Stem cell nano-vesicle capable of being precisely controlled in quality and application of stem cell nano-vesicle

Abstract

The invention relates to a stem cell bioactive nano-vesicle with efficient inflammation regulation and tissue repair effects, the average particle size of the nano-vesicle is 100-350nm, the polydispersity index (PDI) is 0.05-0.40, and the expression rate of phosphatidylserine (PS) on the surface of the nano-vesicle is greater than 40%. According to the preparation method, stem cells are subjected to apoptosis induction, and then a programmed serial extrusion process is adopted to prepare the nano-vesicles. The nano vesicles are moderate in particle size, uniform in distribution and high in production efficiency, the preparation process is easy to operate, the quality is stable and controllable, and the nano vesicles have excellent technical effects in the aspects of repair and regeneration of inflammation injury type tissues and organs.

Description

Technical Field

[0001] The present invention belongs to the field of stem cells, and in particular relates to a stem cell bioactive nanovesicle for treating inflammation. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell derived from the mesodermal matrix of tissues, with multidirectional differentiation and self-renewal potential. They have typical trilineage differentiation potential. Studies in recent years have confirmed that umbilical cord-derived MSCs have the effects of immunomodulation, inflammation suppression, promotion of angiogenesis and reconstruction, and no rejection reaction in allogeneic transplantation. Therefore, they are widely used in preclinical or clinical research on various diseases. However, as a cell therapy method, MSCs have large particle size and certain immunogenicity after subcutaneous or intravenous transplantation, making it difficult to exert excellent homing and therapeutic effects. In addition, the storage and transportation process of MSCs preparations is relatively complicated, and the activity of stem cells decreases significantly from cell culture to clinical application. More importantly, stem cells have a potential risk of tumorigenesis when used in vivo, which greatly limits the development and application of stem cell products.

[0003] In recent years, with the in-depth study of molecular biology and cell therapy, MSC-derived extracellular vesicles (EVs) have garnered widespread attention due to their multiple therapeutic advantages. MSC-EVs are membrane-bound vesicles spontaneously secreted by MSCs. Based on their size and release pattern, they can be broadly divided into three subtypes: exosomes (30-150 nm), microvesicles (100-1000 nm), and apoptotic bodies (1-5 μm). MSC-EVs lack the ability to self-replicate, fundamentally avoiding the tumorigenic risks associated with direct MSC transplantation. Furthermore, MSC-EVs can be stored at -80°C for long-term storage or at 4°C for short-term storage. They can also be freeze-dried for long-term storage at room temperature, significantly reducing the complexity of their clinical application. Furthermore, EVs can effectively address the potential immunogenicity concerns and related ethical concerns associated with MSCs. These advantages make MSC-EVs a highly attractive therapeutic option in the fields of regenerative medicine and cell therapy, offering significant research and application prospects.

[0004] Exosomes (Exo) are currently the most popular type of EV. With diameters ranging from 30 to 150 nm, they appear as round, cup-shaped phospholipid vesicles under an electron microscope. As carriers of paracrine stem cell signals, Exo can deliver proteins, mRNAs, microRNAs, and other signaling molecules to target cells. By modulating diverse intracellular signaling pathways, they participate in processes such as inflammation regulation, cell proliferation and migration, angiogenesis, ECM remodeling, and scar inhibition, thus showing great potential in the treatment of various diseases. Although stem cell exosomes show potential for repairing various types of injury, their clinical application still faces several key technical challenges. First, exosome extraction methods are complex. Common techniques such as ultracentrifugation, ultrafiltration, and immunoaffinity separation are not only cumbersome but also costly. Furthermore, the extraction efficiency of exosomes is generally low, making it difficult to obtain large quantities of exosomes to meet clinical needs. More importantly, stem cell exosomes, in their natural state, often lack specific targeting to inflamed tissues or damaged cells, which to some extent limits their ability to repair and regenerate tissues and organs.

[0005] Apoptotic bodies (ABs) are also EVs that have received widespread attention in recent years. They are special EVs produced during cell apoptosis, with a particle size of more than 1 μm. They contain a rich variety of biological molecules, such as DNA, RNA, proteins, and lipids. They have shown potential roles in mediating intercellular communication, signal transduction, tissue development and regeneration, and disease occurrence and treatment. In addition, the surface of ABs has typical outward-facing phosphatidylserine (PS) molecules, which can induce the rapid recruitment and efficient phagocytosis of macrophages, thereby exerting a strong inflammatory targeting and regulatory effect. Studies have shown that apoptotic bodies have great application value in damage-repair diseases. Although ABs have powerful tissue repair and inflammation regulation capabilities, the current preparation method of ABs is to induce apoptosis in stem cells, and the spontaneously produced ABs have extremely uneven particle size distribution and a large particle size distribution span (1-5μm). These factors, on the one hand, make the preparation process difficult to control, and there are large differences between production batches, making it difficult to establish unified quality control and dosing standards, and unable to achieve unified standard industrial production and transformation application; on the other hand, due to the large particle size distribution of ABs, they are very likely to cause fatal pulmonary thromboembolism after intravenous injection, and their ability to penetrate tissues and organs when used topically is also limited by their large particle size.

[0006] It can be seen that there is an urgent need to develop a stem cell bioactive nanovesicle with a simple preparation process, controllable quality and stronger repair and targeting functions, so as to solve the problem of high difficulty in clinical transformation of stem cell vesicle preparations. Summary of the Invention

[0007] In view of the above-mentioned technical defects, namely, the complex and cumbersome exosome extraction scheme, low extraction efficiency, poor specific targeting effect; the large particle size of apoptotic bodies, extremely uneven distribution and difficulty in automatic control of the preparation process, the purpose of the present invention is to provide a stem cell bioactive nanovesicle (MSC-NVs) that can be programmed, easy to industrialize and have stronger repair and inflammation targeting functions, as well as a preparation method and application thereof.

[0008] To solve the above technical problems, the first aspect of the present invention provides a stem cell bioactive nanovesicle with efficient inflammation regulation and tissue repair effects, wherein the average particle size of the nanovesicle is 100-350nm, the polydispersity index (PDI) is 0.05-0.40, and the phosphatidylserine (PS) expression rate on the surface of the nanovesicle is greater than 40%.

