Therapeutic nanovesicle formulations enriched in mitochondrial function proteins, methods of making and use in treating radiation skin damage

Abstract

The application discloses a therapeutic nano vesicle preparation rich in mitochondrial function proteins and a preparation method and application thereof in treating radioactive skin damage. The preparation method is as follows: umbilical cord mesenchymal stem cells are taken, cultured and expanded, washed and dispersed in a suspension, mechanically extruded into a crude solution through a multi-stage pore size polycarbonate membrane, and then centrifuged, concentrated and purified to obtain the preparation. The preparation has the following advantages: local administration, rapid inhibition of inflammation, reduction of oxidative stress, activation of repair pathways, and application to acute radioactive skin damage treatment; the preparation method is easy to standardize and scale up, has high stability, is convenient for emergency storage and release, and avoids the disadvantages of cell therapy; the preparation can synergistically regulate multiple targets such as immune inflammation and oxidative stress, simulate and partially replace the biological effects of hUMSCs, avoid the limitations caused by cell survival rate and microenvironment dependence, and functionally cover the key roles of hUMSCs, and has higher clinical feasibility, safety and emergency adaptability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a therapeutic nanovesicle formulation rich in mitochondrial functional proteins, its preparation method, and its application in the treatment of radiation-induced skin damage. Background Technology

[0002] Radiation-induced skin injury (RSI) is an inflammatory injury to the skin and mucous membranes caused by ionizing radiation. It is a common and serious adverse reaction during radiotherapy, with an incidence rate as high as 95%. Currently, clinical treatment mainly relies on topical medications and dressings for symptomatic relief, but there is a lack of unified and effective standard protocols, resulting in limited efficacy and numerous side effects. There is an urgent need to develop new intervention methods.

[0003] Studies have shown that mitochondria are a key target of ionizing radiation, and their dysfunction can lead to excessive ROS accumulation, DNA damage, energy metabolism disorders, and apoptosis. Mechanisms maintaining mitochondrial homeostasis (such as mitophagy) play a crucial role in regulating cell fate. Therefore, mitochondrial-targeted repair strategies are considered an effective way to alleviate RSI.

[0004] Human umbilical cord mesenchymal stem cells (hUMSCs) have been extensively studied for radiation injury repair due to their pro-angiogenic, epithelial regeneration, and antioxidant capabilities. However, their clinical translation is limited by issues such as cell proliferation capacity, safety, and quality control. Studies have shown that the therapeutic effects of hUMSCs are mainly achieved through paracrine mechanisms, especially through their secreted extracellular vesicles (EVs). However, EVs have limitations such as low yield, high heterogeneity of contents, and uncontrollable biosynthesis processes, which restrict their standardized clinical application.

[0005] To overcome the aforementioned challenges, existing technologies have developed cell membrane-derived nanovesicles (NVs) as alternatives to EVs. NVs can be rapidly and massively prepared using physical methods such as extrusion or ultrasound, offering advantages such as controllable composition, high reproducibility, and functional stability, making them suitable for large-scale clinical applications. In particular, stem cell-derived NVs not only retain the signaling molecules of their parent cells but are also rich in functional mitochondrial components, enabling them to target and regulate the mitochondrial function of recipient cells.

[0006] Previous studies have preliminarily confirmed the potential of ventricular protein (NV) in promoting wound healing, but its mitochondrial protein loading characteristics and regulatory mechanisms still lack systematic analysis. Therefore, there is an urgent need to develop NVs rich in mitochondrial proteins to regulate mitochondrial homeostasis and alleviate RSI-induced apoptosis. Summary of the Invention

[0007] This invention provides a therapeutic nanovesicle formulation rich in mitochondrial functional proteins, its preparation method, and its application in the treatment of radiation-induced skin damage, thereby overcoming the aforementioned problems.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] This invention provides a method for preparing a therapeutic nanovesicle formulation rich in mitochondrial functional proteins, comprising the following steps:

[0010] Umbilical cord mesenchymal stem cells are cultured, expanded, and washed, then dispersed in a suspension. They are then mechanically squeezed through a multi-pore filtration membrane to form a crude nanovesicle solution. After centrifugation, concentration, and purification, a therapeutic nanovesicle preparation rich in mitochondrial functional proteins is obtained.

[0011] Furthermore, the mitochondrial functional proteins in the therapeutic nanovesicle formulation rich in mitochondrial functional proteins account for more than 56% of the total mitochondrial functional proteins.

[0012] Furthermore, the specific method for mechanically squeezing the multi-pore size filter membrane is as follows: after the umbilical cord mesenchymal stem cells are dispersed in the suspension, they are sequentially passed through polycarbonate membranes with pore sizes of 10μm, 5μm and 1μm, and each type of polycarbonate membrane is squeezed at least 5 times.

[0013] Furthermore, the concentration of umbilical cord mesenchymal stem cells in the suspension is 10. 6 The number of cells / mL; the suspension is phosphate buffer; the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0014] Furthermore, the centrifugation method is low-temperature ultracentrifugation, with a temperature of 4°C, a centrifugal force of 2000-3000g, and a time of 15min.

[0015] Furthermore, the specific method of concentration and purification is as follows: after centrifugation, the crude nanovesicle solution is first filtered using an ultrafiltration membrane, and then subjected to low-temperature ultracentrifugation. The precipitate obtained is the nanovesicle derived from umbilical cord mesenchymal stem cells; it is then suspended in sterile physiological saline and frozen at -80°C for later use.

[0016] Furthermore, in the concentration and purification steps, the low-temperature ultracentrifugation conditions are: temperature 4°C, centrifugal force 10000-20000g, and time 30min.

[0017] Another aspect of the present invention provides a therapeutic nanovesicle formulation rich in mitochondrial functional proteins, wherein the therapeutic nanovesicle formulation rich in mitochondrial functional proteins is prepared by the preparation method described above.

[0018] In another aspect, the present invention provides the application of the therapeutic nanovesicle formulation rich in mitochondrial functional proteins in the treatment of radiation-induced skin damage.

[0019] Furthermore, the concentration of the therapeutic nanovesicle formulation rich in mitochondrial functional proteins is 10-60 μg / mL.

[0020] The beneficial effects of this invention are:

[0021] (1) The nanovesicle preparation prepared by the method of preparing therapeutic nanovesicle preparation rich in mitochondrial functional proteins disclosed in this invention can be directly applied to the damaged area by local injection, which can rapidly inhibit inflammation, reduce oxidative stress, and activate tissue repair signaling pathways, making it suitable for emergency treatment after acute radiation exposure.

[0022] (2) The therapeutic nanovesicle formulation rich in mitochondrial functional proteins provided by the present invention is easy to standardize and prepare on a large scale, has high stability, and is suitable for emergency storage and rapid deployment in the event of sudden radiation accidents. It avoids the problems of large batch differences and harsh storage conditions in cell therapy, and realizes large-scale production and storage.

