Endometrium-targeted mitochondrial function compensation system and application thereof

Abstract

The application discloses an endometrium-targeted mitochondrial function compensation system and application thereof. The endometrium-targeted mitochondrial function compensation system comprises a function unit, a delivery unit and a control unit. The function unit comprises a vesicle preparation formed by wrapping donor mitochondria with red blood cell membranes. The donor mitochondria are isolated mitochondria with complete inner and outer mitochondrial membranes and ridge structures or engineered mitochondria carrying exogenous mitochondrial DNA. The application first constructs a comprehensive system integrating biological active ingredients, targeted delivery and intelligent control, can directly and efficiently repair the mitochondrial function of endometrial cells, promote endometrial proliferation, and provides an innovative solution for treating related reproductive diseases. The system has complete design, clear effect and good clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an endometrial-targeted mitochondrial function compensation system and its application. Background Technology

[0002] The endometrium is a crucial functional layer of the female reproductive system, and its normal proliferation, differentiation, and metabolic functions are fundamental for embryo implantation and maintaining pregnancy. Recent studies have shown that the functional state of mitochondria in endometrial cells directly affects their energy metabolism, proliferative capacity, and receptivity. Mitochondrial dysfunction is considered one of the important pathological bases leading to thin endometrium, recurrent implantation failure, recurrent miscarriage, and various forms of female infertility.

[0003] Currently, clinical treatment options for endometrial dysfunction are limited, mainly including hormone replacement therapy, intrauterine perfusion, and stem cell therapy. However, these methods suffer from unstable efficacy, unclear mechanisms of action, and ethical and safety risks. In particular, existing treatment strategies lack the ability to directly and specifically repair endometrial cell energy metabolism defects.

[0004] Mitochondrial transplantation, as an emerging organelle replacement therapy, theoretically has the potential to directly replenish functional mitochondria and repair cellular energy metabolism. However, this technology faces two major challenges: (1) how to achieve efficient and stable delivery of mitochondria; and (2) how to achieve targeted homing and efficient uptake of mitochondria to specific target tissues (such as the endometrium) in vivo. Currently, there is no mature, clinically applicable endometrial-targeted mitochondrial delivery system.

[0005] Therefore, developing a comprehensive technical system that can efficiently and safely deliver functional mitochondria to the endometrium and effectively repair its cellular function is of great significance for solving the above-mentioned clinical challenges. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides an endometrial-targeted mitochondrial function compensation system and its application. It aims to overcome the technical bottleneck of existing technologies that cannot achieve targeted, efficient delivery and functional integration of functional mitochondria to the endometrium, thereby directly repairing mitochondrial dysfunction in endometrial cells and treating related reproductive diseases.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides an endometrial-targeted mitochondrial function compensation system, the endometrial-targeted mitochondrial function compensation system comprising: a functional unit, a delivery unit, and a control unit; the functional unit comprising a vesicle preparation formed by encapsulating donor mitochondria with a red blood cell membrane; the donor mitochondria being isolated mitochondria with complete inner and outer mitochondrial membranes and cristae structures or engineered mitochondria carrying exogenous mitochondrial DNA.

[0008] This invention is the first to construct a comprehensive system integrating bioactive ingredients, targeted delivery, and intelligent control. It can directly and efficiently repair mitochondrial function in endometrial cells and promote endometrial proliferation, providing an innovative solution for the treatment of related reproductive diseases. The system is well-designed, has clear effects, and possesses promising prospects for clinical translation. A schematic diagram of the preparation process and application of the mitochondrial vesicle formulation of this invention is shown below. Figure 1 As shown.

[0009] Preferably, the diameter of the vesicle formulation is 0.9-1.2 µm, for example, it can be 0.9 µm, 1.0 µm, 1.1 µm or 1.2 µm.

[0010] Preferably, the source of the red blood cell membrane includes autologous red blood cells and / or allogeneic red blood cells derived from type O blood.