[0009] Preferably, the average particle size of the nanovesicles is 150-300 nm. More preferably, the average particle size of the nanovesicles is 150-250 nm. Further preferably, the average particle size of the nanovesicles is 150-200 nm.

[0010] Preferably, the polydispersity index (PDI) of the nanovesicles is 0.05-0.30. More preferably, the polydispersity index (PDI) of the nanovesicles is 0.05-0.25. Further preferably, the polydispersity index (PDI) of the nanovesicles is 0.05-0.20.

[0011] Preferably, the expression rate of phosphatidylserine (PS) on the surface of the nanovesicles is greater than 45%. More preferably, the expression rate of phosphatidylserine (PS) on the surface of the nanovesicles is greater than 50%. Further preferably, the expression rate of phosphatidylserine (PS) on the surface of the nanovesicles is greater than 55%.

[0012] Preferably, the nanovesicles can be terminally sterilized by passing through a 0.22 μm filter membrane.

[0013] Preferably, the nanovesicles are quasi-circular in shape and have a cell membrane-like structure.

[0014] Preferably, the nanovesicles have more powerful immune regulation and tissue repair capabilities than stem cell exosomes and stem cell apoptotic bodies.

[0015] Preferably, the nanovesicles are prepared by first inducing apoptosis of stem cells and then adopting a programmed serial extrusion process.

[0016] Preferably, the apoptosis induction method is chemical induction, physical induction or gene regulation induction.

[0017] Preferably, the apoptosis induction method is hydrogen peroxide induction, staurosporine induction or ultraviolet irradiation induction. More preferably, the apoptosis induction method is 2mM hydrogen peroxide induction for 3 hours, or 2μM staurosporine induction for 12 hours, or irradiation intensity of 100mJ / cm 2 UV irradiation was induced for 20 min.

[0018] Preferably, the programmed serial extrusion process is as follows: under a pressure of 0.1-3.0 MPa, a large-pore porous membrane with a pore size between 1 μm and 10 μm and a small-pore porous membrane with a pore size between 0.1 μm and 0.7 μm are sequentially passed through using a serial extrusion device. More preferably, the programmed serial extrusion process is as follows: under a pressure of 0.5-2.0 MPa, a large-pore porous membrane with a pore size between 2 μm and 5 μm and a small-pore porous membrane with a pore size between 0.2 μm and 0.5 μm are sequentially passed through using a serial extrusion device. Further preferably, the programmed serial extrusion process is as follows: under a pressure of 0.5-1.0 MPa, a large-pore porous membrane with a pore size of 5 μm and a small-pore porous membrane with a pore size of 0.4 μm are sequentially passed through using a serial extrusion device. Most preferably, the programmed serial extrusion process is as follows: under a pressure of 0.5 MPa, a serial extrusion device is used to sequentially pass through a large-pore porous membrane with a pore size of 5 μm, a medium-pore porous membrane with a pore size of 1 μm, and a small-pore porous membrane with a pore size of 0.4 μm.

[0019] A second aspect of the present invention provides a method for preparing the above-mentioned nanovesicles, the preparation method comprising the following steps:

[0020] (1) Cultivating stem cells in a culture medium, and inducing apoptosis when the cells reach a confluence of 70-90%;

[0021] (2) continuing to culture for a period of time, collecting apoptotic cells, and using a programmed serial extrusion process to reorganize the cell membranes of apoptotic cells to form nanovesicles;

[0022] (3) The cell debris is removed by centrifugation, and the resulting supernatant is a suspension containing nanovesicles.

[0023] Preferably, the stem cells in step (1) include but are not limited to unmodified stem cells or stem cells modified by physical, chemical or biological methods.

[0024] Preferably, the types of stem cells in step (1) include, but are not limited to, umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, menstrual blood-derived stem cells, umbilical cord blood-derived stem cells, induced pluripotent stem cells, epidermal stem cells, and endometrial-derived mesenchymal stem cells. More preferably, the type of stem cells is human umbilical cord-derived mesenchymal stem cells.

[0025] Preferably, the culture medium for culturing stem cells is DMEM / F12 medium supplemented with 5% human platelet lysate (HPL).

[0026] Preferably, in step (1), when the cells have grown to a confluence of 80-90%, they are induced to apoptosis. More preferably, in step (1), when the cells have grown to a confluence of 90%, they are induced to apoptosis.

[0027] Preferably, the method of inducing stem cell apoptosis in step (1) is chemical induction, physical induction or gene regulation induction.

[0028] Preferably, the chemical method for inducing stem cell apoptosis in step (1) is hydrogen peroxide induction or staurosporine induction, and the physical method is ultraviolet irradiation induction.

[0029] Preferably, the concentration of hydrogen peroxide induction is 0.2-10 mM. More preferably, the concentration of hydrogen peroxide induction is 0.5-5 mM. Further preferably, the concentration of hydrogen peroxide induction is 1-3 mM. Most preferably, the concentration of hydrogen peroxide induction is 2 mM.

[0030] Preferably, the hydrogen peroxide induction time is 0.5-5 hours. More preferably, the hydrogen peroxide induction time is 1-4 hours. Further preferably, the hydrogen peroxide induction time is 2-3 hours. Most preferably, the hydrogen peroxide induction time is 3 hours.

[0031] Preferably, the concentration of staurosporine induction is 0.1-10 μM. More preferably, the concentration of staurosporine induction is 0.2-5 μM. Further preferably, the concentration of staurosporine induction is 2-3 μM. Most preferably, the concentration of staurosporine induction is 2 μM.

[0032] Preferably, the staurosporine induction time is 4-24 hours. More preferably, the staurosporine induction time is 6-20 hours. Further preferably, the staurosporine induction time is 8-16 hours. Most preferably, the staurosporine induction time is 12 hours.

[0033] Preferably, the intensity of the ultraviolet radiation is 20-1000 mJ / cm 2 More preferably, the intensity of the ultraviolet radiation is 50-500 mJ / cm 2 More preferably, the intensity of the ultraviolet radiation is 100-200 mJ / cm 2 Most preferably, the intensity of the ultraviolet radiation is 100 mJ / cm 2 .

