Butyric acid-loaded exovesicles, methods of preparation, medicaments and uses

Abstract

The application relates to a butyric acid-loaded outer vesicle, a preparation method, a medicine and application, which comprises endothelial cell outer vesicles and butyric acid loaded on the endothelial cell outer vesicles. The application can efficiently deliver butyric acid and specifically improve vascular endothelial cell aging, promote angiogenesis and tissue repair.

Description

Technical Field

[0001] This application relates to the fields of biomedicine and angiogenesis regulation technology, and in particular to a butyric acid-loaded exovesicle, its preparation method, drug and application. Background Technology

[0002] With the accelerating aging of the population, the incidence of vascular aging-related diseases is increasing year by year. Endothelial cell dysfunction is considered one of the important mechanisms leading to decreased angiogenesis, impaired tissue ischemia repair, and slow healing of chronic wounds. Therefore, exploring intervention strategies that can effectively delay endothelial cell aging and promote angiogenesis has become an important research direction in regenerative medicine and the prevention and treatment of vascular diseases.

[0003] Short-chain fatty acid butyrate is an important bioactive molecule produced by gut microbiota metabolism, playing a crucial role in energy metabolism regulation, inflammatory response modulation, and epigenetic modification. Previous studies have found that butyrate can significantly improve the senescent phenotype of vascular endothelial cells and promote angiogenesis by regulating endothelial cell function, thus providing a new target and potential therapeutic strategy for the intervention of vascular aging-related diseases. However, the poor stability of free butyrate in vivo, its short half-life, and the lack of effective targeted delivery capabilities limit its further application in vascular aging and tissue repair-related diseases. Summary of the Invention

[0004] This application provides butyric acid-loaded vesicles, a preparation method, a drug, and an application that can efficiently deliver butyric acid and specifically improve vascular endothelial cell aging, promote angiogenesis, and tissue repair.

[0005] In a first aspect, embodiments of this application provide butyric acid-loaded extravesicles, which include extracellular endothelial vesicles and butyric acid loaded on the extracellular endothelial vesicles.

[0006] In conjunction with the first aspect, in one embodiment, the mass of butyric acid loaded on each milligram of the extracellular vesicles of the endothelial cells is 600 to 1000 μg; And / or, the extracellular vesicles of the endothelial cells are derived from vascular endothelial cells; And / or, the particle size of the extracellular vesicles of the endothelial cells is 50-300 nm; And / or, the extracellular vesicles of the endothelial cells express one or more of the marker proteins CD81, TSG101, and CD9; And / or, the butyric acid enters the extracellular vesicles of the endothelial cells via electroporation.

[0007] Secondly, embodiments of this application provide a method for preparing butyric acid-loaded exovesicles as described above, comprising: Endothelial cell extracellular vesicles were isolated from endothelial cells; Butyric acid was loaded onto the extracellular vesicles of the endothelial cells to obtain butyric acid-loaded extracellular vesicles.

[0008] In conjunction with the second aspect, in one embodiment, loading butyric acid onto the extracellular vesicles comprises: loading the butyric acid into the interior of the extracellular vesicles by electroporation.

[0009] In conjunction with the second aspect, in one embodiment, butyric acid is loaded into the extracellular vesicles of the endothelial cells via electroporation, comprising: Endothelial cell extracellular vesicles were resuspended in butyric acid solution; Electroporation was performed under ice bath conditions using an electroporator with a voltage of 250–500 V, a pulse duration of 10–100 milliseconds, and an interval of 1–2 seconds between each pulse. Centrifugation was performed to remove unloaded butyric acid, yielding butyric acid-loaded exovesicles.

[0010] In conjunction with the second aspect, in one embodiment, after resuspending the extracellular vesicles in butyric acid solution, the concentration of the extracellular vesicles is 1 × 10⁻⁶. 11 ~5×10 11 cells / mL; And / or, the number of pulses is 3-5; And / or, the concentration of butyric acid solution is 1–8 mM; And / or, the butyric acid solution is obtained by dissolving butyric acid in PBS or cell culture medium; And / or, centrifugation conditions are 12000–14000 × g for 10–30 minutes.

[0011] Thirdly, embodiments of this application provide a drug for improving vascular endothelial cell aging or promoting angiogenesis, which includes butyrate-loaded extravesicles as described above.

[0012] In conjunction with the third aspect, in one embodiment, the drug is an injectable formulation; And / or, the drug is used to treat or prevent ischemic tissue injury, peripheral artery disease, or impaired wound healing.

