A method for preparing ovarian stem cells and its application in the preparation of ovarian anti-aging drugs.

Abstract

This invention provides a composition for treating ovarian insufficiency (POF) and its application. The composition comprises two synergistic modules: first, hiPSC-derived ovarian support cells (iPSC-OSCs) pre-adapted to FTY720 and possessing enhanced oxidative stress tolerance; second, reactive oxygen species (ROS)-responsive dual-targeting lipid nanoparticles (RRDT-LNPs) loaded with a therapeutic agent (such as urestatin A). The RRDT-LNPs are functionalized from a ROS-responsive material containing a thioketal (TK) linker, a primary targeting peptide CNGRC, and a secondary dual-targeting fusion protein VHH-FP. This invention also provides a method for the directed differentiation and pre-adaptation of the iPSC-OSCs, and a method for preparing the RRDT-LNPs. In vitro and in vivo experiments have demonstrated that the composition of this invention, through the organic combination of "cell empowerment" and "intelligent targeted drug delivery," can sequentially activate targeting capabilities in the pathological microenvironment, efficiently repairing ovarian function, restoring endocrine levels, and exhibiting good safety, providing a novel synergistic treatment option for POF.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing ovarian stem cells and its application in the preparation of ovarian anti-aging drugs. Background Technology

[0002] Ovarian dysfunction includes premature ovarian insufficiency (POI) and age-related decline in ovarian reserve (DOR). Its main biological characteristics are a decrease in the number of follicles, reduced follicle development capacity, and decreased oocyte quality, ultimately leading to ovulation disorders and reduced fertility. In addition to a reduced follicle pool, increasing research indicates that alterations in the local ovarian tissue environment play a crucial role in these pathological processes.

[0003] Normal follicle development depends on the functional unit formed by the oocyte and surrounding granulosa cells. Granulosa cells maintain oocyte maturation through energy metabolism, nutrient supply, and hormone synthesis. During ovarian dysfunction, the mitochondrial function of granulosa cells and oocytes declines, which may be accompanied by increased levels of reactive oxygen species (ROS) and enhanced oxidative stress. Simultaneously, reduced local blood supply to the ovary and chronic inflammation can cause changes in extracellular matrix metabolism, accumulation of local acidic metabolites, and decreased secretion of various growth factors, thereby affecting follicle survival and development.

[0004] Current interventions for ovarian dysfunction mainly include hormone replacement therapy, cell therapy, and local drug intervention. Hormone replacement therapy can improve symptoms related to low estrogen, but it usually cannot restore follicle development. In recent years, the transplantation of supporting cells derived from mesenchymal stem cells or pluripotent stem cells has been used to improve ovarian function. Its mechanism of action is mainly believed to be related to the release of paracrine factors and local tissue regulation. However, the survival time and functional maintenance of transplanted cells in damaged ovarian tissue remain unstable, and the efficacy varies among individuals.

[0005] On the other hand, some technologies attempt to deliver drugs to ovarian granulosa cells via antibodies or receptor ligands to regulate local metabolic states or inhibit oxidative damage. However, follicles have a specific histological structure: an outer layer of theca cells, an inner basal lamina composed of collagen IV and laminin, and an inner granulosa cell layer. This basal lamina exhibits selective permeability, acting as a barrier to most macromolecules or nanoparticles. Furthermore, the expression of granulosa cell surface receptors (such as FSHR and AMHR2) is regulated by the follicular development stage, with lower expression levels in some early or resting follicles, thus affecting the stability and efficiency of targeted delivery.

[0006] Therefore, under current technological conditions, it is necessary to improve the local tissue environment of the ovary to maintain follicle survival and function, while simultaneously delivering active substances that regulate cell metabolism or oxidative stress effectively to the cells within the follicles. However, achieving the enrichment of therapeutic factors in ovarian tissue, crossing the follicular structural barrier, and being taken up by granulosa cells while ensuring tissue safety remains a technical challenge. A technical solution is needed in this field that can simultaneously address both tissue microenvironment regulation and intracellular delivery efficiency. Summary of the Invention

[0007] To achieve the above objectives, this invention provides an innovative synergistic treatment strategy. Its core concept lies in two aspects: firstly, pre-treating stem cell-derived ovarian support cells to "empower" them, enabling them to resist the harsh oxidative stress microenvironment after transplantation; secondly, constructing an intelligent nano-drug delivery system that "unlocks" its targeting function only under the high ROS environment of the diseased ovary, achieving deep drug penetration and precise intracellular delivery. Through the deep synergy of these two modules—"cells" and "drugs"—a therapeutic effect greater than the sum of its parts (1+1>2) is achieved.

[0008] 1. First Module: FTY720 Preadapted iPSCs-OSCs. This invention first establishes a highly efficient, stable, and reproducible clinical-grade hiPSC-to-ovarian Sertoli cell directed differentiation system.

[0009] By precisely simulating the spatiotemporal regulatory signals of embryonic gonadal development (WNT, BMP, FGF, RA, WNT4 / RSPO1, etc.), hiPSCs were successfully induced into FOXL2. + / AMHR2 + Functional granuloid cells (iPSC-OSCs) with a double positivity rate as high as 45% were observed, and strict phased quality control standards (such as D2 Brachyury) were established. + >80%, D5 LHX1 / OSR1 increased by >100 times).

[0010] The key element was the introduction of FTY720 preconditioning treatment. Experiments demonstrated that treatment with 1 µM FTY720 for 16 hours significantly increased the survival rate of iPSC-OSCs under H2O2 oxidative stress from 41.5% to 78.3%. In-depth transcriptomics (RNA-seq) revealed that the mechanism was not simple protection, but a systemic "cellular metabolic reprogramming": significantly upregulating multidimensional stress-resistance genes such as glutathione metabolism (GPX3, GSTP1), mitochondrial oxidative phosphorylation (NDUFA4, COX7A2), and the HIF-1 signaling pathway. This endowed cells with a durable, "trained immune" resistance to damage.

[0011] 2. Second Module: ROS-Responsive Bipolar Targeting Lipid Nanoparticles (RRDT-LNP). This invention addresses the challenges of ovarian tissue barrier and granulosa cell-specific delivery by designing an environmentally responsive "smart bomb".

[0012] A two-stage sequential targeting design. The first stage of targeting is mediated by a CNGRC peptide embedded under a PEG layer. This peptide is "invisible" in the bloodstream and, upon reaching the high ROS microenvironment of the ovary, the ROS-sensitive thioacetyl (TK) linker breaks, PEG detaches, and CNGRC is exposed, mediating the efficient penetration of nanoparticles into the ovarian stroma / basement membrane barrier (in vitro penetration ability increased by 3.8 times). The second stage of targeting is mediated by the surface-exposed dual-targeting fusion protein VHH-FP. One end of this protein is an anti-AMHR2 single-domain antibody with an affinity of up to 3.2 nM (its amino acid sequence is shown in SEQ ID NO:1), and the other end is an anti-FSHR short peptide, achieving highly efficient dual-receptor-mediated endocytosis of granulosa cells (in vitro uptake positivity rate up to 84.6%, MFI increased by 3.7 times).

[0013] A high-purity, high-activity VHH-FP preparation process was developed. Through codon optimization, low-temperature induced soluble expression, and two-step chromatographic purification, a functional protein with a yield of up to 66 mg / L, purity >95%, and maintaining nanomolar affinity for both targets (AMHR2 KD=3.6 nM, FSHR KD=8.7 nM) was obtained, providing material support for the core material of this invention.

[0014] 3. Synergistic Application: Cell + Nanoparticle Combined Therapy

[0015] This invention organically integrates the above two modules, preferably using the thermosensitive hydrogel Pluronic F-127 as a carrier, and achieves local high concentration and long-lasting sustained release synergistic treatment through precise in situ injection into the ovary under ultrasound guidance.

[0016] Compared with the prior art, the present invention has the following significant and unexpected beneficial effects:

[0017] 1. Significant synergistic effect: In a chemotherapy-induced POF mouse model, the combined treatment group (G7) of this invention showed a synergistic effect that surpassed both single therapy and simple combination therapy in all indicators.

[0018] Endocrine function recovery: 12 weeks after treatment, serum anti-Müllerian hormone (AMH) levels in the combined treatment group recovered to 3.75 ng / mL, close to the normal group (4.10 ng / mL), and significantly higher than the cell-only group (2.45 ng / mL) and nanoparticle-only group (1.92 ng / mL); follicle-stimulating hormone (FSH) levels decreased to 16.3 mIU / mL, significantly lower than other treatment groups. The data indicate that the combined treatment synergistically promoted the fundamental recovery of ovarian reserve function.

[0019] Ovarian structure and reserve repair: The number of primordial follicles (24.8 / section) and the total number of healthy follicles (38.9 / section) in the combined treatment group were significantly better than those in other treatment groups. Histological HE staining showed that the ovarian structure and morphology were closest to the normal group, and the degree of fibrosis was the mildest. This is a synergistic result of cell therapy and targeted drugs rescuing residual follicles and improving the microenvironment.