[0011] Preferably, the donor mitochondria are derived from any one or a combination of at least two of autologous skin fibroblasts, urine-derived cells, or allogeneic umbilical cord mesenchymal stem cells.

[0012] Preferably, the delivery unit includes an intrauterine injection device.

[0013] Preferably, the control unit is connected to the delivery unit and is used to control delivery parameters; the delivery parameters include any one or a combination of at least two of the dosage, rate, or depth of the injected vesicle formulation.

[0014] Preferably, the method for preparing the vesicle formulation includes: mixing and stirring red blood cell membranes and donor mitochondria at 0-4°C (e.g., 0°C, 2°C, or 4°C) to assemble the vesicle formulation.

[0015] Preferably, the mixing speed is 100-500 rpm (e.g., 100 rpm, 300 rpm or 500 rpm), and the mixing time is 20-60 min (e.g., 20 min, 40 min or 60 min).

[0016] In a second aspect, the present invention provides a kit for improving endometrial function, the kit comprising the endometrial-targeted mitochondrial function compensation system described in the first aspect.

[0017] Preferably, the kit for improving endometrial function also includes a pharmaceutically acceptable diluent.

[0018] Thirdly, the present invention provides a method for improving endometrial function, the method comprising: operating a delivery unit using the control unit described in the first aspect to deliver a vesicle preparation to the endometrial region.

[0019] Fourthly, the present invention provides the use of the endometrial-targeted mitochondrial function compensation system described in the first aspect or the kit for improving endometrial function described in the second aspect in the preparation of products for treating spontaneous abortion, recurrent abortion, female infertility or postpartum endometrial incomplete repair.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to organically combine bioactive vesicles (mitochondria wrapped in red blood cell membranes), a dedicated targeted delivery device (intrauterine injection device) with intelligent control technology to build a brand-new and complete treatment platform, solving the systemic problem of mitochondrial targeted delivery; (2) This invention uses the natural red blood cell membrane to wrap mitochondria, protecting them from inactivation. The 1.0 µm mitochondrial capsule can effectively deliver mitochondria into the cell and prevent them from being cleared by autophagy, thereby reducing mitochondrial DNA mutations, restoring function and promoting cell proliferation. By utilizing the natural biocompatibility and homing potential of the red blood cell membrane, combined with precise intrauterine delivery, it achieves efficient and targeted delivery and functional integration of mitochondria to endometrial cells, thus repairing cell metabolism from the root. (3) The present invention achieves precise control of delivery dose, rate and location through control unit, making the treatment process standardized and repeatable, and significantly improving the safety and effectiveness of treatment; (4) This invention provides a novel and well-defined treatment strategy for reproductive diseases that lack effective treatments, such as thin endometrium, recurrent miscarriage, and refractory infertility, and has great potential for clinical translation. Attached Figure Description

[0021] Figure 1 A schematic diagram illustrating the preparation process and application of mitochondrial vesicle formulations; Figure 2 Figure A shows the characterization results of the mitochondrial vesicle formulation, where Figure A is the fluorescence imaging image of the mitochondrial vesicle formulation (scale bar is 2 μm), Figure B is the electron micrograph of the mitochondrial vesicle formulation (scale bar is 200 nm), Figure C is the flow cytometry analysis result, and Figure D is the signal intensity and particle size result. Figure 3The images show the verification results of mitochondrial vesicles being taken up and fused by endometrial cell lines. Image A is a confocal imaging live-cell tracer image (scale bar 10 µm) of the process of donor mitochondria entering HeLa cells derived from cervical epithelium. Image B is a confocal imaging live-cell tracer image (scale bar 10 µm) of the fusion of donor mitochondria and endogenous mitochondria. Image C is the result of flow cytometry detection of donor mitochondrial introduction efficiency. Image D is the result of SNP detection of mtDNA heterogeneity after U2OS donor mitochondria are introduced into HeLa cells. Figure 4 This diagram illustrates the detection results of the effect of mitochondrial vesicles on the function of primary endometrial cells. Figure A shows the result of donor mitochondria entering endometrial epithelial cells and fusing with endogenous mitochondria. Figure B shows the result of mitochondrial transplantation promoting the proliferation of endometrial epithelial cells. Figure C shows the result of mitochondrial transplantation increasing the mitochondrial DNA content of endometrial epithelial cells. Detailed Implementation

[0022] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0023] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0024] Example 1 This embodiment describes the preparation and characterization of mitochondrial vesicle formulations.

