A method for treating premature ovarian insufficiency using a dual-targeting biomimetic nanoparticle based on extracellular vesicles from wolfberry source

Abstract

The application discloses a method for treating premature ovarian insufficiency by using a double-targeting biomimetic nanoparticle based on a wolfberry-derived extracellular vesicle, relates to the technical field of premature ovarian insufficiency, and obtains wolfberry-derived extracellular vesicles, uses the wolfberry-derived extracellular vesicles as a nano delivery carrier, and realizes the treatment effect on the damage related to chemotherapy-induced premature ovarian insufficiency by using active ingredients carried by the wolfberry-derived extracellular vesicles; the wolfberry-derived extracellular vesicles are coated by using homologous ovarian granulosa cell membranes, and a GCM-LBEVs biomimetic nanoparticle vesicle with granulosa cell recognition ability is constructed. The application realizes the accurate delivery of ovarian granulosa cells and mitochondria by constructing a TPP-GCM-LBEVs double-targeting biomimetic nano system, improves the quality of oocytes, ovarian function and fertility, and provides a new nano strategy for the treatment of POI and mitochondrial-related reproductive disorders.

Description

Technical Field

[0001] This application relates to the field of early-onset ovarian insufficiency technology, specifically to a method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry. Background Technology

[0002] Premature ovarian insufficiency (POI) is a disease characterized by ovarian function decline, reduced ovarian reserve, decreased estrogen levels, and impaired fertility in women before the age of 40. Chemotherapy-induced ovarian insufficiency is one of the important complications affecting women's reproductive health after clinical cancer treatment.

[0003] Existing research indicates that chemotherapy drugs can induce oxidative stress, damage mitochondrial DNA (mtDNA), disrupt mitochondrial cristae structure, and inhibit electron transport chain function, leading to mitochondrial dysfunction in granulosa cells (GCs). This results in reduced ATP production, abnormal accumulation of reactive oxygen species (ROS), and further induces granulosa cell apoptosis and senescence. Since granulosa cells play a crucial role in maintaining oocyte nutrition, regulating hormone secretion, and providing metabolic support during follicular development, abnormal granulosa cell function can lead to increased follicular atresia, depletion of ovarian reserve, and ultimately, decreased ovarian endocrine function and reduced fertility.

[0004] Currently, clinical interventions for chemotherapy-induced POI mainly include hormone replacement therapy, antioxidant therapy, and assisted reproductive technologies. However, these methods primarily focus on symptom improvement or fertility assistance, failing to fundamentally repair the damaged ovarian microenvironment and restore mitochondrial function in granulosa cells. In recent years, extracellular vesicles (EVs) have been widely studied as novel delivery carriers for tissue repair and disease treatment due to their natural biocompatibility, low immunogenicity, and ability to carry various bioactive molecules such as proteins, nucleic acids, and lipids. Plant-derived extracellular vesicles (PEVs), in particular, have gradually become an important research direction in the biomedical field due to their abundant sources, low preparation costs, and high safety. Among them, Lycium barbarum (LB), a traditional medicinal and edible plant, has good biocompatibility. Extracellular vesicles derived from Lycium barbarum (LBEVs) have been found to have potential effects such as regulating oxidative stress, improving mitochondrial function, and promoting cellular homeostasis.

[0005] Furthermore, to enhance the targeting capabilities of nanodelivery systems, the construction of biomimetic nanoparticles based on natural cell membrane coating technology has garnered significant attention in recent years. Natural cell membranes retain glycoproteins, glycolipids, and membrane receptor structures from the surface of the source cells, enabling nanocarriers to acquire immune evasion, prolonged circulation time, and homologous cell recognition capabilities. Granulosa cell membranes (GCMs), derived from ovarian function-related cells, theoretically possess the ability to endow delivery systems with recognition and homing capabilities for ovarian granulosa cells. Simultaneously, triphenylphosphonium (TPP), a classic mitochondrial targeting modification group, can utilize mitochondrial membrane potential differences to promote the accumulation of nanomaterials in mitochondria. Therefore, combining plant-derived EVs, cell membrane biomimetic technology, and mitochondrial targeting modification strategies to construct novel delivery systems with dual targeting capabilities at both the tissue and organelle levels has become an important research direction for improving the therapeutic effects of point-of-injection (POI). Figure 1 As shown, Figure 1 This is a schematic diagram illustrating the design principle and treatment mechanism of TPP-GCM-LBEVs in POI treatment.

[0006] However, existing POI-based therapies based on extracellular vesicles still suffer from insufficient targeting efficiency and limited intracellular delivery efficiency. On the one hand, while plant-derived extracellular vesicles possess good biocompatibility and therapeutic potential, they lack specific recognition structures for ovarian tissue, leading to non-specific distribution during in vivo circulation and uptake and clearance by immune cells such as macrophages in the mononuclear phagocytic system, thus limiting their effective accumulation in ovarian tissue. On the other hand, existing EV delivery systems primarily rely on endocytosis for intracellular transport. After entering the cell, a portion of the EV enters the endosome / lysosome degradation pathway, resulting in low delivery efficiency of the active ingredient to functional target organelles such as mitochondria. Since one of the important pathological mechanisms of chemotherapy-induced POI is mitochondrial dysfunction in granulosa cells, improving the precise delivery capability of EVs at the mitochondrial level is crucial for restoring granulosa cell function.

[0007] In addition, existing biomimetic nanodelivery systems based on natural cell membrane coatings mainly enhance recognition capabilities at the cellular or tissue level, but typically lack the ability to further regulate organelle localization.

[0008] Therefore, in the field of POI treatment, there is still a lack of a dual-targeting biomimetic EV platform that combines the ability to target ovarian granulosa cells with the ability to precisely deliver to mitochondria.

[0009] The information disclosed in the background section is only intended to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0010] The purpose of this application is to provide a method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry, in order to solve the problems in the background art mentioned above.

[0011] To achieve the above objectives, this application provides the following technical solution: a method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry, characterized by comprising the following steps: We obtained extracellular vesicles (LBEVs) derived from wolfberry and used them as nano-delivery carriers to achieve therapeutic effects on chemotherapy-induced early-onset ovarian insufficiency-related damage by utilizing the active ingredients carried by the LBEVs. We used homologous ovarian granulosa cell membrane (GCM) to coat extracellular vesicles derived from wolfberry to construct GCM-LBEVs biomimetic nanovesicles with granulosa cell recognition ability. The granulosa cell membrane endowed the GCM-LBEVs biomimetic nanovesicles with ovarian granulosa cell targeting ability. Triphenylphosphine (TPP) was used to functionalize the surface of GCM-LBEVs biomimetic nanovesicles to obtain TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles. The mitochondrial targeting properties of TPP were used to improve the delivery efficiency of TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles to granulocyte mitochondria. TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles were applied to a chemotherapy-induced early-onset ovarian insufficiency model. The selective delivery of TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles to ovarian granulosa cells was achieved through granulosa cell targeting capability. The mitochondrial targeting capability was used to restore the structural integrity and bioenergy function of mitochondria in granulosa cells. By utilizing TPP-GCM-LBEVs dual-targeted biomimetic nanovesicles to repair granulosa cells and maintain ovarian microenvironment stability, the granulosa cells continuously supply ATP and NAD(P)H to damaged oocytes, thereby improving oocyte quality and developmental potential. This approach aims to restore follicle development, ovarian endocrine function, and fertility in patients with chemotherapy-induced early-onset ovarian insufficiency.