[0020] Organelle function repair: The combined treatment group synergistically repaired mitochondrial dysfunction in granulocytes, with JC-1 red-green fluorescence ratio (5.95) and ATP content (11.8 nmol / 10⁻⁶). 4 The levels of mitochondrial ROS (1350 MFI) significantly increased, while those of pre-adapted cells significantly decreased. This reveals the intrinsic mechanism of synergistic effects at the energy metabolism level: pre-adapted cells improved the microenvironment, while targeted delivery of UA directly protected mitochondria in granulocytes, with the two mutually promoting each other and forming a virtuous cycle.

[0021] 2. Precise targeting of the lesion microenvironment

[0022] This invention demonstrates for the first time in a POF animal model that RRDT-LNP can "sensor" the high ROS environment of diseased ovaries in vivo and sequentially activate its dual targeting function. In vivo imaging showed that the accumulation of RRDT-LNP in the ovaries was 2.8 times that of the non-responsive group; co-localization of tissue sections confirmed that the nanoparticles can precisely deliver drugs to the cytoplasm of FSHR⁺ granulosa cells. This "on-demand release, precise recognition" characteristic maximizes efficacy while avoiding side effects caused by insufficient targeting or off-target effects.

[0023] 3. Excellent in vivo safety; a long-term safety evaluation lasting up to 12 weeks confirms the reliability of the present invention:

[0024] Non-tumorigenic: Sensitive methods such as Alu-PCR quantification and OCT4 / Ki67 immunohistochemical double staining were used to detect no teratoma formation or residual pluripotent stem cells in the ovarian tissue of all animals that received iPSC-OSC transplantation, which effectively addresses the most critical safety concerns in the stem cell field.

[0025] Low immunogenicity and biocompatibility: No acute toxicity, hematological abnormalities, or abnormal liver and kidney function were observed. No abnormalities were found in T cell subsets or local CD3⁺ infiltration, indicating that the human cells and humanized nanomaterials constructed in this invention have good immunocompatibility.

[0026] In summary, this invention abandons the traditional single-treatment approach and pioneers a synergistic treatment platform combining "empowered cells + intelligent targeted drugs." This platform not only demonstrates groundbreaking efficacy and synergistic effects in POF treatment, but its design concept (systemic cellular metabolic reprogramming + microenvironment sequential response targeting) also provides a novel technological paradigm for precision cell-based drug therapy in other tissue fibrosis, ischemia-reperfusion injury, and degenerative diseases, possessing high clinical translational value and broad industrialization prospects. Attached Figure Description

[0027] Figure 1 SDS-PAGE analysis results after VHH-FP purification.

[0028] Figure 2 Flow cytometry results of the efficiency of RRDT-LNP uptake by KGN cells. Detailed Implementation

[0029] 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 invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0031] Example 1: Directed differentiation of hiPSCs into ovarian Sertoli cells and FTY720 preconditioning treatment

[0032] 1.1 Targeted Differentiation Process

[0033] 1.1.1 Cells and Reagents

[0034] (1) Cell line: Clinical-grade human induced pluripotent stem cells (hiPSC) were used.

[0035] (2) Main reagents:

[0036] Matrix gel: Recombinant human vitronectin (VTN-N, TransGen Biotech), diluted with DPBS to a working concentration of 0.5 µg / cm before use. 2 .

[0037] Basic culture medium: mTeSR TM Plus(STEMCELL Technologies).

[0038] Differentiation-inducing factors: CHIR99021 (MedChemExpress), prepared as a 10 mM stock solution using DMSO. Recombinant human BMP4 (Huamei Biotechnology), prepared as a 10 µg / mL stock solution using 4 mM HCl containing 0.1% BSA. Recombinant human FGF2 (MedChemExpress), prepared as a 25 µg / mL stock solution using PBS containing 0.1% BSA. All-trans retinoic acid (RA, Sigma), prepared as a 1 mM stock solution using DMSO, stored protected from light. Recombinant human WNT4 (MedChemExpress), prepared as a 50 µg / mL stock solution using PBS containing 0.1% BSA. RSPO1 (MedChemExpress), prepared as a 200 µg / mL stock solution using PBS. SB431542 (MedChemExpress), prepared as a 10 mM stock solution using DMSO.

[0039] Differentiation media: Phase I (Days 0-2): RPMI 1640 medium (Gibco) supplemented with 1×B27 Supplementminus insulin (Gibco). Phase II (Days 3-5): DMEM / F12 medium (Gibco) supplemented with 1×B27 Supplement (Gibco). Phase III (Days 6-12): Customized medium, DMEM / F12 as the base medium supplemented with 1×N2 Supplement (Gibco) and 1×Non-essential amino acids (NEAA). Phase IV (Days 13-20): Ovarian granulosa cell maturation medium (OGCM, ScienCell), supplemented with 50 mIU / mL recombinant human FSH and 10 nM dihydrotestosterone (DHT).

[0040] 1.1.2 Differentiation Steps

[0041] Day -1 (Planting Preparation): Using a 6-well cell culture plate, add 1.5 mL of VTN-N working solution (0.5 µg / cm²) to each well. After coating at room temperature for 1 hour, aspirate the liquid and add 2 mL of mTeSR to each well. TM Plus culture medium is available for use.

[0042] Day 0 (Initiation of Differentiation): When hiPSC confluence reaches 80-85%, discard the old culture medium and gently wash once with DPBS. Add 1 mL of Accutase (STEMCELL Technologies) to each well and incubate at 37°C for 5 minutes until cell edges curl up and intercellular spaces increase. Add 2 mL of mTeSR containing 10 µM Y-27632 (ROCK inhibitor). TM Digestion was terminated with Plus medium, and the cells were gently pipetted to form a single-cell suspension. After cell counting, the cells were cultured at 5.0 × 10⁻⁶ cells / mL. 4 cells / cm 2 The density (i.e., approximately 2.5 × 10⁻⁶ per pore) 5 (Number of cells) were resuspended in Stage I medium (containing 6 µM CHIR99021 and 10 ng / mL BMP4) and seeded into pre-coated 6-well plates, with a final volume of 2 mL per well. This day was marked as Day 0 of differentiation (D0). The culture plates were placed in an incubator at 37°C, 5% CO2, and >95% humidity. The medium was completely changed daily using fresh Stage I induction medium.

[0043] Day 2 (Primitive Streak Identification): One well was randomly selected, and cells were digested with Accutase to form a single-cell suspension. The cells were washed twice with DPBS (FACS buffer) containing 2% FBS. Surface staining was performed using anti-Brachyury(T) antibody (APC conjugate) at the recommended dilution (1:20) and incubated at 4°C in the dark for 30 minutes. After washing twice with FACS buffer, flow cytometry was used for detection. Thresholds were set using unstained cells and isotype control antibody (APC Mouse IgG1κ Isotype Control). Flow cytometry analysis of three independent differentiation experiments (n=3) showed that the proportion of Brachyury(T) positive cells in the D2 cell population was 84.2% in Experiment 1, 81.7% in Experiment 2, and 87.5% in Experiment 3. The average positive rate was (84.5 ± 2.9)%, meeting the quality control standard of >80%.

[0044] Days 3-5 (Intermediate Mesoderm induction): On day 2, completely aspirate stage I medium and add 2 mL of pre-warmed stage II medium (containing 20 ng / mL FGF2 and 0.5 µM RA) to each well. Completely change the medium daily.

[0045] Day 5 (D5) Gene expression identification: Total RNA was extracted using the RNeasy Mini Kit, and its concentration and purity (A260 / A280 > 1.9) were measured. SuperScript was used. TMcDNA was synthesized using IV reverse transcriptase. Quantitative PCR (qPCR) was performed using TaqMan GeneExpression Assays. Target genes: LHX1, OSR1. Internal reference gene: GAPDH. StepOnePlus was used. TM The real-time PCR system was used, with reaction conditions of 95°C for 10 minutes, followed by 95°C for 15 seconds and 60°C for 1 minute, for a total of 40 cycles. Relative expression levels were calculated using the 2^(-ΔΔCt) method, with the expression level of D0 hiPSC as the baseline (set as 1). The results are shown in Table 1. In D5 cells, the mRNA expression levels of the key intermediate mesoderm marker genes LHX1 and OSR1 were significantly upregulated by approximately 155-fold and 135-fold, respectively, compared to D0 hiPSC (p values ​​were both less than 0.0001, one-way ANOVA), demonstrating successful induction of an intermediate mesoderm cell population.

[0046] Table 1 Relative expression fold

[0047]

[0048] Days 6-12 (Gonadal cell fate induction): On D5, completely aspirate the Stage II medium and add 2 mL of pre-warmed Stage III medium (containing 50 ng / mL WNT4, 200 ng / mL RSPO1, and 3 µM SB431542) to each well. Change the medium every other day.

[0049] Days 13-20 (granular cell maturation): On day 12, completely aspirate stage III medium and add 2 mL of OGCM complete medium to each well (with additional 50 mIU / mL recombinant human FSH (MedChemExpress) and 10 nM dihydrotestosterone (DHT, Sigma). Completely change the medium every other day.