[0034] Preferably, the ultraviolet induction time is 5-60 minutes. More preferably, the ultraviolet induction time is 10-40 minutes. Further preferably, the ultraviolet induction time is 20-30 minutes. Most preferably, the ultraviolet induction time is 20 minutes.

[0035] Preferably, the specific steps of step (1) are as follows: the third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) are seeded in a cell culture dish and cultured in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL). When the cells grow to 90% confluence, the culture supernatant is discarded, the cells are washed three times with sterile PBS, serum-free medium is added, and the cells are exposed to ultraviolet light for 20 minutes to induce apoptosis. The irradiation intensity of the ultraviolet light is 100mJ / cm 2 .

[0036] Preferably, the specific steps of step (1) are as follows: the third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) are inoculated in a cell culture dish and cultured in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL). When the cells grow to 90% confluence, the culture supernatant is discarded, the cells are washed three times with sterile PBS, and a serum-free medium containing hydrogen peroxide is added for induction for 3 hours, wherein the hydrogen peroxide concentration is 2mM.

[0037] Preferably, the specific steps of step (1) are as follows: the third generation human umbilical cord-derived mesenchymal stem cells (hU-MSCs) are inoculated in a cell culture dish and cultured in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL). When the cells grow to 90% confluence, the culture supernatant is discarded, the cells are washed three times with sterile PBS, and a serum-free medium containing staurosporine is added for induction for 12 hours, wherein the concentration of staurosporine is 2 μM.

[0038] Preferably, the time for continuing the culture after induction in step (2) is 8-32 hours. More preferably, the time for continuing the culture after induction in step (2) is 12-32 hours. Further preferably, the time for continuing the culture after induction in step (2) is 16-32 hours. Further preferably, the time for continuing the culture after induction in step (2) is 20-32 hours. Most preferably, the time for continuing the culture after induction in step (2) is 24-32 hours.

[0039] Preferably, the programmed serial extrusion process in step (2) is as follows: apoptotic cells are collected and extruded through multiple layers of porous membranes in descending order of pore size using a serial extrusion device under a certain pressure.

[0040] Preferably, the pressure of the series extrusion in step (2) is 0.1-10.0 MPa. More preferably, the pressure of the series extrusion in step (2) is 0.5-5 MPa. Further preferably, the pressure of the series extrusion in step (2) is 0.5-2.0 MPa. Further preferably, the pressure of the series extrusion in step (2) is 0.5-1.5 MPa. Most preferably, the pressure of the series extrusion in step (2) is 0.5-1.0 MPa.

[0041] Preferably, in step (2), the tandem extrusion passes through a large-pore porous membrane and a small-pore porous membrane in sequence, wherein the pore size of the large-pore porous membrane is between 1 μm and 10 μm, and the pore size of the small-pore porous membrane is between 0.1 μm and 0.7 μm. More preferably, the pore size of the large-pore porous membrane is between 2 μm and 5 μm, and the pore size of the small-pore porous membrane is between 0.2 μm and 0.5 μm. Further preferably, the pore size of the large-pore porous membrane is 5 μm, and the pore size of the small-pore porous membrane is 0.4 μm.

[0042] Preferably, the large-pore porous membrane and the small-pore porous membrane further comprise 1-3 medium-pore porous membranes, and more preferably, the large-pore porous membrane and the small-pore porous membrane further comprise 1 medium-pore porous membrane.

[0043] Preferably, the pore size of the mesoporous membrane is between 0.5 μm and 2 μm. More preferably, the pore size of the mesoporous membrane is between 0.8 μm and 1.5 μm. Further preferably, the pore size of the mesoporous membrane is 1 μm.

[0044] Preferably, in step (2), the extrusion is performed in series through a 5 μm large-pore porous membrane, a 1 μm medium-pore porous membrane and a 0.4 μm small-pore porous membrane in sequence.

[0045] Preferably, in step (3), cell debris is removed by centrifugation at 500-2000 g for 5-20 min. More preferably, in step (3), cell debris is removed by centrifugation at 1000-2000 g for 10-20 min. Further preferably, in step (3), cell debris is removed by centrifugation at 1000 g for 10 min.

[0046] The third aspect of the present invention provides the use of the above-mentioned stem cell bioactive nanovesicles in the preparation of drugs for preventing or treating diseases, including genetic diseases, cancer, immune diseases, central nervous system diseases, inflammatory diseases, cardiovascular diseases or infectious diseases.

[0047] The fourth aspect of the present invention provides the use of the above-mentioned stem cell bioactive nanovesicles in the preparation of medical beauty products, wherein the medical beauty products have one or more functions selected from hair growth, whitening, wrinkle removal, firming, freckle removal, improvement of dark circles, and improvement of red blood streaks.

[0048] Beneficial effects of the present invention:

[0049] 1. The stem cell bioactive nanovesicles of the present invention can be stably controlled to have a particle size below 250 nm and are uniform in size. They have a typical cell membrane vesicle structure and highly express phosphatidylserine (PS) >40% on the surface, which can send out a "eat me" signal and are easily phagocytosed by macrophages, thereby inducing the polarization of M1 pro-inflammatory macrophages into M2 anti-inflammatory macrophages and releasing a large amount of repair cytokines, showing a more powerful immune regulation and repair function than exosomes.

[0050] 2. The stem cell bioactive nanovesicles of the present invention are prepared using a programmed method of "first inducing apoptosis, then serial extrusion". The apoptosis induction step not only prompts cells to highly express PS, but also the cells undergo changes in cell membrane structure, cytoskeleton depolymerization, volume reduction and internal pressure reduction during the apoptosis process, which reduces their mechanical strength, makes them softer, and enhances their plasticity, making them easier to reconstitute into nanovesicles with uniform particle size through the subsequent serial extrusion process.

[0051] 3. The preparation process of the stem cell bioactive vesicles of the present invention is simple, highly operable, and the product quality is stable and controllable. The yield is significantly better than that of common vesicles such as stem cell exosomes and stem cell apoptotic bodies, and it has good storage stability.

