Ovary-targeted mRNA liposome nanoparticles, preparation method, drug compound, targeted delivery system and application

Abstract

The invention discloses an ovary-targeting mRNA liposome nanoparticle, a preparation method, a drug compound, a targeting delivery system and application, and relates to the technical field of liposome granules, the ovary-targeting mRNA liposome nanoparticle comprises the following components by mass: 20%-80% of SM-102, 1%-20% of PEG2000, 5%-40% of CHO-HP, 1%-20% of DSCP, and 0.01%-10% of targeting polypeptide, the target polypeptide comprises at least one of the following sequences: LVYKDPARPNTQK, RCGPCRLSSSDCGGPRTQPMACDLP, and CALCRRSTSDCGGPKDHPLT (recombinant human serum albumin), and the target polypeptide comprises at least one of the following sequences. The present invention provides a liposome nanoparticle for targeted ovary and uterus delivery of mRNA. The lipidosome nano-particles are specially researched, developed and designed for mRNA, have a good targeted delivery effect in mRNA in-vivo delivery, and can be applied to the field of targeted drug delivery of ovaries and uteruses. When in use, the kit can be directly prepared with a specific mRNA sequence for use, so that different application requirements can be flexibly met.

Description

Technical Field

[0001] The present invention relates to the technical field of liposome particles, and particularly to an mRNA liposome nanoparticle targeting the ovary, a preparation method, a drug complex, a targeted delivery system and applications thereof. Background Art

[0002] mRNA (messenger RNA) is a novel therapeutic modality that utilizes the genetic information carried by mRNA molecules to induce an immune response in the human body. Once the mRNA enters human cells, it is translated into the corresponding protein by the intracellular machinery, thereby activating the immune system to generate an immune response against specific pathogen proteins. The advantages of mRNA drugs lie in their fast R & D speed, low production cost, strong scalability in the production process, and potential wide application prospects in drug R & D.

[0003] Currently, the delivery formulations of mRNA drugs mainly contain nanoparticles. These nanoparticles are usually composed of liposomes, which are used to encapsulate and protect mRNA molecules and facilitate their entry into target cells. Nanoparticles can also help stabilize mRNA molecules to enhance the persistence and efficacy of the drug. Once the mRNA enters the cell, the nanoparticles fuse with the cell membrane and release the mRNA molecules into the cytoplasm. The mRNA molecules are translated into specific proteins by ribosomes in the cell, thereby exerting their biological functions.

[0004] Currently, although there are already various liposome particles used in the research and application of drug delivery, targeted delivery to the ovary remains difficult. Summary of the Invention

[0005] The technical solution for the present invention to solve the above technical problems is to provide an mRNA liposome nanoparticle targeting the ovary, which is composed of the following components by mass percentage:

[0006]

[0007] Among them, the targeting polypeptide contains at least one of the following sequences: LVYKDPARPNTQK, RCGPCRLSSSDCGGPRTQPMACDLP, CALCRRSTSDCGGPKDHPLT.

[0008] Furthermore, the mass percentages of each component are: SM-102 64%, PEG2000 5%, CHO-HP 18%, DSCP 8%, and targeting polypeptide 10%.

[0009] To solve the above technical problems, the present invention also provides a pharmaceutical complex, comprising the liposome nanoparticles as described above, and the encapsulated mRNA molecules; the mRNA molecules comprise 5'UTR (SEQ ID NO:1), 3'UTR (SEQ ID NO:2) and polyA tail (SEQ ID NO:3).

[0010] To solve the above technical problems, the present invention also provides a method for preparing the pharmaceutical complex, comprising the following steps:

[0011] a) Dissolve each component in absolute ethanol to form a lipid-ethanol solution;

[0012] b) After adding an equal volume of absolute ethanol and mixing, incubate in a water bath at 37°C for 1-5 hours under light protection;

[0013] c) Mix the liposomes and mRNA at a mass ratio of 1:1 - 1:10 through a microfluidic system to form an mRNA-liposome complex.

[0014] Furthermore, the mRNA encodes the α / β subunits of follicle-stimulating hormone (FSH), luteinizing hormone (LH) or equine chorionic gonadotropin (eCG), and their sequences are shown in SEQ ID NO:8, 10, 12, 14 respectively.

