Ovarian targeting peptide for specifically targeting ovarian cells as well as related products and application of ovarian targeting peptide

Abstract

The invention discloses an ovarian targeting peptide specifically targeting ovarian cells and a related product and application thereof, the ovarian targeting peptide is LH-Lamp2b fusion protein or FSH-Lamp2b fusion protein, in addition, the invention further provides an exosome containing the ovarian targeting peptide and specifically targeting the ovarian cells, and the ovarian targeting peptide is an exosome containing the ovarian targeting peptide and specifically targeting the ovarian cells. The ovarian targeting peptide can significantly improve the distribution of exosomes in ovarian tissues, and lays a foundation for the technical field of premature ovarian failure treatment.

Description

[0001] This application is a divisional application of the patent with application date of October 23, 2024, application number 202411482207.7, and invention name “An exosome drug for treating premature ovarian failure, its preparation method and application”. Technical Field

[0002] The present invention belongs to the field of biomedical technology, and in particular relates to an ovarian targeting peptide that specifically targets ovarian cells and related products and uses thereof. Background Art

[0003] Premature ovarian failure (POF), also known as ovarian insufficiency (POI) or premature menopause, is characterized by loss of ovarian function, manifested by cessation of ovarian function and premature follicular depletion before the age of 40. Causes of POF include genetic factors, autoimmune diseases, and unhealthy lifestyle habits. These factors collectively lead to a decrease in the ovarian follicle pool and follicular dysfunction. The incidence of familial POF has been reported to range from 4% to 31% across different populations, with X chromosome abnormalities being the primary cause. Autoimmune diseases are associated with POF, with studies showing that approximately 9% to 40% of POF patients have coexisting autoimmune diseases such as systemic lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis, autoimmune thyroiditis, hypoparathyroidism, and idiopathic thrombocytopenic purpura. The incidence of POF is approximately 1‰ in women under 30 years of age and 1-3% in women under 40 years of age. The incidence is generally higher in economically developed regions and increases with social development. POF causes infertility in women of childbearing age, resulting in significant social and family problems. In addition, decreased estrogen levels cause a series of low estrogen symptoms, such as low libido, hot flashes, sweating, osteoporosis, and urinary system diseases, which reduce the quality of life of patients.

[0004] Exosomes are a type of extracellular vesicle produced by living cells that mediate intercellular communication and the excretion of cellular metabolic products. Numerous studies on naturally occurring exosomes have demonstrated physiological functions such as immunomodulation, inflammation suppression, and tissue repair. These functions may be related to their contents or membrane composition. Exosomes from different cell types exhibit distinct surface features, conferring specific cell-specific tropisms. Exosome contents primarily include proteins, microRNAs, and mRNAs. Recent studies, including transcriptomics, have revealed that the types and amounts of microRNAs in exosomes produced by different cell types or physiological states vary, resulting in distinct regulatory properties for target protein expression. Exosomes, as lipid bilayer-encapsulated vesicles produced by cells, express only a small number of major histocompatibility complex molecules on their surface, along with anti-inflammatory proteins such as CD73. Their excellent tissue compatibility and low immunogenicity have led to their extensive research and development as delivery vehicles for small molecule drugs and gene therapies. At present, the development directions of exosome drug delivery systems mainly include engineering modification to increase the tropism of exosomes for specific tissues and further reduce immunogenicity, engineering modification to increase the intracellular loading of exosomes for target nucleic acids, and in vitro loading of exosomes for target small molecules or nucleic acid drugs through process development.

[0005] Lipid nanoparticles (LNPs) are composed of four different components: cationic lipids, phospholipids, polyethylene glycol (PEG)-lipids, and cholesterol. Various drugs, including small molecules, peptides, and nucleic acids, can be loaded into their cavities through high-flow microfluidics. The nucleic acid-containing phase is the aqueous phase, and the lipid mixture is the organic phase. Each lipid component and its ratio play an important role in LNP encapsulation. Negatively charged nucleic acids and positively charged lipids are electrostatically bound and assembled into LNPs through hydrophobic interactions and van der Waals interactions between the lipid components. LNPs can protect loaded drugs from destructive proteases and assist in the intracellular delivery of mRNA, extending the drug's retention time in the body. In addition, LNPs can support repeated and transient administration, making up for the shortcomings of current gene delivery drug carriers.

[0006] Granulosa cells are crucial cells in the ovarian follicle, providing protection, nourishment, and support. They play a crucial role in follicular development, oocyte maturation, and ovulation through oocyte nutrition, mechanical protection, and the secretion of hormones such as E2 and AMH. POF patients often experience symptoms such as a decrease in follicle number, a decrease in secondary and mature follicles, an increase in atretic follicles, and inability to ovulate. Histological examinations have shown a decrease in granulosa cell number, increased senescence and apoptosis, and decreased secretory capacity in tissues from POF patients and animal models. Therefore, restoring granulosa cell activity and function is an important approach to treating POF.

[0007] Currently, there is no effective treatment for POF, particularly for improving egg quality and restoring fertility. Conventional hormone therapy can only temporarily improve symptoms associated with low hormone levels. Estrogen-progesterone replacement therapy (HRT) is primarily targeted at young women with POF. HRT can alleviate symptoms of low estrogen levels and urogenital atrophy, and prevent long-term complications (osteoporosis, Alzheimer's disease, etc.). However, long-term HRT can lead to the development of endometrial and breast cancer. Progestins are divided into natural and synthetic progestins. Micronized progesterone can be taken orally, but often requires large doses and is expensive. Ovulation induction therapy requires large doses of hMG and long duration, and is less effective for patients with long-term and severe POF. Dehydroepiandrosterone (DHEA) is a key substance in the synthesis of testosterone and E2. DHEA can reduce miscarriage rates to a certain extent in infertile women of older reproductive age with normal ovarian reserve. In addition to the above conventional therapies, immunosuppressive therapy has a certain effect on POF related to autoimmune diseases. There is currently no clear medication standard, and immunosuppression can cause serious side effects, so it is limited in the treatment of POF related to autoimmune diseases. In studies using oral nicotinamide mononucleotide (NMN) to treat rodent models of POF, long-term high-dose oral intake is required. Although good results have been achieved in rodent models, the efficacy of NMN as a supplement in the human population is currently widely questioned. In fact, even oral high-dose NMN still has no significant effect on anti-aging. The reason may be that the efficiency of NMN conversion to nicotinamide adenine dinucleotide (NAD) in the human body is too low. Direct administration of NAD has the problem that NAD has a short half-life in the body and is difficult to enrich in the target tissue. Summary of the Invention

[0008] In view of this, in order to overcome the technical problems existing in the above-mentioned prior art drugs for treating POF, the object of the present invention is to provide an ovarian targeting peptide that specifically targets ovarian cells and related products and uses thereof.