[0050] Terminal flow cytometry identification on day 20 (D20): Digested cells were used as single-cell suspensions and washed with FACS buffer. Surface (AMHR2) and intracellular (FOXL2) staining was performed using antibodies. Procedure: Surface staining was first performed using APC-labeled mouse anti-human AMHR2 antibody (1:20) at 4°C for 30 minutes. After washing, cells were fixed and permeabilized with Foxp3 / Transcription Factor Staining Buffer Set, followed by intracellular staining with PE-labeled mouse anti-human FOXL2 antibody (1:50) at 4°C for 30 minutes. Flow cytometry analysis was performed, and data analysis was conducted using FlowJo v10.8 software. Gating strategy: Live cells (FSC-A / SSC-A) were first selected, while adherent cells (FSC-H / FSC-A) were excluded. Then, the APC and PE double-positive cell populations were analyzed. Results are shown in Table 2. After 20 days of directed differentiation, cell populations using FOXL2 and AMHR2 as markers were successfully obtained. Flow cytometry analysis of 12 samples from three independent replicate experiments confirmed that the average proportion of the double-positive cell population was (45.2±6.8)%, demonstrating the reproducibility and stability of the differentiation process. This cell population was defined as "iPSC-derived ovarian support cells (iPSC-OSCs)" and can be used as the starting material for the cell module of this invention.

[0051] Table 2 FOXL2 + AMHR2 + Percentage of double-positive cells

[0052]

[0053] 1.2 FTY720 Pre-adaptation Processing and Functional Verification

[0054] 1.2.1 Pre-adaptation treatment

[0055] iPSC-OSCs identified after D20 were collected, digested with Accutase, and single-cell suspensions were prepared. After cell counting, the cells were sputtered at 1.0 × 10⁻⁶ cells / mL. 6 The cells / mL density was precisely resuspended in OGCM complete medium.

[0056] Experimental group: FTY720 (Selleck) stock solution (10 mM in DMSO) was added to the cell suspension to bring the final concentration to 1.0 µM.

[0057] Control group: Add an equal volume of DMSO solvent (final concentration 0.01% v / v).

[0058] The cell suspension was aliquoted into 6-well plates with low adsorption (2 mL per well) and incubated at 37°C in a 5% CO2 incubator for 16 hours, with a gentle shake once during the incubation period to ensure that the cells are fully exposed to the drug.

[0059] 1.2.2 Oxidative stress tolerance test

[0060] Cell seeding: After pretreatment, centrifuge the cell suspension (300g, 5 minutes) and wash once with fresh OGCM to remove free FTY720 / DMSO. After resuspending and counting, seed at 1.0 × 10⁻⁶. 4 Cells were seeded at a density of 100 µL per well in black 96-well clear plates coated with Poly-D-Lysine. Cells were cultured for 24 hours to allow them to adhere.

[0061] Oxidative stress induction: The old culture medium was discarded, and the experimental group was replaced with OGCM containing 400 µM H2O2 (freshly prepared); the control group was replaced with ordinary OGCM. The culture plates were returned to the incubator for 6 hours.

[0062] Double staining of live and dead cells: After treatment, add 100 µL of DPBS staining working solution containing 2 µM Calcein-AM (green fluorescent dye for live cells) and 4 µM PI (propidium iodide, red fluorescent dye for dead cells) to each well. Incubate at 37°C in the dark for 30 minutes.

[0063] Quantitative analysis: A PerkinElmer Operetta high-content imaging system with a 20x objective lens was used to automatically capture images of nine fields of view at the center of each well. Excitation / emission wavelengths: Calcein-AM (488 nm / 515-560 nm), PI (561 nm / 570-650 nm). Analysis was performed using Harmony® 4.9 software. The analytical workflow was as follows: ① Identify and count all live cells based on Calcein-AM fluorescence; ② Identify and count all dead cells based on PI fluorescence; ③ Calculate the percentage of live cells: live cell count / (live cell count + dead cell count) × 100%. The experiment was repeated six times (n=6), with three replicates per well. Data are expressed as mean ± standard deviation.

[0064] The results showed that the viable cell percentages in the FTY720 pretreatment group were 79.5%, 76.8%, 81.2%, 75.4%, 77.9%, and 78.8%, with a mean of (78.3 ± 2.1)%. The viable cell percentages in the DMSO control group were 40.1%, 44.5%, 38.2%, 42.8%, 39.7%, and 43.7%, with a mean of (41.5 ± 2.4)%.

[0065] Statistical analysis: Using GraphPad Prism 9.0 software, an unpaired two-tailed Student's t-test was employed to compare the two groups of data. The calculated t=28.67, and the p-value <0.0001.

[0066] In summary, after 16 hours of pretreatment with 1 µM FTY720, the survival rate of iPSC-OSCs under acute oxidative stress of 400 µM H2O2 was significantly higher (78.3%) than that of the untreated control group (41.5%), representing a relative increase of nearly 90%. This demonstrates that FTY720 preconditioning effectively enhances the ability of iPSC-OSCs to resist oxidative damage.

[0067] 1.2.3 Gene expression profiling analysis (RNA-seq)

[0068] Sample preparation and sequencing: iPSC-OSCs (3 biological replicates each) pretreated with FTY720 and DMSO were collected, and total RNA was extracted using the TRIzol method. After passing quality control (RIN value > 9.0), 150 bp paired-end sequencing was performed using the Illumina NovaSeq6000 platform, with a sequencing depth of approximately 40 million reads per sample.

[0069] Bioinformatics analysis: Data underwent FastQC quality control, and clean reads were aligned to the human reference genome (GRCh38.p13) using HISAT2. Gene expression counts were calculated using featureCounts and input into the DESeq2 R package for differential expression analysis. The significance threshold was set as follows: corrected p-value (FDR) < 0.05, and absolute value of the fold change in expression (log2FoldChange) > 1.

[0070] Pathway enrichment results: Differential gene statistics showed that, compared with the control group, 1248 genes were significantly upregulated and 876 genes were significantly downregulated in the FTY720 pretreatment group.

[0071] Table 3 Examples of key upregulated genes (related to antioxidant and stress adaptation)

[0072]

[0073] Pathway enrichment analysis: KEGG pathway enrichment analysis was performed on differentially upregulated genes using clusterProfiler. The most significantly enriched pathways (FDR < 0.01) included: oxidative phosphorylation; glutathione metabolism; HIF-1 signaling pathway; and peroxisome.

[0074] In summary, transcriptome sequencing analysis revealed the role of FTY720 preconditioning at the molecular level. Data showed that preconditioning not only activated classic antioxidant enzyme systems (such as GPX3, SOD2, and HMOX1), but also upregulated the expression of key components of the mitochondrial electron transport chain (such as NDUFA4 and COX7A2) and enhanced cellular preconditioning to hypoxic environments (HIF-1 pathway activation). This systematically explains why cells exhibit stronger functional tolerance to oxidative stress: namely, by enhancing endogenous antioxidant defenses and optimizing the energy metabolism base. These data indicate that preconditioning is a systemic "cellular empowerment" process from phenotype to genome, rather than a non-specific, transient effect.

[0075] Example 2: Construction, characterization and functional verification of ROS-responsive dual-level targeted lipid nanoparticles (RRDT-LNP)

[0076] 2.1 Chemical Synthesis and Characterization of Key Functional Materials

[0077] 2.1.1 Synthesis of ROS-responsive linker-lipid precursor (TK-PEG-LBP)

[0078] (1) Target structure: 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 5000-[thioketal]-cyclic peptide [CNGRC] (abbreviated as TK-PEG-LBP).

[0079] (2) Synthesis steps:

[0080] Synthesis of the thioketal linker (TK-COOH): Under nitrogen protection and an ice bath (0°C), 1,4-dioxa-8-thiaspiro[4.6]undecane (2.08 g, 10.0 mmol) and anhydrous tetrahydrofuran (40 mL) were added dropwise over 30 minutes with vigorous stirring. After the addition was complete, stirring was continued at 0°C for 45 minutes. Subsequently, the reaction mixture was transferred to a pre-cooled (-78°C) container filled with dry carbon dioxide, and the reaction was maintained at this temperature with continuous CO2 purging for 2 hours. After slowly warming to room temperature, the mixture was carefully quenched with 1 M HCl aqueous solution to pH ~3-4. The mixture was extracted with ethyl acetate (3 × 50 mL), the organic phases were combined, washed once with saturated brine, and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation after filtration, yielding a pale yellow oily crude product. Purification was achieved by silica gel column chromatography (eluting gradient: from petroleum ether / ethyl acetate = 10:1 to 4:1, v / v) to give a white solid product, TK-COOH. Yield: 72% (2.12 g). High-resolution mass spectrometry (HR-ESI-MS): m / z [MH]⁻ Calculated value C 10 H 15 O4S2 - , 263.0387; measured value, 263.0383.

[0081] Activation of DSPE-PEG5000-NH2: DSPE-PEG5000-amino (Avanti Polar Lipids, 200 mg, ~33.3 µmol) was dissolved in 5 mL of anhydrous dichloromethane. Under nitrogen protection and an ice bath, N,N'-diisopropylcarbodiimide (DIC, 15.6 µL, 100 µmol) and N-hydroxysuccinimide (NHS, 11.5 mg, 100 µmol) were added sequentially. The ice bath was removed, and the reaction was stirred at room temperature for 5 hours. The reaction progress was monitored by thin-layer chromatography (TLC, developing solvent: chloroform / methanol / water = 65:25:4) (starting agent Rf ≈ 0.1, product agent Rf ≈ 0.7).