[0012] Preferably, the extracellular vesicles derived from wolfberry are obtained through plant tissue isolation and purification, specifically including: washing and treating fresh wolfberry fruits and homogenizing them in ice-pre-cooled phosphate buffer to obtain a mixture containing extracellular vesicles. The mixture is then centrifuged sequentially at 1000×g, 2000×g, and 10000×g to remove tissue residues, cell debris, and non-target particles. The vesicle precipitate is then collected by ultracentrifugation at 150000×g and further purified using 8%, 30%, 45%, and 60% sucrose density gradient systems to collect the wolfberry-derived extracellular vesicle component from the 45% sucrose layer.

[0013] Preferably, the homologous ovarian granulosa cell membrane is obtained by lysis and separation of granulosa cells. After washing the granulosa cells with phosphate buffer, they are subjected to liquid nitrogen freeze-thaw cycles to release the cell membrane structure. The cell nucleus and cell debris are removed by differential centrifugation, and the granulosa cell membrane components containing membrane proteins, glycoproteins and lipid bilayer structures are collected. The obtained granulosa cell membrane is then subjected to extrusion treatment with polycarbonate membranes with pore sizes of 1000 nm, 400 nm and 200 nm in sequence to prepare granulosa cell membrane-derived nanovesicles with uniform particle size.

[0014] Preferably, GCM-LBEVs biomimetic nanovesicles are obtained by fusing nanovesicles derived from granular cell membranes with extracellular vesicles derived from wolfberry. The nanovesicles derived from granular cell membranes and extracellular vesicles derived from wolfberry are mixed in a 1:1 mass ratio and the membrane structure is fused by ice bath ultrasonic treatment. The fusion product is further homogenized by extrusion through a 200nm polycarbonate membrane to obtain a biomimetic nanovesicle structure that combines the ability to deliver active ingredients from plant-derived extracellular vesicles with the ability to recognize homologous components from granular cells.

[0015] Preferably, triphenylphosphine is obtained via cholesterol-PEG. 2000 -TPP functionalized molecules are linked to the surface of GCM-LBEVs biomimetic nanovesicles, where cholesterol structures are embedded in the lipid bilayer of the nanovesicles, and PEG... 2000 Chain segments maintain dispersion stability; triphenylphosphine groups utilize mitochondrial membrane potential differences to achieve subcellular organelle localization regulation; cholesterol-PEG 2000 -TPP was added to the GCM-LBEVs system at a vesicle to modified molecular weight ratio of 1:10, and the surface functionalization modification was completed by gentle shaking incubation at 37°C.

[0016] Preferably, the TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles have selective uptake characteristics of granulosa cells. They enhance the interaction ability with ovarian granulosa cells through membrane proteins, glycoproteins and homology recognition molecules retained on the surface of granulosa cell membranes. When TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles are co-incubated with KGN granulosa cells, they exhibit cellular uptake characteristics and have source cell-related recognition ability compared with 3T3 cells and ID8 cells.

[0017] Preferably, the process of TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles entering granulosa cells involves both caveolin-mediated endocytosis and lipid raft-mediated endocytosis. The relevant endocytosis process is regulated by genistein and methyl-β-cyclodextrin, and the localization relationship between nanovesicles and lysosomes is detected by LysoTracker staining. This reduces the risk of inactivation of nanovesicles after entering the lysosome and improves the effectiveness of vesicle contents transport to the mitochondrial region.

[0018] Preferably, TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles were applied to an animal model of early-onset ovarian insufficiency induced by cyclophosphamide. The administration method was intraperitoneal injection at a dose of 50 mg / kg, administered once every 10 days for three consecutive treatments. The recovery status of ovarian endocrine function was evaluated by detecting ovarian index, estrous cycle, serum anti-Müllerian hormone, estradiol, and follicle-stimulating hormone levels.

[0019] Preferably, TPP-GCM-LBEVs dual-target biomimetic nanovesicles improve the physiological state of oocytes by restoring mitochondrial function in granulosa cells. The restoration process includes regulating mitochondrial reactive oxygen species levels, increasing glutathione content, promoting ATP and NAD(P)H production, improving the number and ultrastructural integrity of oocyte mitochondria, and promoting damaged oocytes to obtain a suitable developmental environment.

[0020] Preferably, the therapeutic effect of TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles was verified through in vitro fertilization experiments and natural fertility evaluation. In vitro fertilization tests included analysis of 2-cell embryo formation rate and blastocyst formation rate. Natural fertility evaluation included detection of pregnancy rate and average litter size in female mice. At the same time, the in vivo safety performance of nanovesicles was evaluated by hematoxylin-eosin staining of heart, liver, spleen, lung and kidney tissues and detection of serum AST, ALT, TBIL, CREA and UREA indicators.

[0021] The technical effects and advantages provided by this application in the above technical solution are as follows: This application constructs a TPP-GCM-LBEVs dual-target biomimetic nanodelivery system, which utilizes the homology recognition properties derived from granulosa cell membranes to enhance the localization ability of extracellular vesicles derived from wolfberry in the ovarian region and strengthen the interaction between nanovesicles and granulosa cells, thereby improving the effective delivery level of therapeutic components in target cells and providing a new delivery method for precise intervention in chemotherapy-induced early-onset ovarian insufficiency.

[0022] This application utilizes the mitochondrial targeting properties of triphenylphosphine to enable TPP-GCM-LBEVs to further accumulate in the mitochondrial region after entering granulosa cells, thereby improving the utilization efficiency of active ingredients in mitochondria, reducing the risk of degradation after vesicles enter lysosomes, and helping to restore the structural stability of damaged mitochondria and improve mitochondrial energy metabolism.

[0023] This application is based on the mitochondrial function repair function of granulosa cells, which promotes the restoration of redox balance and the reconstruction of the metabolic environment, enhances the nutritional support capacity of granulosa cells for oocytes, improves the energy supply status of oocytes, thereby improving the quality of oocytes and the potential for subsequent embryonic development, and achieves multi-level regulation of the ovarian function damage process.

[0024] This application combines plant-derived extracellular vesicles with mammalian cell membrane function through cross-species biomimetic design, achieving follicle development recovery, ovarian endocrine function improvement, and natural fertility enhancement in animal models while maintaining good in vivo safety. This provides a promising new nanotherapy strategy for the treatment of early-onset ovarian insufficiency and mitochondrial-related reproductive disorders. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings.

[0026] Figure 1 A schematic diagram illustrating the overall design principle and mechanism of action of TPP-GCM-LBEVs dual-targeting biomimetic nanoparticles for POI therapy.

[0027] Figure 2 A schematic diagram illustrating the preparation process of extracellular vesicles (LBEVs) derived from wolfberry and their basic therapeutic effects in POI granule cells and mouse models.

[0028] Figure 3 This diagram illustrates the construction process, structural characterization, and in vitro verification of the dual-targeting capabilities of TPP-GCM-LBEVs biomimetic nanoparticles targeting cells and mitochondria.

[0029] Figure 4 This diagram illustrates the ability of TPP-GCM-LBEVs to accumulate in the ovaries, localize to granulosa cells, and target mitochondria in POI mice.

[0030] Figure 5 A schematic diagram illustrating the main endocytic pathway by which TPP-GCM-LBEVs enter granulocytes and the mechanism by which they reduce lysosomal degradation to achieve efficient delivery.

[0031] Figure 6 This diagram illustrates the effects of TPP-GCM-LBEVs treatment on the recovery of mitochondrial structure, improvement of respiratory function, and reconstruction of energy metabolism in POI mouse ovarian granulosa cells.

[0032] Figure 7 This diagram illustrates the effect of TPP-GCM-LBEVs treatment on the redox status, energy metabolism level, and mitochondrial quality improvement of POI mouse oocytes.

[0033] Figure 8 This diagram illustrates the assessment of ovarian reserve function, endocrine levels, and follicular development recovery in POI mice after TPP-GCM-LBEVs treatment.

