Stem cell exosome composition for improving premature ovarian failure and preparation method thereof

Abstract

The invention discloses a stem cell exosome composition for improving premature ovarian failure as well as a preparation method and application of the stem cell exosome composition. The composition is composed of human umbilical cord mesenchymal stem cell exosomes obtained through hypoxia and TGF-beta1 combined pretreatment and resveratrol, and the human umbilical cord mesenchymal stem cell exosomes and the resveratrol are compounded according to a specific mass ratio. The pre-treatment condition is that 1% oxygen concentration and 10 ng / ml TGF-beta1 are co-cultured for 48 hours, and the condition can synergistically up-regulate the expression level of specific miRNA in the exosome. The composition prepared through physical incubation and an ultrafiltration method shows a clear synergistic treatment effect in vitro and in vivo, the combination index of the composition is 0.38-0.45, ovarian endocrine indexes can be effectively recovered, follicular reserve can be protected, and oxidative stress and inflammation states of ovarian tissues can be remarkably improved. The invention provides a novel treatment strategy with a novel mechanism and a remarkable curative effect for premature ovarian failure, especially chemotherapy-induced ovarian function impairment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a stem cell exosome composition for improving premature ovarian failure and its preparation method. Background Technology

[0002] Premature ovarian insufficiency (POI) is an endocrine disorder that seriously affects the reproductive health and quality of life of women of reproductive age. It is characterized by the depletion of ovarian reserve before the age of 40. Clinical diagnostic criteria include oligomenorrhea or amenorrhea, persistently elevated serum follicle-stimulating hormone (FSH) levels (>25 IU / L), and significantly decreased estrogen levels. Among the many causes of POI, iatrogenic factors are particularly prominent, with ovarian damage caused by chemotherapy drugs being the most common. Chemotherapy drugs such as cyclophosphamide and cisplatin can induce mitochondrial dysfunction, generate reactive oxygen species (ROS) bursts, and activate endogenous apoptosis pathways (such as caspase-3) in granulosa cells, directly leading to irreversible depletion of the primordial follicle pool and ovarian stromal fibrosis. Currently, the standard clinical management for this condition is hormone replacement therapy, which aims to alleviate perimenopausal symptoms caused by low estrogen and prevent osteoporosis. However, this therapy is essentially a symptom replacement, unable to reverse lost primordial follicles or restore the patient's natural fertility, and long-term use may be associated with risks such as breast cancer and thrombosis. Therefore, exploring innovative treatment strategies that can intervene at the pathological mechanism level, effectively protect ovarian reserve function, and have the potential to preserve fertility has become a pressing scientific challenge in the field of reproductive medicine.

[0003] In recent years, regenerative medicine strategies based on paracrine effects have brought new perspectives to this field. Small extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs, also known as exosomes), as a class of natural nanocarriers with diameters of approximately 30-150 nanometers, are gradually showing promising potential as tools for "cell-free therapy." sEVs carry bioactive "cargo" such as proteins, lipids, and nucleic acids (especially microRNAs) from the source cells, which can be taken up by recipient cells through membrane fusion or endocytosis, thereby regulating their physiological functions. Studies have shown that MSC-sEVs can deliver miRNAs with cytoprotective effects. For example, miR-21-5p enhances cellular resistance to apoptosis by negatively regulating the target gene PTEN, thereby relieving its inhibition of the PI3K / Akt pro-survival signaling pathway; miR-146a-5p negatively regulates NF-κB-mediated inflammatory responses by inhibiting the formation of the TRAF6 / IRAK1 complex. Nevertheless, natural sEVs without any engineered treatment face significant challenges in practical applications: the composition and abundance of their bioactive components vary considerably between large groups due to the influence of donor cell status and culture conditions; they are easily cleared rapidly by the mononuclear phagocytic system in systemic circulation; and they lack the ability to actively target diseased tissues, resulting in insufficient stability and controllability of their therapeutic effects.

[0004] To overcome the aforementioned limitations, the academic community generally employs a strategy of "priming" MSCs to selectively modify the cargo loading of sEVs, thereby customizing their functional properties. Hypoxia pretreatment simulating the ischemic / injury microenvironment can stably induce the expression of hypoxia-inducible factor-1α (HIF-1α) in MSCs, thereby globally altering their secretome and enriching sEVs with more factors promoting angiogenesis and cell survival. Furthermore, stimulation with specific cytokines (such as pro-inflammatory factors TNF-α and IFN-γ) can programmatically shape the immunomodulatory phenotype of MSCs and confer it upon the secreted sEVs. On the other hand, resveratrol, a plant-derived polyphenol, has attracted widespread attention due to its well-defined cytoprotective activity. Its mechanism of action involves activating endogenous cellular antioxidant defense systems (such as the Nrf2 / ARE pathway), inhibiting NLRP3 inflammasome assembly and activation, and improving mitochondrial function and inhibiting apoptosis by upregulating the deacetylase SIRT1. However, the clinical application of resveratrol is limited by its extremely low water solubility and oral bioavailability, as well as its short half-life due to rapid metabolism in the body.

[0005] Currently, although there are reports on the combination of sEVs and active small molecules, most studies remain at the initial stage of using sEVs as passive carriers or simply applying them together physically. These attempts generally have the following technical gaps: (1) The selection of pretreatment strategies is mostly based on experience, lacking rational design for specific pathological links (such as the coexistence of oxidative stress, apoptosis and fibrosis in chemotherapy-induced ovarian damage), making it difficult to achieve precise enhancement of the functional properties of sEVs; (2) The evaluation of the combined use of sEVs and drugs often stops at the observation of therapeutic effects, lacking rigorous synergistic effect analysis using quantitative pharmacological models such as the Combination Index (CI), and failing to elucidate the complementary mechanisms between the two at the molecular and pathway levels; (3) There are few systematic studies on the sEV subpopulations with unique "molecular signatures" created by different pretreatment methods, and the intrinsic laws of their interaction with specific drugs and the synergistic effect have not yet been revealed. Therefore, developing a mechanism-driven pretreatment scheme to obtain functionally customized sEVs and to form highly efficient synergistic compositions with matching small molecule drugs is of great scientific significance and application value for promoting the development of POI therapy towards high efficiency and precision. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing technologies in the treatment of premature ovarian failure (POI), especially chemotherapy-induced POI. Current monotherapy based on mesenchymal stem cell exosomes (MSC-sEVs) or resveratrol has limited efficacy; while simply combining sEVs with drugs suffers from uncertain sEV function, unclear synergistic effects, and a lack of quantitative assessment and mechanistic elucidation. Therefore, the technical problem this invention seeks to solve is: how to provide an ovarian protection composition with clearly defined components, significant synergistic effects, a clear mechanism, and ease of standardized production.

