 15 minutes. 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 minutes. Finally, DAB was used for color development, and hematoxylin was used for nuclei staining.

[0093] 1.6 Immunohistochemical staining

[0094] The expression of IGF-1 (1:200 HUABIO ER1911-10), VEGF (1:250), Bax (1:800), Bcl-2 (1:250), and cd31 (1:500) was detected by immunohistochemistry. Paraffin-embedded sections were deparaffmized 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 at room temperature for 30 min. Then the sections were incubated with the primary antibody in a humidified chamber at 4°C overnight. The secondary antibody was incubated at room temperature for 1 h, and the DAB color development reaction was performed under a microscope by controlling the color development time. Finally, the sections were stained with hematoxylin. Images were collected using an OLYMPUS BX63 microscope.

[0095] 1.7 Granulosa cell in vitro model construction and intervention

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

[0097] 1.8 β-galactosidase staining

[0098] KGN cells were detected using a senescence β-galactosidase staining kit. KGN cells were seeded into 6-well plates, and after 4 groups of treatment, KGN cells were fixed in 4% paraformaldehyde, washed with PBS 3 times, and then different staining solutions were added according to the manufacturer's instructions. After incubation at 37°C overnight, the cells were observed and photographed under a microscope.

[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 incubation and washing, cells were fixed with 4% paraformaldehyde for 30 minutes, treated with 0.5% Triton X-100 for 10 minutes, and blocked with 2% BSA at 37°C for 1 hour. After washing with PBS, cells were stained with antibodies against Bax (1:500), Bcl-2 (1:200), P21 (1:50), or IL-1-β (1:50) overnight at 4°C. Cells were incubated with a goat anti-rabbit IgG-H&L polyclonal secondary antibody (1:500) at room temperature in the dark for 1.5 hours. Cell nuclei were stained with DAPI. 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 assessed using a cell counting kit. KGN cells were seeded in 96-well plates at a density of 3,000 cells per well. After incubation for approximately 12 hours in DMEM, various concentrations of ZIF-8 (0, 2, 4, 6, 10, 20, 40, and 80 mg / ml) were added sequentially. KGN cells were cultured in this mixed medium for 24 and 48 hours. Subsequently, the KGN cells were incubated with 10% CCK-8 medium for 2 hours according to the manufacturer's instructions. The absorbance of the 96-well plates was measured at 450 nm using a microplate 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 synthesizing ZIF-8-GH, the ZP3 target was added, and the hydrodynamic diameter of ZIF-8-GH@ZP3 was approximately 377 nm ( Figure 2 D). Analyze the surface charge characteristics of the material using a Zeta potential analyzer, see ( Figure 2 C), growth hormone is negatively charged in a 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 a 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 indicated 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 cell compatibility evaluation

[0107] The cell compatibility of ZIF-8 to ovarian granulosa cells was first evaluated. Cells were co-cultured with different concentrations of ZIF-8 (0, 2, 4, 6, 10 mg / ml) and cell viability was quantified after 24 h, 48 h using CCK-8 assay ( Figure 3 C, D). At 24 h, the cell viability reached 98.76% ± 2.47% at 6 mg / mL concentration ( Figure 3 C), indicating good cell compatibility at this concentration. The viability of ovarian granulosa cells at 6 mg / mL concentration was further confirmed by live / dead cell staining experiment ( Figure 3 B). This indicated that ZIF-8 has excellent cell compatibility. Our study showed that ZIF-8 exhibited good cell compatibility within 6 mg / mL concentration.