[0009] The preparation process of the above-mentioned drug for treating POF provided by the present invention involves the acquisition of exosomes, the preparation of LNP-mRNA, the mixing of NAD or LNP-mRNA with exosomes and electroporation treatment. The present invention also involves using NAD as a target for treating POF. Compared with NMN, NAD has the advantage of directly exerting its effect without relying on enzymatic reaction conversion in the body. In ovarian tissue, it improves the physiological functions of eggs, corpus luteum, ovarian stromal cells, and ovarian macrophages by promoting sirt2-dependent spindle-kinetochore assembly, PARP-dependent DNA repair, aerobic respiration and other mechanisms, thereby improving ovarian dysfunction such as premature ovarian failure in the long term. In terms of drugability, the drug preparation method mentioned in this method can be used to produce different dosage forms such as intravenous infusion preparations, in situ injection preparations, and oral preparations, and has good clinical application prospects.

[0010] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0011] A first aspect of the present invention provides a nucleic acid composition for treating premature ovarian failure.

[0012] Furthermore, the nucleic acid composition includes mRNA, DNA or recombinant protein encoding NAMPT, NMNAT3 and COX15.

[0013] In the present invention, the information of the genes NAMPT, NMNAT3, and COX15 is as follows: the Gene ID of the gene NAMPT (nicotinamide phosphoribosyltransferase [Homo sapiens (human)]) is 10135, the Gene ID of the gene NMNAT3 (nicotinamide nucleotide adenylyltransferase 3 [homo sapiens (human)]) is 349565, and the Gene ID of the gene COX15 (cytochrome c oxidase assembly homologCOX15 [Homo sapiens (human)]) is 1355. Detailed information of the genes can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0014] In a specific embodiment of the present invention, the nucleic acid composition is a nucleic acid composition comprising mRNA encoding NAMPT, NMNAT3 and COX15.

[0015] A second aspect of the present invention provides an ovary-targeting peptide.

[0016] Furthermore, the ovary targeting peptide includes LH-Lamp2b fusion protein or FSH-Lamp2b fusion protein;

[0017] The amino acid sequence of the LH-Lamp2b fusion protein is shown in SEQ ID NO: 2;

[0018] The amino acid sequence of the FSH-Lamp2b fusion protein is shown in SEQ ID NO: 4.

[0019] Furthermore, the nucleotide sequence of the LH-Lamp2b fusion protein is shown in SEQ ID NO: 1;

[0020] The nucleotide sequence of the FSH-Lamp2b fusion protein is shown in SEQ ID NO: 3.

[0021] In the present invention, LH refers to luteinizing hormone, a glycoprotein gonadotropin secreted by pituitary cells that promotes the conversion of cholesterol into sex hormones within gonadal cells. In women, it works together with follicle-stimulating hormone (FSH) to promote follicular maturation, estrogen secretion, ovulation, and the formation and maintenance of the corpus luteum, secreting progesterone and estrogen. In men, luteinizing hormone promotes the synthesis and release of testosterone by Leydig cells.

[0022] In the present invention, FSH refers to follicle-stimulating hormone (FSH). FSH is a glycoprotein secreted by basophils in the anterior pituitary gland and primarily functions to promote follicle maturation. FSH promotes the proliferation and differentiation of granulosa cells and the growth of the ovaries. Its action on the seminiferous tubules of the testes promotes spermatogenesis. FSH is secreted in pulses in the human body.

[0023] In the present invention, Lamp2b is one of the key components of the targeted delivery device for exosomes (exosomal membrane protein). Lamp2b is an exosome surface protein commonly used to display targeting motifs. Lamp2b is a member of the lysosomal-associated membrane protein (LAMP) family. It is primarily localized to lysosomes and endosomes within cells and is also abundantly expressed on the surface of exosomes. Target molecules are typically fused to Lamp2b and displayed on the exosome membrane surface via Lamp2b.

[0024] A third aspect of the present invention provides exosomes that specifically target ovarian cells.

[0025] Furthermore, the exosomes contain the ovary-targeting peptide described in the second aspect of the present invention.

[0026] Furthermore, the exosomes are derived from mesenchymal stem cells;

[0027] Optionally, the exosomes contain the FSH-Lamp2b fusion protein described in the second aspect of the present invention.

[0028] In a specific embodiment of the present invention, the exosomes are exosomes comprising the FSH-Lamp2b fusion protein described in the second aspect of the present invention.

[0029] Furthermore, the present invention, through comparative experiments, has demonstrated for the first time that the FSH ovary-targeting peptide structure is significantly superior to the LH ovary-targeting peptide structure. Specifically, the FSH ovary-targeting peptide structure significantly outperforms the LH ovary-targeting peptide structure in improving the delivery efficiency of mesenchymal stem cell-derived exosomes for LNP-encapsulated mRNA in ovarian cells and in improving the ovarian distribution of mesenchymal stem cell-derived exosomes. This technical effect is unexpected by those skilled in the art based on the prior art.