[0052] 4. The stem cell bioactive nanovesicles of the present invention are non-living cell preparations, which can avoid the safety risks caused by the use of exogenous cell transplantation when used in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a flow chart for the preparation of stem cell bioactive nanovesicles of the present invention;

[0054] Figure 2 This is a biological transmission electron microscope image of the stem cell bioactive nanovesicles of the present invention;

[0055] Figure 3 The results of the macrophage quantitative uptake test of stem cell exosomes (Exo), stem cell apoptotic bodies (ABs) and the stem cell bioactive nanovesicles (MSC-NVs) of the present invention;

[0056] Figure 4 The results of the macrophage uptake test of the stem cell bioactive nanovesicles with different PS expression rates of the present invention;

[0057] Figure 5 The results of the inflammation regulation test of stem cell exosomes (Exo), stem cell apoptotic bodies (ABs) and the stem cell bioactive nanovesicles (MSC-NVs) of the present invention;

[0058] Figure 6 The results of the stem cell bioactive nanovesicles promoting cell proliferation test of the present invention;

[0059] Figure 7 These are the test results for the storage stability of the stem cell bioactive nanovesicles of the present invention. DETAILED DESCRIPTION

[0060] The specific embodiments and test examples listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments and test examples described below. For those skilled in the art, any equivalent modifications and substitutions made to the embodiments and test examples described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention. The experimental materials, experimental reagents and instruments used in the embodiments and test examples of the present invention are all commercially available.

[0061] The following test examples use human umbilical cord-derived mesenchymal stem cells as the research objects to illustrate the preparation process and technical effects of the stem cell bioactive nanovesicles of the present invention. The stem cell bioactive nanovesicles prepared using other types of stem cells of the present invention or stem cells modified by chemical, physical, or biological methods all have similar technical effects as those in the following test examples.

[0062] Experimental Example 1: Effect of stem cell apoptosis on cell membrane PS expression and extrusion and reorganization to form nanovesicles

[0063] 1. Test method

[0064] The third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in a cell culture dish and cultured in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL). When the cells grew to 90% confluence, the supernatant was discarded, the cells were washed three times with sterile PBS, serum-free medium was added, and apoptosis was induced by exposure to ultraviolet light for 20 min (100 mJ / cm 2 After the induction, the cells were cultured for 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, and 32 h, respectively. Apoptotic stem cells were collected and resuspended in PBS. The cell suspensions were extruded in series using a tandem extrusion device at a pressure of 0.5 MPa and membrane pore sizes of 5 μm, 1 μm, and 0.4 μm, respectively, to reconstitute nanovesicles. The cells were centrifuged at 1000 g for 10 min to remove cell debris, and the supernatant was collected to obtain a stem cell bioactive nanovesicle suspension (see the preparation flow chart). Figure 1 ).

[0065] Stem cells not induced to apoptosis were separately extruded in series according to the above process to obtain a solution of stem cell vesicles without induced apoptosis. The size and concentration of the vesicles obtained after programmed extrusion and reconstitution of apoptotic and normal stem cells were determined using NTA (nanoparticle tracking analysis), and the protein concentration of the resulting vesicles was determined using BCA protein quantification. The vesicles were specifically stained using the ANNEXIN-V apoptosis kit, and the expression of PS in each vesicle was determined by flow cytometry.

[0066] 2. Test results

[0067] The present invention finds that apoptosis process plays a key role in whether subsequent serial extrusion process can be carried out smoothly. As shown in Table 1, stem cells (i.e. normal stem cells) without apoptosis induction are difficult to form nano-scale vesicles smoothly by serial extrusion process, and porous membrane is very easy to clog, and it is necessary to frequently replace filter membrane, and filtration speed is extremely slow, and it is impossible to achieve mass production, and the number of vesicles obtained is on the small side, and particle size is on the large side, and terminal sterilization cannot be carried out by filtration sterilization process, and safety risks are brought to the clinical application of the product. And the stem cells in apoptosis state can be relatively easily reorganized into nano-scale cell vesicles by serial extrusion process, and it is possible to achieve mass production, and process repeatability is strong, because stem cells have experienced changes in cell membrane structure, depolymerization of cytoskeleton, volume reduction and internal pressure reduction during apoptosis process, so that its mechanical strength declines, becomes softer, plasticity is enhanced, and it is easy to reshape and reorganize under external force to form nano-vesicles. The above results show that in the preparation of stem cell bioactive nano-vesicles of the present invention, apoptosis induction is the key to whether subsequent serial extrusion can be successfully carried out, and it is also one of the key steps whether nano-vesicles can be successfully prepared.

[0068] In addition, the present invention found that the subsequent culture time after the stem cell apoptosis induction treatment will also have a certain impact on its reorganization to form a vesicle structure. As shown in Table 1, after the stem cells are induced to apoptosis and cultured for 16-32 hours, nanovesicles with uniform particle size distribution can be obtained through a series extrusion process, and when the subsequent culture time for stem cell apoptosis induction is 24 hours, it has the highest apoptotic stem cell recombination efficiency, the number of nanovesicle particles formed is the largest, the protein concentration corresponding to the vesicles, and the PS positive rate are also the highest; the culture time after apoptosis is continued to be extended to 28 hours and 32 hours, and no significant changes in particle size, vesicle protein concentration, and particle number are found, and the PS positive rate no longer increases significantly. In summary, the optimal culture time determined in this experimental example is 16-32 hours.

[0069] Table 1 Characterization results of MSC-NVs parameters obtained by serial extrusion of normal and apoptotic MSCs (n=3)

[0070]

[0071]

[0072] Experimental Example 2: Screening of process parameters for serial extrusion of stem cell nanovesicles

[0073] 1. Test method

[0074] The third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in a cell culture dish and cultured in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL). When the cells grew to 90% confluence, the supernatant was discarded, the cells were washed three times with sterile PBS, serum-free medium was added, and apoptosis was induced by exposure to ultraviolet light for 20 min (100 mJ / cm 2 ), after the induction, the cells were cultured for 24 h, and the apoptotic stem cells were collected and resuspended in PBS. The cells were divided into 6 equal parts and extruded in series using a serial extrusion device at different pressures (0.1 MPa, 0.5 MPa, 1.0 MPa, 1.5 MPa, 2.0 MPa, and 2.5 MPa) with membrane pore sizes of 5 μm, 1 μm, and 0.4 μm, respectively, to reconstitute nanovesicles. The cells were centrifuged at 1000 g for 10 min to remove cell debris, and the supernatant was collected to obtain a stem cell bioactive nanovesicle suspension. The protein concentration of the obtained vesicles was quantitatively detected by BCA protein, and the particle size and particle number concentration of the vesicles obtained after programmed extrusion and reconstitution of apoptotic stem cells were detected by NTA (nanoparticle tracking analysis) technology.