[0082] Coupling of the TK-PEG intermediate: The TK-COOH (10.5 mg, 40 µmol) obtained in step 1 was dissolved in 1 mL of anhydrous dimethyl sulfoxide (DMSO) and added dropwise to the reaction solution from step 2. A catalytic amount of 4-dimethylaminopyridine (DMAP, 1.0 mg) was then added. The reaction mixture was stirred overnight (18 hours) at room temperature in the dark. After the reaction was complete, the reaction mixture was added dropwise to 30 mL of cold diethyl ether to precipitate the product. The precipitate was collected by centrifugation (4000 g, 10 min), washed twice with cold diethyl ether, and dried under vacuum overnight to obtain the white waxy solid intermediate DSPE-PEG5000-TK-COOH.

[0083] Conjugation of cyclic peptide CNGRC: Pre-synthesized cyclic peptide CNGRC (sequence: Cys-Asn-Gly-Arg-Cys, Shanghai Sangon Biotech, HPLC purity >98%) was purchased. DSPE-PEG5000-TK-COOH (approximately 33 µmol) and cyclic peptide CNGRC (25 mg, 45 µmol) were dissolved in 3 mL of anhydrous DMSO. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 10 mg, 52 µmol) and N-hydroxythiosuccinimide (sulfo-NHS, 12 mg, 55 µmol) were added. The pH was adjusted to 7.5 with triethylamine, and the reaction was carried out at room temperature in the dark with stirring for 8 hours.

[0084] Purification and identification: The reaction solution was diluted with 0.1 M EDTA·2Na solution (pH 8.0, 30 mL) and transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3.5 kDa. Dialysis was performed against ultrapure water at 4°C for 48 hours (water changed every 8 hours). Lyophilization yielded a white flocculent solid product, TK-PEG-LBP. Final yield (based on DSPE-PEG5000-NH2): ~45% (95 mg).

[0085] (3) Structural verification:

[0086] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS): Using α-cyano-4-hydroxycinnamic acid (CHCA) as the matrix, the main peak was detected in positive ion linear mode. The mass number distribution ranged from 6800 to 7200 Da, with the peak at ~6950 Da, consistent with the target molecular weight (theoretical average molecular weight of DSPE-PEG5000-TK-CNGRC ~6.9 kDa, considering the polydispersity of PEG).

[0087] In summary, a well-defined ROS-responsive functional lipid, TK-PEG-LBP, was synthesized via a three-step chemical coupling process. The optimized synthesis process increased the final yield to 45%, providing a material basis for the large-scale, reproducible preparation of subsequent nanoparticles.

[0088] 2.1.2 Expression and purification of dual-targeting fusion protein (VHH-FP)

[0089] First, using alpaca immunization and phage display technology, a high-affinity single-domain antibody (VHH) specifically targeting human AMHR2 was successfully screened and obtained. Specifically, 6-month-old male alpacas were immunized subcutaneously at multiple sites four times, with two-week intervals, using recombinant human AMHR2 (MedChemExpress) as the immunogen. Peripheral blood was collected on day 7 after the last immunization, and total RNA was extracted from lymphocytes. The VHH gene fragment was amplified by nested PCR, constructing a library with a capacity of 1.2 × 10⁻⁶. 9 A phage display library of CFU was created. Three rounds of solid-phase affinity panning were then performed using a strategy of decreasing coating concentrations (10 μg / mL, 5 μg / mL, 2 μg / mL), with increasing washing intensity in each round. Ninety-four clones were screened by Phage-ELISA, yielding a clone with a highly significant positive signal, named AMHR2-VHH-C6. The full-length VHH sequence of this clone is shown in SEQ ID NO:1. The positive clone was subcloned into the pET25b vector, and after IPTG-induced expression and Ni-NTA purification, soluble VHH protein with a purity >95% was obtained. Affinity was determined using surface plasmon resonance (Biacore T200): AMHR2 protein was immobilized on a CM5 chip to a response value of 500 RU, and VHH concentration gradients (0, 1.25, 2.5, 5, 10, 20 nM) were injected. Kinetic fitting showed a binding rate constant ka = 1.26 × 10⁻⁶. 6 M -1 s -1 The dissociation rate constant kd = 4.03 × 10 -3 s -1 The affinity constant KD = 3.2 × 10⁻⁶ -9 M (3.2 nM). To objectively evaluate the affinity level of the single-domain antibody obtained in this invention, it was compared with existing AMHR2-targeting molecules, among which the anti-AMHR2 monoclonal antibody (AMHR2 antibody (ab197148)) has an affinity Kd of approximately 10 nM. Therefore, our prepared AMHR2-VHH-C6 has a higher affinity.

[0090] Gene construction and plasmids: The fusion gene (VHH-FP) encoding the anti-human AMHR2 single-domain antibody VHH (SEQ ID NO:1), the flexible linker peptide (GGGGS)3, and the FSHR-binding peptide (YTRDLVYKDPARPKIQKTCTF) was codon-optimized (the optimized nucleotide sequence is shown in SEQ ID NO:2), synthesized by GenScript and cloned into the pET-28a(+) expression vector, with a His6 tag and a thrombin cleavage site (LVPRGS) introduced at the N-terminus.

[0091] Protein expression: The validated recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. A single colony was picked and inoculated into 5 mL of LB medium containing 50 µg / mL kanamycin, and cultured overnight at 37°C with shaking at 220 rpm to obtain the seed culture. The seed culture was then transferred at a 1:100 ratio to 500 mL of 2×YT medium (containing 50 µg / mL kanamycin) and cultured at 37°C until OD500. 600 ≈ 0.6-0.8. Add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, lower the culture temperature to 25°C, and induce expression for 16 hours.

[0092] Cell lysis and preparation of soluble supernatant: Collect bacterial cells by centrifugation at 6000 g for 15 minutes at 4°C. Resuspend the cells in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 1 mM PMSF, pH 8.0) and sonicate on ice (300 W, 3 seconds on, 5 seconds off, total 30 minutes). Centrifuge at 12000 g for 45 minutes at 4°C, collect the supernatant, and filter through a 0.45 µm filter membrane.

[0093] Ni-NTA affinity chromatography: The filtered supernatant was loaded into a 5 mL Ni Sepharose High Performance column pre-equilibrated with lysis buffer at a flow rate of 1 mL / min. Contaminating proteins were washed with 10 column volumes (CV) of lysis buffer. Step elution was performed with elution buffer containing 250 mM imidazole (20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 8.0), and the elution peak was collected.

[0094] Thrombin-mediated His tag removal: Immediately replace the eluted protein with a desalting column (HiPrep 26 / 10 Desalting) to the cutting buffer (20 mM Tris-HCl, 150 mM NaCl, 2.5 mM CaCl2, pH 8.0). Add 1 unit of bovine thrombin per milligram of fusion protein and gently cleave at room temperature (22°C) for 4 hours with agitation.

[0095] Reverse Ni-NTA purification and gel filtration: The cleaved mixture was reloaded onto a Ni-NTA column. Unlabeled VHH-FP was present in the flow-through, which was collected. Finally, fine purification was performed using a Superdex 75 Increase 10 / 300 GL gel filtration chromatography column with PBS (pH 7.4) as the buffer. A single symmetrical main peak eluted at approximately 13–14 mL was collected.

[0096] Purity and molecular weight: SDS-PAGE (12% Bis-Tris gel, reduction conditions) showed ( Figure 1 The chromatogram showed a single band at approximately 18 kDa, consistent with the theoretical molecular weight. Grayscale analysis of the Coomassie Brilliant Blue stained gel using ImageJ software showed a purity > 95%.

[0097] Concentration and yield: Using a spectrophotometer, based on absorbance at 280 nm (A0.05). 280 Protein concentration was calculated using the theoretical molar extinction coefficient. On average, approximately 66 mg of purified VHH-FP could be obtained from 1 L of culture.

[0098] Dual-target activity validation (surface plasmon resonance, SPR): A Biacore 8K system was used. Recombinant human AMHR2-Fc and FSHR-Fc were immobilized in CM5 chip channels, respectively. VHH-FP flowed through the chip at a concentration gradient (0-200 nM). Kinetic fitting results showed that the equilibrium dissociation constants (KD) of VHH-FP for AMHR2 and FSHR were (3.6 ± 0.4) nM and (8.7 ± 0.1) nM, respectively, indicating that it maintained high affinity binding to both targets simultaneously.

[0099] In summary, a reproducible expression and purification process for the VHH-FP fusion protein was successfully established. Lowering the induction temperature to 25°C effectively promoted soluble expression; the optimized two-step chromatography strategy (affinity + molecular sieve) achieved the preparation of high-purity, highly active proteins. SPR data confirmed the rationality of the design, laying the foundation for constructing fully functional targeted nanoparticles.

[0100] 2.2 Preparation, characterization and ROS responsiveness verification of RRDT-LNP

[0101] 2.2.1 Nanoparticle Assembly and Surface Modification

[0102] Lipid formulation: The precise lipid molar composition of RRDT-LNP (total lipid concentration fixed at 12.5 mM in ethanol phase) is as follows: DLin-MC3-DMA: 50%; DSPC: 10%; cholesterol: 38.5%; TK-PEG-LBP: 1%; DSPE-PEG2000-Mal: 0.5%.

[0103] Microfluidic assembly parameters: Using Precision NanoSystems' NanoAssemblr® Ignite system and "staggered herringbone" hybrid chip.

[0104] Ethanol phase: Weigh each lipid precisely according to the above formula, dissolve in anhydrous ethanol, vortex and heat briefly in a water bath (40°C) until completely clear.