[0030] A fourth aspect of the present invention provides any of the following products:

[0031] (1) An exosome drug for treating premature ovarian failure, wherein the exosome drug for treating premature ovarian failure is obtained by mixing LNP or TNP encapsulating the nucleic acid composition of the first aspect of the present invention and the exosomes of the third aspect of the present invention;

[0032] (2) An exosome drug encapsulating NAD, wherein the exosome drug encapsulating NAD is an exosome drug obtained by encapsulating NAD drug in exosomes;

[0033] (3) A pharmaceutical composition comprising the exosome drug for treating premature ovarian failure and / or the exosome drug encapsulating NAD;

[0034] (4) A pharmaceutical preparation comprising the exosome drug for treating premature ovarian failure and / or the exosome drug encapsulating NAD.

[0035] Furthermore, the concentration of the nucleic acid composition in the LNP or TNP encapsulating the nucleic acid composition according to the first aspect of the present invention is 10 ng / μL;

[0036] Optionally, the ratio of the mRNAs of NAMPT, NMNAT3 and COX15 in the nucleic acid composition in the LNP or TNP encapsulating the nucleic acid composition according to the first aspect of the present invention is 1:1:1;

[0037] Optionally, the final concentration of the exosome drug for treating premature ovarian failure comprising LNP encapsulating the nucleic acid composition of the first aspect of the present invention is: the exosome NTA of the third aspect of the present invention is 4×10 10 / mL, the LNP encapsulating the nucleic acid composition according to the first aspect of the present invention is 25 μg / mL;

[0038] Optionally, the final concentration of the exosome drug for treating premature ovarian failure comprising TNP encapsulating the nucleic acid composition of the first aspect of the present invention is: the exosome NTA of the third aspect of the present invention is 5×10 10 / mL, the TNP encapsulating the nucleic acid composition according to the first aspect of the present invention is 75 μg / mL;

[0039] Optionally, the exosomes in the NAD-encapsulated exosome drug are 4×10 10 / mL, NAD is 600μg / mL;

[0040] Optionally, the exosomes in the NAD-encapsulated exosome drug are derived from mesenchymal stem cells;

[0041] Optionally, the dosage form of the pharmaceutical preparation includes intravenous infusion preparation, in situ injection preparation, oral preparation, tablet, capsule preparation, granule preparation, sustained-release preparation, powder, and pill.

[0042] In the present invention, NAD (or NAD+) is a biological substance that transfers electrons. Its Chinese name is Nicotinamide Adenine Dinucleotide (NAD), and it is an oxidized coenzyme I. It is a coenzyme in many dehydrogenases in the body, such as alcohol dehydrogenase (ADH), which is used to oxidize ethanol. It plays an irreplaceable role in glycolysis, gluconeogenesis, the tricarboxylic acid cycle, and the respiratory chain. Its function is to connect the tricarboxylic acid cycle and the respiratory chain, transferring hydrogen released during metabolism to flavoproteins. Its reduced forms are NADH and H+.

[0043] In some embodiments, the present invention has no particular limitations on the dosage form of the pharmaceutical preparation, and the dosage forms of the pharmaceutical preparation include, but are not limited to, intravenous infusion preparations, in situ injection preparations, oral preparations, tablets, capsules, granules, sustained-release preparations, powders, and pills. The pharmaceutical preparations can be prepared using methods known to those skilled in the art.

[0044] In some embodiments, the pharmaceutical composition or pharmaceutical formulation further comprises a pharmaceutically acceptable carrier and / or excipient.

[0045] In some embodiments, suitable pharmaceutically acceptable carriers and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995). These substances are used to help the stability of the formulation or to help improve the activity or its biological effectiveness or to produce an acceptable taste or smell when taken orally, as needed. The pharmaceutical composition or pharmaceutical preparation thus formulated can be administered in any appropriate manner known to those skilled in the art as needed. When using the pharmaceutical composition or pharmaceutical preparation provided by the present invention, a safe and effective amount of the pharmaceutical composition or pharmaceutical preparation of the present invention is administered to a human.

[0046] In some embodiments, the present invention has no particular limitations on the pharmaceutically acceptable carriers and / or excipients. Any pharmaceutically acceptable carriers and / or excipients suitable for combination with the active ingredients provided by the present invention (such as the exosome drug for treating premature ovarian failure and the exosome drug encapsulating NAD as described above) to obtain the pharmaceutical composition or pharmaceutical preparation of the present invention are within the scope of protection of the present invention.

[0047] In some embodiments, specific illustrative examples of the pharmaceutically acceptable carriers and / or excipients include, but are not limited to: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc.

[0048] In some embodiments, the appropriate dosage of the pharmaceutical composition or pharmaceutical preparation described in the present invention can be prescribed in a variety of ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. A skilled physician can usually easily determine the prescription and the dosage that is effective for the desired treatment.

[0049] A fifth aspect of the present invention provides any of the following methods:

[0050] (1) A method for preparing the exosome drug for treating premature ovarian failure described in the fourth aspect of the present invention, the preparation method comprising the following steps: mixing LNP or TNP encapsulating the nucleic acid composition described in the first aspect of the present invention and the exosomes described in the third aspect of the present invention to obtain the exosome drug;

[0051] (2) An NAD-encapsulated exosome drug as described in the fourth aspect of the present invention, wherein the NAD-encapsulated exosome drug is an exosome drug obtained by encapsulating the NAD drug in exosomes.

[0052] Furthermore, the LNP or TNP encapsulating the nucleic acid composition of the first aspect of the present invention is prepared by the following method: mixing NAMPT, NMNAT3 and COX15 mRNA to obtain a mixed solution NNC mRNA, loading the NNC mRNA into LNP or TNP to obtain LNP or TNP encapsulating the nucleic acid composition of the first aspect of the present invention;

[0053] Optionally, the final concentration of the mixed solution is 10 ng / μL;

[0054] Optionally, the ratio of NAMPT, NMNAT3 and COX15 mRNA in the mixed solution is 1:1:1;

[0055] Alternatively, the NNC mRNA is loaded into LNPs or TNPs using a high-flow microfluidics method;

[0056] Optionally, in the high-flow microfluidic method, the flow rate ratio of the lipid phase to the aqueous phase is 1:3, and the total flow rate is 20 mL / min;

[0057] Optionally, the exosomes described in the third aspect of the present invention are prepared by the following method: constructing an iMSC-FSH / iMSC-LH cell line by lentiviral infection, collecting and purifying the exosomes secreted by the iMSC-FSH / iMSC-LH cells, thereby obtaining the exosomes described in the third aspect of the present invention;

[0058] Optionally, the NAD-encapsulated exosome drug is an exosome drug obtained by encapsulating the NAD drug in exosomes using an electroporation method;

[0059] Optionally, the exosomes in the NAD-encapsulated exosome drug are 4×10 10 / mL, NAD is 600μg / mL;

[0060] Optionally, the exosomes in the NAD-encapsulated exosome drug are derived from mesenchymal stem cells.