[0007] To address the aforementioned technical problems, this invention provides a stem cell exosome composition for improving premature ovarian failure. The core of this composition lies in a specific combination of a functionally optimized small extracellular vesicle (Op-sEV) and resveratrol.

[0008] The Op-sEVs were obtained through an innovative hypoxia combined with TGF-β1 pretreatment protocol. Specifically, human umbilical cord mesenchymal stem cells were placed in a hypoxic environment of 1% O2 and stimulated with 10 ng / mL transforming growth factor-β1 (TGF-β1) for 48 hours, followed by isolation and purification of sEVs from the culture supernatant. This specific pretreatment was not a simple superposition of known techniques, but was found to synergistically and significantly upregulate the levels of key protective microRNAs (such as miR-21-5p and miR-146a-5p) within sEVs.

[0009] The Op-sEV thus obtained is compounded with resveratrol (Res) at a mass ratio of 1:0.5 to 1:2 (preferably 1:1) by physical incubation combined with ultrafiltration centrifugation to form a stable Op-sEV / Res composition.

[0010] Compared with the prior art, the present invention has the following significant advantages:

[0011] 1. Quantitative Validation of the Synergistic Effect: The composition was demonstrated to have a significant synergistic effect in cell models. Quantitative analysis using the Chou-Talalay Co-excitation Index (CI) method showed that the CI value of the composition of this invention was consistently below 0.5, indicating a strong synergistic effect. In contrast, the synergistic effect of exosomes and resveratrol complexes obtained using conventional single pretreatment (e.g., hypoxia only) was significantly weakened. This demonstrates that the specific co-excitation pretreatment strategy is crucial and not obvious for achieving this excellent synergistic effect.

[0012] 2. Multi-layered therapeutic efficacy: In a chemotherapy-induced premature ovarian failure animal model, the composition of this invention exhibits comprehensive therapeutic effects. It can more effectively restore serum anti-Müllerian hormone levels, which represent ovarian reserve function, and reduce follicle-stimulating hormone levels; histological analysis confirms that its protective effect on primordial follicles and growing follicles is most significant; at the same time, it can also more effectively improve the oxidative stress state of the local ovarian microenvironment and inhibit the activation of inflammatory pathways.

[0013] 3. Clear Mechanism and Controllable Preparation: The mechanism of action of this method is reasonable, namely, the specific microRNA delivered by exosomes complements the pharmacological effects of resveratrol on multiple signaling pathways. Furthermore, its preparation method is based on mature cell culture and physical compounding techniques, with clearly defined process parameters, which helps ensure batch-to-batch consistency and quality control, providing feasibility for subsequent transformation.

[0014] 4. The synergistic effect of this invention stems from the deep complementarity and active dialogue between Op-sEV as a functional biological carrier and resveratrol at the mechanistic level, rather than a simple improvement in physical loading efficiency. First, the "hypoxia combined with TGF-β1" pretreatment does not universally enhance the cargo-carrying capacity of exosomes, but rather specifically and synergistically enriches bioactive molecules with clear functional targets, such as miR-21-5p (targeting the PTEN / PI3K / Akt anti-apoptotic pathway) and miR-146a-5p (inhibiting the TRAF6 / NF-κB inflammatory pathway), which endows Op-sEV with a unique therapeutic molecular phenotype. Second, these endogenous functional molecules and the exogenous antioxidant and anti-inflammatory effects of resveratrol form a non-overlapping and complementary coverage on the signaling pathway. The key quantitative evidence is that the synergistic strength of Op-sEV / Res (CI ~0.4) represents a qualitative leap compared to the control based solely on hypoxia pretreatment (CI ~0.85), and this non-linear synergistic gain cannot be explained by a linear improvement in carrier loading efficiency. In vivo experiments further confirmed that the comprehensive advantages of Op-sEV / Res in improving the ovarian microenvironment (antioxidant, anti-inflammatory) and directly protecting follicular reserve are highly consistent with the functional miRNAs carried by Op-sEV. Therefore, the core value of the "hypoxia combined with TGF-β1" pretreatment in this invention lies in creating a functionally customized exosome, whose binding with resveratrol produces an unexpected, proactive synergistic therapeutic effect based on mechanistic complementarity.

[0015] In summary, this invention, through rational design of a pretreatment scheme, creatively obtained a functionally customized sEV, and combined it with a mechanism-matching small molecule drug, successfully developing an ovarian protection composition with significant synergistic effects, providing a novel solution for the clinical treatment of premature ovarian failure. Attached Figure Description

[0016] Figure 1 Western blotting results for three groups of sEV samples.

[0017] Figure 2 TEM results of the Op-sEV / Res complex. Detailed Implementation

[0018] 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.

[0019] 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.

[0020] Example 1: Preparation, characterization, and miRNA analysis of three different pretreated sEVs

[0021] 1. Cell Culture and Pretreatment

[0022] 1.1 Cells and Reagents: Commercially available human umbilical cord Wharton's colloid mesenchymal stem cells (hUC-MSCs, catalog number: HUXUC-01001, Shanghai Junsheng Biotechnology Co., Ltd.) were used in the experiment. Cells were cultured in α-MEM complete medium containing 10% fetal bovine serum (FBS, exosome-depleted) and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Before use, the cells had been passaged to passages 3-5 (P3-P5) and confirmed to be in good condition.

[0023] 1.2 Preprocessing scheme:

[0024] Op-sEV group (optimized pretreatment group, this invention): When the cells reached 80% confluence in T75 culture flasks, the old culture medium was discarded, and the cells were gently washed twice with pre-warmed PBS. Then, 20 mL of complete culture medium containing 10 ng / mL human recombinant TGF-β1 (TGF-β1) was added. The culture flasks were placed in a tri-gas incubator with adjustable oxygen concentration. The culture conditions were set as follows: 1% O2, 5% CO2, 37°C, for 48 hours.