[0023] (3) The therapeutic nanovesicle formulation rich in mitochondrial functional proteins provided by the present invention can functionally mimic and partially replace the biological effects played by hUMSCs by synergistically regulating multiple targets such as immune inflammatory response, oxidative stress, mitochondrial function and autophagy, thus avoiding the limitations brought about by cell survival rate and microenvironment dependence.

[0024] (4) The therapeutic nanovesicle formulation rich in mitochondrial functional proteins provided by the present invention not only effectively covers the key role of hUMSCs in the repair of radiation-induced skin damage in terms of biological function, but also shows higher clinical feasibility, safety and emergency adaptability in terms of application, providing a more strategically valuable alternative for the treatment of acute radiation-induced skin damage in the future. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a flowchart of a therapeutic nanovesicle (NV) formulation rich in mitochondrial functional proteins disclosed in Example 1 of the present invention;

[0027] Figure 2 This is a graph showing the physical property analysis results of the nanovesicles in the therapeutic nanovesicle formulation rich in mitochondrial functional proteins prepared in Example 1 of the present invention. Figure 2 A is the particle size distribution curve of the nanovesicles. Figure 2B is a bar chart of the zeta potential of nanovesicles;

[0028] Figure 3 Morphological photographs (transmission electron microscope images) of nanovesicles in the therapeutic nanovesicle formulation rich in mitochondrial functional proteins prepared in Example 1 of the present invention.

[0029] Figure 4 This is a Western blot result of the nanovesicles in the therapeutic nanovesicle formulation rich in mitochondrial functional proteins prepared in Example 1 of the present invention.

[0030] Figure 5 This is a proteomic characterization and functional enrichment analysis diagram of NV in the therapeutic nanovesicle formulation rich in mitochondrial functional proteins prepared in Example 1 of this invention, wherein, Figure 5 A shows the subcellular localization distribution of proteins in nanovesicles. Figure 5 B is a diagram showing the proteomic distribution of NV. Figure 5 C is a protein functional classification diagram of NV. Figure 5 D is a graph showing the results of Gene Ontology (GO) and KEGG pathway analysis. Figure 5 E represents the Sankey diagram of nanovesicles;

[0031] Figure 6 This is a flowchart illustrating the construction process of the cell radiation damage model of the present invention.

[0032] Figure 7 This is a graph showing the changes in cell viability under different concentrations of NV treatment in Example 2 of the present invention. Figure 7 A shows the changes in HaCaT cell viability. Figure 7 B shows the changes in HSF cell viability.

[0033] Figure 8 This is a graph showing the effect of NV on cell viability at different time points after radiation in Example 2 of the present invention. Figure 8 A shows the changes in HaCaT cell viability. Figure 8 B shows the changes in HSF cell viability.

[0034] Figure 9 This is an image of PKH26-labeled NV uptake in HaCaT and HSF cells under a laser confocal microscope at the optimal concentration of NV according to the present invention.

[0035] Figure 10 This is a graph showing the changes in cell migration ability in different treatment groups during a cell scratch assay. Figure 10 A shows microscopic images of the scratched areas of HSF cells at different time points in different treatment groups. Figure 10 B is a statistical graph showing the migration area of ​​HSF cells in different treatment groups at different time points;

[0036] Figure 11 The image shows the results of intracellular reactive oxygen species (ROS) detection after treating different cells with the optimal concentration of NV. Figure 11 Figure A shows the ROS detection results for HaCaT. Figure 11 B shows the ROS detection results after processing HSF;

[0037] Figure 12 The image shows the results of HaCaT and HSF cell apoptosis detection. Figure 12 Figure A shows the results of Annexin V / PI staining combined with flow cytometry analysis. Figure 12 B represents the statistical results of HaCaT cell apoptosis rate. Figure 12 C represents the statistical result of HSF cell apoptosis rate;

[0038] Figure 13 The image shows the results of Western blot detection of apoptosis-related proteins (BAX, BCL-2, GAPDH) in HaCaT and HSF cells. Figure 13 A is an electrophoresis diagram of apoptosis-related proteins in HaCaT cells. Figure 13 B represents the relative expression levels of apoptosis-related proteins in HaCaT cells. Figure 13 C is an electrophoresis diagram of apoptosis-related proteins in HSF cells. Figure 13 D represents the relative expression levels of apoptosis-related proteins in HSF cells;

[0039] Figure 14 The image shows the results of Western blot analysis of changes in the p53 signaling pathway in HaCaT and HSF cells. Figure 14 A is a Western blot diagram of p53 signaling pathway proteins in HaCaT cells. Figure 14 Figure B shows the relative expression levels of p53 in HaCaT cells. Figure 14 Figure C shows the relative expression levels of P21 in HaCaT cells. Figure 14 D is a Western blot image of P53 signaling pathway proteins in HSF cells. Figure 14 E shows the relative expression levels of P21 in HSF cells. Figure 14 F shows the relative expression levels of P53 in HSF cells;

[0040] Figure 15 The images show confocal microscopy images and quantitative analysis results of HaCaT (8 Gy) and HSF (16 Gy) cells after X-ray irradiation. Figure 15 A is a confocal microscopy image. Figure 15 B represents the results of the quantitative analysis.

[0041] Figure 16 Super-resolution microscopic images and quantitative morphological analysis of HaCaT and HSF cells after X-ray irradiation and NV treatment. Figure 16 A is a super-resolution micrograph of HaCaT. Figure 16 B is a super-resolution microscopic image of HSF cells; Figure 16 C is the quantitative analysis diagram of the morphology of HaCaT. Figure 16 D is a quantitative morphological analysis diagram of HSF cells;

[0042] Figure 17 The graph shows the mitochondrial membrane potential of HaCaT and HSF cells after X-ray irradiation and NV treatment. Figure 17 Figure A shows the mitochondrial membrane potential detection results for HaCaT. Figure 17 B is the graph showing the HSF mitochondrial membrane potential detection results;

[0043] Figure 18 The results are Western blot analysis of relevant proteins in HSF cells after X-ray irradiation and NV treatment. Figure 18 A is the Western blot plot of PINK1, Parkin, and GAPDH. Figure 18 B shows the relative expression levels of PINK1. Figure 18 C represents the relative expression levels of Parkin. Figure 18 Western blot plots of DRP1, GAPDH, MFN1, MFN2, and GAPDH. Figure 18 E represents the relative expression levels of DRP1. Figure 18 F represents the relative expression levels of MFN1; Figure 18 G represents the relative expression levels of MFN2;

[0044] Figure 19 Flowchart for constructing a rat model of acute radiation-induced skin injury on the lateral buttock;

[0045] Figure 20 Images and simulations of wound healing on the lateral buttocks of rats in different treatment groups;

[0046] Figure 21 Skin lesion scores for different treatment groups;

[0047] Figure 22 For the histopathological and immunohistochemical analysis of skin tissue from different treatment groups, among which, Figure 22 A represents the H&E staining results. Figure 22 B is a fluorescence microscope image of a Masson-stained tissue section. Figure 22 C represents the semi-quantitative analysis results of collagen fraction, hair follicle density, and epidermal thickness;

[0048] Figure 23 The cell activity and angiogenesis of different treatment groups were analyzed. Figure 23 Image A shows the results of immunohistochemical staining. Figure 23 B shows the expression levels of CD31 and α-SMA.