[0061] A sixth aspect of the present invention provides any of the following applications:

[0062] (1) Use of the nucleic acid composition of the first aspect of the present invention, the ovary-targeting peptide of the second aspect of the present invention, and / or the exosomes of the third aspect of the present invention in the preparation of an exosome drug for treating premature ovarian failure;

[0063] (2) Use of the exosomes described in the third aspect of the present invention in targeted delivery of nucleic acid molecules;

[0064] (3) Use of the exosome drug for treating premature ovarian failure and / or the NAD-encapsulated exosome drug described in the fourth aspect of the present invention in the preparation of a pharmaceutical composition or pharmaceutical preparation for treating premature ovarian failure.

[0065] In addition, the present invention also provides a method for treating premature ovarian failure, comprising the steps of administering to a subject in need thereof a therapeutically effective amount of the exosome drug for treating premature ovarian failure, the NAD-encapsulated exosome drug, the pharmaceutical composition and / or the pharmaceutical preparation described in the fourth aspect of the present invention.

[0066] In some embodiments, the subject of the present invention refers to any animal, including humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and any livestock or pets; as well as non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0067] In some embodiments, the treatments described herein generally relate to treatment of humans or animals (e.g., as used by veterinarians) where some desired therapeutic effect is achieved, such as inhibiting the progression of a condition (including slowing or halting progression), ameliorating a condition, and curing a condition. Treatment as a preventative measure (e.g., prophylaxis) is also included. Use in patients who have not yet developed a condition but are at risk of developing the condition is also included within the term treatment as used herein.

[0068] In some embodiments, the effective amount of the present invention refers to an amount having a therapeutic effect or the amount required to produce a therapeutic effect in a subject. For example, a therapeutically or pharmaceutically effective amount of a drug refers to the amount of the drug required to produce the desired therapeutic effect, and the therapeutic effect can be reflected by the results of clinical trials, model animal studies and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to: characteristic factors of the subject (such as height, weight, sex, age and medication history), the severity of the disease, etc.

[0069] In some embodiments, the exosome drugs, NAD-encapsulated exosome drugs, pharmaceutical compositions, and / or pharmaceutical preparations for treating premature ovarian failure described herein may be administered, but are not limited to, intramuscularly, subcutaneously, intradermally, orally, topically, intraperitoneally, intravenously, intranasally, intrapulmonaryly, and intrarectally. When administered orally, a coating may be formulated to protect the active ingredient in the pharmaceutical composition or pharmaceutical preparation from degradation in the stomach. Furthermore, the active ingredient may be administered via any device capable of delivering the active ingredient to the target tissue. In specific embodiments, the pharmaceutical compositions or pharmaceutical preparations provided herein may be formulated into various dosage forms according to actual needs, and a clinician may determine the dosage that is beneficial to the patient based on factors such as the subject's type, age, weight, general disease condition, and route of administration. Administration may be, for example, by injection or any other suitable route known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 The cisplatin levels in the blood, brain and kidney tissues of mice in each group were detected by HPLC-MS at 1 hour and 24 hours after administration.

[0071] Figure 2This is the imaging result of MSC-derived exosomes in various organs of mice after tail vein injection;

[0072] Figure 3 This is a statistical graph showing the changes in the body weight of mice during and after drug administration;

[0073] Figure 4 This is a statistical graph showing changes in food intake of mice during and after drug administration;

[0074] Figure 5 This is a statistical diagram of the changes in body temperature of mice during and after drug administration;

[0075] Figure 6 This is the result chart corresponding to the organ coefficient of ovarian test after 21 days of drug administration;

[0076] Figure 7 This is the result graph showing that the FSH and LH ovarian-targeted peptide structures can improve the ovarian distribution of MSC-derived exosomes, and FSH is superior to LH;

[0077] Figure 8 This is a graph showing that the FSH and LH ovarian targeting peptide structures can improve the delivery efficiency of MSC-derived exosomes to LNP-encapsulated mRNA in ovarian cells, and the FSH ovarian targeting peptide structure is superior to the LH ovarian targeting peptide structure;

[0078] Figure 9 This is the result diagram of the survival status of mice in each group after administration of cisplatin;

[0079] Figure 10 This is the result diagram of the effect of each treatment group on the estrous cycle of mice;

[0080] Figure 11 The results of each treatment group on the volume of mouse ovarian follicles, the number of granulosa cells, the apoptosis of granulosa cells, and the number of atretic follicles;

[0081] Figure 12 The graph shows the results corresponding to the therapeutic effect of exosome-delivered TNP-NNC on mice with premature ovarian failure. DETAILED DESCRIPTION

[0082] The present invention will be further described below with reference to specific embodiments. The specific embodiments are intended only to explain the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and intent of the present invention. The scope of the present invention is defined by the claims and their equivalents.

[0083] The reagents and raw materials used in the present invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions in the present invention are generally performed under conventional conditions or as recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and should not limit the scope of the present invention in any way. Example 1: Exosomes can increase the half-life of small molecule compounds in vivo, thereby increasing long-term drug concentrations.

[0084] 1. Experimental Materials

[0085] The main experimental materials are shown in Table 1 below.

