Engineered nano-vesicle derived from apoptosis mesenchymal stem cells, composite hydrogel as well as preparation method and application of engineered nano-vesicle

Abstract

The invention provides engineering nano vesicles derived from apoptosis mesenchymal stem cells, composite hydrogel as well as a preparation method and application, and particularly belongs to the technical field of biomedical engineering. The invention provides a preparation method of engineered nano vesicles derived from apoptosis mesenchymal stem cells, which comprises the following steps: carrying out apoptosis treatment on mesenchymal stem cells to obtain apoptotic cells; and extruding the apoptotic cells by adopting a microfiltration membrane to obtain the engineered nano-vesicles derived from the apoptotic mesenchymal stem cells. The apoptosis vesicles prepared by the preparation method disclosed by the invention have the advantages of high yield, high efficiency in preparation process, capability of realizing large-scale production and higher transformation potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering, and specifically relates to an engineered nanovesicle derived from apoptotic mesenchymal stem cells, a composite hydrogel, and a preparation method and application thereof. Background Art

[0002] Traditional wound dressings such as gauze and bandages act as physical barriers in wound healing and can provide hemostasis and protection functions, but their effects are limited to isolating wounds and reducing contamination. They cannot provide a suitable moist environment or bioactive substances that promote tissue repair, resulting in slow wound repair and inability to effectively suppress infection risks. In addition, traditional dressings lack active factors related to tissue regeneration, so they are not effective in treating chronic wounds, diabetic ulcers and other difficult-to-heal wounds.

[0003] In recent years, cell therapy has been gradually applied to regenerative medicine and wound repair, especially showing certain advantages in accelerating tissue repair and regulating inflammatory responses. However, the application of cell therapy in the treatment of chronic wounds still faces many challenges, including low cell survival rate after transplantation, difficulty in effectively integrating into damaged tissues, and potential immune rejection reactions. In addition, many cell therapies require the use of non-degradable or potentially toxic carrier materials to support transplanted cells, which not only increases the biocompatibility requirements of the materials, but may also cause side effects. In addition, the process of cell expansion, separation, and transplantation is complicated and costly, which limits its promotion in wound healing treatment.

[0004] In order to overcome the above limitations, cell-derived vesicles (such as exosomes, microvesicles, etc.) have become an alternative that has attracted much attention. These vesicles have good biocompatibility and biological activity, and can carry growth factors, anti-inflammatory factors, microRNAs and other molecules that regulate wound healing, showing broad application potential in chronic wound repair. However, the production of traditional cell-secreted vesicles is limited, and the purification and production process of vesicles is complicated, which cannot meet the needs of large-scale applications. Therefore, it is very important to explore drugs that effectively promote tissue regeneration and are easy to mass produce as a new strategy to promote chronic wound healing. Summary of the invention

[0005] The purpose of the present invention is to provide an engineered nanovesicle derived from apoptotic mesenchymal stem cells, a composite hydrogel, a preparation method and an application thereof. The apoptotic vesicle prepared by the preparation method of the present invention has abundant yield, efficient preparation process, can realize large-scale production, and has more transformation potential.

[0006] The invention provides a method for preparing an engineered nano apoptotic vesicle derived from apoptotic mesenchymal stem cells, comprising the following steps: subjecting mesenchymal stem cells to apoptosis treatment to obtain apoptotic cells; and extruding the apoptotic cells through a microporous filter membrane to obtain an engineered nano apoptotic vesicle derived from apoptotic mesenchymal stem cells.

[0007] Preferably, the apoptotic treatment comprises ultraviolet irradiation.

[0008] Preferably, the wavelength of the ultraviolet irradiation is 280-320 nm and the power is 100 μW / cm 2 ; The ultraviolet irradiation time is 15 to 60 minutes.

[0009] Preferably, after the ultraviolet irradiation, incubation is further included; the incubation conditions are: incubation in a 37° C., 5% CO 2 incubator for 4 to 6 hours.

[0010] Preferably, the mesenchymal stem cells include placental mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.

[0011] Preferably, the microporous filter membrane has pore sizes of 3 μm, 0.6 μm and 0.2 μm.

[0012] The present invention also provides engineered nanovesicles derived from apoptotic mesenchymal stem cells prepared by the preparation method described in the above technical solution.

[0013] The present invention also provides a composite hydrogel, comprising the following raw materials for preparation: the engineered nanovesicles derived from apoptotic mesenchymal stem cells described in the above technical solution and a methacrylic anhydride gelatin solution; the raw materials for preparing the methacrylic anhydride gelatin solution include a LAP photoinitiator and a methacrylic anhydride gelatin.