[0049] Figure 24 The results of cell apoptosis in different treatment groups, among which, Figure 24 A is the TUNEL staining diagram. Figure 24 B shows the results of TUNEL-positive cells;

[0050] Figure 25 Figures showing the results of γ-H2AX immunofluorescence staining in different treatment groups;

[0051] Figure 26 A graph showing the changes in proteins related to apoptosis and DNA damage in different treatment groups;

[0052] Figure 27 This is a graph showing the changes in P53 / P21 pathway factors in different treatment groups. Figure 27 A is the Western blot result graph. Figure 27 B shows the expression levels of P53 and P21.

[0053] Figure 28 The diagram shows the changes in mitochondrial ultrastructure in different treatment groups. Figure 28 A is the TEM image. Figure 28 B is a statistical graph showing the number of mitophagosomes / mitochondria;

[0054] Figure 29 The results of safety (blood compatibility) tests for injections into different treatment groups are shown in the figure.

[0055] Figure 30 The graph shows the toxicity test results after injection in different treatment groups. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0057] The principle of this invention for preparing therapeutic nanovesicle formulations rich in mitochondrial functional proteins is as follows:

[0058] I. This method preserves subcellular organelle membrane structures through mechanical compression.

[0059] Mechanical extrusion through a 10μm→5μm→1μm polycarbonate membrane can generate strong physical shear force on cells, causing the cell membrane and intracellular membrane structures (including the outer mitochondrial membrane) to rupture and reassemble, forming nanoscale vesicles. Mitochondria are abundant in mesenchymal stem cells and are easily released and encapsulated in membrane structures during mechanical shearing, resulting in vesicles containing a large number of mitochondrial-derived membrane proteins and contents (such as PINK1, TOMM20, MFN1, MFN2, etc.).

[0060] Second, since no surface labeling or density gradient separation is used in this preparation method, functional mitochondrial fragments are enriched. Compared with classic exosome extraction (e.g., differential centrifugation or SEC-bound immunoenrichment), this method does not specifically remove mitochondrial fragments, thus making it easier to retain functional mitochondrial proteins and membrane structures, and even encapsulate mitochondrial microparticles or mitovesicles.

[0061] Meanwhile, further removal of fine protein fragments and large molecular debris in the solution through a 100kDa filter membrane also helps to preserve structurally intact and relatively stable mitochondrial-associated nanovesicles.

[0062] Third, the mesenchymal stem cells used in this method have active mitochondria and are rich in natural mitochondrial-related structures. Umbilical cord mesenchymal stem cells can secrete mitochondrial-related extracellular vesicles, such as MitoEVs and Mitochondrial-Derived Vesicles (MDVs), under both homeostatic and stress conditions. These vesicles are naturally rich in mitochondrial membrane proteins and functional enzymes.

[0063] The nanovesicles extracted by this method without the use of special separation steps contain partially decapsulated mitochondria or vesicled fragments of them. Therefore, the proteomic analysis of the final nanovesicles shows a high proportion of mitochondrial proteins (57%).

[0064] Example 1:

[0065] like Figure 1 As shown, a method for preparing a therapeutic nanovesicle (MET-NV, hereinafter referred to as NV) formulation rich in mitochondrial functional proteins includes the following steps:

[0066] Human umbilical cord mesenchymal stem cells were harvested, cultured, and expanded to the desired density. The cells were then washed with phosphate-buffered saline (PBS) and dispersed in PBS, resulting in a suspension concentration of 102 human umbilical cord mesenchymal stem cells. 6Cells / mL of umbilical cord mesenchymal stem cells are then subjected to multi-stage mechanical extrusion through polycarbonate membranes with pore sizes of 10 μm, 5 μm, and 1 μm, with each pore size polycarbonate membrane being extruded 5 times (generally 5-7 times). During the mechanical extrusion process, the cell membranes of umbilical cord mesenchymal stem cells self-assemble to form vesicle-like nanoparticles, forming a crude nanovesicle solution. The crude nanovesicle solution is then subjected to ultracentrifugation at 4°C, 2000g, and 15 min to further filter out unbroken cells and large fragments, and the vesicles are coarsely sieved. Then, it is filtered through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and finally subjected to low-temperature ultracentrifugation at 4°C, 20000g, and 30 min to remove larger particles and impurities. The resulting precipitate is the nanovesicle derived from umbilical cord mesenchymal stem cells. The nanovesicles derived from umbilical cord mesenchymal stem cells are suspended in sterile physiological saline and frozen at -80°C for later use (the nanovesicles, after being suspended in sterile physiological saline, are the therapeutic nanovesicle (NV) preparation rich in mitochondrial functional proteins). The therapeutic nanovesicle formulation rich in mitochondrial functional proteins can eventually be formulated into an injectable or gel formulation for use; in this embodiment, it is an injectable formulation.

[0067] Example 2:

[0068] Screening for optimal concentrations of therapeutic nanovesicle formulations rich in mitochondrial functional proteins

[0069] 1. Materials and culture medium:

[0070] Human umbilical cord mesenchymal stem cells (hUMSCs) were cultured and used. MSC culture medium kit 2.0 (Tongli Haiyuan, Beijing, China) provides the essential nutrients required by the cells. To simulate the full-thickness skin structure, human keratinocytes (HaCaT) and human skin fibroblasts (HSF) were cultured separately, representing the epidermis and dermis, respectively. Both the human skin fibroblast (HSF) and human keratinocyte line HaCaT were purchased from Zhongqiao Xinzhou Biotechnology (Shanghai, China). Both HaCaT and HSF cells were cultured in high-glucose DMEM medium (Zhongqiao Xinzhou), supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin. All cell lines were cultured under standard aseptic conditions at 37°C in a humidified environment with 5% CO2, and passaged when the cell confluence reached 80-90%.

[0071] 2. Construction of a cell radiation damage model:

[0072] Radiation source: Linear accelerator (Elekta Axesse) TM Sweden, 6 MeV X-rays, dose rate 600 cGy / min)

[0073] Dosage: HaCaT: 8 Gy per dose; HSF: 16 ​​Gy per dose;

[0074] The damage model construction process is as follows Figure 6 As shown.

[0075] 3. Experimental grouping: NV formulations with concentrations of 2.5, 10, 15, 20, 40 and 60 μg / mL were prepared using the preparation method described in Example 1.

