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 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 adult stem cells derived from the mesoderm of tissues, with multidirectional differentiation and self-renewal potentials. They have typical trilineage differentiation potentials. 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 of allogeneic transplantation. Therefore, they are widely used in preclinical or clinical studies of various diseases. However, as a cell therapy, MSCs have large particle size and certain immunogenicity after subcutaneous or intravenous transplantation, so it is 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 tumorigenic risk 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, MSCs-derived extracellular vesicles (EVs) have attracted widespread attention due to their multiple therapeutic advantages. MSCs-EVs are membrane-structured vesicles spontaneously secreted by MSCs. According to their size and release mode, they can be roughly divided into three subtypes: exosomes (30-150nm), microvesicles (100-1000nm) and apoptotic bodies (1-5μm). MSCs-EVs themselves do not have the ability to replicate themselves, which fundamentally avoids the tumorigenic risk that may be caused by direct transplantation of MSCs. Secondly, MSCs-EVs can be stored for a long time at -80℃ or for a short time at 4℃, and can also be made into freeze-dried products and stored for a long time at room temperature, which greatly reduces the difficulty of its clinical application. In addition, EVs can effectively solve the immunogenicity problems that MSCs may cause and the related ethical disputes. The above advantages make MSCs-EVs a more attractive treatment option in the field of regenerative medicine and cell therapy, with extremely high research and application prospects.

[0004] Exosomes (Exo) are the most popular type of EVs at present. Their diameter ranges from 30 to 150 nm. They are round cup-shaped phospholipid vesicles under electron microscope. As carriers of stem cell paracrine signals, Exo can deliver proteins, mRNAs, microRNAs and other signal molecules to target cells, and participate in inflammation regulation, cell proliferation and migration, angiogenesis, ECM remodeling and inhibition of scar formation by regulating different intracellular signaling pathways, thus showing great potential in the treatment of various diseases. Although stem cell exosomes show potential application prospects in the repair of various types of injury diseases, they still face several key technical challenges in clinical application. First, the extraction method of exosomes is relatively complicated. Common techniques such as ultracentrifugation, ultrafiltration and immunoaffinity separation are not only cumbersome to operate, but also costly. In addition, the extraction efficiency of exosomes is generally low, and it is difficult to obtain a large number of exosomes that meet clinical needs. More importantly, stem cell exosomes usually lack targeting to specific inflammatory tissues or damaged cells in their natural state, which also limits their tissue and organ repair and regeneration ability to a certain extent.

[0005] Apoptotic bodies (ABs) are also EVs that have received extensive attention in recent years. They are special EVs produced during cell apoptosis, with a particle size of more than 1 μm. They contain a wealth of biological molecules, such as DNA, RNA, proteins, and lipids. They show 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 everted phosphatidylserine (PS) molecules, which send out "eat me" signals and can induce 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 method for preparing ABs is to induce apoptosis of stem cells, so the spontaneously generated ABs have extremely uneven particle size distribution and a large particle size distribution span (1-5μm). These factors, on the one hand, make it difficult to control the preparation process, and there are large differences between production batches, making it difficult to establish unified quality control and dosing standards, and it is impossible to achieve unified standard industrial production and transformation applications; 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, large apoptotic body particle size, extremely uneven distribution and difficult to control the preparation process, the object 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, the average particle size of the nanovesicle is 100-350nm, the polydispersity index (PDI) is 0.05-0.40, and the expression rate of phosphatidylserine (PS) 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 nanovesicle is greater than 45%. More preferably, the expression rate of phosphatidylserine (PS) on the surface of the nanovesicle is greater than 50%. Further preferably, the expression rate of phosphatidylserine (PS) on the surface of the nanovesicle 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 method 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 2 mM hydrogen peroxide induction for 3 hours, or 2 μM staurosporine induction for 12 hours, or irradiation intensity of 100 mJ / cm 2 The cells were induced by ultraviolet irradiation for 20 min.