[0025] H-sEV group (simple hypoxia control group): The treatment steps were exactly the same as those of the Op-sEV group, the only difference being that TGF-β1 was not added to the culture medium, and only complete culture medium was used.

[0026] N-sEV group (normoacid control group): Cells were replaced with complete culture medium without TGF-β1 at the same confluence and cultured in a conventional cell culture incubator (21% O2, 5% CO2, 37°C) for 48 hours.

[0027] 2. sEV separation and purification: After pretreatment, strictly follow the differential centrifugation and ultracentrifugation procedures as follows:

[0028] 2.1 Collect the supernatant: Collect the cell culture supernatant from each group into a 50 mL centrifuge tube.

[0029] 2.2 Removal of cells and debris: Centrifuge at 300 × g for 10 minutes at 4°C, carefully aspirate the supernatant, and discard the cell pellet at the bottom. Transfer the supernatant to a new centrifuge tube, centrifuge at 2,000 × g for 20 minutes at 4°C, aspirate the supernatant, and remove dead cells. Transfer the supernatant again, centrifuge at 10,000 × g for 30 minutes at 4°C to thoroughly remove cell debris and large vesicles.

[0030] 2.3 Filtration: The supernatant after centrifugation was filtered through a PVDF membrane with a pore size of 0.22 μm to remove any possible residual particulate matter.

[0031] 2.4 Ultracentrifugation: Use an ultracentrifuge equipped with a Type 70 Ti fixed-angle rotor. Add the filtrate to an ultracentrifuge tube, centrifuge at 100,000 × g for 70 minutes at 4°C, discard the supernatant, and obtain the sEV precipitate.

[0032] 2.5 Washing: Gently resuspend the precipitate in pre-cooled 1× PBS (phosphate buffer) and wash again under the same conditions (4°C, 100,000 × g for 70 minutes) to remove co-precipitated proteins.

[0033] 2.6 Resuspension and Storage: The final precipitate was resuspended in 200 μL of sterile PBS. The total protein concentration of the sEV suspension was determined using a BCA protein assay kit. All samples were aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles.

[0034] 3. sEV characterization and microRNA expression profiling analysis

[0035] 3.1 Nanoparticle Tracking Analysis (NTA):

[0036] Methods: A NanoSight NS300 system (Malvern Panalytical) and its accompanying software NTA 3.4 were used. Each sEV sample was diluted with sterile PBS to an appropriate concentration (approximately 1 × 10⁻⁶). 8 The particle concentration (particles / mL) was injected into the sample cell at a constant rate (20 μL / s) using a 1 mL sterile syringe. Five 60-second video segments were automatically captured for each sample, with the camera horizontally set to 13 and the shutter speed set to 1000. The software automatically analyzed the Brownian motion trajectory of the particles and calculated the particle concentration and size distribution.

[0037] The results are shown in Table 1: there were no statistically significant differences in the average particle size, peak particle size, and particle concentration among the three groups of sEV samples (P>0.05), and the particle size distribution was concentrated (PDI<0.2), consistent with the typical physical characteristics of sEVs. This indicates that different pretreatment schemes did not change the basic physical properties of sEVs.

[0038] Table 1. Results of NTA analysis

[0039]

[0040] 3.2 Protein Blot Analysis (Western Blot)

[0041] Methods: An equal volume (15 μg) of sEV protein sample was taken, and 5×SDS loading buffer was added. The sample was denatured at 95°C for 5 minutes. Electrophoresis was performed using a 10% Tris-Glycine precast gel, followed by wet transfer to a PVDF membrane. After blocking with 5% skim milk at room temperature for 1 hour, the membrane was incubated overnight at 4°C with primary antibodies CD9 (1:1000), CD63 (1:1000), TSG101 (1:1000), and Calnexin (1:1000), respectively. After washing with TBST, the membrane was incubated with the corresponding HRP-labeled secondary antibody at room temperature for 1 hour. Development was performed using ECL chemiluminescent substrate, and images were acquired using a chemiluminescent imaging system.

[0042] Results: Western blotting results show ( Figure 1 All three groups of sEV samples positively expressed the characteristic membrane protein markers CD9 and CD63 of exosomes and the cytoplasmic protein TSG101, while negatively expressing the endoplasmic reticulum marker Calnexin. This confirms that high-purity sEVs were successfully isolated using the aforementioned ultracentrifugation method, and that pretreatment did not affect the expression of their classic markers.

[0043] 3.3 Quantitative analysis of microRNA (qRT-PCR)

[0044] RNA extraction and quality control: Using the miRNeasy Micro Kit, total RNA was extracted from 20 μg (based on protein) of each sEV sample group, strictly following the instructions. RNA concentration and purity were determined using a NanoDrop One ultra-micro spectrophotometer (A260 / A280 ratios were all between 1.9 and 2.1).

[0045] Reverse transcription: using the miRCURY LNA RT Kit. Take an equal volume of total RNA, add miRCURY LNA RT enzyme and buffer, and reverse transcribe on a PCR instrument according to the program (42°C 60 min, 95°C 5 min) to generate cDNA.

[0046] Real-time quantitative PCR (qPCR): The miRCURY LNA SYBR Green PCR Kit and its matching LNA PCR primers (hsa-miR-21-5p, hsa-miR-146a-5p) and internal reference gene U6 snRNA were used. The reaction volume was 10 μL, and the reaction was performed on a real-time quantitative PCR instrument. The reaction program was: 95°C pre-denaturation for 2 minutes; followed by 95°C for 10 seconds, 56°C for 60 seconds, for 45 cycles. Each sample was tested in triplicate.

[0047] The results are shown in Tables 2 and 3. qRT-PCR quantitative analysis revealed that, compared with the normoxic culture group (N-sEV), hypoxia pretreatment alone (H-sEV) could upregulate the levels of protective miRNAs (miR-21-5p and miR-146a-5p) in sEVs to a certain extent (increasing by approximately 5.1-fold and 2.7-fold, respectively). However, after adopting the optimized pretreatment scheme of "hypoxia combined with TGF-β1" described in this invention, the expression levels of these two key miRNAs in Op-sEVs showed a more significant and synergistic enrichment, with levels 15.6-fold and 10.2-fold higher than those in the N-sEV group, and significantly higher than those in the H-sEV group (P<0.001).