[0076] Treatment method: Two hours before radiation, cells were pretreated with different concentrations of NV preparations, and then subjected to X-ray irradiation;

[0077] Cell viability was assessed after X-ray irradiation using the CCK-8 assay for HaCaT and HSF cells. Cells were cultured at 1 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and treated under different experimental conditions. Treatments included a control group (without nanovesicle (NV) formulation) and treatment groups with different concentrations of NV formulation (2.5, 5, 10, 15, 20, 40, and 60 μg / mL). Evaluation was performed at 24 h, 48 h, and 72 h. 100 μL of 10% CCK-8 working solution (diluted with fetal bovine serum-free medium) was added to each well, and the plates were incubated at 37°C for 1 hour. After incubation, the absorbance of each well was measured at 450 nm using a ThermoScientific, Multiskan MK3 microplate reader. Cell viability was calculated as a percentage against the control group, reflecting changes in cell proliferation and survival.

[0078] 4. Experimental Results: The results are as follows Figure 7 , Figure 8 As shown, the results indicated that NV had no cytotoxicity on HaCaT and HSF cells. In HaCaT cells, a nanovesicle concentration of 5 μg / mL effectively promoted cell proliferation with no statistically significant difference compared to the 10 μg / mL concentration group. However, in HSF fibroblasts, a concentration of 10 μg / mL was required to promote proliferation. Figure 7 A, Figure 7 B. Figure 8 A, Figure 8 Therefore, considering its effects on both cell types, 10 μg / mL was selected as the minimum effective therapeutic concentration. At 48 and 72 hours post-irradiation, NV significantly improved cell viability, indicating that it could alleviate radiation-induced cytotoxicity.

[0079] Test Example 1:

[0080] Testing experiments on therapeutic nanovesicle formulations rich in mitochondrial functional proteins:

[0081] I. Basic Experiments on Therapeutic Nanovesicle Formulations Rich in Mitochondrial Functional Proteins

[0082] 1. Physical and morphological experiments:

[0083] Physical property analysis: using a nanoparticle tracking and analysis system ( PMX-120 (ParticleMetrix) assays were performed on therapeutic nanovesicles (NVs) rich in mitochondrial functional proteins, and the results were as follows: Figure 2 As shown, the particle size of NV is approximately 183.0 ± 7.1 nm. Figure 2 A), consistent with the size range of natural extracellular vesicles; the Zeta potential was detected at -31.93 ± 0.58 mV. Figure 2 B) indicates that its surface carries a negative charge, giving it good dispersibility and the ability to be taken up by target cells.

[0084] Morphological observation: Results are as follows Figure 3 As shown, transmission electron microscopy (TEM) revealed that these vesicles have a typical lipid bilayer capsule and a cup-shaped structure.

[0085] 2. Marker identification:

[0086] Protein fractionation was analyzed by Western blot. Total NV protein was extracted using RIPA lysis buffer (Catalog No. 89901, Thermo Fisher Scientific), with protease inhibitors (Catalog No. 78430) and phosphatase inhibitors (Catalog No. 78420, Thermo Fisher Scientific) added to prevent protein degradation. Protein concentration was determined using a BCA protein quantification kit (Catalog No. P0011, Beyotime). Equal volumes of protein samples were electrophoresed on 4–12% Bis-Tris precast gels (Catalog No. MA0463, Meilun Biotechnology), with pre-stained trichrome protein molecular weight standards (Catalog No. AG11919, AccurateBiology) used as molecular weight references. Separated proteins were transferred to 0.2 μm polyvinylidene fluoride (PVDF) membranes (Millipore) for Western blot analysis. The PVDF membranes were incubated overnight with primary antibody at 4°C, followed by incubation for 2 hours at room temperature with HRP-labeled secondary antibody. The membranes were washed three times with Tris-buffered saline (TBST) containing Tween-20 and then developed using enhanced chemiluminescence (ECL) substrates. Protein signals were detected using a Champ Chemi 610 Plus imaging system. Primary antibodies used included CD9 (ab236630), CD63 (ab134045), HSP70 (ab181606), and GAPDH (ab181602); secondary antibodies were HRP-labeled goat anti-rabbit IgG H&L (ab6721) and goat anti-mouse IgG H&L (ab6789), both purchased from Abcam and diluted 1:1000.

[0087] The results are as follows Figure 4 As shown, the results indicate that Western blotting successfully detected typical exosomal marker proteins such as CD9, CD63, syntenin, hsp70, and Alix, confirming their origin and function.

[0088] 3. Protein composition and function verification:

[0089] Proteins play a crucial role in the function of extracellular vesicles. Through proteomics analysis, the protein composition of therapeutic human umbilical cord mesenchymal stem cell-derived nanovesicles (NVs) rich in mitochondrial proteins was systematically characterized, aiming to identify their potential key bioactive proteins and reveal the underlying mechanisms of their therapeutic effects. Protein function is typically closely related to its subcellular localization; Figure 5 The results of proteomic characterization and functional enrichment analysis of NV are shown. Radar plot analysis revealed that NV contains a variety of subcellular structural proteins from diverse sources, including components of the plasma membrane, mitochondria, and endoplasmic reticulum. Figure 5A). Although plasma membrane proteins account for the largest proportion, the experiment focuses on mitochondrial proteins considering their functional relevance.

[0090] In the context of radiation-induced skin injury, mitochondrial dysfunction plays a central role in the pathological mechanism; therefore, the experiment prioritized the study of mitochondrial-associated proteins carried by radiation-induced skin injuries (NVs). Proteomic analysis identified 638 mitochondrial proteins, representing approximately 56.31% of the mitochondrial protein dataset defined in the MitoCarta 3.0 database. Figure 5 B) suggests that membrane remodeling mechanisms significantly integrate mitochondrial components during nanovesicle formation. Further analysis indicates that although NVs contain proteins from multiple sources, mitochondrial components constitute the largest functional group among shared proteins in the mitochondrial-related proteome. Figure 5 C).

[0091] To further explore the potential biological functions of these shared proteins, functional enrichment analysis was performed. Figure 5 In D, both Gene Ontology (GO) and KEGG pathway analyses showed that these proteins were significantly enriched in mitochondrial-related biological processes, such as oxidative phosphorylation, mitochondrial respiratory chain complexes, bioenergetics, mitochondrial dynamics, and mitophagy. Simultaneously, a Sankey diagram was constructed. Figure 5 E) to demonstrate the functional enrichment of representative proteins. Among these proteins, several key regulatory factors were identified, including OPA1 and mitochondrial fusion protein 2 (MFN2), which regulate mitochondrial fusion and membrane stability; BAX, which is associated with membrane permeability and autophagy; and VDAC1, which is involved in metabolite transport and stress signal regulation. Given the widespread mitochondrial disorders such as impaired respiratory chain function, excessive ROS production, and energy depletion in the pathogenesis of radiation injury, the mitochondrial proteins enriched in NV can help repair mitochondrial function in recipient cells.