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

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

[0020] (1) culturing stem cells using a culture medium, and inducing the cells to enter a state of apoptosis when the cell confluence reaches 70-90%;

[0021] (2) continuing to culture for a period of time, collecting apoptotic cells, and using a programmed tandem 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 medium for culturing stem cells is DMEM / F12 medium supplemented with 5% human platelet lysate (HPL).

[0026] Preferably, in step (1), when the cell confluence reaches 80-90%, the cell is induced to apoptosis. More preferably, in step (1), when the cell confluence reaches 90%, the cell is 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 induced radiation is 20-1000 mJ / cm 2 More preferably, the intensity of the ultraviolet induced radiation is 50-500 mJ / cm 2 Preferably, the intensity of the ultraviolet induced radiation is 100-200 mJ / cm 2 Most preferably, the intensity of the ultraviolet light is 100 mJ / cm 2 .

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

[0035] Preferably, the specific steps of step (1) are as follows: inoculating the third generation human umbilical cord-derived mesenchymal stem cells (hU-MSCs) in a cell culture dish and culturing them in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL), discarding the culture supernatant when the cells grow to 90% confluence, washing with sterile PBS for 3 times, adding serum-free culture medium, and exposing to ultraviolet light for 20 minutes to induce apoptosis, wherein the irradiation intensity of the ultraviolet light is 100mJ / cm 2 .

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

[0037] Preferably, the specific steps of step (1) are as follows: inoculating the third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) in a cell culture dish and culturing them in DMEM / F12 medium supplemented with 5% human platelet lysate (HPL), discarding the culture supernatant when the cells grow to 90% confluence, washing with sterile PBS for 3 times, and adding serum-free medium containing staurosporine for induction for 12 hours, wherein the concentration of staurosporine is 2 μM.

[0038] Preferably, the time for continuing to culture after induction in step (2) is 8-32 hours. More preferably, the time for continuing to culture after induction in step (2) is 12-32 hours. Further preferably, the time for continuing to culture after induction in step (2) is 16-32 hours. Further preferably, the time for continuing to culture after induction in step (2) is 20-32 hours. Most preferably, the time for continuing to 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 passed 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 sequentially passes through a large-pore porous membrane and a small-pore porous membrane, 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 contain 1 to 3 medium-pore porous membranes. More preferably, the large-pore porous membrane and the small-pore porous membrane further contain 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 sequentially carried out through a 5 μm large-pore porous membrane, a 1 μm medium-pore porous membrane and a 0.4 μm small-pore porous membrane.

[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 the group consisting of 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 particle size of the stem cell bioactive nanovesicles of the present invention can be stably controlled below 250nm, and the size is uniform. It has a typical cell membrane vesicle structure, and highly expresses phosphatidylserine (PS)>40% on the surface, which can send out a "eat me" signal and is 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 adopt a programmed preparation method of "first inducing apoptosis, then serial extrusion". The apoptosis induction step not only prompts the cells to highly express PS, but also the cells undergo changes in cell membrane structure, depolymerization of the cytoskeleton, volume reduction and reduced internal pressure during the apoptosis process, which reduces their mechanical strength, makes them softer, and enhances their plasticity, making them easier to reorganize into nanovesicles with uniform particle size through a subsequent serial extrusion process.

[0051] 3. The preparation process of the stem cell bioactive vesicles of the present invention is simple, the operability is strong, the product quality is stable and controllable, the yield is significantly better than 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 potential safety hazards caused by exogenous cell transplantation when used in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The flowchart of 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 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 inflammatory 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 test on promoting cell proliferation by the stem cell bioactive nanovesicles 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 to the embodiments and test examples described below are also within the scope of the present invention. Therefore, the equalization transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. The experimental materials, experimental reagents and instruments used in the embodiments and test examples of the present invention can be obtained commercially.

[0061] The following test examples use human umbilical cord-derived mesenchymal stem cells as the research object 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 to 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 methods

[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 of the culture medium 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, and apoptotic stem cells were collected and resuspended in PBS. The cell suspension was extruded in series using a tandem extrusion device at a pressure of 0.5 MPa and a membrane pore size of 5 μm, 1 μm, and 0.4 μm, respectively, 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 (see the preparation flow chart). Figure 1 ).