[0048] Table 2. Ct values ​​of miRNA qPCR in three independently prepared sEV samples (mean, n=3 batches)

[0049]

[0050] Note: Data are expressed as mean ± standard deviation of three independent experiments.

[0051] Relative expression levels were calculated using the 2^(-ΔΔCt) method. First, using the N-sEV group as the baseline calibrator, ΔCt was calculated for each group as: ΔCt = Ct(target miRNA) - Ct(U6). Then, ΔΔCt was calculated as: ΔCt(experimental group) - ΔCt(N-sEV group). Finally, the relative expression level was calculated as: 2^(-ΔΔCt).

[0052] Table 3. Statistical analysis results of relative miRNA expression levels

[0053]

[0054] 4. Summary

[0055] This study successfully isolated high-purity sEVs (Op-sEV, H-sEV, N-sEV) from the culture supernatant of hUC-MSCs under three different pretreatment conditions using a standardized differential / ultracentrifugation method. Their physical characteristics (particle size approximately 125 nm, uniform distribution) and protein marker expression (CD9+ / CD63+ / TSG101+ / Calnexin-) all met the criteria for extracellular vesicles, confirming the reliability of the preparation method.

[0056] This study demonstrates that hypoxia and TGF-β1 have a synergistic effect on regulating hUC-MSCs exosomal miRNAs, rather than a simple additive effect. This targeted and enhanced modification of the functional "cargo" within sEVs is the material basis and key prerequisite for the subsequent synergistic therapeutic effect of "1+1>2" with resveratrol.

[0057] Example 2: Preparation, characterization and stability study of Op-sEV / Res complex

[0058] 1. Preparation of the complex

[0059] 1.1 Materials

[0060] Op-sEV: Prepared according to the method of Example 1, thawed at 4°C and gently vortexed before use.

[0061] Resveratrol (Res): Purchased from Sigma-Aldrich (catalog number: R5010), purity ≥98% (HPLC). Before use, prepare a 20 mg / mL stock solution with anhydrous dimethyl sulfoxide (DMSO, Sigma) and store at -20°C protected from light. Prepare the working solution fresh with phosphate-buffered saline (PBS, pH 7.4) to ensure the final DMSO concentration is below 0.1% (v / v).

[0062] 1.2 Preparation steps:

[0063] Stock solution preparation and mixing: Accurately measure 100 μL of Op-sEV stock solution (total protein concentration of 1.0 mg / mL, determined by BCA method) (containing 100 μg protein) into a 1.5 mL low-adsorption centrifuge tube. Add 5 μL of DMSO stock solution of Res (containing 100 μg Res), and then immediately add 895 μL of pre-cooled PBS (pH 7.4) to make the total volume 1.0 mL. At this point, the mass ratio of Op-sEV to Res is 1:1.

[0064] Incubation: The mixture was placed on a thermostatic shaker at 37°C and 200 rpm, and incubated in the dark for 30 minutes. These conditions were designed to promote the binding of Res molecules to the lipid bilayer of Op-sEV through hydrophobic interactions.

[0065] Separation, purification, and collection: Immediately after incubation, transfer the entire mixture to pre-cooled 100 kDa ultrafiltration centrifuge tubes. Centrifuge at 4°C, 4000 × g for 15 minutes. After centrifugation, the filtrate contains unbound free Res; carefully collect and record the volume for subsequent drug loading calculations. The retentate contains the Res-loaded Op-sEV / Res complex and a small amount of free Op-sEV that may not have been compressed by centrifugation.

[0066] Washing and final product acquisition: To completely remove free Res, pre-chilled PBS was added to the retentate to the initial volume (approximately 500 μL). After gentle refluxing, the mixture was washed again by centrifugation under the same conditions (4°C, 4000 × g for 15 minutes). Finally, the complex precipitate at the bottom of the tube was gently resuspended in 100 μL of pre-chilled PBS to obtain the Op-sEV / Res complex concentrate. All procedures were performed on ice to maintain sEV activity. Op-sEV + PBS (without Res, treated in the same way) was prepared simultaneously as a blank control.

[0067] 2. Physicochemical characterization of the complex

[0068] 2.1 Determination of drug loading and encapsulation efficiency (HPLC method)

[0069] 2.1.1 Chromatographic conditions: An Agilent 1260 Infinity II HPLC system equipped with a diode array detector (DAD) was used. Column: ZORBAX Eclipse Plus C18 column (4.6 × 150 mm, 5 μm). Mobile phase: Acetonitrile (A) – 0.1% formic acid aqueous solution (B), gradient elution (0–10 min, 45% A; 10–12 min, 45% → 90% A; 12–15 min, 90% A; 15–16 min, 90% → 45% A; 16–20 min, 45% A). Flow rate: 1.0 mL / min. Column temperature: 30°C. Detection wavelength: 306 nm. Injection volume: 20 μL.

[0070] 2.1.2 Standard curve preparation: Accurately weigh Res reference standard, dissolve it in methanol and dilute it stepwise to prepare standard solutions with concentrations of 0.5, 1, 2, 5, 10, and 20 μg / mL for analysis. Perform linear regression of peak area (Y) against concentration (X, μg / mL).

[0071] 2.1.3 Sample Determination and Calculation: The collected filtrate was appropriately diluted with methanol (e.g., 10 times), filtered through a 0.22 μm filter membrane, and then injected. The concentration of free Res in the filtrate (C_free, μg / mL) was calculated based on the standard curve, and then the drug loading parameters were calculated.

[0072] Free Res mass (m_free) = C_free × filtrate dilution factor × total filtrate volume (mL).

[0073] Combined Res mass (m_bound) = Total drug mass (m_total, 100 μg) - m_free.

[0074] Drug Loading Capacity (DLC) = (m_bound / m_Op-sEV) × 100%, where m_Op-sEV is the amount of protein fed into Op-sEV (100 μg).

[0075] Encapsulation Efficiency (EE) = (m_bound / m_total) × 100%.

[0076] 2.1.4 Results

[0077] Standard curve: Res showed good linearity in the range of 0.5-20 μg / mL, with the regression equation being Y = 45.267X + 0.658 (R²). 2 = 0.9999).