[0092] In summary, these results demonstrate that the mitochondrial regulatory capabilities of the NVs prepared in this experiment not only reflect their cellular origin but also constitute one of their key therapeutic mechanisms. Proteomics analysis confirmed that these nanovesicles are rich in mitochondrial-related proteins (such as OPA1, MFN2, VDAC1, and BAX), possessing the potential to regulate oxidative phosphorylation, energy metabolism, mitochondrial dynamics, and autophagy, and can be used to intervene in mitochondrial disorders caused by radiation-induced skin damage.

[0093] 4. PKH26-labeled nanovesicles and cellular uptake experiments:

[0094] NV (10 μg / mL) was labeled with PKH26 fluorescent dye (MIDI26, Sigma-Aldrich) and incubated at room temperature for 5 minutes. The staining reaction was terminated by adding an equal volume of 1% bovine serum albumin (BSA). The cells were then washed three times with PBS and ultracentrifuged (2000×g, 15 minutes) to remove excess dye. Human keratinocytes (HaCaT) and human skin fibroblasts (HSF) were seeded separately in confocal microscope culture dishes (1.5 × 10⁶ cells per cell type). 3 Cells were incubated overnight at 37°C to promote adhesion. The next day, the medium was replaced with high-glucose DMEM containing PKH26-labeled NV, and incubation continued for 2 hours. After incubation, cells were washed with PBS, and the cytoplasm was stained with 1 μM Calcein AM and the nuclei with Hoechst 33342. Imaging was then performed under a laser confocal microscope. Imaging results showed that PKH26-labeled NV exhibited red fluorescence, Calcein AM-labeled cytoplasm showed green fluorescence, and Hoechst 33342-labeled nuclei showed cyan fluorescence. The presence of red fluorescence in the cytoplasm indicated that NV was successfully taken up and internalized by the cells. NV performs its biological functions and delivers mitochondrial proteins through uptake by target cells. Using laser confocal quantitative imaging cell analysis, experiments confirmed that normal human skin cells (HaCaT and HSF) can efficiently internalize PKH26-labeled NV within 2 hours. Figure 9 ).

[0095] 5. Cell scratch healing experiment:

[0096] When the HSF cell monolayer reached 100% confluence, straight scratches were made on the cell layer using a sterile pipette tip. The cells were then gently washed with PBS to remove cell debris and replaced with serum-free medium. Following irradiation, images of the scratched areas were taken using an inverted microscope at 0, 18, 24, and 48 hours, and cell migration was recorded. The percentage of scratch healing was calculated through image analysis to assess cell migration capacity. Results are as follows: Figure 10 As shown, Figure 10 A shows microscopic images of the scratched areas of HSF cells at different time points in different treatment groups. Figure 10 B shows the statistical graph of the migration area of ​​HSF cells at different time points in different treatment groups. The results indicate that radiation significantly inhibits the migration ability of skin cells, while NV treatment can significantly promote the migration potential of HSF cells. Radiation-induced excessive reactive oxygen species (ROS) can aggravate skin damage, forming a vicious cycle of "high-dose radiation - ROS accumulation - tissue damage". Therefore, effective removal of ROS is considered an important step in the treatment of radiation-induced skin damage.

[0097] 6. Detection of intracellular reactive oxygen species (ROS):

[0098] Intracellular ROS levels were detected using the DCFH-DA reactive oxygen species (ROS) detection kit (catalog number: S0033M, Beyotime, China). HaCaT or HSF cells were seeded in 6-well plates. After cell confluence reached 70-80%, cells were treated with NV (10 μg / mL) for 2 hours, followed by X-ray irradiation. Twenty-four hours after irradiation, cells were incubated with 10 μM DCFH-DA working solution in serum-free medium at 37°C in the dark for 20 minutes. After incubation, cells were washed three times with PBS to remove excess probe. The fluorescence intensity of DCFH (excitation wavelength 488 nm, emission wavelength 525 nm) was then detected by fluorescence microscopy or flow cytometry to reflect intracellular ROS levels. Experimental data were analyzed using flow cytometry data analysis software. Flow cytometry results are shown below. Figure 11 As shown, Figure 11 Figure A shows the ROS detection results for HaCaT. Figure 11 Figure B shows the ROS detection results of HSF treatment. The results show that NV can effectively remove excess ROS in HaCaT and HSF cells in the early stage of radiation, demonstrating significant antioxidant capacity.

[0099] 7. Apoptosis detection:

[0100] The apoptosis rate of HaCaT and HSF cells treated with NV was detected using the Annexin V-FITC apoptosis detection kit (catalog number: C1062M, Beyotime). Dye was added to each group of cells according to the kit instructions, and cells were collected after a period of intervention. After centrifugation, the supernatant was discarded, cells were resuspended in binding buffer, and Annexin V-FITC and PI double staining working solution was added and stained in the dark for 30 minutes. Apoptosis was then detected using flow cytometry (SH800, Sony). Annexin V-positive and PI-negative cells (Annexin V-FITC...) were... + / PI-) are considered early apoptotic cells, while Annexin V and PI double-positive cells (Annexin V-FITC) are considered early apoptotic cells. + / PI + These were considered to be late-stage apoptotic cells. Results analysis is as follows: Figure 12 As shown, the results of Annexin V / PI staining combined with flow cytometry analysis ( Figure 12 A) and statistical results of apoptosis rates of HaCaT cells and HSF cells ( Figure 12 B. Figure 12 C) indicates that NV can inhibit radiation-induced apoptosis, suggesting that it plays an important protective role in maintaining cell structural integrity.

[0101] 8. Western blot detection of apoptosis-related proteins:

[0102] Radiation-induced ROS accumulation can activate the pro-apoptotic protein BAX, which is believed to function through the BCL-2 regulatory pathway. BAX activation induces increased mitochondrial outer membrane permeability (MOMP), leading to cytochrome C release and subsequently activating the endogenous apoptosis pathway. BAX also acts as a signaling molecule for mitophagy, promoting mitochondrial fragmentation and clearance. BCL-2, however, has an antagonistic effect, inhibiting increased mitochondrial permeability, allowing for mitochondrial repair and preventing excessive clearance. Figure 13 For Western blot results, Figure 13 A- Figure 13 D-scans showed that the BAX / BCL-2 ratio significantly increased in HaCaT and HSF cells after X-ray irradiation (p<0.01); while NV treatment significantly reversed this change, further validating its anti-apoptotic effect. Furthermore, the role of mitochondria in cell death is not limited to apoptosis, but also involves multiple forms such as pyroptosis, ferroptosis, and necroptosis, suggesting that mitochondria have potential as a therapeutic target. The cross-regulation between these pathways also reflects the complexity of mitochondria in the cell death network, providing a theoretical basis for targeted intervention. To further investigate the effects of NV on the cell cycle and repair pathways, changes in the p53 signaling pathway were detected. Western blot results are shown below. Figure 14 As shown, the expression levels of P53 and its downstream effector molecule P21 in HaCaT and HSF cells were significantly decreased after NV treatment, indicating that it provides cells with more time to complete DNA repair by regulating cell cycle checkpoints. Figure 14 A- Figure 14 F).