[0025] (1) Extraction of red blood cell membrane 10 mL of peripheral blood was collected from healthy volunteers or type O blood donors using heparin sodium anticoagulant tubes. The cells were centrifuged at 800×g for 10 min, and the supernatant was removed, yielding a dark red cell pellet. Pre-chilled PBS solution was added to the cell pellet, and the cells were resuspended by pipetting. The pellet was then filtered through a 0.45 μm filter to remove any aggregated cells. The cell suspension was centrifuged at 800 g for 10 min, the supernatant was removed, and a suitable amount of pre-chilled PBS solution was added before centrifugation. This process was repeated three times. Pre-chilled PBS solution was added to the cell pellet, and the mixture was rotated for 30 min, centrifuged at 800 g for 10 min, and the supernatant was removed. Pre-chilled PBS solution was added again before centrifugation. This process was repeated three times. The membrane pellet was collected, aliquoted, and stored at -80℃ for later use.

[0026] (2) Mitochondrial extraction Umbilical cord mesenchymal stem cells were harvested and cultured in vitro until the cells reached a size of 5 × 10⁶.7 Cells were collected by trypsin digestion, centrifuged at 300 g for 3 min, and the cell pellet was resuspended in mitochondrial extraction buffer (DPBS solution), centrifuged at 300 g for 3 min, and the supernatant was discarded. Mitochondrial extraction solution (Prilele, #C0010) was added to the pellet, and the mixture was incubated on ice for 20 min. Cells were pipetted 35 times on ice using a 5 mL syringe, and centrifuged at 800 g for 5 min. The supernatant was transferred to a new centrifuge tube and centrifuged at 800 g for 5 min. Subsequently, the supernatant was aspirated into a centrifuge tube, centrifuged at 12000 g for 5 min, and the supernatant was discarded. Mitochondria were preliminarily isolated and stored at 4 °C.

[0027] (3) Assembly of vesicle formulations The extracted erythrocyte membranes and mitochondria were resuspended in assembly solution (300 mM sucrose, 0.2% albumin and 1 mM EDTA, pH 7.2). The erythrocyte membrane resuspension and mitochondrial resuspension were mixed thoroughly at a total protein mass ratio of 1:1. The mixture was stirred at 200 rpm for 30 min on ice using a magnetic stirrer. The mixture was centrifuged at 800 g for 5 min. After removing the supernatant, the vesicle preparation was diluted with PBS to allow the erythrocyte membrane to spontaneously encapsulate the mitochondria.

[0028] (4) Characterization Morphology: Vesicles are round or oval, with a clear membrane structure, and are enclosed by typical mitochondrial cristae structures. Figure 2 (Figures A and B in the middle).

[0029] Membrane surface markers: Flow cytometry analysis showed that over 95% of the vesicle formulations expressed the characteristic erythrocyte membrane surface protein CD235a and the immunomodulatory protein CD47. Figure 2 Figure C in the middle section confirms its erythrocyte membrane origin and potential immune escape ability.

[0030] Particle size and fluorescence intensity: Particle size analysis using dynamic optical diffraction showed that the average diameter of the assembled mitochondrial vesicles was approximately 1.1 μm, with a particle size distribution range of 0.9–1.2 μm. Figure 2 (Figure D) The results show that the prepared mitochondrial vesicles have good uniformity.