[0075] 2. Test results

[0076] As shown in Table 2, at an extrusion pressure of 0.1 MPa, the resulting vesicles had a particle size greater than 500 nm, exhibited extremely uneven distribution, and exhibited a slow extrusion speed. As the extrusion pressure increased, the number of vesicles formed by recombining apoptotic stem cells gradually increased. At an extrusion pressure of 0.5 MPa, the harvested vesicles had a particle size of 202 ± 14 nm, exhibited the most uniform distribution (PDI less than 0.2), and possessed the highest protein concentration and the largest number of vesicles. Further increasing the extrusion pressure to 1.0 MPa showed no significant change in the particle size of the nanovesicles formed by recombining apoptotic stem cells, but the PDI increased slightly, and the yield decreased. Further increasing the serial extrusion pressure resulted in a gradual increase in the particle size of the resulting vesicles, an increase in the variability in the particle size distribution, and a significant decrease in the yield. These experimental results indicate that when the serial extrusion pressure is too low, the apoptotic stem cells cannot be effectively extruded to form nanovesicles, while excessive extrusion pressure results in excessively large and highly uneven vesicle size, and a decrease in both protein concentration and vesicle number. This indicates that only when the pressure is within the appropriate range can the formation of nanovesicles be facilitated.

[0077] Table 2 Particle size, PDI, protein concentration, and particle number concentration of MSC-NVs prepared at different tandem extrusion pressures (n=3)

[0078]

[0079]

[0080] Experimental Example 3: Effects of different apoptosis induction methods on cell membrane PS expression and extrusion reorganization to form nanovesicles

[0081] 1. Test method

[0082] (1) Apoptosis process 1

[0083] The third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in 150 mm cell culture dishes and cultured in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL). When the cells grew to 90% confluence, the supernatant was discarded, the cells were washed three times with sterile PBS, serum-free medium was added, and apoptosis was induced by exposure to ultraviolet light for 20 min (100 mJ / cm 2 ), culture was continued for 24 h after the induction, apoptotic stem cells were collected (PS positive rate was 91.47%), and they were resuspended in PBS. The cell suspension was extruded in series in a programmed sequence at a pressure of 0.5 MPa using a series extrusion device according to membrane pore sizes of 5 μm, 1 μm, and 0.4 μm to reconstitute nanovesicles. The cells were centrifuged at 1000 g for 10 min to remove cell debris, and the supernatant was collected to obtain a stem cell bioactive nanovesicle suspension.

[0084] (2) Apoptosis process 2

[0085] The third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in 150 mm cell culture dishes and cultured in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL). When the cells grew to 90% confluence, the supernatant of the culture medium was discarded, the cells were washed three times with sterile PBS, and induced for 3 h in serum-free medium containing hydrogen peroxide (H2O2) (H2O2 concentration was 2 mM). After the induction, the cells were cultured for 24 h. The apoptotic stem cells (PS positive rate was 89.32%) were collected and resuspended in PBS. The cell suspension was extruded in a programmed sequence in series at a pressure of 0.5 MPa using a serial extrusion device according to membrane pore sizes of 5 μm, 1 μm, and 0.4 μm to reconstitute the nanovesicles. The cells were centrifuged at 1000 g for 10 min to remove cell debris, and the supernatant was collected to obtain a stem cell bioactive nanovesicle suspension.

[0086] (3) Apoptosis process 3

[0087] The third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in 150 mm cell culture dishes and cultured in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL). When the cells grew to 90% confluence, the supernatant of the culture medium was discarded, and the cells were washed three times with sterile PBS. Serum-free medium containing staurosporine (STZ) (STZ concentration was 2 μM) was added for induction for 12 h. After the induction, the cells were cultured for 24 h. The apoptotic stem cells (PS positive rate was 87.53%) were collected and resuspended in PBS. The cell suspension was extruded in a programmed sequence in series at a pressure of 0.5 MPa using a serial extrusion device according to membrane pore sizes of 5 μm, 1 μm, and 0.4 μm to reconstitute the nanovesicles. The cells were centrifuged at 1000 g for 10 min to remove cell debris, and the supernatant was collected to obtain a stem cell bioactive nanovesicle suspension.

[0088] (4) Determination of the physicochemical properties of stem cell nanovesicles obtained by different apoptosis processes

[0089] The particle size distribution of stem cell bioactive nanovesicles prepared by three apoptosis processes was detected by NTA (nanoparticle tracking analysis) technology, the protein concentration of the obtained vesicles was detected by BCA protein quantification, the vesicles were specifically stained using the ANNEXIN-V apoptosis kit, and the expression level of PS in each group of vesicles was detected by flow cytometry.

[0090] 2. Test results

[0091] As shown in Table 3, under the preferred tandem extrusion process and apoptosis state, the particle size distribution of the stem cell bioactive nanovesicles prepared by the three apoptosis processes was uniform, with an average particle size between 200-300 nm, a PDI of less than 0.20, and a PS positive rate of greater than 40% with no significant difference, indicating that the apoptosis mode does not affect the formation of nanovesicles.

[0092] Table 3 Particle size and potential characterization of MSC-NVs prepared by different apoptosis induction methods (n=3)

[0093]

[0094] Experimental Example 4: Structural morphology characterization of stem cell bioactive nanovesicles

[0095] Use a dropper to gently pipette 1-2 drops of the stem cell bioactive nanovesicle suspension prepared in the apoptosis process 1 of Experimental Example 3, drop it onto the copper mesh, let it stand at room temperature for 10 minutes to allow it to settle, then rinse it three times with deionized water, use filter paper to absorb the excess floating liquid, add uranyl acetate staining solution for negative staining for 30-40 seconds, rinse it again with deionized water three times, and dry it naturally at room temperature. After drying, place the copper mesh under a 120kV transmission electron microscope to observe the morphology and structure of the stem cell bioactive nanovesicles and take pictures.