[0103] To further elucidate the radiation resistance mechanism mediated by NV, the experiment focused on its impact on mitochondrial homeostasis. Considering the crucial roles of mitophagy and mitochondrial dynamics (dynamic fusion and fission) in responding to oxidative stress and radiation damage, the experiment aimed to assess whether NV maintains mitochondrial integrity by regulating these processes.

[0104] 9. Co-localization and morphological analysis of mitochondria and lysosomes:

[0105] Cells were seeded in confocal microscope culture dishes and, after adhesion, were stained with MitoTracker Deep Red FM (100 nM, Thermo Fisher) and LysoTracker Green DND-26 (75 nM, Thermo Fisher) in serum-free medium at 37°C for 30 minutes. After washing with PBS, super-resolution imaging was performed using a Nikon N-SIM 5.0 super-resolution microscopy system (excitation wavelength: 560 nm; acquisition band: 570-640 nm). Mitochondrial scaffold structure analysis and morphological parameter quantification were performed using the "Mitochondrial Analyzer" plugin in ImageJ. Confocal image acquisition was performed using an Olympus FV1000 system equipped with a 100×, NA 1.44 oil immersion objective (excitation wavelength: 543 nm; acquisition band: 560-660 nm). The colocalization degree between mitochondria and lysosomes was calculated using the colocalization analysis module built into OLYMPUS FLUOVIEW 3.0 software, and quantitative analysis was performed using the Pearson correlation coefficient. To further elucidate the NV-mediated radioprotection mechanism, the experiment will next investigate its impact on mitochondrial homeostasis. Given the crucial roles of mitophagy and mitochondrial dynamics in cellular responses to oxidative stress and radiation damage, this experiment aims to determine whether NV treatment maintains mitochondrial integrity by modulating these processes. Confocal microscopy imaging, such as... Figure 15 A showed that after X-ray irradiation, the colocalization of mitochondria and lysosomes in HaCaT (8 Gy) and HSF (16 Gy) cells significantly increased, suggesting an increase in mitophagy. After NV treatment, this colocalization significantly decreased in both cell types. Quantitative analysis ( Figure 15 B) shows that the Pearson correlation coefficient significantly increased after irradiation (HaCaT: 0.36 vs. 0.18; HSF: 0.52 vs. 0.34), while NV treatment significantly decreased this value (HaCaT: 0.13; HSF: 0.46). These results indicate that ionizing radiation causes mitochondrial damage and activates mitophagy, with HaCaT cells exhibiting a stronger response; while NV effectively mitigates this process, maintaining mitochondrial structural integrity. The difference in response intensity between the two cell types is related to their different metabolic needs and repair mechanisms. Excessive mitophagy leads to a reduction in the number of mitochondria, thereby affecting the cell's energy supply capacity under high metabolic demands such as tissue repair.

[0106] In summary, NVs play a crucial role in maintaining the balance between mitochondrial regeneration and autophagy by regulating mitochondrial quality control mechanisms, thus contributing to the maintenance of cellular energy metabolism homeostasis. Furthermore, during transport, NVs carry mitochondrial components to target cells, facilitating the replenishment of mitochondrial functional proteins, thereby maintaining mitochondrial function, reducing dependence on autophagy, and further promoting the restoration of cellular homeostasis.

[0107] Furthermore, NV can also assist in restoring mitochondrial function and integrity by delivering mitochondrial components to target cells, thereby reducing the cellular stress on autophagy-dependent repair. Figure 16 Super-resolution microscopy (SIM) revealed typical mitochondrial fragmentation in HaCaT and HSF after irradiation, with small, swollen mitochondria predominating. Figure 16 A, Figure 16 B) indicates excessive fission. After NV treatment, the mitochondrial network was restored, with an increase in long and short tubular structures and a significant reduction in fragmented morphology. Quantitative morphological analysis showed that the mitochondrial perimeter (p = 0.0002) and morphological factors (p = 0.00126) of HaCaT cells were significantly increased. Figure 16 C); HSF cells also showed a similar trend ( Figure 16 D) further supports the conclusion that NV promotes mitochondrial fusion and restores network structure. An increased branch number / mitochondrial mass ratio also suggests improved mitochondrial structural integrity.

[0108] 10. Mitochondrial membrane potential (ΔΨm) detection:

[0109] Mitochondrial membrane potential (TMRM) in HaCaT and HSF cells was detected using tetramethylrhodamine methyl ester (TMRM, I34361, Thermo Fisher Scientific). TMRM is a cell-permeable fluorescent probe that selectively accumulates in active mitochondria. After X-ray irradiation for 24 hours, cells were seeded in 6-well plates and cultured to approximately 70% confluence. Following experimental grouping, cells were incubated with 100 nM TMRM working solution (diluted in serum-free medium) at 37°C in the dark for 30 minutes. After staining, cells were washed twice with PBS to remove excess dye. Fluorescence signals were then detected using a flow cytometer (SH800, Sony), and the mean fluorescence intensity was analyzed using flow cytometry data analysis software. A decrease in TMRM fluorescence intensity indicated mitochondrial membrane potential depolarization, suggesting impaired mitochondrial function. Mitochondrial membrane potential (ΔΨm) is a key indicator for maintaining mitochondrial function. Figure 17 As shown, TMRE fluorescence staining combined with flow cytometry revealed that NV treatment significantly restored the ΔΨm level of HaCaT and HSF cells after radiation. Figure 17 A, Figure 17(B) This suggests that it has the ability to maintain mitochondrial activity and bioenergy metabolism. Previous studies have shown that EVs can fuse with the mitochondrial network of recipient cells, thus demonstrating that NVs exert a protective effect by targeting and repairing dysfunctional mitochondria. Mitochondrial function is significantly affected by its morphological changes.

[0110] 11. Research on the correlation between NV treatment and induction of the PINK1 / Parkin-dependent mitophagy pathway:

[0111] Furthermore, radiation induces the PINK1 / Parkin-dependent mitophagy pathway via ROS. PINK1 accumulates on the outer membrane when mitochondrial membrane potential decreases, recruiting Parkin to trigger ubiquitination and labeling damaged mitochondria for autophagic degradation. The investigation into whether NV treatment maintains mitochondrial homeostasis by regulating this pathway yielded the following results: Figure 18 As shown, Figure 18 Western blot and quantitative analysis in A-18C revealed a significant upregulation of PINK1 and Parkin protein expression after radiation, consistent with activation of the PINK1–Parkin pathway and the initiation of mitophagy in response to mitochondrial damage. Conversely, NV treatment significantly suppressed the expression of these two proteins, indicating an inhibitory effect on radiation-induced mitophagy signaling. These results suggest that NV helps maintain mitochondrial integrity by limiting the activation of aberrant autophagy under radiation stress. Therefore, NV-mediated PINK1–Parkin axis regulation could serve as a protective mechanism to maintain mitochondrial homeostasis and mitigate radiation-induced fibroblast damage.