[0078] Table 4. Sample Measurement Results (n=3 batches prepared independently)

[0079]

[0080] HPLC analysis (Table 4) showed that the average encapsulation efficiency of resveratrol in the Op-sEV / Res complex was (68.49 ± 1.66)%, corresponding to a drug loading of (68.49 ± 1.66) μg Res / 100 μg sEV protein. The small standard deviation between batches indicates that the preparation process is stable and reproducible.

[0081] 2.2 Particle size, zeta potential and morphology characterization

[0082] 2.2.1 Dynamic Light Scattering (DLS) and Electrophoretic Light Scattering (ELS): The Op-sEV / Res complex was diluted with PBS to a suitable concentration (protein concentration approximately 50 μg / mL) and injected into a disposable folded capillary sample cell. The temperature was set to 25°C, and the equilibration time was 2 minutes. Each sample was automatically measured 3 times, with each measurement consisting of 11-13 sub-cycles, and the average value was taken.

[0083] 2.2.2 Particle size and polydispersity index (PDI): Measured in DLS mode using NIBS (non-invasive backscattering) optical configuration. The analysis software used the cumulant method to obtain the Z-average particle size (Z-Average) and PDI.

[0084] 2.2.3 Zeta potential: Calculated using the Smoluchowski model with M3-PALS (phase analysis light scattering) technique in ELS mode.

[0085] 2.2.4 Transmission Electron Microscopy (TEM) Observation: Take 10 μL of Op-sEV / Res composite sample and drop it onto a copper grid coated with a carbon film. Incubate at room temperature for 2 minutes. Blot away excess liquid with filter paper, then negatively stain with 2% phosphotungstic acid (pH 7.0) for 1 minute. Blot dry the stain and allow to air dry at room temperature. Observe and photograph using a TEM at an accelerating voltage of 100 kV.

[0086] 2.2.5 Results: As shown in Table 5, compared with the original Op-sEV, the average hydrated particle size of the Op-sEV / Res complex increased significantly (P<0.05), from approximately 122 nm to approximately 137 nm. The PDI increased slightly but remained less than 0.2, indicating that the particle size distribution remained relatively uniform. Simultaneously, its surface Zeta potential shifted significantly negatively (P<0.05), from -22.3 mV to -25.9 mV. The increase in particle size and the change in Zeta potential provide direct physical evidence for the successful binding or embedding of resveratrol into the phospholipid bilayer of Op-sEV.

[0087] Table 5 Physicochemical properties of Op-sEV and its complexes (mean ± SD, n=3)

[0088]

[0089] TEM results showed ( Figure 2 The Op-sEV / Res complex retained its typical saucer-like or cup-shaped vesicle morphology, similar to the original Op-sEV, with no obvious rupture or aggregation observed. The particle size of the complex was consistent with the DLS results under electron microscopy.

[0090] 3. Summary

[0091] This study established a simple and efficient physical incubation-ultrafiltration purification method to successfully synthesize the small molecule drug resveratrol (Res) with functionally optimized exosomes (Op-sEVs) to prepare the Op-sEV / Res complex. This method avoids complex chemical modifications and preserves the natural activities of each component to the greatest extent. Quantitative analysis using optimized HPLC confirmed that the complex has stable drug loading performance, with a resveratrol encapsulation efficiency of (68.49 ± 1.66)% and a clearly defined drug loading amount, providing accurate dosage basis for subsequent pharmacodynamic studies.

[0092] The formation of the complex in this study led to measurable changes in its nanoparticle properties. The moderate increase in particle size was in line with expectations for drug loading. The zeta potential shifted towards a more negative direction, which may be due to the contribution of the resveratrol phenolic hydroxyl group, and a more negative surface charge generally benefits the stability of colloidal dispersions. These changes, from a physicochemical perspective, confirm that Op-sEV and Res are not simply physical coexistences, but rather form a novel, interacting composite nanosystem.

[0093] Example 3: In vitro synergistic effect study

[0094] 1. Cell culture and injury model establishment

[0095] 1.1 Cell Line and Culture: The human ovarian granulosa cell line KGN (preserved in our laboratory) was used in the experiment. Cells were cultured in DMEM / F-12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO2. The cells used in the experiment were in logarithmic growth phase and had been passaged less than 25 times.

[0096] 1.2 Cell Seeding: Cells were collected by digestion with 0.25% trypsin-EDTA and counted using a hemocytometer. The cell suspension was seeded at 5.0 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL per well in 96-well cell culture plates. The plates were then pre-cultured in an incubator for 24 hours to allow the cells to adhere fully and enter the logarithmic growth phase.

[0097] 1.3 Establishment of the cisplatin-induced injury model: After pre-culturing for 24 hours, the old culture medium was discarded. Model group and all treatment groups: 100 μL of complete culture medium containing 4 μM cisplatin (prepared with DMSO stock solution) was added to each well. Control group: An equal volume of complete culture medium containing the same concentration of DMSO (<0.1%) was added. The cell plates were returned to the incubator and cultured for another 24 hours to establish an in vitro chemotherapy-induced ovarian granulosa cell injury model.

[0098] 2. Experimental grouping and drug administration

[0099] After cisplatin treatment for 24 hours, the drug-containing culture medium was discarded, and the cells were gently washed twice with pre-warmed PBS to completely remove residual cisplatin. Then, the culture medium was replaced with complete culture medium (100 μL / well) containing different therapeutic agents, and the cells were cultured for another 48 hours. The experiment was set up with the following 8 groups, each with 6 replicates (n=6):

[0100] Group 1: Blank control group (Control): Normal complete culture medium.

[0101] Group 2: Model group: Normal complete culture medium.

[0102] Group 3: Op-sEV monotherapy group (Op-sEV): Culture medium containing 20 μg / mL (based on sEV protein concentration) of Op-sEV.

[0103] Group 4: Resveratrol monotherapy group (Res): Culture medium containing 20 μg / mL resveratrol (Res) (preparation method as in Example 2).

[0104] Group 5: N-sEV / Res complex group (N-sEV / Res): Culture medium containing N-sEV and Res complex, wherein the final concentration of both sEV and Res is 20 μg / mL. The complex was prepared ad hoc before administration according to the method in Example 2.

[0105] Group 6: H-sEV / Res complex group (H-sEV / Res): Culture medium containing H-sEV and Res complex, with a final concentration of 20 μg / mL for both sEV and Res. Preparation method is the same as Group 5.