[0031] Mitochondrial content: The copy number of mitochondrial DNA (mtDNA) in the vesicle formulation was quantified by qPCR, and the formulation concentration was adjusted accordingly to ensure that each dose contained approximately 1 × 10⁻⁶ mtDNA. 8 Copy of mtDNA.

[0032] Example 2 This embodiment verifies the effect of vesicle formulations on endometrial cell lines in vitro.

[0033] (1) After counting, HeLa cells were seeded into 35 mm glass-bottomed cell culture dishes, with 2 × 10⁶ cells seeded per well. 5 Cells were cultured for 24 h to allow for full cell adhesion. Encapsulated mitochondria were added to culture dishes, and the mitochondrial entry process was recorded using a Zeiss LSM 900 laser confocal microscope. Cells with mitochondria labeled with EGFP and DsRed were expanded and added to the mitochondrial transplantation system. After 12 h of culture, the cells were washed three times with DPBS buffer and cultured again in medium containing penicillin and streptomycin. The mitochondrial transplantation process was dynamically recorded using a laser confocal microscope. Simultaneously, cell samples were collected, filtered, and sorted using a Fortessa flow cytometer. Mitochondrial transplantation efficiency was calculated, and the mitochondrial genome was extracted. Next-generation sequencing was performed to detect single nucleotide polymorphisms (SNPs) in mitochondrial DNA and to assess the long-term survival of donor mitochondrial DNA in recipient cells.

[0034] Mitochondria were labeled with mt-DsRed, and after cell expansion and culture, fluorescent mitochondria were extracted. After transplanting the fluorescent mitochondria into cells, live-cell imaging was used to detect whether the exogenous mitochondria could survive within the cells. To verify whether the mitochondrial transplantation system successfully transferred mitochondria into cells, the DsRed-labeled mitochondrial transplantation system was co-incubated with cells, and the process of mitochondrial entry into the cells was recorded using laser confocal microscopy. Figure 3 As shown in Figure A, in HeLa cells, the mitochondrial transplantation system completely entered the cells approximately 60 minutes after contact. Simultaneously, the DsRed fluorescently labeled mitochondrial transplantation system was co-incubated with EGFP-labeled HeLa cells, and the dynamic changes of the transplanted mitochondria were recorded. The transplanted mitochondria branched and extended from a spherical shape within the cell. Furthermore, fusion between the transplanted mitochondria and the mitochondria of the recipient cells was observed. Figure 3 (Figure B). Flow cytometry was used to detect the transplantation efficiency of exogenous mitochondria. DsRed-labeled HeLa cell mitochondria were transplanted into EGFP-labeled HeLa cells, and the proportion of cells containing DsRed-labeled mitochondria in the total cell count was detected using flow cytometry. Figure 3 As shown in Figure C, after mitochondrial transplantation, the proportion of cells containing DsRed-labeled mitochondria was over 79.8% of the total cells, indicating that the mitochondrial transplantation system can transplant mitochondria into a large number of cells in a short period of time.

[0035] To further demonstrate the successful integration of exogenous mitochondria into cells, mtDNA from different cell types was sequenced. This revealed single nucleotide polymorphisms (SNPs) in the mitochondrial genomes of U2OS and HeLa cells. Mitochondrial DNA was extracted from both cell types and subjected to next-generation sequencing. Figure 3 As shown in Figure D, after mitochondrial transplantation, SNP sites of exogenous mitochondria were detected at the SNP sites, indicating that the mitochondrial transplantation system can transplant mitochondria into the cell.

[0036] Example 3 This embodiment verifies the effect of vesicle preparations on the endometrial epithelial cells of patients.

[0037] After counting, human endometrial epithelial cells were seeded into 35 mm glass-bottomed cell culture dishes, with 2 × 10⁶ cells per well. 5 Cells were cultured for 24 h to allow for full cell adhesion, and the coated mitochondria were then added to the culture dish. Mitochondria were labeled with the fluorescent dye MitoTracker Deep Red, and live-cell imaging was performed using an LSM 900 laser confocal microscope. After 72 h of cell growth, cells were digested with trypsin and counted. Simultaneously, genomic DNA was extracted, and mitochondrial DNA copy number was detected by qPCR.