[0015] To solve the above technical problems, the present invention also provides a targeted delivery system, which administers the above pharmaceutical complex to a mammal through the intraperitoneal injection route to achieve ovarian-specific mRNA delivery and protein expression.

[0016] To solve the above technical problems, the present invention also provides the application of the liposome nanoparticles as described above in the preparation of drugs for treating ovarian diseases, and the diseases include follicular development disorders.

[0017] The advantages and positive effects of the technical solution to be protected by the present invention are as follows:

[0018] First, the present invention provides a liposome nanoparticle, which can encapsulate and adsorb mRNA, improving the effectiveness of existing mRNA vaccines. The liposome nanoparticles are safe and non-toxic, improving the applicability to existing mRNA vaccines. The liposome nanoparticles are simple and rapid to produce, with good effects, and have broad application prospects in the research and development of mRNA vaccines, antibodies and drugs.

[0019] Second, the present invention provides a liposome nanoparticle, which binds to receptors on the ovary or uterus through a targeting peptide, thereby achieving targeted delivery of mRNA.

[0020] Third, as the creative auxiliary evidence of the claims of the present invention, it is also reflected in the following important aspects:

[0021] (1) The expected benefits and commercial value after the transformation of the technical solution of the present invention are as follows:

[0022] The present invention provides a novel liposome nanoparticle, which can be prepared on a large scale relying on an in vitro reaction, and is significantly superior to traditional delivery preparations in terms of production cost, production speed, safety, protection scope, etc.

[0023] (2) Whether the technical solution of the novel liposome nanoparticle of the present invention solves the technical problems that people have been eager to solve but have never been successful in:

[0024] The present invention provides a novel liposome nanoparticle for mRNA delivery, which improves the uptake efficiency of ovarian cells for mRNA, thereby improving the effect of mRNA.

[0025] (3) Whether the technical solution of the present invention overcomes the technical prejudice:

[0026] This technical solution overcomes the problem that existing mRNA drugs cannot be targeted for delivery. This technical solution overcomes the problem of low cell uptake efficiency.

[0027] Fourth, the liposome nanoparticle provided by the present invention comprises the following technical features and innovations:

[0028] (1) The synthesis method of the liposome nanoparticle is simple: The synthesis strategy of the liposome nanoparticle is simple and easy to implement, suitable for large-scale preparation, and provides a feasible way for large-scale mRNA vaccine production.

[0029] (2) The liposome nanoparticle improves the stability of mRNA: The liposome nanoparticle can effectively protect mRNA molecules from being degraded in vivo, thereby significantly improving the stability of mRNA and prolonging its existence time in vivo, providing strong support for the effective delivery of vaccines.

[0030] (3) The liposome nanoparticle enables targeted delivery of mRNA: After being mixed with mRNA, the liposome nanoparticle significantly improves the targeted delivery to the ovary or uterus, and improves the efficacy and reliability of mRNA drugs.

[0031] (4) The liposome nanoparticle can be used for the delivery of various mRNAs: The liposome nanoparticle can be applied to mRNAs of different proteins, and this feature makes it widely applicable in meeting the treatment needs of different ovarian or uterine diseases.

[0032] Generally speaking, these technical features and innovations have brought significant technological progress, can improve the delivery effect of mRNA, and also facilitate the storage and use of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Fluorescence results of different parts of live mice after immunization by different methods (from left to right are the intraperitoneal injection group, intravenous injection group, and intramuscular injection group);

[0034] Figure 2 Fluorescence results of different organs after live imaging after immunization by different methods;

[0035] Figure 3 Results of imaging different doses of polypeptides in rats;

[0036] Figure 4 Fluorescence imaging result map of the ovary-containing (from left to right, the three organs are the liver, spleen, and ovary. The upper left is the blank control group, the upper right is the non-targeted group, the lower left is the group containing 0.05% polypeptide, and the lower right is the group containing 10% polypeptide);

[0037] Figure 5 Ovary imaging results of different doses of polypeptides (from left to right are the blank control group, non-targeted group, group containing 0.05% polypeptide, and group containing 10% polypeptide);

[0038] Figure 6 Tissue staining results of peptide 1 targeting ovarian delivery at different concentrations;

[0039] Figure 7 Statistical results of different oocytes for different concentrations of peptide 1 (*: compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001);

[0040] Figure 8 Tissue staining results of peptide 2 targeting ovarian delivery at different concentrations;

[0041] Figure 9 Statistical results of different oocytes for different concentrations of peptide 2 (*: compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001);

[0042] Figure 10 Tissue staining results of peptide 3 targeting ovarian delivery at different concentrations;

[0043] Figure 11 Statistical results of different oocytes for different concentrations of peptide 3 (*: compared with the blank control group, *P<0.05, **P<0.01, ***P<0.001). Specific implementation manner

[0044] The present invention provides an mRNA liposome nanoparticle targeting the ovary, a preparation method, a drug complex, a targeted delivery system and an application, aiming to achieve targeted delivery to ovarian cells by modifying specific targeting peptides on the surface of liposome particles.