[0086] Table 1 Main experimental materials

[0087] name factory Item No. Normal saline Shandong Qidu Pharmaceutical Co., Ltd. 2A21102602 75% alcohol disinfectant Shandong Lierkang Medical Technology Co., Ltd. 20221127A Eye scissors Shanghai Medical Equipment (Group) Co., Ltd. Surgical Instrument Factory 201102 ophthalmic forceps Shanghai Medical Equipment (Group) Co., Ltd. Surgical Instrument Factory 210602 syringe Wuxi Yushou Medical Equipment Co., Ltd. 2208133

[0088] 2. Experimental methods

[0089] SPF grade C57BL / 6J male mice, 6-8 weeks old, were used. The supplier is Beijing Weitong Lihua Co., Ltd., and the production license number is SCXK (Su) 20211-0006. The animals were housed in intelligent independently ventilated animal cages (IVC) with cage specifications of 362mm×154mm×135mm, with no more than 5 animals per box. Artificial lighting, light: dark = 12h:12h. The animals were allowed to eat and move freely during the entire breeding process. SPF rat growth and breeding feed, produced by Beijing Keao Xieli Feed Co., Ltd. The batch number is 200663313, the production license number is SCXK (Beijing) 2019-0003, and the quality certificate number is 1112622000023997. Full feeding is carried out 1-2 times a week. The drinking water bottle (Watsons) is replaced 1-2 times a week. Use wood shavings litter from the Chenfu Eden Litter Processing Plant in Dachang Hui Autonomous County, batch number 20200622, production license number SCXK(Hebei)2017-001. Change the litter weekly and immediately if any abnormalities such as leaks or contamination occur. Mark animals with ear numbers, and display the ear numbers on cage cards. Cage Labeling: Before grouping, label the cages containing the animals using temporary cage cards. After grouping, indicate the study number, cage number, dose, animal group, breed, sex, animal number, study leader, and the start and end dates of the experiment on color-coded cage cards.

[0090] Exosome (EXO)-cisplatin: Prepare 1 mg / mL cisplatin solution with normal saline, take 0.5 mL of cisplatin solution and 0.5 mL of NTA to form 9×10 9 / mL exosomes were slowly and thoroughly mixed to prepare a cisplatin concentration of 0.5 mg / mL and an exosome NTA of 4.5×10 9The mixture was transferred to a Celtrix electroporation cuvette for electroporation. The electroporation conditions were 1 mL, -4 program.

[0091] The groups and dosing conditions are shown in Table 2. Blood, brain, and kidney tissues of the mice were collected 1 hour and 24 hours after administration, and the cisplatin content in the above tissues was determined by HPLC-MS using Zhonglian (Beijing) Pharmaceutical Technology Co., Ltd.

[0092] Table 2 Grouping and medication

[0093]

[0094] Note: Before sampling, normal saline needs to be perfused to remove blood from the brain parenchyma to prevent cisplatin in the blood from interfering with the test results.

[0095] 3. Experimental results

[0096] The results are as follows Figure 1 As shown in the results, cisplatin was detected in the blood 1 hour and 24 hours after tail vein injection of cisplatin and exosome-cisplatin, and the cisplatin content in the exosome-cisplatin group was higher than that in the cisplatin group. In the brain tissue, cisplatin was detected only 1 hour after tail vein injection of exosome-cisplatin, while no cisplatin was detected in the cisplatin alone group. In the kidney, cisplatin was detected 1 hour and 24 hours after tail vein injection of cisplatin and exosome-cisplatin, and the cisplatin content in the cisplatin + EXO group was higher than that in the cisplatin group. These results indicate that exosome-encapsulated cisplatin can allow cisplatin to cross the blood-brain barrier, increase cisplatin concentrations in the blood and tissues, and prolong its half-life.

[0097] Example 2: Exosomes derived from mesenchymal stem cells (MSCs) have ovarian enrichment characteristics

[0098] 1. Experimental Materials

[0099] The main experimental materials are shown in Table 3 below.

[0100] Table 3 Main experimental materials

[0101]

[0102] 2. Experimental methods

[0103] SPF-grade Balb / c nude female mice, 6-8 weeks old, were obtained from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a production license number of SCXK(Beijing)2021-0006. The animals were acclimated for 5 days and then injected with DIR-stained MSC-derived exosomes (Chengnuo Medical) via the tail vein at a dose of 6×10 10vg / animal. 48 hours after tail vein administration, the animals were dissected and the heart, liver, spleen, lung, kidney and ovary were taken for organ imaging. All data were obtained by After Lumina III ROI analysis, the data were imported into Excel worksheets for organization and visualized using GraphPad Prism 8.0.

[0104] 3. Experimental results

[0105] The results are as follows Figure 2 As shown in the results, after tail vein injection, MSC-derived exosomes were mainly distributed in the liver, with some distribution in the lungs, spleen, and ovaries, but no distribution in the heart or kidneys. These results indicate that MSC-derived exosomes have the characteristic of ovarian enrichment.

[0106] Example 3 Therapeutic Effect of NAD-Encapsulated Exosomes on Animal Models of Premature Ovarian Failure

[0107] 1. Experimental Materials

[0108] The main experimental materials are shown in Table 4 below.

[0109] Table 4 Main experimental materials

[0110]

[0111]

[0112] 2. Experimental methods

[0113] The test drug of the present invention is an exosome encapsulated with NAD. The specific preparation method is: the required NAD drug is encapsulated into the exosome by electroporation. The prepared exosome-NAD is used for animal and clinical experiments. The final concentration of the drug used is: exosome NTA (the concentration of exosomes is detected by nanoparticle tracking analysis technology) is 4×10 10 / mL, NAD was 600μg / mL. The exosomes used were derived from mesenchymal stem cells (MSC).

[0114] The specific drug preparation method is as follows: (1) Dissolve the NAD drug in DPBS to a 10 mg / mL stock solution. (2) Take the required NAD stock solution and slowly add it to the exosomes. After thorough mixing, incubate the mixture at room temperature for 15 minutes. (3) Electroporate the exosome-NAD mixture using a cellcyte electroporator. The optimal electroporation program for a 1mL system electroporation cuvette is "-4"; the optimal electroporation program for a 10mL system electroporation cuvette is "-6". (4) Let the electroporated exosome-NAD mixture stand on ice for 30 minutes to complete the preparation.