[0096] 2. Test results

[0097] The TEM image of the stem cell bioactive nanovesicles in this experimental example is as follows Figure 2 As shown, these vesicle-like particles exhibit a typical spherical, cup-shaped morphology with clear boundaries and a membrane structure.

[0098] Experimental Example 5: Comparison of the extraction efficiency of stem cell nanovesicles, stem cell exosomes, and stem cell apoptotic bodies of the present invention

[0099] 1. Test method

[0100] (1) Stem cell nanovesicles

[0101] By the method described in the apoptosis process 1 of Experimental Example 3, 5×10 6 After inducing apoptosis of individual stem cells, stem cell nanovesicles were prepared by serial extrusion.

[0102] (2) Preparation of stem cell exosomes

[0103] The main methods for preparing exosomes include ultracentrifugation, density gradient centrifugation, ultrafiltration, precipitation reagent method, immunoaffinity capture method and microfluidics technology. Ultracentrifugation is the most commonly used basic method. Therefore, the present invention uses ultracentrifugation to extract and prepare stem cell exosomes as a control. The specific method is as follows: 5×10 6The culture supernatant of each stem cell was centrifuged at 300g and 2000g for 10 minutes to remove dead cells. The supernatant was taken and centrifuged at 10000g for 30 minutes to remove large vesicles. The treated cell supernatant was collected and passed through a 0.22μm filter membrane. The treated cell supernatant was transferred to an ultracentrifuge tube and centrifuged at 100000g for 70 minutes at 4°C. The supernatant was removed and the exosome pellet was resuspended with an appropriate amount of PBS buffer.

[0104] (3) Preparation of stem cell apoptotic bodies

[0105] Differential centrifugation was used to extract and prepare apoptotic bodies of stem cells as a control. The specific method was as follows: 5×10 6 After inducing apoptosis of each stem cell, centrifuge at 300g for 10 min, take the supernatant and centrifuge at 12000g for 30 min at 4°C, remove the supernatant, and resuspend the exosome pellet with an appropriate amount of PBS buffer.

[0106] (4) Determination of extraction efficiency of stem cell nanovesicles, exosomes, and apoptotic bodies

[0107] The same number of the above (5×10 6 The concentrations of stem cell exosomes, stem cell apoptotic bodies and stem cell bioactive nanovesicles extracted from individual (100) stem cells were detected to detect the extraction efficiency of stem cell bioactive nanovesicles; the particle size and particle number concentration of stem cell exosomes, stem cell apoptotic bodies and stem cell bioactive nanovesicles were detected by NTA (nanoparticle tracking analysis) technology; the stem cell exosomes, stem cell apoptotic bodies and stem cell bioactive nanovesicles were specifically stained using the ANNEXIN-V apoptosis kit, and the PS expression level of each group of samples was detected by flow cytometry.

[0108] 2. Test results

[0109] As shown in Table 4, the extraction efficiency of the stem cell bioactive nanovesicles prepared in the present invention was significantly superior to that of both stem cell exosomes and stem cell apoptotic bodies, with a yield approximately 15 times that of exosomes and over 6 times that of apoptotic bodies. Furthermore, the stem cell bioactive nanovesicles prepared in the present invention exhibited a PS-positive rate exceeding 50%, while stem cell exosomes showed no PS expression. These experimental results demonstrate that the stem cell bioactive nanovesicles prepared in the present invention exhibited a much higher extraction efficiency than exosomes and apoptotic bodies, demonstrating their potential for industrialization. Furthermore, the high PS expression rate of the stem cell bioactive nanovesicles demonstrates their ability to specifically target inflammation.

[0110] Table 4 Comparison of extraction efficiency of stem cell exosomes, apoptotic bodies, and stem cell nanovesicles (n=3)

[0111]

[0112] Experimental Example 6: Macrophage Uptake of Stem Cell Bioactive Nanovesicles

[0113] Take the stem cell exosomes (Exo), stem cell apoptotic bodies (ABs) and stem cell bioactive nanovesicles (MSC-NVs) prepared in Experimental Example 5, and use the cell membrane green fluorescent probe DiO (concentration of 10 μg / mL) to co-incubate with the above three cell vesicles at 37 ° C in the dark for 30 minutes. After the incubation is completed, centrifuge at 40000g for 40 minutes at 4 ° C, discard the supernatant, add pre-cooled PBS to wash to remove excess dye, continue to centrifuge at 4 ° C for 40 minutes, collect the precipitate, and use a certain amount of PBS to resuspend the corresponding vesicle precipitate to obtain three DiO-labeled vesicles. The protein content is determined by BCA kit to determine its concentration. Take mouse mononuclear macrophages (RAW264.7) cells that are in logarithmic growth phase and in good condition, and use 1×10 5 Cells were seeded at a density of 100 μg / well in a 12-well cell culture plate. After the cells adhered, the culture medium was discarded and 1 mL of serum-free culture medium containing stem cell exosomes, stem cell apoptotic bodies, and stem cell bioactive nanovesicles (at a concentration of 40 μg / mL) was added to each well. The cells were incubated at 37°C for 4 hours. After incubation, the cells were collected from each group, and the uptake efficiency of stem cell exosomes, stem cell apoptotic bodies, and stem cell bioactive nanovesicles was quantitatively determined by flow cytometry.

[0114] 2. Test results

[0115] The effective uptake of MSC-NVs by macrophages is crucial for their biological function. To investigate the uptake of MSC-NVs by RAW264.7 macrophages, this study used the cell membrane green fluorescent probe DiO to stain exosomes, apoptotic bodies, and MSC-NVs for tracing. Flow cytometry was then used to measure the fluorescence distribution of DiO within macrophages after 4 hours of incubation.

[0116] The test results show that (see Figure 3 ), the macrophage uptake efficiency of bioactive nanovesicles (MSC-NVs) was much higher than that of exosomes (Exo), and comparable to that of apoptotic bodies (ABs), indicating that smaller and more uniform bioactive nanovesicles have natural advantages in intracellular delivery and tissue penetration, enabling them to maintain macrophage phagocytic properties comparable to those of apoptotic bodies at relatively low PS expression levels.