[0014] Preferably, the number of engineered nanovesicles derived from apoptotic mesenchymal stem cells in the methacrylic anhydride gelatin solution is (1-9)×10 8 Pieces / mL.

[0015] The present invention also provides the use of the engineered nanovesicles derived from apoptotic mesenchymal stem cells described in the above technical solution or the composite hydrogel described in the above technical solution in the preparation of wound healing drugs.

[0016] The present invention provides a method for preparing engineered nanovesicles derived from apoptotic mesenchymal stem cells. The engineered nanovesicles prepared by the preparation method of the present invention have abundant yields, efficient preparation processes, can be mass-produced, and have greater transformation potential. The engineered nanovesicles of the present invention can promote the proliferation and migration of a variety of cells, accelerate angiogenesis, and regulate the phenotype of macrophages, thereby promoting wound healing. The experimental results show that the engineered nanovesicles derived from apoptotic mesenchymal stem cells prepared by the preparation method of the present invention can significantly promote the proliferation and migration of vascular endothelial cells, while regulating the polarization state of macrophages, and effectively improve the inflammatory microenvironment of chronic diabetic wounds. The engineered nanovesicles derived from apoptotic mesenchymal stem cells show excellent immunomodulation and tissue repair potential, providing a novel and highly potential clinical solution for the treatment of chronic wounds.

[0017] The composite hydrogel provided by the present invention is a new type of wound dressing, which not only has good biocompatibility and extremely low cytotoxicity, but also can simulate a moist wound environment, provide more suitable conditions to promote cell migration and tissue regeneration, and can achieve the continuous release of engineered nanovesicles derived from apoptotic mesenchymal stem cells, regulate the inflammatory environment, and promote angiogenesis well. The use of composite hydrogel loaded with engineered nanovesicles derived from apoptotic mesenchymal stem cells provides a more efficient and controllable solution for the treatment of chronic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] Figure 1 This is a transmission electron microscope image of the engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 1 of the present invention;

[0020] Figure 2 The particle size distribution diagram of the engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 1 of the present invention;

[0021] Figure 3 This is a graph showing the results of polyacrylamide gel electrophoresis of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 1 of the present invention;

[0022] Figure 4 This is a scanning electron microscope image of the composite hydrogel provided in Example 2 of the present invention;

[0023] Figure 5 A graph showing the rate of apoptotic vesicle release from the composite hydrogel provided in Example 2 of the present invention;

[0024] Figure 6 This is an immunofluorescence image of a tissue section showing that the composite hydrogel provided in Example 2 of the present invention is used to promote diabetic wound healing in vivo;

[0025] Figure 7 This is a graph showing the results of the detection of HaCaT cell proliferation by the engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 3 of the present invention;

[0026] Figure 8 This is a graph showing the results of the detection of HaCaT cell proliferation by engineered nanovesicles derived from apoptotic bone marrow mesenchymal stem cells provided in Example 3 of the present invention;

[0027] Fig. 9 This is a graph showing the experimental results of uptake of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 3 of the present invention;

[0028] Fig.10 A diagram showing that engineered nanovesicles derived from apoptotic placental mesenchymal stem cells promote HaCaT cell migration as provided in Example 3 of the present invention;

[0029] Fig.11 A quantitative graph showing the area of ​​HaCaT cell migration promoted by engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 3 of the present invention;

[0030] Fig.12 This is a flow cytometry result diagram of the engineered nanovesicles derived from apoptotic placental mesenchymal stem cells reprogramming macrophage phenotypes provided in Example 3 of the present invention;

[0031] Fig.13 This is a Transwell result diagram of the engineered nanovesicles derived from apoptotic placental mesenchymal stem cells inducing HUVEC migration provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] The present invention provides a method for preparing engineered nano vesicles derived from apoptotic mesenchymal stem cells (abbreviated as apoptotic vesicles), comprising the following steps: subjecting mesenchymal stem cells to apoptosis treatment to obtain apoptotic cells; and extruding the apoptotic cells through a microporous filter membrane to obtain engineered nano apoptotic vesicles derived from apoptotic mesenchymal stem cells.