[0115] POF animal model establishment: 8-week-old C57B / L6 female mice were intraperitoneally injected with cisplatin (2 mg / kg / day) for 7 consecutive days. The experimental animals were divided into five groups, with five mice in each group. Group 1 served as the healthy control group, and Groups 2-5 served as cisplatin-induced POF models.

[0116] The test drug was administered once every three days for a total of three times. Group 1 (the healthy control group) and Group 2 (the vehicle group) received normal saline; Group 3 received exosomes without NAD encapsulation, Group 4 received NAD alone, and Group 5 received exosomes-NAD. Administration was via tail vein injection. Key physiological parameters, such as body weight, diet, and body temperature, were recorded during and after dosing to assess safety. Ovaries were harvested 21 days after dosing to measure organ function.

[0117] 3. Experimental results

[0118] The results are as follows Figure 3-6 As shown in the results, cisplatin modeling caused the ovarian organ coefficient to decrease by about 50%. The use of exosome-NAD via tail vein injection can restore the organ coefficient, while tail vein injection of NAD alone has no significant effect ( Figure 6 ). The above results show that exosomes encapsulating NAD (exosome-NAD) have a significant therapeutic effect on the animal model of premature ovarian failure. Example 4 Engineering exosomes to achieve ovarian targeted delivery of drugs

[0119] 1. Experimental Materials

[0120] The main experimental materials are shown in Table 5 below.

[0121] Table 5 Main experimental materials

[0122]

[0123]

[0124] 2. Experimental methods

[0125] The inventors designed FSH and LH ovarian targeting peptide structures, which were connected to the extracellular end of the exosome membrane protein Lamp2b and targeted to ovarian tissue by relying on ligand-receptor binding. iMSC-FSH / iMSC-LH cell lines were constructed by lentiviral infection, specifically as follows: (1) constructing lentiviral vectors overexpressing FSH-Lamp2b and LH-Lamp2b; (2) using HEK293T to package the corresponding lentivirus: 3 million cells were seeded into a 10 cm culture dish. After 24 h, the cell confluence was 80%, and the cells were transfected with lentiviral packaging plasmids, 10 μg of FSH-Lamp2b or LH-Lamp2b, 7 μg of PMD-2G, 5 μg of VSV-G, and 4 μg of REV. The cells were then cultured in a 5% CO2, 37°C incubator. After 48 and 72 h, the supernatant was collected into a 50 mL centrifuge tube, centrifuged at 3000 rpm for 5 min to remove cell debris, and the supernatant was collected with a 10 mL syringe and filtered using a 0.45 μm filter membrane. 5X PEG8000+NaCl, 10000g at 4℃ overnight, then centrifuged at 4℃ for 1.5h, the supernatant was removed, and the precipitate was gently washed with 1mL DPBS until the virus precipitate was completely dissolved. The virus titer was detected by qPCR; (3) Lentivirus infection of cells: The target cells in good condition were inoculated into a 6-well plate to make the cell concentration 10 6 cells / well with a cell density of 30%. Before infection, the original culture medium of the cells was aspirated, 2 mL of new culture medium was added, and the virus suspension was added to the cell culture supernatant at a ratio of 5000 viruses / cell. The culture was then continued for 72 hours, and puromycin was added at a final concentration of 10 ng / mL for screening for 72 hours. Total RNA was extracted from the obtained cells, and the expression of the target gene was detected by RT-qPCR. Exosomes secreted by iMSC-FSH / iMSC-LH (i.e., iPSC-derived MSCs stably transfected with FSH-Lamp2b or LH-Lamp2b) cells were collected and purified using the PEG8000 concentration method. The obtained exosomes were stained with DIR dye and injected into the tail vein of mice at an injection dose of 2.68*10 9 Particles / mouse. 48 hours after tail vein administration, the animals were dissected and the heart, liver, spleen, lungs, kidneys, and ovaries were removed for organ imaging. SPF-grade Balb / c nude female mice, 6-8 weeks old, were supplied by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with production license number SCXK(Beijing)2021-0006.

[0126] The LH-Lamp2b fusion protein DNA sequence, LH-Lamp2b fusion protein amino acid sequence, FSH-Lamp2b fusion protein DNA sequence, and FSH-Lamp2b fusion protein amino acid sequence are shown in Table 6 below.

[0127] Table 6 DNA sequence and amino acid sequence of LH-Lamp2b fusion protein and FSH-Lamp2b fusion protein

[0128]

[0129]

[0130] In addition to animal level, the present invention also verified the targeting of FSH and LH to ovarian cells at the cellular level. Rats were intraperitoneally injected with pregnant mare serum gonadotropin at a dose of 40 IU. After 48 hours of administration, rat ovarian granulosa cells (GC) were isolated and the granulosa cells were plated at 1×10 5 The cells were seeded at a density of 100 cells / well in a 12-well cell culture plate. Twelve hours later, 2 μg of GFP mRNA encapsulated in different ways were added to the cell culture supernatant, namely LNP-GFP, LNP-GFP+iMSC-EXO, LNP-GFP+iMSC-FSH-EXO, and LNP-GFP+iMSC-LH-EXO. Fluorescence imaging of the cells was performed at 24 and 48 hours.

[0131] The LNP-mRNA preparation method is as follows: (1) GFP mRNA solution with a concentration of 110 ng / μL was prepared with citric acid buffer; (2) the prepared GFP mRNA was loaded into LNP using a high-flow microfluidic method, with a lipid phase to aqueous phase flow rate ratio of 1:3 and a total flow rate of 20 mL / min; (3) the encapsulated LNP-GFP was centrifuged at 3000 g for 10 min using a Milipore 30KD ultrafiltration tube and ultrafiltered to 1 / 4 of the original volume, and then PBS buffer was added and the sample was ultrafiltered. This was repeated three times to obtain a concentrated sample; (4) the obtained LNP-GFP was mixed with the above-mentioned different exosomes and allowed to stand at 4°C for 30 min. The amount of exosomes used was 1×10 9 Particles / 2μg mRNA.