[0112] like Figure 18 As shown in D and 18E, Western blot results revealed that radiation significantly upregulated the expression of the fission protein Drp1 (p<0.01), suggesting that radiation leads to enhanced mitochondrial fission and increased mitochondrial fragmentation. However, after NV intervention, Drp1 expression decreased, indicating that NV can inhibit excessive mitochondrial fission and maintain mitochondrial stability. Meanwhile, the fusion proteins MFN1 and MFN2 are substrates of Parkin.

[0113] When radiation damages mitochondria, it simultaneously inhibits Parkin activity, preventing it from functioning properly as an E3 ubiquitin ligase. This hinders Parkin's ubiquitination and degradation of MFN1 and MFN2, leading to the accumulation of these two fusion proteins due to reduced degradation and increased expression. Abnormal expression of Mfn1 and Mfn2 results in over-fusion, triggering the formation of "giant mitochondria" and initiating autophagy for clearance. NV downregulates Drp1 expression to inhibit excessive fission, and the increased degradation of MFN1 and MFN2 reduces their expression, preventing over-fusion and the formation of giant mitochondria, ultimately maintaining the normal structure and function of the mitochondrial network. Figure 18 F, Figure 18 G).

[0114] In summary, this invention reveals the multiple mechanisms by which therapeutic human umbilical cord mesenchymal stem cell-derived nanovesicles (NVs) rich in mitochondrial proteins alleviate X-ray-induced skin damage. NVs significantly scavenge ROS, inhibit p53 / p21 pathway activation, reduce apoptosis, and enhance the survival and migration of HaCaT and HSF cells in the early stages of radiation exposure, thus promoting wound repair. Simultaneously, NVs effectively alleviate radiation-induced mitochondrial dysfunction and maintain mitochondrial structure and metabolic homeostasis by restoring mitochondrial membrane potential, regulating mitochondrial fission and fusion dynamics (Drp1, MFN1, MFN2), and intervening in PINK1 / Parkin-mediated mitophagy. These results demonstrate that NVs not only exhibit significant antioxidant, repair-promoting, and anti-apoptotic functions at the cellular level but also play a core radioprotective role at the subcellular level by regulating mitochondrial quality control mechanisms. This invention provides a solid theoretical foundation and experimental evidence for NVs as a novel, controllable, and promising therapeutic strategy for radiation-induced skin damage.

[0115] II. Animal experiments on therapeutic nanovesicle formulations rich in mitochondrial functional proteins

[0116] 1. Animal experiment design:

[0117] To evaluate the potential and safety of nanovesicles (NVs) in treating radiation-induced skin injury in vivo, an acute radiation-induced skin injury rat model was established. SD rats (6-8 weeks old, approximately 200g) used in this study were purchased from Liaoning Changsheng Biotechnology Co., Ltd. All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Laboratory Animal Ethics Committee of Dalian Medical University (ethics approval number: AEE24145). Husbandry conditions included a temperature of 25±2℃, relative humidity of 50%, a 12-hour light / dark cycle, and free access to standard feed and purified water. A total of 18 rats were randomly divided into 3 groups (6 rats per group): (1) negative control group (NC); (2) X-ray irradiation group (IR); and (3) nanovesicle treatment group (NV).

[0118] Rats were acclimatized for one week prior to the experiment and weighed one day before the experiment. Sodium pentobarbital (50 mg / kg, intraperitoneal injection) was used as the anesthetic, followed by shaving the hair on the lateral right rump. In the IR and NV groups, rats were placed in a linear accelerator (Elekta Axesse). TMThe patient was irradiated with 6MeV X-rays in Sweden, with unirradiated areas protected by lead plates. Irradiation parameters were set as follows: 3cm × 3cm electron beam irradiation area, dose rate 600 cGy / min, total dose 35Gy. Nine days post-irradiation, redness, swelling, and desquamation were observed in the irradiated area, confirming the successful establishment of an acute radiation-induced skin injury model on the lateral buttock. The establishment process is as follows: Figure 19 As shown in the figure. The NV group and the IR group were injected with NV (10 μg / mL) or PBS (1 mL) every 3 days starting from day 9, and skin tissue was collected for analysis on day 28. Rats were weighed on days 9, 12, 15, 18, 21, and 28, and the irradiated areas were visually observed and photographed to dynamically monitor the wound healing process.

[0119] Gross observation results showed that in the IR+PBS group, the wounds persisted in ulceration, crusting, and failed to heal for a long time; while the IR+NV group showed significant improvement, with faster wound closure and reduced skin lesions. In particular, starting from day 15, the wound surface in the NV treatment group was smoother, with a significant reduction in erythema, and the wound area was smaller by day 28. Figure 20 Body weight change data showed that, compared with the PBS group, the NV-treated group rats experienced a significant increase in body weight after treatment. Skin lesion scoring results showed that NV significantly reduced the degree of trauma ( Figure 21 ).

[0120] 2. Skin tissue pathology and immunohistochemical analysis:

[0121] Histopathological analysis was used to evaluate skin structure and treatment efficacy. Six rats were sacrificed in each group, and skin tissue samples were collected and fixed in 4% paraformaldehyde. The tissues were then routinely embedded in paraffin and stained with hematoxylin and eosin (H&E) for histopathological analysis. Masson's trichrome staining was performed using a commercial kit (Servicebio, China) according to the manufacturer's instructions to assess the distribution and arrangement of collagen fibers. Images of the stained tissue sections were acquired using an inverted fluorescence microscope. Results are shown below. Figure 22 As shown. H&E staining revealed significant epidermal thickening in the IR+PBS group ( Figure 22 A) Significant crusting, reduced dermal appendages, and marked inflammatory infiltration; while the IR+NV group showed more intact skin structure, reduced epidermal thickness, and increased number of hair follicles. Figure 22 B). Masson staining results showed that the collagen fibers in the IR+PBS group were disordered, while the collagen structure in the IR+NV group was more ordered and the content was significantly increased. Figure 22 C).

[0122] Immunofluorescence staining was used to assess cell viability and angiogenesis. Immunohistochemical staining for CD31 (ab281583, Abcam) and α-SMA (19245S, Cell Signaling Technology) was performed, and the average optical density (AOD) was used to assess the level of angiogenesis in regenerated tissue. Results are as follows: Figure 23 As shown, Figure 23 Image A shows the results of immunohistochemical staining. Figure 23 Figure B shows the expression levels of CD31 and α-SMA. The results indicate that the expression levels of CD31 and α-SMA in the IR+PBS group were significantly lower than those in the NC group, suggesting impaired angiogenesis after radiation. In contrast, the expression levels of these two vascular markers were significantly upregulated in the IR+NV group, and the vascular density was close to that of the normal control group, suggesting that NV treatment helps promote angiogenesis after radiation-induced skin injury.