[0065] Stem cells that were not induced to undergo apoptosis were separately taken and extruded in series according to the above process to obtain a stem cell vesicle solution that was not induced to undergo apoptosis. The vesicle size and vesicle concentration obtained after programmed extrusion and reorganization of apoptotic stem cells and normal stem cells were detected by NTA (nanoparticle tracking analysis) technology, and 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 of PS in each group of vesicles was detected by flow cytometry.

[0066] 2. Test results

[0067] The present invention finds that apoptosis process plays a key role in whether the subsequent series extrusion process can proceed smoothly. As shown in Table 1, stem cells (i.e. normal stem cells) that have not undergone apoptosis induction are difficult to form nano-scale vesicles smoothly through the series extrusion process, and the porous membrane is very easy to clog, and the filter membrane needs to be frequently replaced, and the filtration speed is extremely slow, and it is impossible to achieve mass production, and the number of vesicles obtained is relatively small, and the particle size is relatively large, and terminal sterilization cannot be performed by the filtration sterilization process, which brings safety risks to the clinical application of the product. And the stem cells in the state of apoptosis can be relatively easily reorganized into nano-scale cell vesicles through the series extrusion process, which can achieve mass production, and the process repeatability is strong, because the stem cells experience changes in cell membrane structure, depolymerization of the cytoskeleton, volume reduction and internal pressure reduction during the apoptosis process, so that its mechanical strength decreases, becomes softer, and plasticity is enhanced, and it is easy to reshape and reorganize under the action of external forces to form nano-vesicles. The above results show that in the preparation of the biologically active nano-vesicles of stem cells of the present invention, apoptosis induction is the key to whether the subsequent series extrusion can be successfully completed, and it is also one of the key steps for whether the nano-vesicles can be successfully prepared.

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

[0069] Table 1 Characterization results of parameters of MSC-NVs 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 methods

[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 of the culture medium 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, apoptotic stem cells were collected, resuspended in PBS, and divided into 6 equal parts. The cell suspensions were extruded in series using a series of extrusion devices 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 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, when the extrusion pressure is 0.1MPa, the obtained vesicle particle size is greater than 500nm, the distribution is extremely uneven, and the extrusion speed is slow. With the increase of extrusion pressure, the number of vesicles formed by the reorganization of apoptotic stem cells gradually increases. When the extrusion pressure is 0.5MPa, the particle size of the harvested cell vesicles is 202±14nm, the distribution is the most uniform (PDI is less than 0.2), with the highest protein concentration and the largest number of vesicles. When the extrusion pressure is further increased to 1.0MPa, the particle size of the nanovesicles formed by the reorganization of apoptotic stem cells does not change significantly, but the PDI increases slightly and the yield also decreases. After continuing to increase the series extrusion pressure, the obtained vesicle particle size gradually increases, and the difference in particle size distribution increases, and the yield is greatly reduced. The above experimental results show that when the series extrusion pressure is too small, the apoptotic stem cells cannot be effectively extruded to reorganize into nanovesicles, and excessive extrusion pressure will cause the vesicle particle size to be too large and very uneven, and the protein concentration and the number of vesicles are also reduced. It can be seen that only when the pressure is within the appropriate range can it help the formation of nanovesicles.

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

[0078]

[0079] Experimental Example 3: Effects of different apoptosis induction methods on cell membrane PS expression and extrusion reorganization to form nanovesicles

[0080] 1. Test methods

[0081] (1) Apoptosis process 1

[0082] The third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in a 150 mm 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 of the culture medium was discarded, and 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 positivity was 91.47%), resuspended in PBS, and extruded in series using a series extrusion device at a pressure of 0.5 MPa and a membrane pore size 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.

[0083] (2) Apoptosis process 2

[0084] The third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in a 150 mm 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 of the culture medium was discarded, and the cells were washed three times with sterile PBS and added with hydrogen peroxide (H 2 O 2 ) serum-free medium (H 2 O 2 The cells were induced with 5 μm, 1 μm, and 0.4 μm membrane pore sizes at a pressure of 0.5 MPa and extruded in series to form nanovesicles. The cells were centrifuged at 1000 g for 10 min to remove cell debris, and the supernatant was collected to obtain a bioactive nanovesicle suspension of stem cells.