[0013] Fourthly, embodiments of this application provide the use of butyric acid-loaded vesicles as described above in the preparation of a drug for improving vascular endothelial cell aging or promoting angiogenesis.

[0014] In conjunction with the fourth aspect, in one embodiment, the drug is an injectable formulation; And / or, the drug is used to treat or prevent ischemic tissue injury, peripheral artery disease, or impaired wound healing.

[0015] The beneficial effects of the technical solution provided in this application include: Butyrate was loaded into extracellular vesicles of endothelial cells to obtain butyrate-loaded extracellular vesicles with intact structure, uniform particle size, and expression of typical marker proteins. These butyrate-loaded extracellular vesicles can be effectively taken up by vascular endothelial cells, promoting the proliferation, migration, and tube formation of senescent endothelial cells, reducing the activity of senescence-related β-galactosidase and the expression of senescence markers such as p53, p21, and p16, thereby improving vascular endothelial cell senescence, promoting angiogenesis, and tissue repair. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The results of gas chromatography-mass spectrometry (GC-MS) detection and quantitative analysis of butyric acid content in butyric acid-loaded exovesicles provided in the embodiments of this application; Figure 2 Butyric acid-loaded exovesicle morphology diagram observed by transmission electron microscopy in the embodiments of this application; Figure 3 The results of butyric acid-loaded exovesicle particle size distribution obtained by nanoparticle tracking analysis provided in the embodiments of this application; Figure 4 The Western blot results for the extracellular vesicle marker proteins CD81, TSG101, and CD9 provided in the embodiments of this application; Figure 5 The results of the analysis of membrane integrity and butyrate retention of butyrate-vesicles in PBS under different time conditions provided in the embodiments of this application; Figure 6 Confocal microscopy images of butyrate-loaded vesicles taken up by vascular endothelial cells, provided in the embodiments of this application; Figure 7 The experimental results provided in the embodiments of this application show that butyric acid-loaded vesicles improve the ability of vascular endothelial cells to form. Figure 8 The experimental results of butyrate-loaded extravesicles promoting vascular endothelial cell proliferation provided in the embodiments of this application; Figure 9 The experimental results provided in the embodiments of this application demonstrate the ability of butyric acid-loaded vesicles to enhance the migration capacity of vascular endothelial cells. Figure 10 The detection results of butyrate-loaded exovesicles reducing aging-related β-galactosidase activity provided in the embodiments of this application; Figure 11 The results of detecting the expression levels of butyrate-loaded exovesicles regulating aging-related molecules p53, p21, and p16 provided in the embodiments of this application; Figure 12 The results of the detection of butyric acid-loaded exovesicles promoting blood flow recovery in ischemic limbs provided in the embodiments of this application; Figure 13 The butyric acid-loaded vesicles provided in this application increase the vascular density of ischemic limbs. Immunofluorescence staining and quantitative analysis of CD31 in the gastrocnemius muscle showed a significant increase in vascular density (n = 5). Figure 14 The detection results of butyrate-loaded extravesicles promoting matrix gel embolization angiogenesis provided in the embodiments of this application (n = 5). Figure 15 The test results (n=5) of butyric acid-loaded exovesicles promoting skin wound healing provided in the embodiments of this application. Figure 16 Histological test results of butyric acid-loaded vesicles promoting capillary formation in wound edge tissue, as provided in the embodiments of this application. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0019] To address the lack of effective intervention methods in the existing technology that can improve vascular endothelial cell senescence with good stability and in vivo delivery efficiency, the purpose of this application is to provide an application of butyrate-loaded exovesicles in improving vascular endothelial cell senescence and its preparation method. Furthermore, the improvement in vascular endothelial cell senescence is accompanied by an enhancement of angiogenesis capacity, thereby providing a new technical approach for the prevention and treatment of vascular aging-related diseases.

[0020] Specifically, this application provides a butyric acid-loaded extravesicle, which includes an extracellular endothelial cell vesicle and butyric acid loaded on the extracellular endothelial cell vesicle.

[0021] The mass of butyric acid loaded on each milligram of the extracellular vesicles of the endothelial cells is 600–1000 μg.

[0022] The extracellular vesicles of the endothelial cells are derived from vascular endothelial cells.

[0023] The particle size of the extracellular vesicles of the endothelial cells is 50-300 nm.

[0024] The extracellular vesicles of the endothelial cells express one or more of the marker proteins CD81, TSG101, and CD9.