[0108] 2.3 In vivo treatment effect on primary ovarian insufficiency

[0109] In this study, cisplatin was chosen 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), ZIF-8-GH@ZP3 (100 mg / kg) were injected subcutaneously daily for 7 consecutive days. The body weight of mice was measured at 0, 4, 7, 11, 14 d after cisplatin injection; the body weight of mice in each group decreased significantly at 4 d and 8 d 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 due to the slow release and targeting effect of ZIF8@ZP3, the effect of GH on weight gain is delayed ( Figure 5 A). After 7 consecutive days of intraperitoneal injection of cisplatin, the morphology of the ovary was observed under a microscope. Compared with the NC group, the ovary of the cisplatin group was obviously atrophic. The ovary of the CIS group was white in color and smooth in surface ( Figure 4 B). While the ovary of the NC group mouse was pink in color and the normal ovary surface was rough. This is because the normal group of mice has normal-looking follicles in the ovary ( Figure 4 A). The morphology of the ovary in different intervention groups also improved. ZIF-8-GH@ZP3 had obvious improvement ( Figure 4 C, D, E, F). The study also showed that ZIF-8 has a clear benefit in improving the morphology of the ovary. Next, the ovaries of mice were surgically removed and placed on a microscope slide ( Figure 5B). The ovary of the treatment group was significantly larger than that of the CIS group. Compared with the CIS group (3.27 ± 0.32) mg, the ovary mass of the ZIF-8 group (4.03 ± 0.71) mg, the GH group (5.53 ± 0.35) mg, the ZIF-8-GH group (6.27 ± 0.15) mg, and the ZIF-8-GH@ZP3 group (7.03 ± 0.15) mg was significantly increased (p < 0.05) Figure 4 C). We speculate that the addition of ZIF-8 and ZP3 makes the drug act locally on the ovary, and the ovary weight is significantly increased compared with the GH group. Compared with the GH group, the body weight of the ZIF-8-GH@ZP3 group mice (7.03 ± 0.15 mg) was significantly increased (p < 0.01). The ovary / body weight index was calculated by dividing the ovary weight by the body weight. The ovary / 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 restores the ovary weight and reduces the impact of GH on other parts of the mouse.

[0110] The ovary section was stained with hematoxylin-eosin (HE staining) Figure 7The NC group had larger ovaries and the largest number of follicles. The CIS group showed significant ovarian atrophy, with disordered cortical and medullary structures, reduced ovarian size, the 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. Follicles were classified and counted. The true ovarian reserve is reflected by the number of follicles. Follicles were classified using the following criteria: Primordial follicle: oocyte surrounded by a layer of flat granulosa cells; Primary follicle: oocyte surrounded by 1 to 6 layers of cuboidal granulosa cells; Secondary follicle: oocyte surrounded by at least 2 layers of cuboidal granulosa cells, with an antral cavity between the follicular cells; Mature follicle: ovarian follicle diameter significantly enlarged, protruding above the ovarian surface; Atretic follicle: zona pellucida and oocyte deformed. The number of primordial follicles, primary follicles, secondary follicles, and mature follicles was counted in serial ovarian sections. HE staining showed a significant decrease in the number of follicles in the CIS group. 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. Compared with the GH group, subcutaneous injection of ZIF-8-GH@ZP3 significantly restored the development of ovarian follicles. The number of mature follicles in the ZIF-8-GH@ZP3 group was significantly higher than that in the ZIF-8-GH group. 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. Therefore, 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 had better recovery of follicle development than the CIS group. This suggests that ZIF-8 is also effective in treating 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) treatment, changes in serum sex hormone levels in mice in different treatment groups showed a significant recovery effect. The ZIF-8-GH@ZP3 group was the most significant, especially the estrogen level, which was comparable to the NC group (p<0.05). It is worth noting that the use of ZIF-8 alone also had a certain restorative effect on ovarian hormone secretion. Compared with the CIS group, serum E2 levels increased (p<0.05) and serum FSH levels decreased (p<0.05). Serum FSH levels in the treatment groups were significantly higher than those in the CIS group, and the differences were statistically significant, with the ZIF-8-GH@ZP3 group (0.74±0.06pg / ml) being the most significant. Compared with the CIS group, serum E2 levels were significantly increased in the ZIF-8 (8.60±0.26 pg / ml), GH (9.36±0.28 pg / ml), ZIF-8-GH (10.48±0.51 pg / ml), and ZIF-8-GH@ZP3 (12.28±1.08 pg / ml) groups, with the ZIF-8-GH@ZP3 group showing the most significant increase (p<0.01). Compared with the CIS group, serum LH levels (4.34±0.21 mIU / ml) were significantly decreased in the ZIF-8-GH@ZP3 group (p<0.001). Compared with the GH group, serum FSH levels were significantly decreased in the ZIF-8-GH@ZP3 group (p<0.0001). As shown below, P levels were significantly higher in the ZIF-8-GH@ZP3 group (9.49 ± 0.26) than in the CIS group (4.00 ± 1.51 ng / ml) (P < 0.001). ZIF-8-GH@ZP3 treatment significantly restored serum P levels compared to the GH group (7.12 ± 0.63 ng / ml) (P < 0.01). In addition to the increased P levels, serum E2 levels were statistically significantly different between the two groups. In summary, subcutaneous injection of ZIF-8-GH@ZP3 significantly restored ovarian function compared to the GH group. ZIF-8 treatment significantly restored serum estrogen levels (P < 0.05) and reduced serum FSH levels (1.53 ± 0.08 mIU / ml) compared to the CIS group, with statistically significant differences (P < 0.05). These data demonstrate that both ZIF8-GH@ZP3 and ZIF-8 can restore ovarian endocrine function in POI mice.