[0105] Aqueous phase: Dissolve urostatin A (UA) or the lipophilic fluorescent dye DiD in 25 mM sodium citrate buffer (pH 4.0). For drug-loaded LNPs, the UA concentration is 1 mg / mL; for fluorescently labeled LNPs, the DiD concentration is 0.02 mg / mL.

[0106] Mixing parameters: The flow rate ratio of the ethanol phase to the water phase was set to 1:3 (v / v), and the total flow rate was 12 mL / min. The two phases were instantaneously mixed in the chip mixing chamber at 22-25°C (total contact time <10 ms). The eluent was immediately collected into a glass bottle.

[0107] LNP formation and initial purification: The collected solution was incubated at room temperature for 30 minutes to allow the lipid bilayer to close spontaneously and the vesicles to mature. Subsequently, the solution was dialyzed and concentrated using a KrosFlo® tangential flow filtration system equipped with a 100 kDa MWCO hollow fiber column against 10 times the volume of PBS (pH 7.4) to completely remove ethanol, free drugs / dyes, and buffer salts, and then replaced with neutral PBS.

[0108] (1) Targeted ligand conjugation and final purification:

[0109] Site-directed thiolization of VHH-FP: Purified VHH-FP was reacted with 15 molar excess of Traut's Reagent (2-iminothiocyclopentane hydrochloride) in PBS containing 1 mM EDTA at room temperature for 1 hour. Immediately after reaction with Zeba... TMExcess reagents were removed by a Spin desalting column (7K MWCO) to obtain thiolated VHH-FP (an average of 1.2 thiol groups were introduced into each protein molecule, as determined by Ellman's reagent).

[0110] Coupling reaction: Thiolized VHH-FP was mixed with the purified "pre-coupled LNP" (with maleimide groups on the surface) at a ligand:Mal-lipid molar ratio of 0.8:1. The mixture was reacted with gentle stirring on a rotary mixer at 4°C under nitrogen protection for 16 hours.

[0111] Final purification and sterilization: Purification was performed again using tangential flow filtration against PBS to remove unreacted protein. Finally, the product RRDT-LNP was obtained by filtration through a 0.22 µm polyethersulfone sterile filter and stored at 4°C protected from light.

[0112] (2) Preparation of control LNP:

[0113] NR-ST-LNP: Replace TK-PEG-LBP in the formulation with an equimolar amount of the non-cuttable control material DSPE-PEG5000-CNGRC (where PEG and CNGRC are linked by a stable amide bond and have no thioketal structure), and the other steps are exactly the same.

[0114] NT-LNP: Replace both TK-PEG-LBP and DSPE-PEG2000-Mal in the formula with an equimolar amount of DSPE-PEG2000, and skip the protein coupling step.

[0115] 2.2.2 Physicochemical Characterization

[0116] (1) Particle size, polydispersity index (PDI), and zeta potential: These were determined using a dynamic / static light scattering instrument. Data for RRDT-LNP (n=5 independent batches): average hydrated particle size (Z-average) was 105.3 ± 3.2 nm; polydispersity index (PDI) was 0.078 ± 0.015; and zeta potential was +2.1 ± 0.8 mV. The particle sizes of NR-ST-LNP and NT-LNP were 106.8 ± 2.9 nm and 104.5 ± 4.1 nm, respectively, with no statistically significant difference in PDI and potential (p>0.05).

[0117] (2) Determination of encapsulation efficiency (EE%) and drug loading (DL%):

[0118] Total drug content determination: Take 100 µL of LNP suspension, add 900 µL of methanol, vortex thoroughly to break the emulsion, let stand at room temperature for 15 minutes, centrifuge at 15000 g for 10 minutes, collect the supernatant, and filter through a 0.22 µm filter membrane. HPLC analysis was performed. Chromatographic conditions: C18 column (4.6 × 150 mm, 5 µm); mobile phase: acetonitrile / water (containing 0.1% formic acid) = 55:45; flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 280 nm. The total drug content (W) was calculated based on the UA standard curve. total ).

[0119] Determination of free drug content: Take another 100 µL of LNP suspension and place it in a 100 kDa ultrafiltration centrifuge tube, centrifuge at 14000 g for 30 minutes. Collect the filtrate and determine the free drug content (Wf) using the same method. ree ).

[0120] Total lipid mass determination: The total phospholipid content in LNP suspension was determined using a phospholipase D / choline oxidase colorimetric assay kit and converted to total lipid mass (W). lipi d).

[0121] Calculation and Result: EE% = (W total - Wf ree ) / W total × 100%; DL% = (W total - Wf ree / W lipi d×100%. RRDT-LNP data (n=3): EE% = 91.8 ± 2.1%; DL% = 4.5 ± 0.3%.

[0122] 2.2.3 Deep Verification of ROS Responsiveness

[0123] (1) Simulation of response conditions: To simulate the high oxidative stress environment of diseased tissue, a Fenton reaction system was established: H2O2 (final concentration 1.0 mM) and FeSO4 (final concentration 100 µM) were added to PBS (pH 7.4). RRDT-LNP and NR-ST-LNP were added to this ROS system or ordinary PBS (control), respectively, with a final lipid concentration of 0.2 mg / mL, and incubated at 37°C with constant temperature shaking.

[0124] (2) Monitoring of characterization changes:

[0125] Particle size and PDI kinetic monitoring: Samples were taken at 0, 15, 30, 60, and 120 minutes of incubation and measured immediately using DLS. Results showed that after 30 minutes of incubation in the ROS system, the average particle size of RRDT-LNP significantly decreased from 105.3 nm to 82.6 ± 2.8 nm (a decrease of approximately 21.5%), while the PDI increased from 0.078 to 0.121 ± 0.020. In ordinary PBS, the particle size remained stable (103.8–106.5 nm) over 120 minutes. The particle size of NR-ST-LNP showed no significant change under both conditions (p>0.05).

[0126] Direct chemical evidence of ligand exposure (gel permeation chromatography, GPC): A TSKgel G3000SWXL column was used with PBS as the mobile phase at a flow rate of 0.5 mL / min. The detectors were a refractive index detector (RID) and a fluorescence detector (Ex / Em = 280 / 350 nm, used to track tryptophan-containing VHH-FP). The elution curves of RRDT-LNP before and after ROS treatment were compared. The results showed that the RID signal of the treated sample exhibited a new, independent peak at a short retention time (corresponding to the large molecular weight PEG fragment), and the fluorescence detector also detected the VHH-FP signal at the corresponding retention time. This directly demonstrates that ROS triggers the dissociation of the high molecular weight PEG-VHH-FP fragment from the LNP.

[0127] Functional validation of ligand exposure (ELISA): 96-well plates were coated with streptavidin. LNP samples with different treatments (pre-incubated with biotinylated goat anti-VHH polyclonal antibody) were added to the wells, washed, and then HRP-streptavidin was added. TMB was used for color development, and the absorbance at 450 nm (OD) was measured. 450 The results showed that the OD of the ROS-treated RRDT-LNP sample... 450 The value was 1.25 ± 0.15, which was about 7 times that of the untreated group (0.18 ± 0.05) (p < 0.001) and also significantly higher than that of the ROS-treated NR-ST-LNP group (0.22 ± 0.06).

[0128] 2.3 Validation of in vitro sequential targeting and cellular uptake

[0129] 2.3.1 Establishment and optimization of in vitro follicular microenvironment simulation model

[0130] Human ovarian stromal fibroblasts (HOSFs): isolated from surgically obtained human ovarian cortical tissue, purified by CD90⁺ immunomagnetic bead sorting (Miltenyi Biotec), and cultured in DMEM / F12 (containing 10% FBS and 1% penicillin-streptomycin). Cells from passages 3-6 were used in the experiments.

[0131] Human ovarian granulosa cell line (KGN): Routinely cultured using DMEM / F12 (containing 10% FBS). AMHR2 and FSHR expression rates were periodically assessed by flow cytometry (both >90%).

[0132] Establishment of a laminin-coated Transwell co-culture model: To more realistically simulate the follicular basement membrane, the Transwell nest (polycarbonate membrane, 3.0 µm pore size) was pretreated: the bottom surface of the upper chamber membrane was coated overnight at 4°C with 10 µg / mL recombinant human laminin (Lamining-511). KGN cells (2 × 10⁶ cells / well) were seeded in the lower chamber (24-well plate substrate). 4 Cells / well), cultured overnight for adhesion. The next day, the coated Transwell was inserted into the upper chamber, and HOSF cells (1×10⁶) were seeded in the upper chamber. 5 The co-culture system was maintained in a 37°C incubator containing 5% CO2 for 48 hours to allow the cells to secrete a more complex interface. Six hours before nanoparticle treatment, 100 µM H2O2 was added to the lower chamber medium to stimulate oxidation and simulate the oxidative stress microenvironment of diseased ovaries ("+ROS" group); a "-ROS" control group without H2O2 was also established.

[0133] 2.3.2 Primary Targeting: Assessment of Nanoparticle Penetration Ability

[0134] Procedure: DiD-labeled RRDT-LNP, NR-ST-LNP, and NT-LNP were added to the upper chamber medium of the co-culture system (final DiD concentration: 1 µM, corresponding to a lipid concentration of ~20 µg / mL). Co-culture was continued at 37°C for 4 hours.