[0045] The following will illustrate a kind of mRNA liposome nanoparticles targeting the ovary, a preparation method, a drug complex, a targeted delivery system and an application proposed by the present invention in specific embodiments:

[0046] Example 1:

[0047] A kind of mRNA liposome nanoparticles targeting the ovary, which is composed of the following components by mass percentage:

[0048]

[0049] Among them, the targeting polypeptide contains at least one of the following sequences: LVYKDPARPNTQK, RCGPCRLSSSDCGGPRTQPMACDLP, CALCRRSTSDCGGPKDHPLT.

[0050] Among them, SM-102 is a synthetic ionizable cationic lipid, and its chemical name is 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]-octanoate; the molecular formula of PEG2000 is: H(OCH2CH2) n OH; CHO-HP is injection-grade high-purity cholesterol; DSCP is distearoyl choline phosphate;

[0051] The average particle size distribution of the immune complex formed by the liposome nanoparticles and mRNA is between 100 nanometers and 140 nanometers; the final concentration of mRNA in the immune complex is 100 micrograms per milliliter (including the mRNA adsorbed by the liposome nanoparticles and the free mRNA not adsorbed), and by measuring the mRNA of the immune complex, the encapsulation rate of the liposome nanoparticles for mRNA is calculated to be greater than 80%.

[0052] Example 2:

[0053] A kind of mRNA liposome nanoparticles targeting the ovary, the mass percentages of each component are: SM-102 64%, PEG2000 5%, CHO-HP 18%, DSCP 8%, targeting polypeptide 10%; the targeting polypeptide contains at least one of the following sequences: LVYKDPARPNTQK, RCGPCRLSSSDCGGPRTQPMACDLP, CALCRRSTSDCGGPKDHPLT.

[0054] Example 3:

[0055] A pharmaceutical complex comprising the liposome nanoparticles of Example 1 and encapsulated mRNA molecules; the mRNA molecules comprise a 5'UTR (SEQ ID NO:1), a 3'UTR (SEQ ID NO:2) and a polyA tail (SEQ ID NO:3).

[0056] Example 4:

[0057] A method for preparing a pharmaceutical complex, comprising the following steps:

[0058] a) Dissolve each component in absolute ethanol to form a lipid-ethanol solution;

[0059] b) Add an equal volume of absolute ethanol, mix, and incubate in a water bath at 37 °C for 1-5 hours in the dark;

[0060] c) Mix the liposomes and mRNA at a mass ratio of 1:1 - 1:10 through a microfluidic system to form an mRNA-liposome complex.

[0061] The method for preparing mRNA includes: synthesizing Luciferase mRNA by in vitro transcription, but not limited to this method. Insert the gene encoding the epitope protein sequence into the pUC57 vector, but not limited to this vector, and then transform it into competent Escherichia coli for expression, perform bacterial amplification, and harvest the bacteria when the bacteria are in the stationary phase. Subsequently, mRNA can be obtained through steps such as plasmid extraction, enzymatic digestion and linearization, in vitro transcription, and chromatography purification; the DNA sequence of the 5' untranslated region (5'UTR) is as shown in SEQ ID NO.1; the DNA sequence of the 3' untranslated region (3'UTR) is as shown in SEQ ID NO.2; the sequence of polyadenylic acid (poly A) is as shown in SEQ ID NO.3, containing 104 base As; the DNA sequence of the T7 promoter is as shown in SEQ ID NO.4; the amino acid sequence of Luciferase is as shown in SEQ ID NO.5; the sequence of Luciferase mRNA is as shown in SEQ ID NO.6.

[0062] Furthermore, the mRNA encodes the α / β subunits of follicle-stimulating hormone (FSH), luteinizing hormone (LH), or equine chorionic gonadotropin (eCG), and their sequences are as shown in SEQ ID NO:8, 10, 12, 14 respectively.