[0117] Experimental Example 7: Effects of different PS expression rates on macrophage uptake of stem cell bioactive nanovesicles

[0118] 1. Test method

[0119] Take the stem cell vesicles prepared by extrusion of uninduced apoptotic stem cells and the stem cell bioactive nanovesicles with different PS expression rates prepared in Experimental Example 1 (i.e., continue to culture for 8h, 12h, 16h, 20h, 24h, 28h, 32h after induction, and prepare the nanovesicles by the serial extrusion process of the present invention), respectively recorded as samples 1-8, and use the cell membrane green fluorescent probe DiO (concentration of 10μg / mL) to co-incubate with the above vesicles at 37°C in the dark for 30min. After the incubation is completed, centrifuge at 40000g for 40min at 4°C, discard the supernatant, add pre-cooled PBS to wash to remove excess dye, continue to centrifuge at 4°C for 40min to collect the precipitate, use a certain amount of PBS to resuspend the corresponding vesicle precipitate to obtain DiO-labeled vesicles, and determine its concentration by protein content determination using the BCA kit. Take mouse mononuclear macrophages (RAW264.7) cells in the logarithmic growth phase and in good condition, and use 1×10 5 Cells were seeded at a density of 100 μg / well in a 12-well cell culture plate. After the cells attached, the culture medium was discarded and 1 mL of serum-free culture medium containing the aforementioned stem cell bioactive nanovesicles expressing different PS (at a concentration of 40 μg / mL) was added to each well. The cells were incubated at 37°C for 4 hours. After incubation, the cells were collected from each group, and the uptake efficiency of the stem cell bioactive nanovesicles expressing different PS was quantitatively determined by flow cytometry.

[0120] 2. Test results

[0121] The test results show that (see Figure 4 ), the nanovesicles prepared by serial extrusion of stem cells without inducing apoptosis (i.e., nanovesicles with a PS expression rate of 0.4%) had a poor uptake efficiency of only 31%. As the PS positivity rate increased, the macrophage uptake rate of the stem cell bioactive nanovesicles prepared by the present invention also increased, up to a maximum of more than 80%. This shows that the effect of the stem cell bioactive nanovesicles of the present invention is highly correlated with its PS expression. When the PS positivity rate is greater than 50%, the stem cell bioactive nanovesicles have the strongest macrophage uptake efficiency. This result further confirms that the apoptosis induction process induces an increase in vesicle PS expression, which can significantly enhance the ability of nanovesicles to be taken up by target cells. It can be seen that the apoptosis induction step before the programmed serial extrusion process is not only a key link in the vesicle preparation process, but also an indispensable key process in enhancing the biological function of the vesicles.

[0122] Experimental Example 8: Inflammation-regulating effect of stem cell bioactive nanovesicles

[0123] 1. Test method

[0124] Take RAW264.7 cells in the logarithmic growth phase and in good condition, blow down the semi-adherent RAW264.7 cells directly with pre-cooled PBS, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the bottom cells with 1 mL of culture medium and count them, and use 1×10 5 The cells were seeded in a 12-well cell culture plate at a density of 1 μg / mL per well. After the cells adhered to the wall, the culture medium was discarded, and a serum-free DMEM high-glucose medium with a lipopolysaccharide (LPS) concentration of 1 μg / mL was added to each well. The culture was continued for 24 hours to polarize the macrophages to an M1 phenotype. The culture medium containing LPS was aspirated and replaced with a fresh complete culture medium. The stem cell exosomes Exo, stem cell apoptotic bodies ABs and stem cell nanovesicles MSC-NVs prepared in Test Example 5 were added for administration at a concentration of 40 μg / mL and incubated at 37°C for 24 hours. After the incubation, the culture medium was collected and centrifuged at 1500 rpm for 10 minutes to remove the cell precipitate. The contents of cytokines such as IL-4, IL-10 and VEGF in the supernatant of the macrophages were determined by enzyme-linked immunosorbent assay (ELISA) kits.

[0125] 2. Test results

[0126] Macrophages of different phenotypes can secrete different types of cytokines. M1 macrophages primarily secrete pro-inflammatory cytokines, promoting inflammatory responses; whereas M2 macrophages can secrete anti-inflammatory cytokines and reparative growth factors, such as IL-4, IL-10, and VEGF, which promote cell proliferation, improve immune tolerance, and thus promote the repair of tissue damage. To further confirm the effective conversion and maintenance of macrophage phenotype after MSC-NVs treatment, this study used an enzyme-linked immunosorbent assay (ELISA) to detect the levels of anti-inflammatory cytokines and growth factors (IL-4, IL-10, and VEGF) in the macrophage culture supernatant.

[0127] The test results show that (see Figure 5 ), LPS was used to induce macrophages into the inflammatory M1 phenotype. After co-culture with Exo, ABs, and MSC-NVs for 24 hours, compared with Exo and ABs, M1 macrophages co-incubated with MSC-NVs for 24 hours could effectively promote the secretion of anti-inflammatory cytokines and growth factors, confirming that MSC-NVs have the best inflammation regulation ability, which may be related to their smaller particle size and higher PS expression.

[0128] Experimental Example 9: Effect of Stem Cell Bioactive Nanovesicles on Promoting Cell Proliferation

[0129] 1. Test method

[0130] HUVEC cells in the logarithmic growth phase and in good condition were taken, and the adherent cells were digested with 0.25% trypsin for 5 minutes, and complete culture medium was added to stop the digestion. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells at the bottom were resuspended with 1 mL of culture medium and counted. 2×10 3 The cells were seeded in a 96-well cell culture plate at a density of 100 μL / well. After the cells adhered to the wall, the culture medium was discarded, and the stem cell exosomes Exo, stem cell apoptotic bodies ABs and stem cell nanovesicles MSC-NVs prepared in Test Example 5 were added for administration, all at a concentration of 40 μg / mL, and placed in a 37 ° C incubator for continued culture. At 0 h, 12 h, 24 h and 48 h after the addition of the preparation, the culture medium was removed, and 100 μL of fresh culture medium containing 10 μL CCK-8 reagent was gently added to each well to avoid the generation of bubbles. The culture was continued at 37 ° C for 4 h, and the absorbance (OD) of each well sample at 450 nm was measured using a multifunctional microplate reader.