[0106] Group 7: Op-sEV / Res complex group (Op-sEV / Res, the core group of this invention): Culture medium containing Op-sEV and Res complex, with a final concentration of 20 μg / mL for both sEV and Res. Preparation method is the same as in Example 2.

[0107] Group 8: Physical Mixture Control Group (Op-sEV+Res Mix): A physical mixture containing 20 μg / mL Op-sEV and 20 μg / mL Res. The mixture was simply vortexed in the culture medium immediately before use, without incubation or ultrafiltration.

[0108] 3. Detection indicators, raw data and result analysis

[0109] 3.1 Cell viability assay (CCK-8 assay)

[0110] Methods: After culturing for 48 hours, 10 μL of CCK-8 solution was added directly to each well, gently shaken to mix, and then returned to the incubator for another 2 hours of incubation. After incubation, the absorbance (OD) of each well was measured at 450 nm using a full-wavelength microplate reader. Wells containing only culture medium and CCK-8 were used as blanks for zeroing. Cell viability (%) was calculated using the formula: [(OD_experimental group - OD_blank) / (OD_control group - OD_blank)] × 100%.

[0111] The results are shown in Table 6: the cell survival rate in the cisplatin model group (Model) decreased to approximately 41%, indicating that the injury model was successfully established. All treatment groups showed some protective effect. The key finding was that the Op-sEV / Res complex group (Group 7) exhibited the strongest protective effect, with a cell survival rate of 86.7%, significantly higher than the Op-sEV monotherapy group (70.5%, P<0.01), the Res monotherapy group (63.5%, P<0.001), and the equivalent dose physical mixture group (76.1%, P<0.05). More importantly, its effect was significantly superior to other exosome complex groups, including the H-sEV / Res group (73.4%, P<0.001) and the N-sEV / Res group (67.4%, P<0.001). This preliminarily suggests that the binding of Op-sEV and Res produces an effect beyond simple addition, and that this effect depends on specific sEV pretreatment.

[0112] Table 6. OD450 values ​​and cell viability of each group as measured by CCK-8 assay (mean ± SD, n=6)

[0113]

[0114] Note: One-way ANOVA and Tukey's multiple comparison test were used.

[0115] 3.2 Quantitative analysis of synergistic effects (Chou-Talalay joint index method)

[0116] Methods: To accurately quantify the synergistic effect, a multi-concentration combination analysis was performed on the combination of Op-sEV and Res. Concentration gradients of Op-sEV and Res were set (sEV protein:Res mass ratio fixed at 1:1): (5+5), (10+10), (20+20), (40+40) μg / mL. The inhibitory rates (Fa) of the three complexes N-sEV / Res, H-sEV / Res, and Op-sEV / Res on cisplatin-damaged KGN cells at each concentration were tested. Using CompuSyn 1.0 software, the combination index (CI) for each effect point (Fraction Affected, Fa) was calculated based on the median-effect principle. CI < 1, = 1, and > 1 represent synergistic, additive, and antagonistic effects, respectively.

[0117] The results are shown in Table 7: Quantitative CI analysis provided conclusive evidence. The CI values ​​of the N-sEV / Res complex were in the ED... 90 A CI greater than 1 indicates an antagonistic tendency, even in the high-effect region. The CI values ​​of the H-sEV / Res complex are between 0.8 and 0.9, showing a weak synergistic or approximately additive effect. In contrast, the Op-sEV / Res complex exhibits a weak synergistic or approximately additive effect at all tested effect points (ED). 50 ED 75 ED 90 The CI values ​​of all samples were much less than 1 (0.38-0.45), clearly confirming the strong synergistic effect. This directly proves that the Op-sEV obtained by "hypoxia combined with TGF-β1" pretreatment is the key and irreplaceable factor that enables it to produce a strong synergistic effect with Res.

[0118] Table 7. Co-existing indices (CI) of the three sEV / Res complexes at different effect sites (Fa).

[0119]

[0120] Note: ED xx This represents the dose required to achieve the xx% effect. The CI value is the average of three independent experiments.

[0121] 3.3 Mechanism Exploration: Apoptosis and Related Pathway Protein Expression (Western Blot)

[0122] Methods: To investigate the molecular mechanism of the synergistic effect, cells were seeded in parallel in 6-well plates, and the above group treatment was repeated (Group 2, 3, 4, 6, 7). After 48 hours of treatment, cells were washed with pre-cooled PBS and collected. Total protein was extracted using RIPA lysis buffer (containing protease and phosphatase inhibitors) and quantified using the BCA method. An equal amount of protein (30 μg) was subjected to SDS-PAGE electrophoresis, transferred to a membrane, and blocked. The membranes were incubated overnight at 4°C with the following primary antibodies: p-Akt (Ser473) (1:2000), Total Akt (1:2000), Cleaved Caspase-3 (1:1000), Pro-caspase-3 (1:1000), Bcl-2 (1:1000), and β-actin (1:5000). After incubation with HRP-labeled secondary antibody at room temperature for 1 hour, ECL was used for imaging. ImageJ software was used to analyze the grayscale values ​​of the bands, and the target protein level was expressed as a ratio to the internal control (β-actin) or total protein.

[0123] The results are shown in Table 8. Western blotting results supported the functional data at the molecular mechanism level. Compared with the model group, all treatment groups upregulated the pro-survival signaling protein (p-Akt) and the anti-apoptotic protein Bcl-2 to varying degrees, and downregulated the apoptosis-executing protein Cleaved Caspase-3. However, the Op-sEV / Res complex group (Group 7) showed the strongest regulatory effect across all indicators: it had the highest p-Akt / Akt ratio and Bcl-2 expression level, and the lowest Cleaved Caspase-3 level. Compared with the H-sEV / Res group, the Op-sEV / Res group showed statistically significant p-Akt activation and Caspase-3 inhibition effects (P<0.05). This indicates that the synergy between Op-sEV and Res is not only reflected in cell viability, but also, more profoundly, in the synergistic enhancement of the PI3K / Akt survival signaling pathway and the synergistic inhibition of the mitochondrial apoptosis pathway.