[0123] After confirming that hUMSC-derived nanovesicles (NVs) have good therapeutic effects and safety in a rat model of radiation-induced skin injury, the experiment further explored their potential mechanism of action.

[0124] Skin samples from three rats in each group were subjected to TUNEL staining to detect cell apoptosis in the tissues. The TUNEL staining results are shown below. Figure 24 As shown, Figure 24 A is the TUNEL staining diagram. Figure 24 Figure B shows the results of TUNEL-positive cells. The results show that the number of TUNEL-positive cells in the IR+PBS group increased significantly, indicating that radiation induced significant apoptosis; while the number of TUNEL-positive cells in the IR+NV group decreased significantly, indicating that NV treatment effectively reduced radiation-induced apoptosis and improved cell survival.

[0125] Similarly, γ-H2AX immunofluorescence staining (a classic marker of DNA double-strand breaks) showed enhanced expression after irradiation, while its positive signal was significantly weakened in the NV treatment group, suggesting improved cellular DNA repair capacity. Figure 25 Western blot analysis further validated the above results, showing that the expression of proteins related to apoptosis and DNA damage underwent regulatory changes. Figure 26 ).

[0126] As is well known, radiation-induced DNA damage activates the p53 protein, which plays a central role in maintaining genome stability by inducing cell cycle arrest, promoting DNA repair, and inducing apoptosis. Furthermore, p53 can bind to and activate the p21 promoter, which subsequently inhibits cyclin-dependent kinases (CDKs), particularly during the G1-S phase transition. Statistical analysis of Western blotting data is as follows: Figure 27 As shown, Figure 27 A is the Western blot result graph. Figure 27 Figure B shows the expression levels of P53 and P21. The results indicate that, compared with the IR+PBS group, the expression levels of P53 and P21 were significantly reduced in the NV treatment group. This result suggests that NV can promote tissue regeneration by regulating the P53 / P21 pathway, alleviating radiation-induced cell cycle arrest and cellular senescence, enhancing DNA repair and cell survival.

[0127] In addition, transmission electron microscopy (TEM) was used to observe the ultrastructural changes of mitochondria in rats two weeks after skin irradiation. Figure 28 ). Figure 28 In group A, swollen mitochondria and damaged cristae structures were observed in the skin of the IR+PBS group (green arrows in the figure), accompanied by autophagosomes containing mitochondrial fragments (yellow arrows in the figure), which is a typical morphology of mitophagy, indicating that the cells are under stress. In contrast, in the IR+NV group, mitochondrial structure was significantly improved, with intact membrane structure and clear cristae (green arrows in the figure), and mitophagy was reduced. The number of mitophagosomes / mitochondria in the field of view also showed that, compared with the PBS group, the level of mitophagy was significantly reduced after NV treatment. Figure 28 B). Desmosome structure was also observed in the NV treatment group. Figure 28 The number of desmosomes (indicated by the red arrow in section A) increases. Desmosomes not only enhance cell adhesion under mechanical stress, but also participate in signaling pathways related to cell behavior and tissue morphogenesis, suggesting that NVs play a positive role in maintaining the structural integrity of skin tissue.

[0128] In conclusion, NV therapy can improve the tissue microenvironment and promote the repair of radiation-induced skin damage by restoring mitochondrial homeostasis and alleviating X-ray-induced mitochondrial structural damage.

[0129] 3. Verify the in vivo safety of NV:

[0130] To assess the blood compatibility of nanovesicles (NVs), a hemolysis assay was performed. Whole blood was collected from three healthy male SD rats, anticoagulated with sodium citrate, and then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was discarded, and the erythrocyte pellet was collected. The erythrocytes were then repeatedly washed with physiological saline to prepare a 2% erythrocyte suspension. NV at concentrations of 10 μg / mL and 100 μg / mL were added to the erythrocyte suspension, respectively; PBS was used as a negative control, and distilled water as a positive control. After incubation at 37°C for 1 hour, the suspension was centrifuged again, and the absorbance of the supernatant was measured at 540 nm. The hemolysis rate was calculated using the following formula:

[0131] Hemolysis rate (%) = (OD sample - OD negative control) / (OD positive control - OD negative control) × 100%

[0132] When the hemolysis rate is less than 5%, it indicates that NV has good blood compatibility. Results are as follows... Figure 29 As shown, both low concentrations (10 μg / mL) and high concentrations (100 μg / mL) of NV resulted in hemolysis rates below the safety threshold of 5%, indicating good blood compatibility. H&E staining results also showed no significant toxic reactions observed in major organs (heart, liver, lungs, kidneys) after NV injection. Figure 30 In vitro hemolysis experiments and in vivo tissue toxicity analyses revealed no adverse reactions or tissue damage, indicating that the nanovesicles at this concentration possess excellent biocompatibility and application safety.

[0133] In summary, 10 μg / mL nanovesicles represent the optimal therapeutic dose, combining efficacy, safety, and cytoprotective function, thus providing a solid foundation for their subsequent application in acute radiation-induced skin injuries.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of therapeutic nanovesicles rich in mitochondrial functional proteins in the preparation of agents for treating radiation-induced skin damage, characterized in that, The concentration of therapeutic nanovesicle formulations rich in mitochondrial functional proteins is 10-60 μg / mL; A method for preparing a therapeutic nanovesicle formulation rich in mitochondrial functional proteins includes the following steps: Umbilical cord mesenchymal stem cells were cultured, expanded, and washed, then dispersed in a suspension. The suspension was then sequentially passed through polycarbonate membranes with pore sizes of 10 μm, 5 μm, and 1 μm, with each membrane size being repeatedly squeezed at least five times to form a crude nanovesicle solution. This solution was then subjected to low-temperature ultracentrifugation at 4°C, 2000-3000 g for 15 min. The solution was first filtered using an ultrafiltration membrane with a molecular weight cutoff of not less than 100 kDa, followed by low-temperature ultracentrifugation at 4°C, 10000-20000 g for 30 min. The resulting precipitate was the nanovesicle derived from umbilical cord mesenchymal stem cells. A therapeutic nanovesicle formulation rich in mitochondrial functional proteins was thus prepared. The mitochondrial functional proteins in this therapeutic nanovesicle formulation accounted for more than 56% of the total mitochondrial functional proteins.

2. The application according to claim 1, characterized in that, The concentration of human umbilical cord mesenchymal stem cells in the suspension was 10. 6 The number of cells / mL; the suspension is phosphate buffer; the umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

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