[0132] 3. Experimental results

[0133] The results are as follows Figure 7-8 As shown, the results showed that FSH and LH ovarian targeting peptide structures can improve the ovarian distribution of MSC-derived exosomes, and the effect of FSH is better than that of LH ( Figure 7 ). FSH and LH ovarian targeting peptide structures can improve the delivery efficiency of MSC-derived exosomes to LNP-encapsulated mRNA in ovarian cells, and the FSH ovarian targeting peptide structure is superior to the LH ovarian targeting peptide structure ( Figure 8 ).

[0134] Example 5 Therapeutic effect of FSH ovary-targeting peptide-modified exosomes delivering nucleic acid drugs (LNPs encapsulating NAMPT, NMNAT3, and COX15 mRNA) on mice with premature ovarian failure

[0135] 1. Experimental Materials

[0136] The main experimental materials are shown in Table 7 below.

[0137] Table 7 Main experimental materials

[0138] name factory Item No. EP tube 1.5mL (QSP) Thermo 509-GRD-Q 0.22μm filter pall PALL 4612 SPL 10mL pipette SPL SPL91010 Boxed pipette tips 1250μL (QSP) Thermo T112NXLRS-Q 50mL centrifuge tube QSP Thermo 339652 Cisplatin for injection (dry powder) Qilu Pharmaceutical SM-102 Ai Weituo 2089251-47-6 Distearoylphosphatidylcholine DSPC Ai Weituo S01005 CHO-HP high purity cholesterol Ai Weituo O01001 PEG2000-DMG Ai Weituo O02005

[0139] 2. Experimental methods

[0140] The test drug of the present invention is a mixture of LNPs (lipid nanoparticles) and exosomes encapsulating NAMPT, NMNAT3, and COX15 mRNA (abbreviated as NNC mRNA).

[0141] The specific preparation method of LNP-NNC is as follows: (1) the three mRNAs mentioned above are mixed with citric acid buffer to prepare a solution (NNC) with a final concentration of 10 ng / μL, with a ratio of 1:1:1; (2) the prepared NNC is placed in a high-flow microfluidic system. mRNA was loaded into LNP (SM-102, Aiweituo, 2089251-47-6; distearoylphosphatidylcholine DSPC, Aiweituo, S01005; CHO-HP high-purity cholesterol, Aiweituo, O01001; PEG2000-DMG, Aiweituo, O02005), with the lipid phase and aqueous phase flow rate ratio of 1:3 and the total flow rate of 20 mL / min; (3) the encapsulated LNP-NNC was centrifuged at 3000 g for 10 min using Milipore30KD ultrafiltration tube, ultrafiltered to 1 / 4 of the original volume, and then PBS buffer was added and the ultrafiltration sample was repeated three times to obtain a concentrated sample; (4) the obtained LNP-NNC was mixed with FSH ovarian targeting peptide modified exosomes (FSH-EXO) at a volume ratio of 1:1, and the concentration of the FSH-EXO was 8×10 10 / mL, and FSH-EXO-LNP-NNC was obtained. The final concentration of the drug FSH-EXO-LNP-NNC used was: exosome (FSH-EXO) NTA was 4×10 10 / mL, and LNP-NNC was 25μg / mL.

[0142] POF animal model: 8-week-old C57B / L6 female mice were intraperitoneally injected with cisplatin (2 mg / kg / day) for 7 consecutive days. The experimental animals were divided into four groups: Group 1 was a healthy control group (12 mice); Groups 2-4 were cisplatin-treated mice; the vehicle group was 12 mice; the estradiol E2 group was 4 mice; and the NNC group was 15 mice.

[0143] Grouping and drug administration: Group 1 is the healthy control group; Group 2 is the solvent group, which is injected with normal saline through the tail vein; Group 3 is the positive drug estradiol E2 group, which is injected subcutaneously with E2, 8 μg / kg / time, for 3 consecutive days, and then stopped for one day until the end of the experiment; Group 4 is FSH-EXO-LNP-NNC, with a dose of exosome NTA of 8×10 9 The drug was administered three times per mouse, with NNC mRNA at a dose of 5 μg per mouse per dose, on Days 1, 3, and 5, via a combination of tail vein and intraperitoneal injection. Key physiological parameters, such as body weight, diet, and body temperature, were recorded during and after dosing to assess safety. Vaginal smears were performed daily, fixed with anhydrous ethanol, and stained with methylene blue solution to analyze the estrous cycle of the mice. Fifteen days after dosing, the mice were dissected, and organ coefficients were measured and ovarian tissue was stained with hematoxylin and eosin.

[0144] 3. Experimental results

[0145] The results showed that after cisplatin administration, one animal died in the MC group on DAY8 and DAY9, one animal died in the E2 group on DAY9, and the HC and NNC groups were in good condition ( Figure 9 ).

[0146] Observation of mouse vaginal cell sections showed that the estrous cycle of the HC group was normal. After POF modeling, the estrous cycles of all mice in the NC and E2 groups began to become disordered to varying degrees on DAY 5. Three-quarters of the mice in the NNC group were at normal levels ( Figure 10 ), the above results indicate that FSH-modified exosomes delivered LNP-NNC have a therapeutic effect on the premature ovarian failure model caused by cisplatin.

[0147] The results of observation of mouse ovarian sections showed that compared with the HC group, the volume of ovarian follicles in animals after POF modeling was significantly reduced, the number of granulosa cells decreased, the apoptosis of granulosa cells increased, and the number of atretic follicles increased. FSH-modified exosomes delivered LNP-NNCs could significantly improve the above phenotypes ( Figure 11 The above results once again demonstrated that FSH-modified exosomes delivered LNP-NNC have a significant effect in treating premature ovarian failure.

[0148] Example 6 Therapeutic Effect of Exosome-Delivered Nucleic Acids (TNP Encapsulating NAMPT, NMNAT3, and COX15 mRNA) on Mice with Premature Ovarian Failure

[0149] 1. Experimental Materials

[0150] The main experimental materials are shown in Table 8 below.

[0151] Table 8 Main experimental materials

[0152]

[0153]

[0154] 2. Experimental methods

[0155] The test drug of the present invention is a mixture of TNP (novel mRNA delivery system) encapsulating NAMPT, NMNAT3, COX15 mRNA (abbreviated as NNC mRNA) and exosomes.