[0124] Table 8. Relative quantification of key protein expression in Western Blot (grayscale ratio, mean ± SD, n=3)

[0125]

[0126] 4. Summary

[0127] This study, through in vitro experiments, fully verified the superior performance and unique inventiveness of the composition (Op-sEV / Res) of this invention from three levels: cell function recovery, quantitative analysis of synergistic effects, and molecular mechanisms. Functional experiments showed that Op-sEV / Res had the strongest protective effect against cisplatin-damaged ovarian granulosa cells, and its effect was significantly better than that of each single-use group, the physical mixture group, and the sEV complex group from other pretreatment sources (P<0.05). Key quantitative analysis showed that, calculated using the Chou-Talalay method, Op-sEV / Res had the highest effective rate at all tested effect points (EDS). 50 ED 75 ED 90 The combined index (CI) of sEV / Res and N-sEV / Res ranged from 0.38 to 0.45, clearly indicating a strong synergistic effect; while the CI values ​​of the control groups H-sEV / Res and N-sEV / Res only showed a weak synergistic or additive effect. This significant quantitative difference, combined with the stronger synergistic regulatory ability on the PI3K / Akt pro-survival pathway and the Caspase-3 apoptosis pathway revealed by Western blotting, jointly confirms that the specific pretreatment process of "hypoxia combined with TGF-β1" is the fundamental reason for the unexpected strong synergistic effect between sEV and resveratrol.

[0128] Example 4: In vivo pharmacodynamic study

[0129] 1. Animal model establishment, grouping, and drug administration

[0130] 1.1 Experimental animals: Thirty-two 6-8 week old, 18-22 g SPF-grade female C57BL / 6 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected. All mice were acclimatized for one week in a standard environment (temperature 22±2°C, humidity 50±10%, 12-hour light-dark cycle) with free access to food and water.

[0131] 1.2 Establishment of Chemotherapy-Induced Point-of-Impact (POI) Model: After adaptive feeding, except for the sham-operated group, the remaining mice were injected intraperitoneally with a mixture of chemotherapy drugs to establish the model. Preparation of the mixture: Cyclophosphamide (CTX) and cisplatin (DDP) were dissolved separately in physiological saline and mixed at a dose of 50 mg / kg CTX and 2.5 mg / kg DDP, adjusting the injection volume to 10 mL / kg body weight. The sham-operated group (Sham) was injected with an equal volume of physiological saline.

[0132] 1.3 Grouping and Dosing Regimen: 24 hours after modeling, mice were randomly divided into 5 groups (n=8):

[0133] Group 1: Sham surgery group: Sterile PBS (10 mL / kg) was injected via the tail vein every 3 days for a total of 4 times.

[0134] Group 2: Model group: Same as the Sham group, but with sterile PBS injected via tail vein.

[0135] Group 3: N-sEV / Res treatment group: N-sEV / Res complex preparation was administered via tail vein injection. The preparation was freshly formulated according to the method in Example 2, with an injection dose of 5 mg / kg sEV protein + 5 mg / kg Res, and an injection volume of 10 mL / kg, administered once every 3 days for a total of 4 times.

[0136] Group 4: H-sEV / Res treatment group: H-sEV / Res complex preparation was injected via tail vein, with the same dosage and regimen as Group 3.

[0137] Group 5: Op-sEV / Res treatment group (core group of this invention): Op-sEV / Res complex preparation is injected via tail vein, with the same dosage and regimen as Group 3.

[0138] All therapeutic preparations were prepared on ice within 2 hours prior to administration and sterilized by filtration through a 0.22 μm filter.

[0139] 2. Sample collection and processing: Samples were collected from mice on day 7 after the last administration.

[0140] 2.1 Blood Collection: Collect approximately 0.8-1.0 mL of whole blood via the retro-orbital venous plexus. After allowing the blood to stand at room temperature for 2 hours, centrifuge at 3000×g for 15 minutes at 4°C to separate the serum, aliquot it, and store it at -80°C for hormone detection.

[0141] 2.2 Ovarian Tissue Collection: After blood collection, mice were euthanized by cervical dislocation. Both ovaries were quickly removed via laparotomy, rinsed in pre-cooled physiological saline, and blotted dry with filter paper. The left ovary was immediately flash-frozen in liquid nitrogen and then transferred to -80°C for storage for molecular biological analysis; the right ovary was placed in Bouin's fixative (24 hours) for histological analysis.

[0142] 3. Detection indicators, raw data and result analysis

[0143] 3.1 Serum hormone level detection (ELISA)

[0144] Methods: The procedure was strictly performed according to the instructions of the mouse anti-Müllerian hormone (AMH) and mouse follicle-stimulating hormone (FSH) enzyme-linked immunosorbent assay (ELISA) kits. Absorbance (OD) was read at 450 nm using an ELISA reader. The concentration of each sample was calculated based on the standard curve.

[0145] The results are shown in Table 9: After chemotherapy, the serum AMH level in the Model group mice decreased sharply, while the FSH level increased significantly, indicating severe ovarian dysfunction and a successful model. All treatment groups were able to reverse this trend to varying degrees. The Op-sEV / Res group showed the most significant effect: its AMH level recovered to 79.6% of the Sham group, significantly higher than the N-sEV / Res group (42.6%) and the H-sEV / Res group (57.0%); its FSH level was also closest to normal, significantly lower than the other two treatment groups. This directly demonstrates that the composition of this invention has the optimal effect in regulating ovarian endocrine function.

[0146] Table 9. Serum AMH and FSH levels in mice of each group (mean ± SD, n=8)

[0147]

[0148] Note: Compared with the Model group, P < 0.01; compared with the N-sEV / Res group, ##P < 0.01; compared with the H-sEV / Res group, &&P < 0.01. One-way ANOVA, Tukey test.

[0149] 3.2 Ovarian histology and follicle count

[0150] Methods: Ovarian tissue fixed with Bouin's fixative was dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin. Serial sections (5 μm thick) were prepared along the largest diameter of the ovary, with sections taken at 50 μm intervals, for a total of 5 sections, and stained with hematoxylin and eosin (HE). Follicles were counted in all sections. Follicle classification criteria: primordial follicles (a single layer of flattened granulosa cells surrounding the oocyte) and growing follicles (including primary and secondary follicles, with increased granulosa cell layers or the presence of sinus cavities). The average number of follicles in both ovaries of each mouse was calculated.