[0085] (3) Apoptosis process 3

[0086] The third generation of human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in a 150 mm 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 of the culture medium was discarded, the cells were washed three times with sterile PBS, and a serum-free medium containing staurosporine (STZ) (STZ concentration was 2 μM) was added to induce for 12 h. After the induction, the cells were cultured for 24 h, and 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 series extrusion device according to the membrane pore size of 5 μm, 1 μm, and 0.4 μm to reorganize into 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.

[0087] (4) Determination of the physicochemical properties of stem cell nanovesicles obtained by different apoptosis processes

[0088] 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.

[0089] 2. Test results

[0090] As shown in Table 3, under the preferred tandem extrusion process and apoptosis state, the particle size distribution of stem cell bioactive nanovesicles prepared by the three apoptosis processes was uniform, with an average particle size between 200-300 nm, PDI was less than 0.20, and the PS positive rate was greater than 40% with no significant difference, indicating that the apoptosis mode did not affect the formation of nanovesicles.

[0091] Table 3 Particle size and potential characterization of MSC-NVs prepared by different apoptosis induction methods (n=3)

[0092]

[0093] Experimental Example 4: Structural morphology characterization of stem cell bioactive nanovesicles

[0094] Use a dropper to gently pipette 1-2 drops of the stem cell bioactive nanovesicle suspension prepared in apoptosis process 1 in Experimental Example 3, drop it on the copper mesh, let it stand at room temperature for 10 minutes to settle, wash it three times with deionized water, use filter paper to absorb excess floating liquid, add uranyl acetate staining solution for negative staining for 30-40 seconds, wash it again with deionized water three times, dry it naturally at room temperature, and 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.

[0095] 2. Test results

[0096] The TEM image of the stem cell bioactive nanovesicles in this test example is shown in Figure 2 As shown, these vesicle-like particles present a typical spherical, cup-shaped morphology with clear boundaries and a membrane structure.

[0097] Experimental Example 5: Comparison of the extraction efficiency of stem cell nanovesicles, stem cell exosomes, and stem cell apoptotic bodies of the present invention

[0098] 1. Test methods

[0099] (1) Stem Cell Nanovesicles

[0100] By the method described in the apoptosis process 1 in Experimental Example 3, 5×10 6 The stem cell nanovesicles were prepared by serial extrusion of individual stem cells after inducing apoptosis.

[0101] (2) Preparation of stem cell exosomes

[0102] The preparation methods of exosomes mainly 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 reference substance. The specific method is as follows: 5×10 6The culture supernatant of each stem cell was centrifuged at 300g and 2000g for 10 min to remove dead cells, and the supernatant was centrifuged at 10000g for 30 min to remove large vesicles. The treated cell supernatant was collected and filtered through a 0.22μm filter membrane. The treated cell supernatant was transferred to an ultracentrifuge tube and centrifuged at 4°C and 100000g for 70 min. The supernatant was removed and the exosome precipitate was resuspended with an appropriate amount of PBS buffer.

[0103] (3) Preparation of stem cell apoptotic bodies

[0104] 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 10min, take the supernatant and centrifuge at 12000g for 30min at 4°C, remove the supernatant, and resuspend the exosome precipitate with an appropriate amount of PBS buffer.

[0105] (4) Determination of extraction efficiency of stem cell nanovesicles, exosomes, and apoptotic bodies

[0106] 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) 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 ANNEXIN-V apoptosis kit was used to specifically stain stem cell exosomes, stem cell apoptotic bodies and stem cell bioactive nanovesicles, and the expression level of PS in each group of samples was detected by flow cytometry.

[0107] 2. Test results

[0108] The test results are shown in Table 4. The extraction efficiency of the stem cell bioactive nanovesicles in the present invention is significantly better than that of stem cell exosomes and stem cell apoptotic bodies, and its yield is about 15 times that of exosomes and more than 6 times that of apoptotic bodies. In addition, the PS positive rate of the stem cell bioactive nanovesicles prepared by the present invention is more than 50%, while the stem cell exosomes have no PS expression. The above test results show that the extraction efficiency of the stem cell bioactive nanovesicles prepared by the present invention is much higher than that of exosomes and apoptotic bodies, and has industrial prospects, and the PS expression rate of the stem cell bioactive nanovesicles is relatively high, indicating that it has inflammation-specific targeting capabilities.