[0131] 2. Test results

[0132] In this experiment, stem cell exosomes Exo and stem cell apoptotic bodies ABs were used as positive controls. HUVEC cells were co-incubated with equal protein concentrations of Exo, ABs, MSC-NVs or equal volumes of PBS. At different time points, CCK-8 reagent was used to investigate the proliferation-promoting effects of ABs and MSC-NVs on HUVEC cells.

[0133] The test results show that (see Figure 6 ), compared with the control group, the Exo group and the ABs group could promote HUVEC cell proliferation to a certain extent, and there was no significant difference between the two groups, but the cell proliferation-promoting ability of MSC-NVs was significantly better than those of the two groups, indicating that MSC-NVs had excellent cell proliferation-promoting effect, which may be related to its smaller particle size and higher PS expression.

[0134] Experimental Example 10: Stability Study of Stem Cell Bioactive Nanovesicles

[0135] 1. Test method

[0136] The stem cell exosomes Exo, stem cell apoptotic bodies ABs and stem cell bioactive nanovesicles MSC-NVs with the same protein concentration prepared in Experimental Example 5 were stored at -20°C for one month, and the storage stability of each sample was investigated by detecting the change in particle size.

[0137] 2. Test results

[0138] In this experiment, stem cell exosomes Exo and stem cell apoptotic bodies ABs were used as controls. Exo, ABs, and MSC-NVs samples with equal protein concentrations were placed at -20°C. The storage stability of each group of samples was investigated by continuously observing the changes in particle size. Figure 7 ), MSC-NVs and Exo showed similar stability, with almost no significant change in particle size after storage at -20°C for 28 days, while the particle size of stem cell apoptotic bodies increased significantly with increasing storage time, indicating that the MSC-NVs prepared by the present invention not only possess some of the advantages of apoptotic bodies but also have storage stability comparable to that of stem cell exosomes, showing great potential for industrial development and clinical application.

[0139] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A stem cell bioactive nanovesicle with inflammation regulation and tissue repair effects, wherein the nanovesicles have an average particle size of 100-350 nm, a polydispersity coefficient of 0.05-0.40, and a phosphatidylserine expression rate on the surface of the nanovesicles greater than 40%. The preparation method of the stem cell bioactive nanovesicles comprises the following steps: (1) Cultivating stem cells in a culture medium, and when the cells reach a confluence of 70-90%, inducing them to apoptosis so that the PS expression rate is greater than 85%; (2) continuing to culture for a period of time, collecting apoptotic cells, and using a programmed serial extrusion process to reorganize the cell membranes of apoptotic cells to form nanovesicles; (3) Cell debris is removed by centrifugation, and the resulting supernatant is a suspension containing nanovesicles.

2. The nanovesicle according to claim 1, characterized in that The type of stem cells in step (1) is selected from umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, menstrual blood-derived stem cells, umbilical cord blood-derived stem cells, induced pluripotent stem cells, epidermal stem cells, and endometrial-derived mesenchymal stem cells.

3. The nanovesicle according to claim 1, characterized in that The type of stem cells in step (1) is mesenchymal stem cells derived from human umbilical cord.

4. The nanovesicle according to claim 1, characterized in that The method of inducing stem cell apoptosis in step (1) is chemical induction, physical induction or gene regulation induction.

5. The nanovesicle according to claim 1, characterized in that The pressure of the serial extrusion in step (2) is 0.1-10.0 MPa.

6. The nanovesicle according to claim 1, characterized in that In the step (2), the series extrusion passes through a large-pore porous membrane and a small-pore porous membrane in sequence, wherein the pore size of the large-pore porous membrane is between 1 μm and 10 μm, and the pore size of the small-pore porous membrane is between 0.1 μm and 0.7 μm.

7. The nanovesicle according to claim 1, characterized in that The nanovesicles are quasi-circular in shape and have a cell membrane structure.

8. The method for preparing the nanovesicles according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Cultivating stem cells in a culture medium, and when the cells reach a confluence of 70-90%, inducing them to apoptosis so that the PS expression rate is greater than 85%; (2) continuing to culture for a period of time, collecting apoptotic cells, and using a programmed serial extrusion process to reorganize the cell membranes of apoptotic cells to form nanovesicles; (3) Cell debris is removed by centrifugation, and the resulting supernatant is a suspension containing nanovesicles.

9. The method for preparing nanovesicles according to claim 8, characterized in that: The stem cells in step (1) are selected from unmodified stem cells or stem cells modified by physical, chemical or biological methods, and the types of the stem cells are selected from umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, menstrual blood-derived stem cells, umbilical cord blood-derived stem cells, induced pluripotent stem cells, epidermal stem cells, and endometrial-derived mesenchymal stem cells.

10. The method for preparing nanovesicles according to claim 8, characterized in that: The method of inducing stem cell apoptosis in step (1) is chemical induction, physical induction or gene regulation induction.

11. The method for preparing nanovesicles according to claim 8, characterized in that: The pressure of the serial extrusion in step (2) is 0.1-10.0 MPa.

12. The method for preparing nanovesicles according to claim 8, characterized in that: In the step (2), the series extrusion passes through a large-pore porous membrane and a small-pore porous membrane in sequence, wherein the pore size of the large-pore porous membrane is between 1 μm and 10 μm, and the pore size of the small-pore porous membrane is between 0.1 μm and 0.7 μm.

13. Use of the stem cell bioactive nanovesicles according to any one of claims 1 to 7 in the preparation of a medicament for preventing or treating a disease selected from genetic diseases, cancer, immune diseases, central nervous system diseases, inflammatory diseases, cardiovascular diseases or infectious diseases.

14. Use of the stem cell bioactive nanovesicles according to any one of claims 1 to 7 in the preparation of medical cosmetic products, wherein the medical cosmetic products have one or more effects selected from the group consisting of hair growth, whitening, wrinkle removal, firming, freckle removal, improvement of dark circles, and improvement of red blood streaks.

Images