[0135] Sample detection: After culture, carefully remove the Transwell insert. Collect the lower chamber culture medium and centrifuge at 1500 g for 5 minutes to remove any cell debris that may have detached. Take 200 µL of supernatant and measure its fluorescence intensity (Ex / Em = 644 / 665 nm) using a multi-mode microplate reader to quantify the nanoparticles that have penetrated from the HOSFs layer to the KGN side and are free in the culture medium.

[0136] The results are shown in Table 4: In the absence of ROS stimulation, there was no significant difference in the penetration ability of the three LNPs (penetration fold 1.0-1.3 times), indicating that the penetration ability of the simple CNGRC peptide (NR-ST-LNP) or the unactivated RRDT-LNP is limited. However, under the simulated pathological microenvironment conditions in the presence of ROS, the penetration amount of RRDT-LNP surged to 3.8 times that of NT-LNP, while NR-ST-LNP did not exhibit this effect. This suggests that ROS-triggered PEG deshielding and exposure of the CNGRC peptide (primary target) are key steps for RRDT-LNP to achieve efficient penetration of the cell layer / simulated basement membrane barrier, consistent with the designed "sequential targeting" logic.

[0137] Table 4 Summary and Analysis of Sample Test Results

[0138]

[0139] Statistical analysis: Two-way ANOVA was used, followed by Tukey's multiple comparison test. ** indicates that compared with the "-ROS" group of the same nanoparticles, p < 0.001; compared with the other two nanoparticles under the "+ROS" condition, p < 0.001.

[0140] 2.3.3 Secondary Targeting: Assessment of Cell-Specific Uptake

[0141] Procedure: After completing the penetration experiment, KGN cells in the lower chamber were digested with 0.25% trypsin-EDTA and collected. The cells were washed twice with PBS containing 2% FBS and resuspended in 200 µL PBS.

[0142] Flow cytometry analysis: Detection was performed using a flow cytometer. First, viable cell populations were gating using forward scattered light (FSC-A) and side scattered light (SSC-A), then adherent cells were excluded using FSC-H vs FSC-A. 10,000 single cells were analyzed in the DiD channel (using a 660 / 20 nm filter), and the percentage of DiD-positive cells and the mean fluorescence intensity (MFI) of the cell population were recorded.

[0143] The results are shown in Table 5 and Figure 2 As shown, in the ROS-triggered pathological microenvironment, RRDT-LNP was taken up by KGN cells at an extremely high efficiency (positive rate 84.6%, MFI 12450), which was approximately 2.6 times (positive rate) and 3.7 times (MFI) higher than that of the single-target NR-ST-LNP. Crucially, in the absence of ROS, the uptake level of RRDT-LNP was comparable to that of NR-ST-LNP, once again irrefutably demonstrating its "conditional activation" characteristic.

[0144] By combining "penetration" and "uptake" experiments, in vitro data validated the design logic of RRDT-LNP: it remains "invisible" under normal conditions; under diseased (high ROS) conditions, it sequentially performs "ROS response demasking -> CNGRC-mediated penetration / enrichment -> VHH-FP-mediated high-efficiency memory".

[0145] Table 5 Summary of Cell-Specific Uptake Results

[0146]

[0147] Statistical analysis: ** indicates that compared with the "-ROS" group of the same nanoparticles, p < 0.001; compared with the other two nanoparticles under the "+ROS" condition, p < 0.001.

[0148] Example 3: Evaluation of synergistic therapeutic effects, mechanisms and long-term safety in animals

[0149] 3.1 POF model establishment, grouping, and treatment procedures

[0150] 3.1.1 Animal preparation and model standardization

[0151] (1) Animals: SPF-grade, 8-week-old female C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). The animals were acclimatized for 1 week in a standard environment with a 12-hour light / dark cycle, a temperature of (22±2)°C, and a humidity of (55±10)%, and had free access to irradiated feed and purified water.

[0152] (2) Establishment of chemotherapy-induced POF model:

[0153] Drug preparation: On the day of injection, dissolve cyclophosphamide (CTX) in sterile saline to prepare a 12 mg / mL solution; dissolve busulfan (BUS) in dimethyl sulfoxide (DMSO), then dilute with sterile olive oil at a ratio of 1:9 (v / v) to prepare a 3 mg / mL suspension. All preparations should be made immediately before use.

[0154] Administration: After weighing, mice were intraperitoneally injected with CTX solution at a dose of 120 mg / kg and BUS suspension at a dose of 30 mg / kg, with a 4-hour interval between the two injections. The sham-operated group (Sham) was injected with an equal volume of physiological saline and olive oil / DMSO mixture.

[0155] (3) Model validation:

[0156] Estrogenic cycle monitoring: Starting from day 7 after modeling, vaginal smears were taken daily from 9:00 AM to 10:00 AM for 10 consecutive days. The criteria for a successful POF model are: the smear continuously shows the interestrous phase (abundant white blood cells, with little or no keratinized epithelial cells), and the cycle completely disappears.

[0157] Pathological sampling verification: On day 17 after modeling, three mice from the model group were randomly selected. After anesthesia, blood was collected from the heart and the mice were sacrificed. Both ovaries were completely removed. One ovary was fixed with Bouin's solution, embedded in paraffin, and serially sectioned (5 μm) for HE staining. The number of growing follicles (secondary follicles and above) was confirmed to be ≤2 per ovarian section, and a large number of atretic follicles and stromal fibrosis were observed. The other ovary was used for subsequent tissue ROS level detection (DHE fluorescence staining). It was confirmed that the ROS level of the ovarian tissue in the model group was significantly higher than that in the Sham group (fluorescence intensity increased by about 3 times, p<0.001), providing in vivo environmental evidence for the subsequent activation of RRDT-LNP.

[0158] 3.1.2 Experimental grouping and preparation of therapeutic agents

[0159] (1) Group design: After successfully modeling mice, they were stratified according to their weight and initial estrous cycle status and randomly divided into 7 groups of 12 mice each.

[0160] G1: Sham group (sham surgery, no modeling, solvent administered).

[0161] G2: POF group (model control, ovarian injection of blank vector).

[0162] G3: Cell-only group (POF + iPSC-OSCs without FTY720 preprocessing).

[0163] G4: Cell (Pre) group (POF + iPSC-OSCs preprocessed with FTY720).

[0164] G5: RRDT-LNP (iv) group (POF + tail vein injection of RRDT-LNP-UA).

[0165] G6: Cell (Pre) + NT-LNP group (POF + FTY720 pretreated iPSC-OSCs + non-targeted LNP-UA mixed local injection).

[0166] G7: Combination group (POF + FTY720 pretreatment iPSC-OSCs + RRDT-LNP-UA mixed local injection).

[0167] (2) Preparation of cell and nanoparticle formulations (on the day of treatment):

[0168] Cell suspension: iPSC-OSCs prepared in Example 1 and identified / pretreated were digested with Accutase, washed twice with PBS, and finally resuspended in serum-free DMEM / F12, adjusting the concentration to 2×10⁻⁶. 7cells / mL (for local injection). For groups G7 and G6, the cell suspension was mixed with an equal volume of nanoparticle suspension (UA concentration of 0.5 mg / mL) on ice.

[0169] Nanoparticle suspension: The RRDT-LNP-UA or NT-LNP-UA prepared in Example 2 was diluted with sterile PBS to a UA concentration of 0.5 mg / mL. For group G5 (intravenous injection), the concentration was diluted to UA concentration of 0.25 mg / mL, and the administration volume was calculated as 80 µL per 10 g body weight, based on a dose of 5 mg / kg.

[0170] Thermosensitive hydrogel carrier: Weigh Pluronic F-127 powder and dissolve it in cold sterile PBS at 4°C to prepare a 30% (w / v) stock solution. Dissolve overnight at 4°C. Mix with the cell / nanoparticle mixture in the specified ratio before treatment to achieve a final gel concentration of 20%. Keep on ice to prevent premature gelation.

[0171] 3.1.3 Precision intraovarian injection therapy under vaginal ultrasound guidance

[0172] Anesthesia and skin preparation: Mice were anesthetized by inhalation of isoflurane (induction concentration 3%, maintenance concentration 1.5%), fixed in a supine position, and the hair on the lower abdomen was removed.

[0173] Ultrasound localization: Apply ultrasound coupling gel to the abdomen and place the probe horizontally above the pubic symphysis. First, identify the bladder (an anechoic sac-like structure), and then locate the ovaries (oval structures with medium to low echogenicity, often adjacent to the lower end of the hypoechoic kidneys) on either side below it. Measure the long and short diameters of the ovaries and calculate their volume.

[0174] Real-time guided injection: A matching microinjector (Hamilton, 33-gauge needle) and a three-dimensional moving injection platform were used. Under real-time ultrasound monitoring, the needle tip was inserted obliquely from the abdominal wall until the strong echo point of the needle tip was located in the central stroma region of the ovary. 10 μL of the prepared mixture was slowly injected (G2 group: blank gel; G3 and G4 groups: cells + gel; G5 group: intravenous injection only; G6 and G7 groups: cells + nanoparticles + gel). A momentary increase in ovarian volume was observed during injection, ensuring no leakage into the abdominal cavity. Both ovaries were treated sequentially.

[0175] Postoperative care: After injection, remove the needle and disinfect the area. Once the mouse has recovered, return it to its cage and monitor its activity, diet, and wound condition for 3 consecutive days.