[0109] Table 4 Comparison of extraction efficiency of stem cell exosomes, apoptotic bodies, and stem cell nanovesicles (n=3)

[0110]

[0111] Experimental Example 6: Macrophage Uptake of Stem Cell Bioactive Nanovesicles

[0112] 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, use a certain amount of PBS to resuspend the corresponding vesicle precipitate, and obtain three DiO-labeled vesicles. The protein content is determined by the BCA kit to determine its concentration. Take mouse mononuclear macrophages (RAW264.7) cells that are in the logarithmic growth phase and in good condition, and use 1×10 5 The cells were inoculated at a density of 100 cells / well in a 12-well cell culture plate. After the cells adhered to the wall, 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 and incubated at 37°C for 4 hours. After the incubation, the cells in each group were collected, and the uptake efficiency of stem cell exosomes, stem cell apoptotic bodies, and stem cell bioactive nanovesicles was quantitatively detected by flow cytometry.

[0113] 2. Test results

[0114] The effective uptake of stem cell bioactive nanovesicles MSC-NVs by macrophages is an important prerequisite for their biological functions. In order to investigate the uptake ability of macrophages RAW264.7 for stem cell bioactive nanovesicles, this experiment used the cell membrane green fluorescent probe DiO to stain stem cell exosomes, stem cell apoptotic bodies and stem cell bioactive nanovesicles for tracing experiments, and detected the fluorescence distribution of DiO in macrophages after incubation with macrophages for 4 hours by flow cytometry.

[0115] 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 was comparable to that of apoptotic bodies (ABs), indicating that smaller and more uniform bioactive nanovesicles have natural advantages in intracellular delivery and tissue penetration, allowing them to maintain macrophage phagocytic properties that are no less than those of apoptotic bodies at relatively low PS expression levels.

[0116] Experimental Example 7: Effects of different PS expression rates on macrophage uptake of stem cell bioactive nanovesicles

[0117] 1. Test methods

[0118] 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, 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 The cells were inoculated at a density of 100 μg / well in a 12-well cell culture plate. After the cells adhered to the wall, the culture medium was discarded, and 1 mL of serum-free culture medium containing the above-mentioned stem cell bioactive nanovesicles with different PS expressions (concentration of 40 μg / mL) was added to each well and incubated at 37°C for 4 hours. After the incubation, the cells of each group were collected, and the uptake efficiency of stem cell bioactive nanovesicles with different PS expressions was quantitatively detected by flow cytometry.

[0119] 2. Test results

[0120] 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 the expression of its PS. 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.

[0121] Experimental Example 8: Inflammatory regulation effect of stem cell bioactive nanovesicles

[0122] 1. Test methods

[0123] Take RAW264.7 cells in the logarithmic growth phase and in good condition, blow down the semi-adherent RAW264.7 with pre-cooled PBS, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the bottom cells with 1 mL of culture medium and count them, and count them as 1×10 5 The cells were inoculated in a 12-well cell culture plate at a density of 1 μg / 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 the M1 phenotype, and 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, all at a concentration of 40 μg / mL, and incubated at 37 ° C for 24 hours. After the incubation, the culture medium was collected, centrifuged at 1500 rpm for 10 minutes to remove the cell precipitate, and the contents of cytokines such as IL-4, IL-10, and VEGF in the supernatant of macrophages were determined by enzyme-linked immunosorbent assay (ELISA) kits.