[0034] Figure 9 This diagram illustrates the effect of TPP-GCM-LBEVs treatment on the recovery of in vitro embryonic development and natural fertility in POI mice.

[0035] Figure 10This diagram illustrates the evaluation of granulocyte proliferation activity and cell safety under different concentrations of LBEVs treatment.

[0036] Figure 11 For cholesterol-PEG 2000 -Schematic diagram of chemical structure characterization and synthesis verification of TPP functionalized modified molecules.

[0037] Figure 12 This is a schematic diagram of the morphology of major organs and tissues and the safety evaluation of H&E staining after TPP-GCM-LBEVs treatment of POI mice.

[0038] Figure 13 This diagram illustrates the detection of blood biochemical parameters and in vivo safety evaluation in POI mice after treatment with TPP-GCM-LBEVs. Detailed Implementation

[0039] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the examples set forth herein; rather, they are provided so that the description of this disclosure will be more complete and fully convey the concept of the exemplary embodiments to those skilled in the art.

[0040] like Figures 2 to 13 As shown, this application provides a method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry, comprising the following steps: We obtained extracellular vesicles (LBEVs) derived from wolfberry and used them as nano-delivery carriers to achieve therapeutic effects on chemotherapy-induced early-onset ovarian insufficiency-related damage by utilizing the active ingredients carried by the LBEVs. We used homologous ovarian granulosa cell membrane (GCM) to coat extracellular vesicles derived from wolfberry to construct GCM-LBEVs biomimetic nanovesicles with granulosa cell recognition ability. The granulosa cell membrane endowed the GCM-LBEVs biomimetic nanovesicles with ovarian granulosa cell targeting ability. Triphenylphosphine (TPP) was used to functionalize the surface of GCM-LBEVs biomimetic nanovesicles to obtain TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles. The mitochondrial targeting properties of TPP were used to improve the delivery efficiency of TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles to granulocyte mitochondria. TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles were applied to a chemotherapy-induced early-onset ovarian insufficiency model. The selective delivery of TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles to ovarian granulosa cells was achieved through granulosa cell targeting capability. The mitochondrial targeting capability was used to restore the structural integrity and bioenergy function of mitochondria in granulosa cells. By utilizing TPP-GCM-LBEVs dual-targeted biomimetic nanovesicles to repair granulosa cells and maintain ovarian microenvironment stability, the granulosa cells continuously supply ATP and NAD(P)H to damaged oocytes, thereby improving oocyte quality and developmental potential. This approach aims to restore follicle development, ovarian endocrine function, and fertility in patients with chemotherapy-induced early-onset ovarian insufficiency.

[0041] Specific implementation method 1: Preparation of extracellular vesicles (LBEVs) derived from wolfberry and their basic therapeutic verification on chemotherapy-induced early-onset ovarian insufficiency; This embodiment provides a basic nanocarrier preparation process and preliminary therapeutic efficacy verification method involved in the treatment of early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on Lycium barbarum-derived extracellular vesicles. This embodiment first prepares biocompatible Lycium barbarum-derived extracellular vesicles (LBEVs), using natural plant-derived nanovesicles as the core component of the subsequent biomimetic nanodelivery system. Specifically, fresh Lycium barbarum fruits with stable sources are selected as raw materials. After thoroughly washing away impurities from the surface of the Lycium barbarum fruits, they are pretreated in ice-cold phosphate-buffered saline (PBS) to reduce the impact of temperature increases during processing on the integrity of the vesicle structure. Subsequently, mechanical homogenization is used to disrupt the plant tissue structure, allowing the full release of cell contents to obtain a mixed juice containing cell debris, protein aggregates, organelles, and extracellular vesicles.

[0042] The obtained wolfberry juice was first subjected to low-speed gradient centrifugation to gradually remove larger tissue debris and non-target particles. Centrifugation at 1000×g for 20 minutes removed undisturbed tissue and large cell fragments; further centrifugation at 2000×g for 20 minutes removed smaller cell debris and aggregated impurities; subsequently, centrifugation at 10000×g for 60 minutes removed organelle residues, large vesicle structures, and other non-specific precipitates, yielding a supernatant rich in plant-derived extracellular vesicles. This supernatant was then further ultracentrifuged at 150000×g for 120 minutes, causing smaller, membrane-structured wolfberry-derived extracellular vesicles to deposit at the bottom of the centrifuge tube. The precipitate was collected and gently resuspended in PBS to obtain a preliminarily purified LBEV sample.

[0043] To further improve the purity of LBEVs and reduce interference from protein aggregates and non-vesicle structures, a discontinuous sucrose density gradient ultracentrifugation method was used for secondary purification. Specifically, the initially obtained LBEVs were sequentially loaded into sucrose gradient systems of 8%, 30%, 45%, and 60%, and centrifuged at 150,000 × g for 1.5 hours. The difference between the different density components was used to further separate the vesicle structures. After density gradient separation, the target vesicle components located in the 45% sucrose layer were collected and washed with PBS to remove residual sucrose and low-molecular-weight impurities, finally obtaining high-purity LBEV samples, which were then stored at low temperature for later use.

[0044] like Figure 2 Figure A illustrates the complete preparation process of LBEVs, including key steps such as wolfberry raw material processing, juice extraction, filtration and impurity removal, ultracentrifugation, and density gradient purification. The LBEVs obtained through this method exhibit a stable membrane structure and good morphological integrity. Figure 2 As shown in Figure B, the morphology of LBEVs was observed using transmission electron microscopy (TEM). The results showed that the obtained nanovesicles exhibited typical extracellular vesicle morphology, mainly showing regular spherical or cup-shaped double membrane structures. The particle size distribution conformed to the characteristics of natural extracellular vesicles, indicating that the above separation and purification method can effectively obtain structurally intact plant-derived extracellular vesicles.

[0045] The in vitro biosafety of LBEVs was further evaluated. Ovarian granulosa cells were used as the evaluation subject. Different concentrations of LBEVs were added to the cell culture system, including 25 μg / mL, 50 μg / mL, 100 μg / mL, and 150 μg / mL. The effect of LBEVs on granulosa cell viability was evaluated using cell proliferation assays. Figure 10 As shown, within the above concentration range, no significant inhibition of cell proliferation was observed in granulocytes treated with LBEVs, and cell activity remained at a high level across different treatment groups. This indicates that LBEVs have good cell compatibility and low cytotoxicity, and can be used as a natural carrier material for subsequent biomimetic nanodelivery systems.

[0046] Based on the confirmed good biocompatibility of LBEVs, a chemotherapy-induced early-onset ovarian insufficiency (POI) extracellular cell model was further established to evaluate the repair effect of LBEVs on mitochondrial damage in granulosa cells. Specifically, ovarian granulosa cells were treated with 4-hydroxycyclophosphamide (4-OH-CTX) to establish a POI cell damage model by simulating chemotherapy-induced oxidative stress. Subsequently, a certain concentration of LBEVs was added to the damage model for intervention, and mitochondrial function-related indicators of granulosa cells were detected.

[0047] Experimental results showed that 4-OH-CTX treatment caused significant mitochondrial dysfunction in granulosa cells, manifested as a significant increase in mitochondrial reactive oxygen species (mitoROS) levels, a decrease in mitochondrial membrane potential (MMP), and reduced intracellular ATP production. This indicates that chemotherapy damage disrupts redox homeostasis and leads to abnormal energy metabolism in granulosa cells. After LBEV intervention, intracellular mitoROS levels in granulosa cells significantly decreased, mitochondrial membrane potential was restored, and ATP production significantly improved. Figure 2 C to Figure 2 As shown in H, LBEVs can effectively alleviate chemotherapy-induced mitochondrial oxidative damage and improve the energy metabolism level of granulocytes, indicating that the natural active ingredients carried by LBEVs can protect damaged mitochondria.