[0063] FSH, LH, and eCG can all act on the ovary to promote the development of egg cells. All three proteins contain two subunits, α and β, and the α subunits of the three proteins are the same. Therefore, by synthesizing 4 mRNAs, three protein structures can be assembled. Peptide 1 (the targeting peptide sequence is LVYKDPARPNTQK) uses the mRNA sequence encoding FSH, as shown in SEQ ID NO.8 and SEQ ID NO.10. Peptide 2 (the targeting peptide sequence is RCGPCRLSSSDCGGPRTQPMACDL) uses the mRNA sequence encoding LH, as shown in SEQ ID NO.8 and SEQ ID NO.12. Peptide 3 (the targeting peptide sequence is CALCRRSTSDCGGPKDHPLT) uses the mRNA sequence encoding eCG, as shown in SEQ ID NO.8 and SEQ ID NO.14.

[0064] Among them, the amino acid sequence of the α subunit is as shown in SEQ ID NO.7; the mRNA sequence of the α subunit is as shown in SEQ ID NO.8; the amino acid sequence of the FSHβ subunit is as shown in SEQ ID NO.9; the mRNA sequence of the FSHβ subunit is as shown in SEQ ID NO.10; the amino acid sequence of the LHβ subunit is as shown in SEQ ID NO.11; the mRNA sequence of the LHβ subunit is as shown in SEQ ID NO.12; the amino acid sequence of the eCGβ subunit is as shown in SEQ ID NO.13; the mRNA sequence of the eCGβ subunit is as shown in SEQ ID NO.14;

[0065] Groups with peptide contents of 0%, 0.05%, and 10% were respectively selected as the non-targeting group, low-dose targeting peptide group, and high-dose targeting peptide group. The LNPs synthesized with Peptide 1 formula encapsulated the mRNAs of the α subunit and the FSHβ subunit respectively, and were subsequently mixed in a 1:1 ratio. The LNPs synthesized with Peptide 2 formula encapsulated the mRNAs of the α subunit and the LHβ subunit respectively, and were subsequently mixed in a 1:1 ratio. The LNPs synthesized with Peptide 3 formula encapsulated the mRNAs of the α subunit and the eCGβ subunit respectively, and were subsequently mixed in a 1:1 ratio. Sucrose with a final concentration of 10% was added to the mixed LNP-mRNA, and it was stored at -20°C. Targeting effects of liposome nanoparticles and mRNA complexes with different Peptide 1 contents in mice

[0066] The prepared polypeptide 1 (FSH mRNA-LNP) was used to immunize mice to observe its in vivo targeted delivery effect. Twenty 8-week-old female SPF Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and randomly divided into 4 groups with 5 mice in each group. They were immunized by intraperitoneal injection with 10 μg of non-targeting peptide 1 group, 0.05% peptide group (peptide 1, 10 μg, targeting polypeptide with 0.05% content), and 10% peptide group (peptide 1, 10 μg, targeting polypeptide with 10% content), and the blank control group (intraperitoneally injected with 100 μL of PBS).

[0067] An increase in the content of FSH in mice can promote the development of mouse oocytes. Therefore, on the 3rd day after immunization, the mice were sacrificed, and their ovarian tissues were isolated, sectioned for observation, and the number of follicles at each stage was counted. The results showed that the non-targeting group did not cause the development of mouse ovarian cells, while the two targeting groups (0.05% peptide group, 10% peptide group) of mRNA-LNP could significantly promote the development of oocytes in the ovaries of mice, manifested as an increase in the number of growing follicles and cystic follicles, and an increase in the number of corpora lutea (as Figure 6 - 7 shown).

[0068] Targeting effects of liposome nanoparticles and mRNA complexes with different contents of polypeptide 2 in mice

[0069] The prepared polypeptide 2 (LH mRNA-LNP) was used to immunize mice to observe its in vivo targeted delivery effect. Twenty 8-week-old female SPF Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and randomly divided into 4 groups with 5 mice in each group. They were immunized by intraperitoneal injection with 10 μg of non-targeting peptide 2 group, 0.05% peptide group (peptide 2, 10 μg, targeting polypeptide with 0.05% content), and 10% peptide group (peptide 2, 10 μg, targeting polypeptide with 10% content), and the blank control group was intraperitoneally injected with 100 μL of PBS.