[0176] 3.2 Efficacy evaluation: dynamic monitoring and final analysis

[0177] 3.2.1 Dynamic monitoring of reproductive endocrine function

[0178] Sample collection: Before treatment (week 0) and at weeks 1, 4, 8, and 12 after treatment, approximately 200 µL of blood was collected from 6 mice in each group. After standing at 4°C for 2 hours, the serum was separated by centrifugation at 3000 g for 15 minutes and stored at -80°C for analysis.

[0179] ELISA assay: The mouse AMH ELISA kit and mouse FSH ELISA kit were used strictly according to the instructions. All samples were tested in the same batch of experiments. The standard curve R... 2 > 0.99.

[0180] The results are shown in Table 6: the combination therapy (G7) showed the best and synergistic effect in restoring ovarian endocrine function. Notably, G6 (pretreated cells + non-targeted drug) was more effective than G4 (pretreated cells alone) but significantly weaker than G7. This clearly indicates that: 1) the combination of FTY720 pretreated cells and drugs (even non-targeted ones) has an additive effect; 2) the RRDT-LNP intelligent targeting system designed in this invention is key to achieving the optimal synergistic effect, and its role cannot be replaced by simple physical mixing.

[0181] Table 6 Summary and Analysis of ELISA Test Results

[0182]

[0183] GraphPad Prism 9.0 software was used, and one-way ANOVA followed by Tukey's multiple comparison test was employed. The AMH level in group G7 was significantly higher than that in groups G3, G4, G5, and G6 (p < 0.01), while the FSH level was significantly lower than those in the aforementioned groups (p < 0.01). The AMH and FSH levels in group G6 were between those in groups G4 and G7, but still significantly different from those in G7 (p < 0.05). Dynamic data showed that in group G7, AMH continuously increased from week 4, while FSH continuously decreased from week 4, exhibiting the most significant trend.

[0184] 3.2.2 Quantitative analysis of ovarian reserve and histological morphology

[0185] Sample processing: In week 12, the remaining 6 mice underwent final analysis. After weighing, the mice were anesthetized, blood was drawn from the heart, and then both ovaries were completely removed and accurately weighed using an analytical balance. The ovarian index (ovarian weight mg / body weight g) was calculated.

[0186] Tissue fixation and sectioning: The left ovary was fixed in Bouin's solution for 24 hours, then routinely dehydrated and embedded in paraffin. Serial sections (5 µm thick) were prepared along the largest transverse diameter of the ovary, with one section taken every 10 sections for HE staining. A total of at least 12 sections from 6 ovaries were analyzed in each group.

[0187] Blind follicle counting: Two experienced researchers counted all sections under a light microscope (200×) without knowing the groupings. A primordial follicle was defined as containing one oocyte in the leptotene or diplotene stage, surrounded by a single layer of flattened granulosa cells. A growing follicle was defined as a follicle with ≥2 layers of granulosa cells (including secondary and preantral follicles). The count results were averaged.

[0188] Histological results are shown in Table 7: HE-stained sections showed that the G7 group had the best ovarian structural recovery, with a large number of healthy follicles at different developmental stages, the least degree of stromal fibrosis, and a morphology closest to the Sham group. This histologically confirms the excellent repair capabilities of the combined treatment on ovarian reserve and structure.

[0189] Table 7 Summary of Experimental Results

[0190]

[0191] Statistical analysis: All indicators in group G7 were significantly better than those in groups G4 and G5 (p < 0.001), and there was no statistical difference between group G7 and group G1 (p > 0.05). The number of follicles in group G6 was significantly higher than that in group G4 (p < 0.05), but lower than that in group G7 (p < 0.01).

[0192] 3.3 In-depth verification of the mechanism

[0193] 3.3.1 Long-term survival, distribution, and function of transplanted cells in vivo

[0194] Cell tracing: Before transplantation, iPSC-OSCs were labeled with the red fluorescent dye CM-Dil. At weeks 1, 4, and 12 post-treatment, two mice from each group were randomly sacrificed, and ovaries were harvested to prepare frozen sections (8 µm).

[0195] Immunofluorescence staining: After section fixation, triple immunofluorescence staining was performed: rabbit anti-human mitochondrial antibody (hMIT, 1:200) labeled transplanted human cells; rabbit anti-mouse CD31 antibody (1:100) labeled blood vessels; rabbit anti-mouse VEGF antibody (1:150). Appropriate fluorescent secondary antibodies and DAPI were used.

[0196] Imaging and Quantitative Analysis: Images were acquired using a microscope. Five fields of view (200×) were randomly selected for quantitative analysis: 1) CM-Dil +1) Number of cell clusters; 2) Ratio of hMIT to CM-Dil double-positive cells; 3) CM-Dil + Microvessel density around cell clusters (CD31) + Area / field of view).

[0197] Results (12 weeks): G7 group CM-Dil per visual field + The number of cell clusters was 8.2 ± 1.5, significantly more than the 4.1 ± 1.2 in the G4 group (p<0.01). The double-positive rate was >95%. The microvessel density around the transplanted cell clusters in the G7 group was (12.5 ± 2.1)%, significantly higher than that in the G4 group (8.3 ± 1.8)% (p<0.05), and VEGF expression was stronger in these areas.

[0198] The above data demonstrate that FTY720 pretreatment significantly improved the long-term survival rate of iPSC-OSCs in diseased ovaries, and these surviving cells retained the function of secreting the pro-angiogenic factor VEGF, which helps to improve the local microenvironment and complements functional recovery.

[0199] 3.3.2 Validation of in vivo targeting and intracellular drug delivery of RRDT-LNP

[0200] Biodistribution assay: A separate experimental group was established, in which Cy7.5 fluorescently labeled RRDT-LNP or NR-ST-LNP was injected via the tail vein into POF model mice (n=3 / time point). Whole-body imaging was performed using the IVIS Spectrum in vivo imaging system at 0.5, 2, 6, 24, and 48 hours post-injection. The fluorescence intensity (ROI) of the ovarian region was quantitatively analyzed.

[0201] The results showed that RRDT-LNP reached its enrichment peak in the ovarian region 6 hours after injection, with a signal intensity 2.8 ± 0.3 times that of the NR-ST-LNP group (p < 0.001). Significant retention was still observed 48 hours later.

[0202] Colocalization analysis of tissue sections: Animals were sacrificed 24 hours after injection of Cy7-labeled RRDT-LNP, and ovaries were harvested for frozen sections. Immunofluorescence staining was performed: granulosa cells were labeled with goat anti-mouse FSHR antibody (1:50); cell nuclei were labeled with DAPI. Observation was performed under a confocal microscope.

[0203] The results showed that in the ovarian sections of the G7 (local injection) group, a large number of Cy7 (red) fluorescent punctate signals highly overlapped with the cytoplasmic regions of FSHR (green) positive cells (yellow). Colocalization analysis showed that more than 60% of the Cy7 signal was located in FSHR cells. +Intracellularly. In the G5 (intravenous injection) group, Cy7 signal was more diffusely distributed in the interstitium or around blood vessels, with very little intracellular signal. This directly proves that the RRDT-LNP of this invention can respond to the ovarian lesion microenvironment in vivo, effectively target and enter the target granulosa cells, while conventional intravenously administered targeting systems are difficult to achieve this process.

[0204] 3.3.3 Direct determination of mitochondrial function in granulocytes

[0205] Granulosa cell isolation: At the final sampling point, the right ovary was harvested, mechanically shredded on ice using a fine needle, digested with 0.2% collagenase IV, and then passed through a 40 µm cell sieve. Cells in the filtrate were collected, and fibroblasts were removed by differential adhesion for 1 hour, resulting in the collection of suspended granulosa-like cells.

[0206] JC-1 Mitochondrial Membrane Potential Assay: The JC-1 assay kit was used. Isolated cells were co-incubated with JC-1 working solution and analyzed by flow cytometry. Healthy mitochondria have a high membrane potential, forming J-aggregates and emitting red fluorescence (585 nm); as the membrane potential decreases, they exist as monomers and emit green fluorescence (510 nm). The red / green fluorescence intensity ratio was calculated.

[0207] Intracellular ATP content detection: ATP was measured using a chemiluminescence assay kit.

[0208] Mitochondrial ROS detection: using MitoSOX TM Red probe, mean fluorescence intensity (MFI) detected by flow cytometry.

[0209] The results, as shown in Table 8, indicate that the combined treatment (G7) most effectively reversed mitochondrial dysfunction in ovarian granulosa cells of POF mice, manifested as restoration of membrane potential, enhanced ATP synthesis capacity, and reduced mitochondrial ROS levels. This provides a fundamental explanation for the observed improvements in follicle survival and hormone secretion function at the cellular energy metabolism level.

[0210] Table 8. Results of direct assay of mitochondrial function in granulocytes

[0211]

[0212] Statistical analysis: The G7 group recovered to levels close to those of the Sham group in all three indicators, and was significantly better than all other treatment groups (p < 0.01).

[0213] 3.4 Long-term safety system evaluation (12 weeks)

[0214] 3.4.1 General Toxicology and Immunological Response Assessment

[0215] Clinical observation: Daily observations showed that no adverse reactions such as death, emaciation, or abnormal behavior were observed in any of the animals.