[0048] Further analysis of the subcellular localization of LBEVs after entering granulosa cells was conducted using fluorescent labeling combined with confocal microscopy. Colocalization analysis of LBEV fluorescence signals and mitochondrial labeling signals revealed that although LBEVs could enter granulosa cells and exert a certain mitochondrial protective effect, the overlap between LBEVs and mitochondrial regions was low. Figure 2 G and Figure 2 As shown in Figure I, natural LBEVs lack active mitochondrial targeting capabilities, and their therapeutic efficacy may be limited by intracellular transport efficiency.

[0049] In verifying the in vivo therapeutic effect, a cyclophosphamide-induced POI mouse model was further constructed. Specifically, female C57BL / 6J mice were used as experimental subjects, and a chemotherapy-related ovarian function impairment model was established by intraperitoneal injection of cyclophosphamide. After the model was established, LBEVs were administered intraperitoneally at a dose of 50 mg / kg, and the effect of LBEVs on improving ovarian function was comprehensively evaluated using multiple indicators, including ovarian index, estrous cycle changes, serum hormone levels, and follicular development status.

[0050] like Figure 2 As shown in Figure J, the POI mouse model construction and LBEVs treatment process are illustrated. Further testing revealed that LBEVs treatment could improve ovarian function abnormalities in POI mice to some extent. Specifically, serum anti-Müllerian hormone (AMH) levels recovered, estradiol (E2) levels increased, follicle-stimulating hormone (FSH) levels decreased, and abnormal follicle activation and atresia were partially improved. Figure 2 K to Figure 2As shown in Figure 1, some follicle development indicators improved after LBEVs treatment, indicating that LBEVs have a certain protective effect on the ovaries.

[0051] However, further analysis revealed that LBEVs alone still have certain limitations. Although LBEVs can improve mitochondrial function in granulosa cells and alleviate oxidative stress damage, natural LBEVs lack active recognition structures targeting ovarian tissue and have limited mitochondrial localization capabilities. Therefore, their local accumulation in the ovary and intracellular mitochondrial delivery efficiency are still limited, and thus they cannot fully restore ovarian reserve and fertility in POI mice.

[0052] Therefore, this embodiment verifies the application potential of Lycium barbarum-derived extracellular vesicles as a natural therapeutic carrier in the treatment of chemotherapy-induced early-onset ovarian insufficiency. At the same time, it clarifies the problems of insufficient ovarian targeting ability and limited mitochondrial delivery efficiency of natural LBEVs, providing a technical basis for the subsequent construction of TPP-GCM-LBEVs dual-targeting biomimetic nanoparticles through granule cell membrane biomimetic modification and triphenylphosphine mitochondrial targeting modification.

[0053] Specific Implementation Method 2: Construction and structural performance verification of TPP-GCM-LBEVs dual-targeted biomimetic nanoparticles; This embodiment provides a method for constructing dual-targeting biomimetic nanoparticles TPP-GCM-LBEVs based on Lycium barbarum-derived extracellular vesicles. By introducing biomimetic modifications to the ovarian granulosa cell membrane and mitochondrial-targeting functional modifications, the nanovesicles achieve dual precise delivery to ovarian granulosa cells and their mitochondria. This embodiment uses Lycium barbarum-derived extracellular vesicles (LBEVs) obtained in Embodiment 1 as the basic nanocarrier. By encapsulating the vesicles with homologous granulosa cell membranes, tissue and cell recognition capabilities are acquired. Then, by functional modification with triphenylphosphonium (TPP), the subcellular organelle localization capability is improved, ultimately constructing TPP-GCM-LBEVs biomimetic nanoparticles with both cell-targeting and mitochondrial-targeting characteristics.

[0054] Specifically, granulosa cell membranes (GCMs) derived from ovarian granulosa cells were first prepared. Well-cultured granulosa cells were selected, thoroughly washed with PBS buffer, and collected. The collected cells were resuspended in pre-cooled PBS, and the cell membrane integrity was disrupted through multiple liquid nitrogen freeze-thaw cycles, allowing for the full release of cytoplasmic contents while retaining cell membrane components characterized by membrane proteins, glycoproteins, and lipid bilayer structures. Subsequently, differential centrifugation was used to separate the cell lysis products. First, cell nuclei, large cell debris, and incompletely lysed cell structures were removed, followed by high-speed centrifugation to collect membrane components rich in granulosa cell membrane structures. The GCMs obtained through this method retain characteristic membrane proteins, receptor structures, and homology recognition molecules from the surface of the derived granulosa cells, providing a foundation for the subsequent construction of a biomimetic nanodelivery system with granulosa cell homing capabilities.

[0055] After obtaining the granulocyte cell membrane components, GCM-derived nanovesicles were further prepared using membrane extrusion technology. Specifically, purified GCM was sequentially extruded through polycarbonate membranes with pore sizes of 1000 nm, 400 nm, and 200 nm, gradually transforming the large, non-uniform membrane structure into uniformly sized and structurally stable GCM nanovesicles. By controlling the number of extrusion cycles, pore size, and operating conditions, the obtained GCM nanovesicles exhibited good particle size distribution and membrane structural integrity, while preserving the natural recognition molecules on the granulocyte cell membrane surface to the greatest extent possible.

[0056] Subsequently, the obtained GCM nanovesicles were fused with the LBEVs prepared in Example 1. Specifically, GCM nanovesicles and LBEVs were mixed at a mass ratio of 1:1 and treated with low-intensity ultrasound under ice bath conditions. The mechanical energy generated by ultrasound promoted the contact and fusion between the two vesicle membrane structures, integrating the plant-derived extracellular vesicle membrane structure with the granular cell membrane structure. After ultrasound treatment, the mixture was further homogenized by extrusion through a 200nm polycarbonate membrane to remove larger fused particles and insufficiently fused membrane structures, obtaining GCM-LBEVs biomimetic nanovesicles with uniform particle size.

[0057] Through the membrane hybridization process described above, GCM-LBEVs simultaneously retain the natural active ingredient delivery capability derived from LBEVs and the homologous cell recognition capability derived from GCM. LBEVs, as the core carrier, can provide antioxidant and mitochondrial protection-related active molecules; the GCM membrane structure can provide granulosa cell-specific recognition information, making it easier for nanovesicles to locate to the ovarian granulosa cell region during in vivo circulation, thus improving local delivery efficiency.

[0058] Based on the obtained GCM-LBEVs, further mitochondrial-targeted functionalization modifications were performed. To improve the mitochondrial localization efficiency of nanovesicles after entering granulocytes, cholesterol-PEG was used. 2000 -TPP is used as a functionalized molecule. Cholesterol has a strong affinity for lipid bilayers and can be inserted into nanovesicle membrane structures; PEG... 2000 The chain segment can improve the water solubility stability of the modified molecule and reduce non-specific aggregation; the TPP group has positive charge properties and can utilize the difference in membrane potential between the inner and outer mitochondria to promote the enrichment of nanoparticles into the mitochondrial region under the driving effect of negative potential.

[0059] In the specific modification process, cholesterol-PEG is used. 2000 -TPP was added to the GCM-LBEVs system at a vesicle to modified molecular weight ratio of 1:10, and the mixture was gently incubated with shaking at 37°C to allow the cholesterol structure to gradually embed into the GCM-LBEVs lipid bilayer, while simultaneously introducing the TPP functional groups stably onto the vesicle surface. After modification, unbound cholesterol-PEG was removed by ultracentrifugation at 100,000 × g. 2000 -TPP molecules and free membrane fragments were resuspended in PBS to obtain the final product, TPP-GCM-LBEVs dual-target biomimetic nanoparticles.