[0070] An increase in the content of LH in mice can promote the development of mouse oocytes. Therefore, on the 3rd day after immunization, the mice were sacrificed, and their ovarian tissues were isolated, sectioned for observation, and the number of follicles at each stage was counted. The results showed that the non-targeting group did not cause the development of mouse ovarian cells, while the two targeting groups of mRNA-LNP could significantly promote the development of oocytes in the ovaries of mice, manifested as an increase in the number of growing follicles and cystic follicles, and an increase in the number of corpora lutea (as Figure 8 - 9 described).

[0071] Targeting effects of liposome nanoparticles and mRNA complexes with different contents of polypeptide 3 in mice

[0072] The prepared polypeptide 3 (eCG mRNA-LNP) was used to immunize mice to observe its in vivo targeted delivery effect. Twenty 8-week-old female SPF Kunming mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. and randomly divided into 4 groups with 5 mice in each group. They were immunized by intraperitoneal injection with 10 μg of non-targeted peptide group 3, 0.05% peptide group (peptide 3, 10 μg, targeted polypeptide with 0.05% content), and 10% peptide group (peptide 3, 10 μg, targeted polypeptide with 10% content). The blank control group was intraperitoneally injected with 100 μL of PBS.

[0073] The increase in the content of eCG in mice can promote the development of mouse oocytes. Therefore, on the 3rd day after immunization, the mice were sacrificed, and the ovarian tissues were isolated, sectioned and observed, and the number of follicles at each stage was counted. The results showed that the non-targeted group did not cause the development of mouse ovarian cells, while the two targeted groups of mRNA-LNP could significantly promote the development of oocytes in the ovaries of mice, manifested as an increase in the number of growing follicles and cystic follicles, and an increase in the number of corpora lutea (as Figure 10 - 11 shown).

[0074] Verification experiment: 1. Liposome nanoparticle characterization:

[0075] The detection of the encapsulation rate was carried out using the Quant-iT™ RiboGreen detection kit. The results showed that when the polypeptide concentration was between 0.01% and 10%, the encapsulation rate was above 80%. The particle size analysis was carried out using a particle size analyzer. The particle size analysis showed that when the polypeptide content was between 0.05% and 10%, the particle size of the drug complex (including the liposome nanoparticles in Example 2, with the polypeptide content as the variable) was mainly distributed around 130 nm (Table 1-2). When the polypeptide content was more than 20%, the LNP particle size increased significantly and the encapsulation rate decreased significantly. Based on this result, the polypeptide range of 0.01% - 10% was selected.

[0076] Table 1 Statistical results of particle size and encapsulation rate of LNP encapsulated with polypeptides with 0.01% - 0.1% content:

[0077] Polypeptide content 0％ 0.01％ 0.02％ 0.03％ 0.04％ 0.05％ 0.06％ 0.07％ 0.08％ 0.09％ Particle size 131nm 179nm 182nm 154nm 145nm 135nm 133nm 131nm 136nm 132nm Entrapment efficiency 84.2％ 85.3％ 83.4％ 82.1％ 83.9％ 81.7％ 92.3％ 91.3％ 87.9％ 85.6％

[0078] Table 2 Statistical results of particle size and encapsulation rate of LNP encapsulated with polypeptides with 0.1% - 20% content:

[0079] Polypeptide content 0.1％ 0.2％ 0.4％ 0.8％ 1％ 2％ 5％ 10％ 20％ Particle size 134nm 126nm 132nm 126nm 131nm 124nm 133nm 132nm 402nm Entrapment efficiency 87.5％ 83.6％ 88.6％ 92.3％ 85.7％ 85.2％ 91.2％ 92.3％ 31.4％

[0080] 2. Expression of liposome nanoparticles and mRNA complexes in mice through different routes:

[0081] The drug complex containing 10% polypeptide (including the liposome nanoparticles of Example 2) was intraperitoneally, intravenously and intramuscularly injected into 6- to 8-week-old female BALB / c mice at a dose of 100 μL. At the same time, a blank control group without polypeptide (non-targeted group) was set up, and 100 μL of LucferasemRNA-LNP was intraperitoneally, intravenously and intramuscularly injected. After 2-4 hours of immunization, the Luciferase substrate was intraperitoneally injected. After 10 minutes, the mice were sacrificed for in vivo imaging.