[0025] The butyric acid enters the extracellular vesicles of the endothelial cells via electroporation.

[0026] Butyrate was loaded into extracellular vesicles of endothelial cells to obtain structurally intact, uniformly sized butyrate-loaded extracellular vesicles that expressed typical marker proteins. These butyrate-loaded extracellular vesicles could be effectively taken up by vascular endothelial cells, promoting the proliferation, migration, and tube-forming ability of senescent endothelial cells, while reducing the activity of senescence-related β-galactosidase and the expression of senescence markers such as p53, p21, and p16. In hindlimb ischemia, streptavidin embolism, and skin wound healing models in aged mice, administration promoted blood flow restoration, enhanced angiogenesis, and accelerated tissue repair. This application provides a strategy for vascular aging intervention based on extracellular vesicle delivery of butyrate, which can be used for the prevention and treatment of ischemic diseases and age-related vascular dysfunction.

[0027] This application employs electroporation instead of other loading methods because it achieves a better balance between efficiency, controllability, and impact on the extracellular vesicle structure. Extracellular vesicles are essentially nanoscale vesicles composed of a lipid bilayer membrane. While small molecules such as butyric acid have small molecular weights, they exist in an ionic state under physiological pH conditions. Simply relying on passive incubation or concentration gradient diffusion to enter the vesicle lumen is typically inefficient and difficult to guarantee stable loading. Electroporation creates reversible nanopores on the lipid bilayer using instantaneous high-intensity electrical pulses, allowing butyric acid in solution to cross the membrane and enter the vesicle lumen in a short time. Subsequently, the electric field is removed, and the membrane structure re-closes, thus achieving "active loading." This physical loading method does not rely on carrier chemical modification or alter the chemical structure of butyric acid.

[0028] Compared to simple co-incubation, electroporation significantly improves loading efficiency and reproducibility. Co-incubation typically relies on long-duration diffusion, and the loading rate is greatly affected by temperature, pH, and membrane permeability, making it difficult to achieve high internal content. Compared to sonication or repeated freeze-thaw cycles, electroporation provides more controllable disruption to membrane structure. Sonication and freeze-thaw cycles often lead to extracellular vesicle membrane rupture, aggregation, or protein conformational changes, thus affecting their biological activity and targeting properties. Compared to chemical transfection reagents or liposome fusion methods, electroporation does not introduce additional chemical components, avoids the subsequent removal of residual reagents, and reduces the risk of potential toxicity and contamination, making it particularly suitable for formulations requiring high purity for in vivo experiments or clinical translation studies.

[0029] Furthermore, electroporation parameters (voltage, pulse width, pulse count, and temperature) can be finely adjusted, thereby optimizing loading while maintaining vesicle integrity. This "engineerable controllability" is difficult to achieve with many other methods. For nanoscale vesicle systems, electroporation has been widely validated in RNA, miRNA, and small molecule loading. The process is mature, reproducible, and the equipment is highly standardized, facilitating scale-up and process validation. Therefore, when it is necessary to increase the proportion of butyrate entering the extracellular vesicle lumen while maintaining vesicle structural integrity and functional stability, electroporation is a more advantageous choice.

[0030] Loading butyrate inside extracellular vesicles rather than on their outer surface is primarily aimed at improving stability, enhancing intracellular delivery efficiency, optimizing pharmacokinetic behavior, and maintaining the integrity of the extracellular vesicles' biological functions. Butyrate is a small-molecule, short-chain fatty acid that exists in ionic form in the physiological environment. If it is only adsorbed or bound to the surface of extracellular vesicles, it is easily dissociated after entering the bloodstream or cell culture system, and is rapidly diluted or metabolically eliminated, resulting in a significant decrease in the effective dose actually delivered to the target cells. Encapsulating it inside a lipid bilayer structure, however, can utilize the membrane barrier effect to reduce its loss during circulation, thereby improving in vivo stability and local concentration.

[0031] Furthermore, butyrate's mechanism of action is not solely dependent on cell membrane surface receptor signaling pathways. While it can indeed exert some signaling regulation by activating short-chain fatty acid receptors such as FFAR2 and FFAR3 on the membrane, one of its more important biological effects is as a histone deacetylase (HDAC) inhibitor, regulating gene expression in the cell nucleus. This epigenetic regulation requires its entry into the cell interior. If butyrate is only located on the surface of external vesicles, even if it comes into contact with the cell membrane, its effect is mainly limited to transient receptor binding. However, when it is encapsulated inside external vesicles and enters the cell via membrane fusion or endocytosis, it can be released into the cytoplasm and even the nucleus, thus producing a more persistent and profound regulatory effect.