[0111] Improvement of reproductive capacity of POI mice. We statistically analyzed the reproductive 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 birth was recorded. On day 28, 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 of new blood vessels and can promote angiogenesis. CD31 is a marker of 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 ( Figure 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 ( Figure 9 B). The anti-apoptotic protein Bcl-2 can inhibit the intrinsic apoptotic pathway. The pro-apoptotic protein Bax can initiate cell death. Therefore, we used Bcl-2 and Bax immunohistochemistry to detect apoptosis in 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 ( Figure 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 ( Figure 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 the other glycoproteins. In addition, we found through immunofluorescence analysis that the expression of ZP3 gradually increases with the maturation of follicles ( Figure 2 J) Theoretical feasibility of targeting preferential ovarian follicles. ZIF-8-GH, conjugated to the ZP3 antibody branch, enhances the interaction between the drug delivery system and the ovary. ZIF-8-GH actively targets the ovary, increasing GH accumulation there and improving drug delivery efficiency. ZIF-8-GH was first labeled with Cy5 and then loaded with ZP3. ZIF-8-GH(Cy5)@ZP3 was then injected intraperitoneally. Twelve hours later, mice were sacrificed and their organs dissected. To enhance fluorescence intensity, the ovaries, heart, liver, spleen, lungs, and kidneys of each group were isolated and placed on culture dishes. In vivo targeting of the ZIF-8-GH@ZP3 group was monitored using an in vivo imaging system (IVIS). The fluorescence intensity generated by Cy5 can be used as an indicator of ovarian GH content. Ovarian fluorescence intensity in the ZIF-8-GH(Cy5)@ZP3 group was significantly higher than in the non-targeted group, confirming that ZP3-mediated ovarian targeting and drug delivery enhance the efficacy of GH therapy. Therefore, ZIF-8-GH(Cy5)@ZP3 can accumulate more GH in the ovary, thus providing better treatment ( Figure 2 G, H, I).

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

[0116] The KGN cells treated with the above four groups were stained for β-galactosidase activity, and three independent experiments were performed for each group. When the cisplatin concentration was 1.5 mg / ml, the expression of senescence-related β-galactosidase in KGN cells was significantly increased (37.02±3.66). After GH treatment, the senescence of KGN cells was significantly reduced (14.37±0.40) ( Figure 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). ( Figure 11In 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 and GH groups was significantly lower than that in the CIS group ( Figure 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 and GH groups was significantly reduced ( Figure 12 C), indicating that both ZIF-8 and GH treatment inhibited inflammation and senescence in 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 thus 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 alleviating 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 treatment.

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

[0124] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A drug carrier targeting ovary, characterized in that: The drug carrier at least comprises a zeolite imidazole framework ZIF-8, and the ZIF-8 is loaded with growth hormone and zona pellucida antibody; The zona pellucida antibody is specifically the zona pellucida 3 antibody; The volume-to-mass ratio of the zona pellucida antibody to growth hormone is 2-6 μl:3 μg.

2. The ovary-targeting drug carrier according to claim 1, characterized in that The growth hormone is specifically human recombinant growth hormone.

3. The ovary-targeted drug carrier according to claim 1, wherein The drug carrier is a nanogel preparation.

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

5. The preparation method according to claim 4, wherein In step S1, the concentration ratio of 2-methylimidazole, growth hormone and zinc nitrate hexahydrate is 200-300:1-5:50-150, and 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, wherein Synthesize growth hormone-loaded ZIF-8 and dilute it with PBS buffer for later use.

7. The preparation method according to claim 4, wherein In step S2, stirring is performed during the process of adding the zona pellucida antibody, and the stirring speed is 500-1000 rpm.

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 ovary-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) preparation of products for reversing reduced fertility; (e) Preparation of products for the prevention and / or treatment of primary ovarian insufficiency.

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