[0060] like Figure 3 As shown in Figure A, the complete construction process of TPP-GCM-LBEVs is illustrated, including the preparation of extracellular vesicles derived from wolfberry, extraction of granule cell membranes, membrane hybridization to form GCM-LBEVs, and TPP functionalization modification to form the final dual-targeting nanoparticles. This construction strategy achieves a continuous delivery process from "ovarian granulosa cell recognition" to "intracellular mitochondrial localization" through the superposition of different levels of targeting structures.

[0061] Furthermore, various characterization methods were employed to evaluate the structural performance of the constructed TPP-GCM-LBEVs. For example... Figure 3 As shown in Figure B, transmission electron microscopy revealed that both GCM-LBEVs and TPP-GCM-LBEVs maintained typical extracellular vesicle morphology, exhibiting complete spherical or cup-shaped membrane structures without obvious membrane rupture or structural collapse, indicating that the membrane fusion and surface modification process did not damage the overall vesicle structure.

[0062] Nanoparticle tracking analysis (NTA) was used to detect changes in nanoparticle size at different stages. For example... Figure 3As shown in Figure C, the average particle size of unmodified LBEVs was approximately 132.5 nm. After GCM membrane hybridization, the particle size increased, and after further TPP functionalization, the average particle size increased to approximately 192.5 nm. This indicates that both cell membrane coating and the introduction of surface functional molecules can alter the size characteristics of nanoparticles while maintaining their nanoscale delivery range. Further analysis of surface charge changes was performed using Zeta potential detection, such as... Figure 3 D and Figure 3 As shown in E, the initial Zeta potential of LBEVs is approximately -22 mV. After GCM film fusion and TPP modification, the surface potential of TPP-GCM-LBEVs changes to approximately +10 mV, indicating that the positively charged TPP groups are successfully exposed on the nanoparticle surface, achieving surface property regulation.

[0063] To further verify the functionalization effect of TPP, Fourier transform infrared spectroscopy (FTIR) was used for structural analysis. Figure 3 As shown in Figure F, TPP-GCM-LBEVs exhibited characteristic absorption peaks derived from the aromatic ring structure of TPP, showing a significant change compared to unmodified LBEVs, demonstrating the successful introduction of TPP functional molecules into the nanovesicle structure. Simultaneously, cholesterol-PEG... 2000 -TPP-functionalized molecules were detected by proton nuclear magnetic resonance (¹H NMR) spectroscopy, such as... Figure 11 As shown in the results, the detection results indicate the presence of characteristic peaks corresponding to the TPP group, PEG segment, and cholesterol backbone, suggesting the presence of cholesterol-PEG. 2000 -TPP has a complete chemical structure, providing a stable material basis for subsequent functionalization modification of nanoparticles.

[0064] The membrane fusion between GCM and LBEVs was further verified using a dual-fluorescence labeling method. Specifically, before membrane fusion, LBEVs membrane structures were labeled with Dil-Red, and GCM nanovesicle structures were labeled with DiO-Green. After fusion, the fluorescence distribution was observed using a confocal laser scanning microscope. Figure 3 As shown in G, the red and green fluorescence signals in TPP-GCM-LBEVs show significant overlap, forming a yellow fusion signal region, indicating that the LBEVs membrane structure and the GCM membrane structure are effectively fused, rather than simply physically mixed.

[0065] In summary, this embodiment successfully constructed a TPP-GCM-LBEVs dual-targeting biomimetic nanoparticle based on extracellular vesicles derived from Lycium barbarum. This nanoparticle uses LBEVs as a natural therapeutic carrier, the granulosa cell membrane as a homologous targeting recognition structure, and TPP as a mitochondrial localization functional unit. Through multi-level functional design, it achieves continuous regulation from ovarian granulosa cell targeting to precise mitochondrial delivery, providing a structural basis for subsequently improving mitochondrial damage and restoring ovarian function in chemotherapy-induced early-onset ovarian insufficiency.

[0066] Specific Implementation Method 3: Verification of the Dual-Targeted Delivery Capability and Mitochondrial Function Recovery of TPP-GCM-LBEVs; This embodiment further evaluates the in vitro and in vivo targeted delivery performance of the TPP-GCM-LBEVs dual-targeting biomimetic nanoparticles constructed in Embodiment 2, and verifies the molecular mechanism by which they achieve POI therapeutic effects through granule cell targeting and mitochondrial targeting. This embodiment mainly conducts a systematic evaluation from aspects such as cellular selective uptake ability, mitochondrial subcellular localization ability, in vivo ovarian enrichment ability, regulation of endocytosis pathway, and mitochondrial function repair effect, to clarify that TPP-GCM-LBEVs have superior delivery efficiency and therapeutic potential compared to natural LBEVs and GCM-LBEVs alone.

[0067] First, the granulocyte-targeting ability of TPP-GCM-LBEVs in vitro was verified. Specifically, LBEVs obtained in Example 1, GCM-LBEVs prepared in Example 2, and TPP-GCM-LBEVs were labeled with Dil fluorescently to give the nanovesicles a detectable red fluorescent signal. Subsequently, the nanovesicles with different fluorescent labels were added to the KGN cell culture system for co-incubation. The fluorescence distribution of different nanovesicles after entering granulocytes was observed by confocal laser scanning microscopy, and the cell uptake efficiency of different treatment groups was quantitatively analyzed by fluorescence intensity.

[0068] like Figure 3 H and Figure 3 As shown in Figure K, unmodified LBEVs exhibited relatively weak fluorescence signals after entering KGN cells, while GCM-LBEVs and TPP-GCM-LBEVs, modified with granule cell membrane biomimetic technology, both showed enhanced cellular uptake levels, with TPP-GCM-LBEVs maintaining higher intracellular fluorescence accumulation. These results indicate that membrane proteins, glycoproteins, and related recognition molecules derived from granule cell membranes can endow nanovesicles with homologous granule cell recognition capabilities, improving the interaction efficiency between nanoparticles and target cells, and transforming the LBEV-based delivery system from non-specific entry into a cell-selective active delivery process.

[0069] To further verify whether TPP-GCM-LBEVs possess source-dependent granulosa cell selection ability, TPP-GCM-LBEVs were co-cultured with different cell types, including 3T3 cells derived from mouse fibroblasts, ID8 ovarian tumor cells, and KGN cells, an ovarian granulosa cell model. Differences in nanoparticle uptake were evaluated by detecting intracellular fluorescence signals in different cells. Figure 3 J and Figure 3 As shown in Figure L, the uptake efficiency of TPP-GCM-LBEVs in KGN cells was significantly higher than that in 3T3 cells and ID8 cells, indicating that the biomimetic structure of the granulosa cell membrane can retain the homology recognition characteristics of the source cells, enabling nanoparticles to preferentially enter granulosa cells with the same membrane recognition characteristics, thereby achieving selective delivery to target cells related to ovarian function.

[0070] Building upon the achievement of granulocyte-mediated targeting, the subcellular localization capability of TPP-GCM-LBEVs was further evaluated. Specifically, different nanovesicles were co-incubated with KGN cells, and the mitochondria were fluorescently labeled with MitoTracker dye. The spatial relationship between the fluorescence signals of nanovesicles and mitochondrial signals was observed using confocal microscopy, and the degree of co-localization of the two fluorescence signals was analyzed using Pearson correlation coefficient.