[0032] From an engineering and formulation perspective, surface loading is often a reversible adsorption process, significantly affected by ionic strength, pH, and competition from plasma proteins, resulting in substantial batch-to-batch variations and hindering quality control and precise dosage. Internal encapsulation, on the other hand, allows for more quantitative control through metrics such as encapsulation efficiency and drug loading. Furthermore, the surface of external vesicles naturally contains various membrane proteins and recognition molecules, whose structures determine their targeting and cellular uptake capabilities. Immobilizing butyrate on the surface may obscure or interfere with these key structures, affecting its biodistribution and targeting efficacy. In summary, loading butyrate inside external vesicles can improve delivery efficiency and effective intracellular concentration while maintaining structural stability and targeting function.

[0033] This application also provides a method for preparing butyric acid-loaded exovesicles, which includes: 101: Endothelial cell extracellular vesicles were isolated from endothelial cells.

[0034] Specifically, vascular endothelial cells are cultured and their culture supernatant is collected. Endothelial cell extracellular vesicles are obtained by differential centrifugation, ultracentrifugation, density gradient centrifugation, or equivalent separation and purification methods.

[0035] 102: Butyric acid is loaded onto the extracellular vesicles of the endothelial cells to obtain butyric acid-loaded extracellular vesicles.

[0036] Specifically, butyric acid is loaded into the extracellular vesicles of the endothelial cells using an electroporation method.

[0037] Extracellular vesicles, as natural nanoscale lipid bilayer structures derived from the cell membrane system, possess excellent biocompatibility, low immunogenicity, and strong in vivo stability. They can participate in intercellular substance and signal transduction under physiological conditions, thus being considered ideal biological delivery carriers. Encapsulating butyrate in extracellular vesicles offers several advantages. First, the lipid bilayer structure provides physical protection, reducing its rapid metabolism and clearance in the bloodstream, thereby improving its in vivo stability and tissue exposure time. Second, extracellular vesicles can directly deliver their loaded molecules to the cytoplasm or even near the nucleus of recipient cells via membrane fusion or endocytosis, increasing the effective intracellular concentration. Compared to free butyrate's reliance on monocarboxylic acid transporters for cell entry or activation of G protein-coupled receptors (such as FFAR3 / FFAR2)-mediated signaling pathways, vesicle delivery is more conducive to targeted intracellular release. Furthermore, extracellular vesicles often exhibit tissue tropism in inflammatory or ischemic microenvironments, which helps improve the accumulation efficiency of butyrate in vascular injury sites or senescent endothelial cells. Therefore, using extracellular vesicles as butyrate delivery carriers can theoretically balance the advantages of stability, intracellular delivery efficiency, and safety, providing a more promising strategy for the intervention of vascular endothelial aging and ischemic repair-related diseases.

[0038] Loading butyric acid into the extracellular vesicles of endothelial cells via electroporation includes: Endothelial extracellular vesicles were resuspended in butyric acid solution to achieve a concentration of 1 × 10⁻⁶ vesicles. 11 ~5×10 11 Cells / mL, preferably 2×10 11 The concentration of butyric acid solution is 1-8 mM, preferably 4 mM, and the butyric acid solution is obtained by dissolving butyric acid in PBS or cell culture medium. Electroporation was performed under ice bath conditions using an electroporator with a voltage of 250–500 V, a pulse duration of 10–100 milliseconds, an interval of 1–2 seconds between each pulse, and 3–5 pulses. Centrifuge to remove unloaded butyric acid and obtain butyric acid-loaded exovesicles. Centrifugation conditions are 12000–14000 × g for 10–30 minutes.

[0039] This application also provides a drug for improving vascular endothelial cell aging or promoting angiogenesis, which includes the butyrate-loaded extravesicles.

[0040] The drug is an injectable preparation.

[0041] The drug is used to treat or prevent ischemic tissue injury, peripheral artery disease, or impaired wound healing.

[0042] This application also provides the use of butyric acid-loaded vesicles in the preparation of drugs for improving vascular endothelial cell aging or promoting angiogenesis.

[0043] It can inhibit the increase in aging-related β-galactosidase activity, downregulate the expression of aging-related molecules such as p53, p21, and p16, restore the proliferation, migration, and angiogenesis capabilities of endothelial cells, and improve the ability to repair ischemic tissues.