[0151] The results are shown in Table 10: Histological analysis directly reflects the treatment effect. In the Model group, ovarian volume atrophy was observed, with numerous atretic follicles and stromal fibrosis. The treatment groups showed varying degrees of improvement in ovarian structure. Quantitative follicle counting is the gold standard for assessing ovarian reserve function. Data showed that the Op-sEV / Res group was significantly superior to other treatment groups in both the number of primordial follicles (key to ovarian reserve) and the number of growing follicles preserved. Its primordial follicle count recovered to 80.7% of the Sham group, while the H-sEV / Res and N-sEV / Res groups only achieved 58.5% and 43.5%, respectively. This strongly demonstrates that the composition of this invention can most effectively protect the ovarian primordial follicle pool damaged by chemotherapy and maintain its reproductive potential.

[0152] Table 10. Ovarian follicle count results of mice in each group (follicles / ovary, mean ± SD, n=8)

[0153]

[0154] Note: Compared with the Model group, P < 0.01; compared with the N-sEV / Res group, ##P < 0.01; compared with the H-sEV / Res group, &&P < 0.01.

[0155] 3.3 Assessment of Ovarian Tissue Oxidative Stress and Inflammatory Status

[0156] Methods: Ovarian tissue stored at -80°C was taken and pre-cooled physiological saline was added at a weight (mg):volume (mL) ratio of 1:9. A 10% tissue homogenate was prepared in an ice bath using a homogenizer. The homogenate was centrifuged at 3000×g for 10 minutes at 4°C, and the supernatant was collected.

[0157] Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content: The SOD and MDA were determined using commercial kits (SOD, A001-3; MDA, A003-1) strictly following the instructions, employing the xanthine oxidase method and the thiobarbituric acid method.

[0158] NF-κB p65 nuclear protein expression (Western Blot): Nuclear protein was isolated from another portion of ovarian tissue using a nuclear protein extraction kit. The method was the same as in Example 3, with NF-κB p65 (1:1000) as the primary antibody and Lamin B1 (1:1000) as the internal control.

[0159] The results are shown in Table 11: Chemotherapy caused an imbalance in the oxidative-antioxidant system of ovarian tissue (decreased SOD activity and increased MDA) and activated inflammatory pathways (increased NF-κB p65 nuclear translocation). Op-sEV / Res treatment most effectively reversed these pathological changes: its effects in increasing SOD activity, reducing MDA content, and inhibiting NF-κB p65 nuclear translocation were significantly better than those in the N-sEV / Res and H-sEV / Res groups. This reveals the deep mechanism by which the composition of the present invention exerts its superior protective effect at the tissue microenvironment level, namely, by creating a more favorable local environment for follicle survival through stronger antioxidant and anti-inflammatory capabilities.

[0160] Table 11. Relative expression of oxidative stress markers and NF-κB p65 nuclear protein in ovarian tissue (mean ± SD, n=6)

[0161]

[0162] Note: Compared with the Model group, P < 0.01; compared with the N-sEV / Res group, ##P < 0.01; compared with the H-sEV / Res group, &&P < 0.01.

[0163] 4. Summary

[0164] This study, through systematic animal experiments, demonstrated that the composition of this invention (Op-sEV / Res) exhibited remarkable synergistic therapeutic effects in a mouse model of chemotherapy-induced premature ovarian failure. It most significantly restored ovarian endocrine function (significantly increasing AMH and decreasing FSH levels) and maximally protected ovarian follicular reserve (especially primordial follicles), with effects significantly superior to the control groups treated with N-sEV / Res and H-sEV / Res. This superior in vivo efficacy stems from the synergistic and strongest antioxidant (increasing SOD and decreasing MDA) and anti-inflammatory (inhibiting NF-κB activation) effects of this composition in ovarian tissue. These results not only perfectly confirm the strong synergistic effect observed in vitro but also extend the key value of the specific pretreatment process of "hypoxia combined with TGF-β1" from the cellular level to the level of complete organ function repair, providing solid in vivo evidence for the inventiveness and practicality of this study.

[0165] 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 composition for improving premature ovarian failure, characterized in that, The composition consists of small extracellular vesicles derived from functionally optimized human umbilical cord mesenchymal stem cells and resveratrol; The functionally optimized small extracellular vesicles are obtained by treating human umbilical cord mesenchymal stem cells with 5-15 ng / mL TGF-β1 for 36-60 hours under 1% oxygen concentration conditions, and then separating them from their culture supernatant. The mass ratio of the functionally optimized small extracellular vesicles to resveratrol is 1:0.5 to 1:

2.

2. The composition according to claim 1, characterized in that, The treatment concentration of TGF-β1 was 10 ng / mL, and the treatment time was 48 hours.

3. The composition according to claim 1, characterized in that, The mass ratio of the functionally optimized small extracellular vesicles to resveratrol is 1:

1.

4. A method for preparing the composition according to any one of claims 1-3, characterized in that, The process includes the following steps: (1) Under hypoxic conditions with an oxygen concentration of 1%, human umbilical cord mesenchymal stem cells were cultured in a medium containing 10 ng / mLTGF-β1 for 48 hours, and the supernatant of the medium was collected. (2) The conditioned medium from step (1) was subjected to differential centrifugation and ultracentrifugation to purify and obtain functionally optimized small extracellular vesicles; (3) The purified functionally optimized small extracellular vesicles and resveratrol were dissolved together in a buffer solution at a certain mass ratio and incubated at 37°C for 30 minutes. (4) The incubation solution from step (3) is subjected to ultrafiltration and centrifugation to remove unbound free resveratrol, thereby obtaining the composition.

5. The method according to claim 4, characterized in that, The mass ratio of the functionally optimized small extracellular vesicles to resveratrol in step (3) is 1:

1.

6. The method according to claim 4, characterized in that, The buffer solution in step (3) is a pre-cooled PBS solution with a pH of 7.

4.

7. Use of the composition according to any one of claims 1-3 in the preparation of a medicament for the prevention or treatment of premature ovarian failure; wherein the premature ovarian failure is ovarian dysfunction induced by chemotherapy drugs.

8. Use of the composition according to any one of claims 1-3 in the preparation of a medicament for protecting ovarian follicular reserve.

9. Use of the composition according to any one of claims 1-3 in the preparation of a medicament for reducing oxidative stress levels in ovarian tissue.

10. Use of the composition according to any one of claims 1-3 in the preparation of a medicament for reducing inflammatory response in ovarian tissue.

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