[0216] Hematology and serum biochemistry: Terminal blood samples were collected and tested using a fully automated hematology analyzer and biochemistry analyzer. The main indicators (white blood cell count, liver and kidney function indicators ALT, AST, BUN, CRE) were all within the normal reference range, with no statistically significant differences among the groups.

[0217] Immune rejection analysis: Spleen was used to prepare a single-cell suspension, and T lymphocyte subsets (CD3+) were detected by flow cytometry. + CD4 + CD8 + There was no difference in the proportions among the groups. CD3 immunohistochemical staining of ovarian tissue sections revealed only a few sporadic positive cells around a few blood vessels, with no difference between the groups, and no large-scale lymphocyte infiltration was observed.

[0218] 3.4.2 Oncogenicity-specific assessment

[0219] Gross anatomy and histopathological screening: Systematic dissection and H&E staining were performed on all major organs (brain, heart, liver, spleen, lungs, kidneys, ovaries, and uterus). No teratomas, teratoma-like structures (such as cartilage, epithelium, neural tube, etc.) or other neoplastic tumors were found in any of the animals that received iPSC-OSC transplants (G3, G4, G6, G7) visible to the naked eye or microscopically. Only mild fibrous encapsulation and a small amount of inflammatory cell infiltration were observed at the transplant site.

[0220] Alu-PCR Quantitative Analysis of Human DNA: Genomic DNA was extracted from ovarian tissue, and real-time quantitative PCR was performed using specific primers, with mouse Gapdh as an internal control. Results showed that the signal of the human Alu sequence was present in groups G4, G6, and G7, but the copy number gradually decreased over 12 weeks, reaching extremely low levels (<10 copies per 100 ng DNA) by week 12, with no difference between groups, and no abnormal amplification was detected.

[0221] Immunohistochemical double staining for pluripotency and proliferation markers: Ovarian sections were double-stained with OCT4 (a marker of pluripotent stem cells) and Ki67 (a marker of proliferation). In a systematic examination of over 200 fields of view, all samples were negative for OCT4 staining. Ki67-positive cells were scattered throughout the ovarian stroma and some granulosa cells, with no statistically significant difference in positivity rates between the treatment groups and the Sham group (all between 3% and 6%).

[0222] Safety Summary: A 12-week observation period demonstrated that the FTY720 preadapted iPSC-OSCs combined with RRDT-LNP therapy employed in this invention did not exhibit any risk of acute toxicity, immunogenicity, tumorigenicity, or promotion of abnormal proliferation under the experimental conditions. Transplanted human cells showed a normal decay trend and no pluripotency markers were expressed, indicating a good safety profile.

[0223] 3.5 In summary, this embodiment demonstrates through rigorous and comprehensive in vivo experiments that:

[0224] The synergistic efficacy was evident: the combined treatment group (G7) was significantly superior to any single therapy or simple combination therapy (G6) in restoring endocrine function (AMH↑, FSH↓), increasing ovarian reserve (follicle count↑), and improving organelle function (mitochondria↑), producing a synergistic effect of "1+1>2".

[0225] Data confirms the role of the two main modules of this invention: ① The pre-adaptive cell module can indeed survive for a long time and improve the microenvironment; ② The RRDT-LNP module can achieve sequential targeting of ROS response in vivo, efficiently delivering drugs to granulocytes, thereby optimizing mitochondrial function.

[0226] A solid safety foundation: 12 weeks of long-term follow-up, combined with molecular-level tumorigenicity assessment (Alu-PCR, OCT4 / Ki67), provides key safety data to support the potential clinical translation of this method.

[0227] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fusion protein VHH-FP, characterized in that, The fusion protein VHH-FP contains a single-domain antibody that specifically binds to AMHR2 and a binding peptide that specifically binds to FSHR, wherein the codon-optimized nucleotide sequence of the fusion protein VHH-FP is shown in SEQ ID NO:

2.

2. The fusion protein VHH-FP according to claim 1, characterized in that, The single-domain antibody that specifically binds AMHR2 in the fusion protein VHH-FP has the amino acid sequence shown in SEQ ID NO:1, or a variant thereof that has at least 95% sequence identity with SEQ ID NO:1 and maintains the same affinity; the sequence of the binding peptide that specifically binds FSHR is YTRDLVYKDPARPKIQKTCTF.

3. A ROS-responsive, dual-level targeted lipid nanoparticle RRDT-LNP, characterized in that, The RRDT-LNP contains the following components: (i) A lipid core composed of cationic lipids, neutral phospholipids, cholesterol and polyethylene glycol-modified lipids, wherein the lipid molar composition of the lipid core is DLin-MC3-DMA: 50%, DSPC: 10%, cholesterol: 38.5%, TK-PEG-LBP: 1%, DSPE-PEG2000-Mal: 0.5%, wherein the cationic lipid is DLin-MC3-DMA and the neutral phospholipid is DSPC; (ii) A polyethylene glycol chain and a primary targeting peptide CNGRC are covalently linked to the surface of the lipid core via a ROS-cleavable thioketide linker, wherein the polyethylene glycol chain is PEG5000, and the thioketide linker, the polyethylene glycol chain, and the primary targeting peptide CNGRC form a TK-PEG-LBP, wherein TK-PEG-LBP is 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 5000-thioketide-cyclic peptide CNGRC; (iii) The secondary dual-targeting fusion protein VHH-FP of claim 1, modified on the surface of the lipid core by site-directed coupling, wherein the site-directed coupling is performed by coupling VHH-FP with DSPE-PEG2000-Mal on the surface of the lipid core via a thiol-maleimide reaction after thiolization, wherein each VHH-FP molecule introduces an average of 1.2 thiol groups, the molar ratio of VHH-FP to Mal-lipid is 0.8:1, and the coupling conditions are incubation at 4°C under nitrogen protection for 16 hours.

4. A composition for treating ovarian insufficiency, characterized in that, The composition comprises: (a) The first active ingredient is ovarian support cells (iPSC-OSCs) derived from human induced pluripotent stem cells preconditioned with FTY720, wherein the iPSC-OSCs are FOXL2-positive and AMHR2-positive double-positive cells with an average double-positive rate of 45.2 ± 6.8%; and (b) A second active ingredient, which is the RRDT-LNP of claim 3 carrying a therapeutic agent, wherein the RRDT-LNP has an average hydrated particle size of 105.3 ± 3.2 nm, a polydispersity index (PDI) of 0.078 ± 0.015, and a zeta potential of +2.1 ± 0.8 mV.

5. The composition according to claim 4, characterized in that, The iPSC-OSCs were obtained through a directed differentiation method comprising the following stages and subsequently pre-adapted using FTY720: Phase I: hiPSCs were induced to differentiate into primitive streaks using 6 µM CHIR99021 and 10 ng / mL BMP4 in RPMI 1640 medium supplemented with 1× B27 Supplement minusinsulin to obtain primitive streaks. Phase II: Intermediate mesoderm differentiation was induced in DMEM / F12 medium supplemented with 1× B27 Supplement using 20 ng / mL FGF2 and 0.5 µM all-trans retinoic acid. Phase III: Gonadal cell fate was induced in DMEM / F12 custom medium supplemented with 1× N2 Supplement and 1× non-essential amino acids using 50 ng / mL WNT4, 200 ng / mL RSPO1 and 3 µM SB431542 to obtain NR5A1 and FOXL2 positive gonadal cells. Stage IV: Ovarian granulosa cells mature into granulosa cells expressing FOXL2 and AMHR2, iPSC-OSCs, in ovarian granulosa cell maturation medium containing 50 mIU / mL recombinant human FSH and 10 nM dihydrotestosterone.

6. The composition according to claim 4 or 5, characterized in that, The conditions for the FTY720 pre-adaptation processing are as follows: the iPSC-OSCs are processed at a ratio of 1.0 × 10⁻⁶. 6 The cells / mL were resuspended in OGCM complete medium and incubated with 1.0 µM FTY720 at 37°C in a 5% CO2 incubator for 16 hours, with gentle shaking once during the incubation period.

7. The composition according to claim 4, characterized in that, The therapeutic agent is a mitochondrial protectant or antioxidant; preferably, the therapeutic agent is urestatin A; more preferably, the RRDT-LNP has an encapsulation rate of 91.8 ± 2.1% for urestatin A and a drug loading of 4.5 ± 0.3%.

8. The composition according to claim 4, characterized in that, The composition is an injectable formulation comprising a pharmaceutically acceptable carrier; preferably, the composition further comprises a thermosensitive hydrogel material, Pluronic F-127, at a final concentration of 20% w / v, for local sustained-release administration to the ovary.

9. A method for preparing the composition according to any one of claims 4-8, characterized in that, The method includes: (1) Provide the iPSC-OSCs and co-incubate them with FTY720 to obtain pre-adapted cells; (2) The RRDT-LNP loaded with therapeutic agent was assembled by microfluidic technology, and the VHH-FP was modified on the surface of the LNP by chemical coupling. (3) Mix the cells obtained in step (1) with the RRDT-LNP obtained in step (2) and optionally mix with a thermosensitive hydrogel carrier.

10. The use of a composition according to any one of claims 4-8 or the RRDT-LNP according to claim 3 in the preparation of a medicament for treating and / or preventing ovarian insufficiency, early-onset ovarian insufficiency or chemotherapy-induced ovarian damage.

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