[0044] The technical solutions provided in this application will be described in detail below with reference to the embodiments.

[0045] The experimental animals used were SPF-grade C57BL / 6J mice, purchased from the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. The experimental animal production license number complied with relevant national regulations. The aged mice were over 18 months old.

[0046] Human umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cords, cultured using conventional methods at 37°C and 5% CO2, and passaged for expansion according to standard procedures.

[0047] The main reagents used included: sodium butyrate (Sigma-Aldrich, USA); endothelial cell culture medium (ScienCell, USA); matrix gel (Corning, USA); hemoglobin (Hb) assay kit (Solarbio, China); SA-β-gal staining kit and EdU cell proliferation assay kit (Beyotime Biotechnology, China); and extracellular vesicle marker antibodies CD81, TSG101, and CD9, and aging marker antibodies p53, p21, and p16 (Abclonal, China). All other chemical reagents not specifically mentioned were commercially available analytical grade products.

[0048] The main instruments and equipment used included: an ultracentrifuge (Beckman Coulter, USA), a nanoparticle tracking analyzer (NTA, Malvern Panalytical, UK), a transmission electron microscope (TEM, JEOL, Japan), a laser Doppler blood flow imaging system (Perimed, Sweden), a fluorescence microscope (Olympus, Japan), and an electroporation system (Bio-Rad, USA). Other routine experimental equipment was standard laboratory equipment.

[0049] Example 1: Preparation of butyric acid-loaded exovesicles Human umbilical vein endothelial cells (HUVECs) were cultured in complete medium containing 10% serum to remove extracellular vesicles at 37°C and 5% CO2 until 70%–80% confluence. The medium was then replaced with extracellular vesicle-free medium and cultured for another 48 hours. The cell culture supernatant was collected. The supernatant was then centrifuged sequentially at 200×g for 10 minutes to remove cells, at 2700×g for 10 minutes to remove cell debris, and at 14000×g for 1 hour to separate extracellular vesicles. The purified endothelial extracellular vesicles were resuspended in sterile PBS.

[0050] The purified extracellular vesicles of endothelial cells were mixed with butyric acid solutions of different concentrations (0 mM, 1 mM, 2 mM, 4 mM, and 8 mM) to achieve a concentration of 2 × 10⁻⁶ endothelial cell extracellular vesicles. 11 Butyric acid was injected at a concentration of 1 / mL into an electroporation vessel and subjected to transient electroporation at 270V, a pulse duration of 20 ms, a pulse interval of 1 sec, and a pulse count of 3 times to allow butyric acid to enter the extracellular vesicles of endothelial cells. The vesicles were then immediately annealed on ice and centrifuged again at 14000×g for 10 min to remove unencapsulated butyric acid, resulting in butyric acid-loaded extracellular vesicles. The butyric acid content of the electroporated extracellular vesicle samples was determined by gas chromatography-mass spectrometry (GC-MS / MS) for quantitative analysis. The experiment was repeated three times. See [link to relevant documentation]. Figure 1 As shown, the results indicated that butyric acid was successfully encapsulated in extracellular vesicles of endothelial cells, with the butyric acid loading increasing in a concentration-dependent manner and approaching saturation at approximately 4 mM. Therefore, all subsequent in vitro and in vivo experiments used butyric acid-loaded extracellular vesicles prepared with 4 mM butyric acid as the standard experimental material.

[0051] For ease of understanding, in the following text, butyrate-loaded extracellular vesicles will be referred to as butyrate-vesicles, and extracellular vesicles of endothelial cells will be referred to as empty-loaded vesicles.

[0052] Example 2: Identification of the physicochemical properties of butyric acid-loaded exovesicles The empty vesicles and butyrate-vesicles derived from HUVECs obtained in Example 1 were observed using transmission electron microscopy. See [link to original text]. Figure 2 and Figure 3 As shown, the vesicles exhibit a near-circular bilayer membrane structure, and their particle size distribution was determined using nanoparticle tracking analysis, with an average particle size of approximately 200 nm.

[0053] See Figure 4 As shown, Western blot analysis revealed positive expression of extracellular vesicle marker proteins CD81, TSG101, and CD9. The control group consisted of whole-cell proteins extracted from HUVECs.