[0082] The mice were observed with different immunization methods. The results showed that in the non-targeted group, through intraperitoneal injection, intravenous injection and intramuscular injection respectively, fluorescence was visible in the spleen in the intraperitoneal injection group, in the liver in the intravenous injection group, and at the intramuscular injection site in the intramuscular injection group. In the targeted group, through intraperitoneal injection, intravenous injection and intramuscular injection respectively, fluorescence was visible in the spleen and uterus in the intraperitoneal injection group, in the liver in the intravenous injection group, and at the intramuscular injection site in the intramuscular injection group (as Figure 1 described). The mice were dissected, different organs were isolated, and imaging observations were carried out. The observation results of the mice were consistent with those in vivo (as Figure 2 shown).

[0083] 3. Verification of the effects of liposome nanoparticles and drug complexes with different polypeptide contents in mice:

[0084] Eight-week-old female SD rats were intraperitoneally injected with 100 μL of LNP-mRNA complex. Among them, the blank group was injected with PBS, the negative control group was injected with non-targeted Luciferase, and the polypeptide groups selected low dose 0.05% and high dose 10%. After 2-4 hours of injecting the LNP-mRNA complex, the substrate was intraperitoneally injected for color development. Compared with the non-targeted group by intraperitoneal injection, fluorescence expression was visible in the targeted group, and the fluorescence intensity after injecting 10% polypeptide was higher than that of the 0.05% polypeptide group ( Figure 3 ). After the liver, spleen and ovaries were taken out for observation, the results were consistent with those in mice, and the fluorescence intensity after injecting 10% polypeptide was higher than that of the 0.05% polypeptide group (as Figure 4 described). When observing the ovaries alone, the fluorescence intensity after injecting 10% polypeptide was higher than that of the 0.05% polypeptide group (as Figure 5 shown).

[0085] Example 5:

[0086] A targeted delivery system, in which the drug complex of Example 3 is administered to a mammal through the intraperitoneal injection route to achieve ovarian-specific mRNA delivery and protein expression.

[0087] Example 6:

[0088] Use of the liposome nanoparticles according to Example 1 in the preparation of a therapeutic drug for ovarian diseases, said diseases including follicular development disorders.

[0089] The diseases may also include ovarian function decline or polycystic ovary syndrome.

Claims

1. An ovarian-targeted mRNA liposome nanoparticle, characterized in that: It is composed of the following components by mass percentage: SM-102 20%-80%, PEG2000 1%-20%, CHO-HP 5%-40%, DSCP 1%-20%, Targeted peptide 0.01%-10%, Wherein, the targeting polypeptide comprises at least one of the following sequences: LVYKDPARPNTQK, RCGPCRLSSSDCGGPRTQPMACDLP, CALCRRSTSDCGGPKDHPLT.

2. The liposome nanoparticle according to claim 1, characterized in that The mass percentage of each component is: SM-102 64%, PEG2000 5%, CHO-HP 18%, DSCP 8%, and targeting peptide 10%.

3. A drug complex, characterized in that The method comprises the liposome nanoparticle as claimed in claim 1 or 2, and an encapsulated mRNA molecule; the mRNA molecule comprises a 5'UTR (SEQ ID NO: 1), a 3'UTR (SEQ ID NO: 2) and a polyA tail (SEQ ID NO: 3).

4. The method for preparing the drug complex according to claim 3, characterized in that: The following steps are involved: Dissolving each component in anhydrous ethanol to form a lipid-ethanol solution; After adding an equal volume of anhydrous ethanol and mixing, incubate in a 37°C water bath for 1-5 hours in the dark; Liposomes and mRNA were mixed at a mass ratio of 1:1-1:10 through a microfluidic system to form an mRNA-liposome complex.

5. The method for preparing the drug complex according to claim 4, characterized in that: The mRNA encodes the α / β subunit of follicle-stimulating hormone, luteinizing hormone or equine chorionic gonadotropin, and the sequences thereof are shown in SEQ ID NOs: 8, 10, 12 and 14, respectively.

6. A targeted delivery system, characterized in that: The drug complex of claim 3 is administered to mammals via intraperitoneal injection to achieve ovarian-specific mRNA delivery and protein expression.

7. Use of the liposome nanoparticles according to any one of claims 1-2 in the preparation of a drug for treating ovarian diseases, wherein the disease comprises follicular development disorder.

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