[0038] After transplanting EGFP-labeled mitochondria into MitoTracker Deep Red-labeled patient endometrial epithelial cells and co-incubating them, the fusion of the transplanted mitochondria with the recipient mitochondria was observed using an LSM900 laser confocal microscope. Figure 4 (Figure A). Simultaneously, an experiment was conducted to detect cell proliferation after mitochondrial transplantation; the results are as follows. Figure 4 As shown in Figure B, cell proliferation increased after mitochondrial transplantation. Furthermore, cell samples were collected after mitochondrial transplantation, and genomic DNA was extracted. Changes in mtDNA copy number were detected using qPCR. Figure 4 As shown in Figure C, the intracellular mtDNA copy number increased after mitochondrial transplantation. The experimental results indicate that the mitochondrial transplantation system can function within cells and improve mitochondrial function.

[0039] In summary, this invention is the first to construct a comprehensive system integrating bioactive ingredients, targeted delivery, and intelligent control. This system can directly and efficiently repair mitochondrial function in endometrial cells and promote endometrial proliferation, providing an innovative solution for the treatment of related reproductive diseases. The system is well-designed, has clear effects, and possesses promising prospects for clinical translation.

[0040] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A mitochondrial function compensation system targeting the endometrium, characterized in that, The endometrial-targeted mitochondrial function compensation system includes a functional unit, a delivery unit, and a control unit; the functional unit includes a vesicle preparation formed by encapsulating donor mitochondria with a red blood cell membrane; the donor mitochondria are isolated mitochondria with complete inner and outer mitochondrial membranes and cristae structures or engineered mitochondria carrying exogenous mitochondrial DNA.

2. The endometrial-targeted mitochondrial function compensation system according to claim 1, characterized in that, The diameter of the vesicle formulation is 0.9-1.2 µm.

3. The endometrial-targeted mitochondrial function compensation system according to claim 1 or 2, characterized in that, The red blood cell membranes are derived from autologous red blood cells and / or allogeneic red blood cells derived from type O blood.

4. The endometrial-targeted mitochondrial function compensation system according to any one of claims 1-3, characterized in that, The donor mitochondria are derived from any one or a combination of at least two of the following: autologous skin fibroblasts, urine-derived cells, or allogeneic umbilical cord mesenchymal stem cells.

5. The endometrial-targeted mitochondrial function compensation system according to any one of claims 1-4, characterized in that, The delivery unit includes an intrauterine injection device.

6. The endometrial-targeted mitochondrial function compensation system according to any one of claims 1-5, characterized in that, The control unit is connected to the delivery unit and is used to control delivery parameters; the delivery parameters include any one or a combination of at least two of the dosage, rate or depth of the injected vesicle formulation.

7. The endometrial-targeted mitochondrial function compensation system according to any one of claims 1-6, characterized in that, The method for preparing the vesicle formulation includes: mixing and stirring red blood cell membranes and donor mitochondria at 0-4°C to assemble them into a vesicle formulation; Preferably, the mixing speed is 100-500 rpm, and the mixing time is 20-60 min.

8. A kit for improving endometrial function, characterized in that, The kit for improving endometrial function includes the endometrial-targeted mitochondrial function compensation system according to any one of claims 1-7; Preferably, the kit for improving endometrial function also includes a pharmaceutically acceptable diluent.

9. A method for improving endometrial function, characterized in that, The method for improving endometrial function includes: operating a delivery unit using a control unit according to any one of claims 1-7 to deliver a vesicle preparation to the endometrial region.

10. The endometrial-targeted mitochondrial function compensation system of any one of claims 1-7 or the kit for improving endometrial function as described in claim 8, in the preparation of products for the treatment of spontaneous abortion, recurrent miscarriage, female infertility or postpartum endometrial incomplete repair.

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