[0124] 2. Test results

[0125] Macrophages of different phenotypes can secrete different types of cytokines. M1 macrophages mainly secrete pro-inflammatory cytokines to promote inflammatory responses, while M2 macrophages can secrete anti-inflammatory cytokines and repair growth factors, such as IL-4, IL-10, and VEGF, to promote cell proliferation, improve the body's immune tolerance, and thus promote the repair of tissue damage. In order to further confirm the effective conversion and maintenance of macrophage phenotypes after MSC-NVs treatment, this experimental example used an enzyme-linked immunosorbent assay (ELISA) to detect the content of anti-inflammatory cytokines and growth factors (IL-4, IL-10, and VEGF) in the supernatant of macrophage culture.

[0126] The test results show that (see Figure 5 ), LPS was used to induce macrophages into inflammatory M1 phenotype. After 24h of co-culture with Exo, ABs, and MSC-NVs, compared with Exo and ABs, M1 macrophages co-incubated with MSC-NVs for 24h 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.

[0127] Experimental Example 9: Effect of Stem Cell Bioactive Nanovesicles on Promoting Cell Proliferation

[0128] 1. Test methods

[0129] 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 min, and complete medium was added to stop digestion. The cells were centrifuged at 1000 rpm for 5 min, 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 inoculated 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 0h, 12h, 24h and 48h 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 4h, and the absorbance value (OD) of each well sample at 450nm was measured using a multifunctional microplate reader.

[0130] 2. Test results

[0131] In this experiment, stem cell exosomes Exo and stem cell apoptotic bodies ABs were used as positive controls. HUVEC cells were co-incubated with Exo, ABs, MSC-NVs or an equal volume of PBS at equal protein concentrations. At different time points, CCK-8 reagent was used to investigate the proliferation-promoting effects of ABs and MSC-NVs on HUVEC cells.

[0132] The test results show that (see Figure 6 ), compared with the control group, the Exo group and the ABs group were able to promote HUVEC cell proliferation to a certain extent, and there was no significant difference between the two groups, but the ability of MSC-NVs to promote cell proliferation was significantly better than those of the two groups, indicating that MSC-NVs had excellent cell proliferation-promoting effects, which may be related to its smaller particle size and higher PS expression.

[0133] Experimental Example 10: Stability Study of Stem Cell Bioactive Nanovesicles

[0134] 1. Test methods

[0135] 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.

[0136] 2. Test results

[0137] In this test, 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, and the storage stability of each group of samples was investigated by continuously observing the changes in the particle size of each group of samples. The test results showed (see Figure 7 ), MSC-NVs and Exo had similar stability, and there was 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 the increase of storage time, indicating that the MSC-NVs prepared by the present invention not only have some advantages of apoptotic bodies, but also have storage stability that is no less than that of stem cell exosomes, showing great potential for industrial development and clinical application.

[0138] 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 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 expression rate of phosphatidylserine (PS) on the surface of the nanovesicle is greater than 40%.

2. The nanovesicle according to claim 1, characterized in that The nanovesicle is quasi-circular in shape and has a cell membrane structure.

3. The nanovesicle according to claim 1 or 2, characterized in that The nanovesicles are prepared by first inducing apoptosis of stem cells and then adopting a programmed serial extrusion process.

4. The method for preparing the nanovesicles according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) culturing stem cells using a culture medium, and when the cell confluence reaches 70-90%, inducing the cells to enter a state of 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 tandem extrusion process to reorganize the cell membrane of the apoptotic cells to form nanovesicles; (3) The cell debris is removed by centrifugation, and the resulting supernatant is a suspension containing nanovesicles.

5. The method for preparing nanovesicles according to claim 4, characterized in that: 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, and the types of stem cells 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.

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

7. The method for preparing nanovesicles according to claim 4, characterized in that: The programmed serial extrusion process in step (2) is as follows: apoptotic cells are collected and passed through multiple layers of porous membranes in descending order of pore size using serial extrusion equipment under a certain pressure.

8. The method for preparing nanovesicles according to claim 4, 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.

9. Use of the stem cell bioactive nanovesicles according to any one of claims 1 to 3 in the preparation of a drug for preventing or treating a disease, wherein the disease comprises a genetic disease, cancer, an immune disease, a central nervous system disease, an inflammatory disease, a cardiovascular disease or an infectious disease.

10. Use of the stem cell bioactive nanovesicles according to any one of claims 1 to 3 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.

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