[0054] To verify the integrity of the butyrate-vesicle membrane after electroporation, the retention of butyrate in butyrate-loaded vesicles was analyzed. Butyrate-vesicles were resuspended in PBS (approximately 2 mg / mL) and kept at room temperature. Samples were taken at 0, 24, 48, 72, 96, 120, 144, and 168 hours. After sampling, vesicle residues were removed by centrifugation at 12000×g for 10 minutes, and the supernatant was collected to detect the content of leaked butyrate. Precipitated vesicles were thoroughly lysed with RIPA lysis buffer, and the butyrate content inside the vesicles was detected. Subsequently, butyrate in the supernatant and lysis buffer was quantitatively analyzed using GC-MS / MS, and the integrity of the vesicle membrane at different time points was assessed by calculating the mass ratio of butyrate in the supernatant to total butyrate. The experiment was conducted in triplicate, with results expressed as mean ± SD, to determine the stability of the vesicle membrane structure and the butyrate encapsulation effect after electroporation. See [link to relevant documentation]. Figure 5 As shown, the test results prove that structurally intact and clearly derived extracellular vesicles of endothelial cells were successfully obtained and butyrate was effectively loaded.

[0055] Example 3: Cellular uptake and in vitro anti-aging effects of butyrate-loaded exovesicles Butyrate-vesicles labeled with the fluorescent dye PKH26 were co-cultured with PKH67-labeled HUVECs at 37℃ and 5% CO2 for 2 h. Co-localization was observed using a confocal microscope. (See [link to documentation]). Figure 6 As shown, fluorescence microscopy revealed that vesicles can be effectively internalized by endothelial cells and distributed within the cytoplasm.

[0056] Starting from passage P2 of HUVEC cells, butyrate-vesicles (concentration 2 μg / mL) were added to the culture medium for HUVEC cells every 2 days and the treatment continued until the P9 stage (i.e., 14 days of culture) to establish a replicative senescence model as the butyrate-vesicle group.

[0057] Starting from passage P2 of HUVEC cells, empty vesicles (concentration 2 μg / mL) were added to the culture medium for HUVEC cells every 2 days and the treatment continued until the P9 stage (i.e., 14 days of culture) to establish the empty vesicle group.

[0058] Starting from passage P2 of HUVEC cells, the cells were continuously treated until the P9 stage (i.e., cultured for 14 days), and a control group was established.

[0059] Functional tests were performed on the butyrate-vesicle group, empty vesicle group, and control group, including the ability to form angioid structures, cell proliferation capacity, cell migration capacity, and aging degree.

[0060] The ability to form blood vessel-like structures was evaluated using a matrix gelation tube formation assay: HUVECs from the butyrate-vesicle group, empty vesicle group, and control group were divided into groups of 1×10⁻⁶. 4 Each well was inoculated onto the solidified substrate and incubated for 6 hours. Images were then taken under an inverted microscope, and the total tube length was calculated using image analysis software. See [link to documentation]. Figure 7 As shown, butyrate-loaded exovesicles significantly enhanced the angiogenesis capacity of senescent HUVECs compared with the control group, where ns indicates no significant difference.

[0061] Cell proliferation capacity was detected using the EdU incorporation method: HUVECs from the butyrate-vesicle group, empty vesicle group, and control group were seeded in 96-well plates, respectively. EdU working solution at a final concentration of 10 μM was added and incubated for 2 h. After fixation and permeabilization, a click reaction was performed, and the nuclei were counterstained with DAPI. Five fields of view were randomly photographed to determine the proportion of EdU-positive cells among the total cells. (See also...) Figure 8 As shown, butyrate-loaded exovesicles significantly enhanced the proliferation capacity of senescent HUVECs compared with the control group.

[0062] Cell migration ability was assessed using a Transwell assay: 3 × 10⁻⁶ cells were collected from the butyrate-vesicle group, the empty vesicle group, and the control group. 4 HUVECs were added to the upper chamber with serum-free medium and serum-containing endothelial culture medium to the lower chamber. After incubation for 6 h, unmigrated cells from the upper chamber were removed, and the cells were fixed, stained, and randomly photographed to count the number of cells that had migrated through the membrane. See also Figure 9 As shown, butyrate-loaded exovesicles significantly enhanced the cell migration ability of senescent HUVECs compared with the control group.

[0063] The degree of aging was assessed using SA-β-gal staining: HUVECs from the butyrate-vesicle group, empty vesicle group, and control group were fixed and incubated with β-gal staining solution overnight at 37°C in the dark. The proportion of blue-positive cells was counted. Simultaneously, total cellular protein was extracted, and the expression levels of aging-related molecules such as p16, p21, and p53 were detected by Western blot. See also... Figure 10 and Figure 11 As shown, compared with the control group, butyric acid-loaded vesicles significantly reduced the positive rate of SA-β-gal and the expression levels of p16, p21, and p53.