[0156] Preparation method of TNP-NNC:

[0157] (1) The three mRNAs were mixed with RNase-free ultrapure water to prepare a solution (NNC) with a final concentration of 1 mg / mL, with a ratio of 1:1:1; (2) The prepared mRNA was loaded into TNP (Deng Hongzhang Laboratory, Xidian University) using a high-flow microfluidic method, with the lipid phase and water phase flow rates of 0.1 mL / min and 0.3 mL / min, respectively; (3) The encapsulated TNP-NNC was ultrafiltered to 1 / 4 of the original volume using a Milipore 30KD ultrafiltration tube, centrifuged at 3000 g for 10 min, and then ultrafiltered to 1 / 4 of the original volume. PBS buffer was then added and the sample was ultrafiltered. This was repeated three times to obtain a concentrated sample; (4) The obtained TNP-NNC was mixed with exosomes (the exosomes used were from wild-type mesenchymal stem cells (MSC)) at a volume ratio of 1:1, and the concentration of the exosomes was 10 11 / mL, and let it stand at 4℃ for 30min to obtain EXO-TNP-NNC. The final concentration of the drug EXO-TNP-NNC used is: exosome (EXO)NTA is 5×10 10 / mL, TNP-NNC was 75μg / mL.

[0158] POF animal model: 8-week-old C57B / L6 female mice were intraperitoneally injected with 4.5 mg / (kg·d) of cisplatin for 4 consecutive days. The experimental animals were divided into four groups: Group 1 served as the healthy control group, and Groups 2-4 served as cisplatin-induced POF models.

[0159] The test drugs were administered as follows: Group 1 was the healthy control group; Group 2 was the vehicle group, which was injected with normal saline via the tail vein; Group 3 was the positive drug estradiol E2 group, which was injected subcutaneously at 8 μg / kg / time; Group 4 was EXO-TNP-NNC, which was injected with 15 μg / mouse via the tail vein. The exosome NTA was 10 10 The drug was administered to mice on days 2, 3, 4, 5, and 7. Key physiological parameters, such as body weight, diet, and temperature, were recorded during and after dosing to assess safety. Vaginal smears were performed daily, fixed with anhydrous ethanol, and stained with methylene blue solution to analyze the estrous cycle of the mice. Ten days after dosing, the mice were dissected, and organ coefficients were measured and ovarian tissue was stained with hematoxylin and eosin.

[0160] 3. Experimental results

[0161] Observation of mouse vaginal cells and statistics of estrus on DAY15 showed that the estrus cycle of mice in the HC group was in different cycles, and the estrus cycle was in accordance with normal rules; after POF modeling, most mice in the NaCl group were in proestrus and could not enter estrus; all mice in the E2 group were in estrus; the estrus cycle of mice in the NNC group was similar to that of HC and in accordance with normal rules ( Figure 12 ), indicating that exosome-delivered TNP-NNC has a therapeutic effect on mice with premature ovarian failure.

Claims

1. An ovary-targeting peptide, characterized in that: The ovary targeting peptide includes LH-Lamp2b fusion protein or FSH-Lamp2b fusion protein; The amino acid sequence of the LH-Lamp2b fusion protein is shown in SEQ ID NO: 2; The amino acid sequence of the FSH-Lamp2b fusion protein is shown in SEQ ID NO:

4.

2. The ovary targeting peptide according to claim 1, characterized in that The nucleotide sequence of the LH-Lamp2b fusion protein is shown in SEQ ID NO: 1; The nucleotide sequence of the FSH-Lamp2b fusion protein is shown in SEQ ID NO:

3.

3. An exosome specifically targeting ovarian cells, characterized in that: The exosomes contain the ovary-targeting peptide according to claim 1 or 2.

4. The exosome according to claim 3, characterized in that The exosomes are derived from mesenchymal stem cells.

5. The exosome according to claim 3, characterized in that The exosomes contain the FSH-Lamp2b fusion protein according to claim 1 or 2.

6. An exosome drug encapsulating NAD, characterized in that The NAD-encapsulated exosome drug is an exosome drug obtained by encapsulating the NAD drug in exosomes; Optionally, the exosomes in the NAD-encapsulated exosome drug are 4×10 10 / mL, NAD is 600μg / mL; Optionally, the exosomes in the NAD-encapsulated exosome drug are derived from mesenchymal stem cells.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the exosomes according to any one of claims 3 to 5 and / or the NAD-encapsulated exosome drug according to claim 6.

8. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the exosomes according to any one of claims 3 to 5 and / or the NAD-encapsulated exosome drug according to claim 6; Optionally, the dosage form of the pharmaceutical preparation is an intravenous infusion preparation, an in situ injection preparation, an oral preparation, a tablet, a capsule preparation, a granule preparation, a sustained-release preparation, a powder or a pill.

9. A method for preparing the NAD-encapsulated exosome drug according to claim 6, characterized in that: The NAD-encapsulated exosome drug is an exosome drug obtained by encapsulating the NAD drug in exosomes; Optionally, the NAD-encapsulated exosome drug is an exosome drug obtained by encapsulating the NAD drug in exosomes using an electroporation method; Optionally, the exosomes in the NAD-encapsulated exosome drug are 4×10 10 / mL, NAD is 600μg / mL; Optionally, the exosomes in the NAD-encapsulated exosome drug are derived from mesenchymal stem cells.

10. Any of the following applications: (1) Use of the ovarian targeting peptide according to claim 1 or 2 and / or the exosomes according to any one of claims 3 to 5 in the preparation of an exosome drug for treating premature ovarian failure; (2) Use of the ovarian targeting peptide according to claim 1 or 2 and / or the exosomes according to any one of claims 3 to 5 in the preparation of a drug for delivering nucleic acid molecules to ovarian tissue; (3) Use of the NAD-encapsulated exosome drug according to claim 6 in the preparation of a pharmaceutical composition or pharmaceutical preparation for treating premature ovarian failure.

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