[0071] like Figure 3 I, Figure 3 M and Figure 3 As shown in Figure N, although the LBEVs and GCM-LBEVs groups could enter granulosa cells, their fluorescence signals showed low overlap with the mitochondrial region. However, in the TPP-GCM-LBEVs treatment group, the fluorescence signals of the nanovesicles significantly overlapped with the MitoTracker-labeled mitochondrial signals, forming numerous yellow fusion regions, and the Pearson colocalization coefficient was significantly increased. These results indicate that TPP functionalization can utilize the difference in membrane potential between the inner and outer mitochondria to enable TPP-GCM-LBEVs to be further directed towards the mitochondrial region after entering granulosa cells, achieving a further enhancement from cell-level targeting to organelle-level targeting.

[0072] Further in vivo delivery and distribution experiments were conducted to evaluate the ovarian targeting ability of TPP-GCM-LBEVs in a POI animal model. Specifically, Dil fluorescently labeled LBEVs, GCM-LBEVs, and TPP-GCM-LBEVs were administered intraperitoneally at a dose of 50 mg / kg to cyclophosphamide-induced POI mouse models. At 24, 48, 72 hours, day 5, and day 10 post-administration, changes in fluorescence signals in the isolated ovarian and uterine regions were detected using an in vivo fluorescence imaging system to dynamically analyze the retention and tissue distribution characteristics of different nanovesicles in vivo.

[0073] like Figure 4 A and Figure 4 As shown in Figure B, the fluorescence signal in the LBEVs group gradually decreased early after drug administration and rapidly decreased over time, indicating that the natural plant-derived EVs exhibited rapid clearance during in vivo circulation and had limited local accumulation capacity in the ovary. In contrast, the GCM-LBEVs and TPP-GCM-LBEVs groups maintained higher levels of fluorescence signal in the ovarian region, with a significantly prolonged fluorescence duration. Specifically, the TPP-GCM-LBEVs group maintained a high level of ovarian retention even after 48 hours and still showed a significant fluorescence signal on day 10, indicating that the granule cell membrane biomimetic structure improved the ovarian tissue homing ability of the nanovesicles, while TPP modification further enhanced local delivery stability.

[0074] To further confirm whether TPP-GCM-LBEVs can simultaneously achieve granulosa cell and mitochondrial localization in vivo, frozen section analysis was performed on ovarian tissue from treated mice. Granulosa cell regions were labeled with anti-AMH antibody, and mitochondrial structures were labeled with MitoTracker. The relationship between nanovesicle fluorescence signals and cell and organelle labeling signals was observed using confocal microscopy. Figure 4 C and Figure 4 As shown in Figure D, the LBEVs group exhibited weak fluorescence signals in ovarian tissue, while both the GCM-LBEVs and TPP-GCM-LBEVs groups showed significantly enhanced enrichment in the ovarian region. Among them, TPP-GCM-LBEVs not only highly overlapped with AMH-positive granulosa cell regions but also showed significant co-localization with mitochondrial signals, further demonstrating its dual-targeted delivery to both granulosa cells and mitochondria in vivo.

[0075] Further investigation was conducted into the transport mechanism of TPP-GCM-LBEVs after entering granulosa cells. Since extracellular vesicles mainly enter recipient cells via different endocytic pathways, this study employed various pharmacological inhibitors to analyze the endocytic process. These included amiloride inhibiting macropinocytosis, genistein inhibiting caveolin-mediated endocytosis, chlorpromazine inhibiting clathrin-mediated endocytosis, and methyl-β-cyclodextrin disrupting lipid raft structure. By comparing the efficiency of nanovesicles entering KGN cells under different inhibitory conditions, the main transport pathway was determined.

[0076] like Figure 5 A to Figure 5 As shown in Figure J, the cellular uptake of TPP-GCM-LBEVs is significantly influenced by caveolin-mediated endocytosis and lipid raft-mediated endocytosis pathways, while other endocytic pathways have a relatively weaker impact. This indicates that the membrane protein structure derived from the granule cell membrane and TPP modification jointly regulate the way nanoparticles enter the cell. Furthermore, LysoTracker staining analysis revealed that the co-localization between nanovesicles and lysosomes showed lower overlap between TPP-GCM-LBEVs and lysosomal labeling signals compared to LBEVs and GCM-LBEVs. This suggests that after entering the cell, TPP-GCM-LBEVs can reduce the probability of lysosomal degradation after traditional endocytosis and increase the likelihood of transporting active ingredients to the mitochondrial region.

[0077] After confirming the dual-target delivery capability, the repair effect of TPP-GCM-LBEVs on mitochondrial function in POI ovaries was further evaluated. A cyclophosphamide-induced POI mouse model was established. After treatment with TPP-GCM-LBEVs, ovarian tissue was observed by transmission electron microscopy, mitochondrial respiratory chain complex activity was detected, and redox status was analyzed.

[0078] like Figure 6 As shown in Figure A, the number of mitochondria in granulocytes of the POI model group was significantly reduced, and obvious ultrastructural abnormalities such as mitochondrial swelling, cristae breakage, and membrane structure damage were observed. After TPP-GCM-LBEVs treatment, the number of mitochondria in granulocytes increased, the mitochondrial membrane structure was restored to integrity, and the cristae arrangement was more regular, indicating that nanoparticles can effectively improve mitochondrial structural damage caused by chemotherapy.

[0079] Further examination of functional indicators related to the mitochondrial electron transport chain was conducted. For example... Figure 6 B to Figure 6 As shown in Figure F, the activities of mitochondrial complexes I, III, IV, and V in the POI model group were significantly reduced. However, after treatment with TPP-GCM-LBEVs, the activities of each complex were significantly restored, with complexes I, III, and V showing particularly significant recovery. This indicates that TPP-GCM-LBEVs can promote the recovery of oxidative phosphorylation and improve mitochondrial energy production capacity.

[0080] Simultaneously, oxidative stress levels and energy metabolism indicators in ovarian tissue were measured. For example... Figure 6 G to Figure 6 As shown in Figure I, the POI model group exhibited elevated ROS levels, decreased GSH levels, and insufficient ATP production. However, after TPP-GCM-LBEVs treatment, ROS levels significantly decreased, the content of the antioxidant molecule GSH increased, and ATP levels recovered, indicating that these dual-targeting nanoparticles can re-establish the redox balance in ovarian tissue and improve the energy supply capacity of granulosa cells. Furthermore, as... Figure 6 J and Figure 6 As shown in K, Ki67 immunohistochemical staining results showed that TPP-GCM-LBEVs treatment enhanced granulocyte proliferation activity, further demonstrating that mitochondrial function recovery can promote granulocyte function reconstruction.

[0081] In summary, this embodiment demonstrates that TPP-GCM-LBEVs dual-targeting biomimetic nanoparticles can achieve selective delivery to ovarian granulosa cells through homologous targeting mediated by the granulosa cell membrane, improve subcellular organelle delivery efficiency through TPP-mediated mitochondrial localization, and repair chemotherapy-induced granulosa cell damage through multiple mechanisms such as improving mitochondrial structure, restoring respiratory chain function, reducing oxidative stress, and enhancing ATP production, thus providing an effective technical basis for subsequent restoration of ovarian function and improvement of fertility.

[0082] Specific Implementation Method 4: TPP-GCM-LBEVs promote ovarian function recovery, improve fertility, and evaluate biosafety; This embodiment further verifies the therapeutic efficacy, reproductive function recovery ability, and in vivo biosafety of TPP-GCM-LBEVs, a dual-targeting biomimetic nanoparticle based on Lycium barbarum extracellular vesicles, in a chemotherapy-induced early ovarian insufficiency (POI) animal model. This embodiment constructs a chemotherapy-induced POI mouse model and compares treatments using different types of nanodelivery materials, including natural LBEVs, GCM-LBEVs, and TPP-GCM-LBEVs. The therapeutic advantages of TPP-GCM-LBEVs are systematically evaluated across multiple dimensions, including ovarian function indicators, oocyte quality, fertility, and tissue safety.