[0064] Experimental results show that butyric acid-loaded vesicles have a clear effect on delaying endothelial cell aging in vitro.

[0065] Example 4: Butyrate-loaded exovesicles promote the recovery of blood flow perfusion in ischemic lower limbs. Naturally aged C57BL / 6 mice were selected. The butyrate-vesicle group received butyrate-vesicles via tail vein injection at a dose of 100 μg / mouse twice weekly for 8 weeks. The control group received an equal volume of PBS. A hindlimb ischemia model was then established. Under anesthesia, the femoral artery was exposed and segmentally ligated and resected from the iliofemoral artery down to below the knee, ensuring a significant reduction in blood flow to the ischemic limb. Appropriate analgesia and resuscitation were administered immediately postoperatively, maintaining body temperature and fluid balance. Postoperative blood flow recovery was assessed using laser Doppler flow imaging at 0, 3, 7, and 14 days post-surgery. Under anesthesia, the ischemic limb and contralateral limb were scanned using laser Doppler flow imaging to obtain blood perfusion images, and the ischemic / unaffected side blood perfusion ratio was calculated to quantitatively analyze the rate and level of blood flow recovery. See [link to relevant documentation]. Figure 12 As shown, the blood flow recovery rate and perfusion level in the butyrate-vesicle group were significantly higher than those in the control group.

[0066] Control / Non-ischemic lower limb: Naturally aged C57BL / 6 mice were selected. The control group was injected with an equal volume of PBS. Eight weeks later, the mice underwent lower limb ischemia surgery. Fourteen days after the surgery, the non-ischemic lower limb was harvested for CD31 immunofluorescence staining to detect vascular density.

[0067] Control / Ischemic Lower Limb: Naturally aged C57BL / 6 mice were selected. The control group was injected with an equal volume of PBS. Eight weeks later, the mice underwent lower limb ischemia surgery. Fourteen days after the surgery, the ischemic lower limb was harvested for CD31 immunofluorescence staining to detect vascular density.

[0068] Butyrate-vesicle / non-ischemic lower limb: Naturally aged C57BL / 6 mice were selected and administered butyrate-vesicles via tail vein injection at a dose of 100 μg / mouse twice a week for 8 consecutive weeks. Lower limb ischemia surgery was then performed, and the non-ischemic lower limb was harvested after surgery for CD31 immunofluorescence staining to detect vascular density.

[0069] Butyrate-vesicles / ischemic lower limbs: Naturally aged C57BL / 6 mice were selected and administered butyrate-vesicles via tail vein injection at a dose of 100 μg / mouse twice a week for 8 consecutive weeks. Lower limb ischemia surgery was then performed, and the ischemic lower limb was harvested after surgery for CD31 immunofluorescence staining to detect vascular density.

[0070] See Figure 13 The results showed that the vascular density of butyrate-vesicles in the ischemic lower limb was significantly increased compared with that in the control / ischemic lower limb, suggesting that butyrate-vesicles can effectively improve the angiogenesis capacity of elderly individuals after ischemia.

[0071] Example 5: Butyrate-loaded exovesicles promote angiogenesis in aged mice Naturally aged C57BL / 6 mice were selected. The butyrate-vesicle group was administered butyrate-vesicles via tail vein injection at a dose of 100 μg / mouse twice weekly for 8 weeks. The control group received an equal volume of PBS. After 8 weeks of butyrate-vesicle administration, five aged C57BL / 6 mice were selected per group. Under anesthesia, approximately 0.5 mL / mouse of Corning gel (containing 50 U / mL heparin sodium and 20 μg / L recombinant VEGF) was injected subcutaneously into the abdomen to form a subcutaneous embolization. The mice were kept in a normal feeding environment post-surgery. Seven days later, the mice were sacrificed, and the Corning gel emboli were removed, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned.

[0072] Neovascularization was observed using HE staining. Embolism hemoglobin content was measured: the matrix gel embolization block was weighed, placed in 1 mL of deionized water, and ground evenly. After repeated freeze-thaw cycles to disrupt the cell tissue, the hemoglobin (Hb) concentration was measured using a hemoglobin (Hb) content detection kit according to the instructions. The absorbance (OD value) was read at a specified wavelength on a spectrophotometer, and the hemoglobin concentration (μg / mg tissue) was calculated using a standard curve to quantitatively compare the levels of intraembolic blood perfusion and angiogenesis in different treatment groups. See also... Figure 14 As shown in the figure, the experimental results showed that the embolized hemoglobin content in the butyrate-vesicle group was significantly higher than that in the control group, further proving that butyrate-loaded vesicles can promote angiogenesis in vivo.

[0073] Example 6: Butyrate-loaded exovesicles promote wound healing in aged mice Naturally aged C57BL / 6 mice were selected. The butyrate-vesicle group received butyrate-vesicles via tail vein injection at a dose of 100 μg / mouse twice weekly for 8 weeks. The control group received an equal volume of PBS. After 8 weeks of butyrate-vesicle administration, a full-thickness skin defect of approximately 6 mm in diameter was created on the back of the mice, exposing the subcutaneous fascia layer. Postoperatively, changes in wound area were recorded daily by photograph, and wound closure speed was measured using image analysis software on days 0, 3, and 7. Mice were sacrificed at the end of day 7, and wound edge tissue was collected for fixation in 4% paraformaldehyde, paraffin embedding, and CD31 immunohistochemical staining to observe capillary density. (See also...) Figure 15 As shown, the wound closure speed was significantly faster in the butyrate-vesicle group. (See [reference]) Figure 16 As shown, the capillary density in the wound edge area was higher than that in the control group, further demonstrating that butyrate-loaded vesicles have a stable and significant pro-angiogenic and tissue repair effect in vivo.

[0074] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A butyric acid-loaded exovesicle, characterized in that, It includes extracellular endothelial vesicles and butyric acid loaded on the extracellular endothelial vesicles. The butyric acid enters the extracellular endothelial vesicles through electroporation. The voltage of the electroporation is 250-500 V, the pulse time is 10-100 milliseconds, and the interval between each pulse is 1-2 seconds.

2. The butyric acid-loaded exovesicles as described in claim 1, characterized in that: The mass of butyric acid loaded on each milligram of the extracellular vesicles of the endothelial cells is 600–1000 μg; And / or, the extracellular vesicles of the endothelial cells are derived from vascular endothelial cells; And / or, the particle size of the extracellular vesicles of the endothelial cells is 50-300 nm; And / or, the extracellular vesicles of the endothelial cells express one or more of the marker proteins CD81, TSG101, and CD9.

3. A method for preparing butyric acid-loaded exovesicles as described in claim 1 or 2, characterized in that, It includes: Endothelial cell extracellular vesicles were isolated from endothelial cells; Butyric acid was loaded onto the extracellular vesicles of the endothelial cells to obtain butyric acid-loaded extracellular vesicles; Loading butyric acid onto the extracellular vesicles includes loading butyric acid into the interior of the extracellular vesicles by electroporation.

4. The method for preparing butyric acid-loaded exovesicles as described in claim 3, characterized in that, Loading butyric acid into the extracellular vesicles of endothelial cells via electroporation includes: Endothelial cell extracellular vesicles were resuspended in butyric acid solution; Electroporation was performed using an electroporator under ice bath conditions. Centrifugation was performed to remove unloaded butyric acid, yielding butyric acid-loaded exovesicles.

5. The method for preparing butyric acid-loaded exovesicles as described in claim 4, characterized in that: After resuspending the extracellular vesicles in butyric acid solution, the concentration of the extracellular vesicles was 1 × 10⁻⁶. 11 ~5×10 11 cells / mL; And / or, the number of pulses is 3-5; And / or, the concentration of butyric acid solution is 1–8 mM; And / or, the butyric acid solution is obtained by dissolving butyric acid in PBS or cell culture medium; And / or, centrifugation conditions are 12000–14000 × g for 10–30 minutes.

6. A drug for improving vascular endothelial cell senescence or promoting angiogenesis, characterized in that: It includes butyric acid-loaded exovesicles as described in claim 1 or 2.

7. The medicament for improving vascular endothelial cell senescence or promoting angiogenesis as described in claim 6, characterized in that: The drug is an injectable preparation; And / or, the drug is used to treat or prevent ischemic tissue injury, peripheral artery disease, or impaired wound healing.

8. The use of butyric acid-loaded exovesicles as described in claim 1 or 2 in the preparation of a medicament for improving vascular endothelial cell senescence or promoting angiogenesis.

9. The application as described in claim 8, characterized in that: The drug is an injectable preparation; And / or, the drug is used to treat or prevent ischemic tissue injury, peripheral artery disease, or impaired wound healing.

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