[0083] Specifically, a chemotherapy-induced POI animal model was established using female C57BL / 6J mice. First, ovarian damage was induced by intraperitoneal injection of cyclophosphamide (CTX) to simulate the ovarian dysfunction experienced by clinical cancer patients after chemotherapy. After entering the body, CTX induces enhanced oxidative stress in ovarian tissue, leading to mitochondrial damage in granulosa cells, increased apoptosis levels, and decreased follicular reserve, thus forming a mouse model with typical POI pathological characteristics. After model establishment, the experimental animals were randomly divided into four groups: PBS control, LBEVs treatment, GCM-LBEVs treatment, and TPP-GCM-LBEVs treatment. The recovery effects of different treatment groups were compared to analyze the role of dual modification targeting granulosa cells and mitochondria in improving treatment efficiency.

[0084] During the treatment regimen design, the dosing frequency of TPP-GCM-LBEVs was optimized based on the results of previous in vivo distribution experiments. Compared to natural LBEVs, which require repeated dosing at higher frequencies, TPP-GCM-LBEVs, due to their stronger ovarian tissue accumulation capacity and longer local retention time, were used in animal experiments with a treatment regimen of 50 mg / kg, administered once every 10 days for three consecutive times. After the treatment cycle, systematic ovarian function tests were performed on mice in each group, including ovarian index calculation, vaginal smear analysis of estrous cycle changes, serum reproductive hormone level analysis, and evaluation of follicular development status.

[0085] like Figure 8 As shown in Figure A, the overall experimental procedure for treating POI mice with TPP-GCM-LBEVs is illustrated, including the establishment of the CTX-induced POI model, nanoparticle therapy, and subsequent ovarian function testing. Figure 8 B and Figure 8 As shown in Figure C, compared with the untreated POI model group, the ovarian index of mice in the TPP-GCM-LBEVs treatment group was significantly increased, the estrous cycle disorder was improved, and the reproductive cycle characteristics were closer to those of normal mice, indicating that the dual-targeting nanoparticles can improve chemotherapy-induced ovarian tissue atrophy and endocrine dysfunction.

[0086] Further testing of serum hormone levels is needed to evaluate the recovery of ovarian endocrine function. Figure 8 D to Figure 8As shown in Figure F, the POI model group mice exhibited typical hormonal imbalance characteristics, including decreased anti-Müllerian hormone (AMH) levels, decreased estradiol (E2) levels, and increased follicle-stimulating hormone (FSH) levels. However, after TPP-GCM-LBEVs treatment, serum AMH and E2 levels significantly recovered, and FSH levels significantly decreased, with the improvement effect being superior to that of the LBEVs and GCM-LBEVs treatment groups. These results indicate that TPP-GCM-LBEVs can effectively improve the ovarian endocrine environment and enhance the recovery of ovarian function.

[0087] To further evaluate the effects of TPP-GCM-LBEVs on ovarian reserve and follicular development, histological analysis of ovarian tissue from each group of mice and statistical analysis of the number of follicles at different stages were performed. Figure 8 As shown in G, hematoxylin and eosin (H&E) staining revealed a significant decrease in the number of normally developing follicles in the ovarian tissue of the POI model group, with a large number of follicles exhibiting atresia and degeneration. However, after TPP-GCM-LBEVs treatment, the ovarian tissue structure was significantly improved, and more healthy follicle structures at different developmental stages could be observed. Figure 8 H to Figure 8 As shown in M, further statistical analysis of the number of primordial follicles, primary follicles, secondary follicles, antral follicles, and atretic follicles revealed that TPP-GCM-LBEVs significantly increased the proportion of normally developing follicles and reduced the number of abnormal atretic follicles, indicating that these dual-targeting nanoparticles can effectively protect ovarian reserve and promote the recovery and development of damaged follicles.

[0088] Further research was conducted on the effects of TPP-GCM-LBEVs on oocyte quality and embryonic developmental potential. Since the mitochondrial maturation of oocytes is limited, their normal growth and development are highly dependent on ATP, NAD(P)H, and metabolic support provided by surrounding granulosa cells. Therefore, the restoration of mitochondrial function in granulosa cells can further improve oocyte quality. In this implementation method, after treatment, mouse oocytes were obtained from each group via superovulation, and the oxidative stress level and energy metabolism status of the oocytes were detected.

[0089] like Figure 7 A to Figure 7As shown in Figure G, oocytes in the POI model group exhibited significant metabolic abnormalities, including elevated ROS levels, decreased NAD(P)H levels, reduced GSH reserves, and insufficient ATP production, accompanied by a decrease in mitochondrial number and ultrastructural damage. After TPP-GCM-LBEVs treatment, ROS levels in oocytes decreased significantly, while NAD(P)H, GSH, and ATP levels recovered. Transmission electron microscopy revealed an increase in mitochondrial number and improved mitochondrial membrane and cristae structures. These results indicate that TPP-GCM-LBEVs improve the internal energy supply environment of oocytes and enhance their developmental potential by restoring the metabolic support capacity of granulosa cells.

[0090] To verify the effect of TPP-GCM-LBEVs on improving actual reproductive capacity, further in vitro fertilization experiments and natural fertility evaluations were conducted. During the in vitro fertilization experiment, oocytes from treated mice were collected and fertilized with sperm from normal male mice. Embryo development after fertilization was continuously observed. Figure 9 A to Figure 9 As shown in Figure D, compared with the POI model group and other treatment groups, the TPP-GCM-LBEVs treatment group achieved a higher rate of 2-cell embryo formation and blastocyst formation, indicating that the nanoparticles can improve oocyte quality and enhance early embryonic development.

[0091] Further natural mating experiments were conducted to evaluate long-term reproductive outcomes. After treatment, female mice were paired with male C57 mice of normal fertility, and reproductive recovery was evaluated by recording pregnancy status and average litter size. Figure 9 E and Figure 9 As shown in F, the POI model group showed a significant decrease in pregnancy rate and litter size due to decreased ovarian reserve and reduced oocyte quality, while the TPP-GCM-LBEVs treatment group showed a significant increase in pregnancy rate and average litter size. This indicates that the dual-targeting biomimetic nanoparticles can not only improve ovarian physiological indicators, but also further restore actual fertility.

[0092] Furthermore, to evaluate the in vivo safety of TPP-GCM-LBEVs during long-term use, this implementation method further conducted a systems toxicology analysis. After treatment, histological examinations were performed on the major organs of the experimental animals, including the heart, liver, spleen, lungs, and kidneys. H&E staining was used to observe changes in the structure of each tissue, such as… Figure 12 As shown, all major organs maintained normal tissue morphology, and no obvious abnormal pathological manifestations such as inflammatory cell infiltration, tissue necrosis, or structural destruction were found, indicating that TPP-GCM-LBEVs have good tissue tolerance in vivo.

[0093] Simultaneously, blood biochemical indicators of the mice after treatment were measured, including important indicators reflecting liver and kidney function such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), creatinine (CREA), and urea (UREA). Figure 13 As shown, all serum biochemical indicators remained within the normal range, and no significant abnormal changes were observed compared with the control group, indicating that TPP-GCM-LBEVs did not cause significant liver or kidney toxicity and had good biocompatibility in vivo.

[0094] In summary, this embodiment demonstrates that the TPP-GCM-LBEVs dual-targeting biomimetic nanoparticles constructed based on Lycium barbarum-derived extracellular vesicles can achieve multi-level treatment of chemotherapy-induced POI through granulosa cell membrane-mediated ovarian granulosa cell targeting and TPP-mediated precise mitochondrial delivery. These nanoparticles can restore granulosa cell and oocyte mitochondrial function, improve the ovarian endocrine environment, promote follicle development, enhance embryonic development and natural fertility, while exhibiting good in vivo safety. This provides a novel biomimetic nanodelivery strategy with clinical translational potential for the treatment of early-onset ovarian insufficiency.

[0095] The foregoing has only described certain exemplary embodiments of this application by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of this application. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of this application.

Claims

1. A method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry, characterized in that, Includes the following steps: Goji berry-derived extracellular vesicles (LBEVs) were obtained and used as nano-delivery carriers. The active ingredients carried by the goji berry-derived extracellular vesicles were used to achieve a therapeutic effect on chemotherapy-induced early-onset ovarian insufficiency-related damage. The extracellular vesicles derived from wolfberry were coated with homologous ovarian granulosa cell membrane (GCM) to construct GCM-LBEVs biomimetic nanovesicles with granulosa cell recognition ability. The granulosa cell membrane was used to endow the GCM-LBEVs biomimetic nanovesicles with ovarian granulosa cell targeting ability. The surface of the GCM-LBEVs biomimetic nanovesicles was functionalized using triphenylphosphine (TPP) to obtain TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles. The mitochondrial targeting properties of the triphenylphosphine were used to improve the delivery efficiency of the TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles to the mitochondria of granulocytes. The TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles were applied to a chemotherapy-induced early-onset ovarian insufficiency model. The TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles were selectively delivered to ovarian granulosa cells through the granulosa cell targeting capability. The mitochondrial targeting capability was used to restore the mitochondrial structural integrity and bioenergy function of granulosa cells. The TPP-GCM-LBEVs dual-targeted biomimetic nanovesicles are used to repair granulosa cells, maintain the stability of the ovarian microenvironment, promote the continuous supply of ATP and NAD(P)H from granulosa cells to damaged oocytes, improve oocyte quality and developmental potential, and achieve the recovery of follicle development, ovarian endocrine function and fertility in chemotherapy-induced early-onset ovarian insufficiency.

2. The method for treating early-onset ovarian insufficiency based on dual-targeting biomimetic nanoparticles derived from wolfberry extracellular vesicles according to claim 1, characterized in that, The extracellular vesicles derived from wolfberry were obtained through plant tissue isolation and purification, specifically including: washing and processing fresh wolfberry fruits and homogenizing them in ice-pre-cooled phosphate buffer to obtain a mixture containing extracellular vesicles. The mixture was then centrifuged sequentially at 1000×g, 2000×g, and 10000×g to remove tissue residues, cell debris, and non-target particles. The vesicle precipitate was then collected by ultracentrifugation at 150000×g and further purified using a sucrose density gradient system of 8%, 30%, 45%, and 60%, collecting the wolfberry-derived extracellular vesicle component from the 45% sucrose layer.

3. The method for treating early-onset ovarian insufficiency based on dual-targeting biomimetic nanoparticles derived from wolfberry extracellular vesicles according to claim 1, characterized in that, The homologous ovarian granulosa cell membrane was obtained by lysis and separation of granulosa cells. After washing the granulosa cells with phosphate buffer, they were subjected to liquid nitrogen freeze-thaw cycles. The cell nucleus and cell debris were removed by differential centrifugation. Granular cell membrane components containing membrane proteins, glycoproteins and lipid bilayer structures were collected. The obtained granulosa cell membrane was then subjected to extrusion treatment with polycarbonate membranes with pore sizes of 1000 nm, 400 nm and 200 nm to prepare granulosa cell membrane-derived nanovesicles with uniform particle size.

4. The method for treating early-onset ovarian insufficiency based on dual-targeting biomimetic nanoparticles derived from wolfberry extracellular vesicles according to claim 1, characterized in that, The GCM-LBEVs biomimetic nanovesicles are obtained by hybridization and fusion of nanovesicles derived from granular cell membranes and extracellular vesicle membranes derived from wolfberry. The nanovesicles derived from granular cell membranes and extracellular vesicles derived from wolfberry are mixed in a 1:1 mass ratio and the membrane structure is fused by ice bath ultrasonic treatment. The fusion product is further homogenized by extrusion through a 200nm polycarbonate membrane to obtain a biomimetic nanovesicle structure that combines the ability to deliver active ingredients from plant-derived extracellular vesicles with the ability to recognize homologous components from granular cells.

5. The method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry, as described in claim 4, is characterized in that... triphenylphosphine via cholesterol-PEG 2000 -TPP functionalized molecules are linked to the surface of GCM-LBEVs biomimetic nanovesicles, where cholesterol structures are embedded in the lipid bilayer of the nanovesicles, and PEG... 2000 Chain segments maintain dispersion stability; triphenylphosphine groups utilize mitochondrial membrane potential differences to achieve subcellular organelle localization regulation; cholesterol-PEG 2000 -TPP was added to the GCM-LBEVs system at a vesicle to modified molecular weight ratio of 1:10, and the surface functionalization modification was completed by gentle shaking incubation at 37°C.

6. The method for treating early-onset ovarian insufficiency based on dual-targeting biomimetic nanoparticles derived from wolfberry extracellular vesicles according to claim 1, characterized in that, The TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles exhibit selective uptake characteristics by granulosa cells. They enhance the interaction with ovarian granulosa cells by retaining membrane proteins, glycoproteins, and homology recognition molecules on the surface of granulosa cell membranes. When co-incubated with KGN granulosa cells, the TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles show cellular uptake characteristics.

7. The method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry, as described in claim 6, is characterized in that... The process by which the TPP-GCM-LBEVs dual-target biomimetic nanovesicles enter granulosa cells involves the caveolin-mediated endocytosis pathway and the lipid raft-mediated endocytosis pathway. The relevant endocytosis process is regulated by genistein and methyl-β-cyclodextrin, and the localization relationship between nanovesicles and lysosomes is detected by LysoTracker staining.

8. A method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry, as described in claim 7, is characterized in that... The TPP-GCM-LBEVs dual-targeting biomimetic nanovesicles were applied to an animal model of early-onset ovarian insufficiency induced by cyclophosphamide. The administration method was intraperitoneal injection at a dose of 50 mg / kg, administered once every 10 days for three consecutive treatments. The recovery status of ovarian endocrine function was evaluated by detecting ovarian index, estrous cycle, serum anti-Müllerian hormone, estradiol, and follicle-stimulating hormone levels.

9. A method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry, as described in claim 8, characterized in that... The TPP-GCM-LBEVs dual-target biomimetic nanovesicles improve the physiological state of oocytes by restoring mitochondrial function in granulosa cells. The restoration process includes regulating mitochondrial reactive oxygen species levels, increasing glutathione content, promoting ATP and NAD(P)H production, improving the number and ultrastructural integrity of oocyte mitochondria, and promoting damaged oocytes to obtain a suitable developmental environment.

10. A method for treating early-onset ovarian insufficiency using dual-targeting biomimetic nanoparticles based on extracellular vesicles derived from wolfberry, as described in claim 9, is characterized in that... The TPP-GCM-LBEVs dual-target biomimetic nanovesicles were validated for their therapeutic efficacy through in vitro fertilization experiments and natural fertility evaluation. In vitro fertilization assays included analysis of 2-cell embryo formation rate and blastocyst formation rate. Natural fertility evaluation included detection of pregnancy rate and average litter size in female mice. Simultaneously, the in vivo safety of the nanovesicles was evaluated through hematoxylin-eosin staining of heart, liver, spleen, lung, and kidney tissues, as well as detection of serum AST, ALT, TBIL, CREA, and UREA indicators.

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