[0033] The present invention performs apoptosis treatment on mesenchymal stem cells to obtain apoptotic cells. In a specific embodiment, the mesenchymal stem cells include placental mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells. In a specific embodiment, the apoptosis treatment includes ultraviolet irradiation. In a specific embodiment, the ultraviolet irradiation is performed when the fusion rate of the mesenchymal stem cells is 70-95%, and further can be 85%-90%. In a specific embodiment, before the apoptosis treatment, the mesenchymal stem cells can be cultured in an MSCM culture medium containing 10% FBS. The present invention does not specifically limit the sources of the FBS, MSCM culture medium, and stem cell growth additives, and commercially available products in the art can be used. In a specific embodiment, the MSCM culture medium is a special culture medium containing mesenchymal stem cell growth additives (the culture medium brand is Science Cell). In a specific embodiment, the ultraviolet irradiation is in situ irradiation of the mesenchymal stem cells. In a specific embodiment, the wavelength of the ultraviolet irradiation is 280-320nm (UVB), and the power of the ultraviolet irradiation is 100μW / cm 2 Compared with UVA, UVB is more damaging to DNA, which can easily cause direct cell damage and obtain more apoptotic cells; the ultraviolet irradiation time is 15 to 60 minutes, specifically 60 minutes. Appropriate ultraviolet irradiation can cause cells to obtain a certain degree of DNA damage, thereby stimulating programmed cell death. If the ultraviolet irradiation time is too short, the degree of cell apoptosis is not high, and if the irradiation time is too long, the cell apoptosis is excessive, which may lead to cell necrosis. In a specific embodiment, after the ultraviolet irradiation, incubation is also included; the incubation conditions are: incubation in a 37°C, 5% CO2 incubator for 4 to 6 hours, specifically 4 hours. Ultraviolet irradiation can cause apoptosis in large quantities, improve efficiency, and the obtained engineered nanovesicles are pure. The engineered nanovesicles derived from apoptotic mesenchymal stem cells of the present invention are rich in source, and more apoptotic vesicles can be obtained by engineering extrusion, so that the yield of apoptotic vesicles is increased.

[0034] After obtaining the apoptotic cells, the present invention uses a microporous filter membrane to extrude the apoptotic cells to obtain engineered nano apoptotic vesicles derived from apoptotic mesenchymal stem cells. In a specific embodiment, the present invention uses PBS buffer to resuspend the apoptotic cells to obtain a single cell suspension, and then uses a microporous filter membrane to extrude the single cell suspension to obtain engineered nano vesicles. In a specific embodiment, the microporous filter membrane is a microporous filter membrane with a pore size of 3μm, 0.6μm and 0.2μm. In a specific embodiment, when a single cell suspension is extruded using a microporous filter membrane, the single cell suspension can be filtered and extruded with a microporous filter membrane with a pore size of 3μm, 0.6μm and 0.2μm in turn. In a specific embodiment, the number of extrusions is 2 to 4 times, and can further be 3 times.

[0035] The present invention also provides engineered nanovesicles derived from apoptotic mesenchymal stem cells prepared by the preparation method described in the above technical solution. The engineered nanovesicles derived from apoptotic mesenchymal stem cells contain bioactive molecules such as miRNA, growth factors, and proteins. Apoptotic vesicles play an important role in tissue repair and immune regulation. They can transmit signal molecules, accurately target lesions, regulate the behavior of surrounding cells, promote the transformation of M1 (pro-inflammatory) macrophages to M2 (anti-inflammatory) macrophages, and promote wound healing. The present invention constructs engineered nanovesicles derived from apoptotic mesenchymal stem cells to replace cell autocrine vesicles, which can not only obtain the biological advantages derived from stem cells, especially in immune regulation, wound repair and targeted therapy, but also realize a more stable and efficient preparation process.

[0036] The present invention also provides a composite hydrogel, comprising the following raw materials for preparation: the engineered nanovesicles derived from apoptotic mesenchymal stem cells described in the above technical solution and a methacrylic anhydride gelatin solution; the raw materials for preparing the methacrylic anhydride gelatin solution include LAP photoinitiator and methacrylic anhydride gelatin. In a specific embodiment, the number of engineered nanovesicles derived from apoptotic mesenchymal stem cells in the methacrylic anhydride gelatin is (1 to 9)×10 8 / mL, specifically 8×10 8 Pieces / mL.

[0037] The preparation method of the composite hydrogel of the present invention comprises the following steps: mixing the engineered nanovesicles derived from apoptotic mesenchymal stem cells and the methacrylic anhydride gelatin solution, irradiating the gelatin solution with a light source to obtain a composite hydrogel. In a specific embodiment, the irradiation wavelength of the light source is 405nm. In a specific embodiment, the irradiation time is 10 to 40s, which can be 20 to 30s. In the present invention, the preparation method of the methacrylic anhydride gelatin solution comprises: mixing the LAP photoinitiator and the methacrylic anhydride gelatin (brand: EFL), and heating and dissolving. In a specific embodiment, the LAP photoinitiator is mixed with the methacrylic anhydride gelatin in the form of a solution (initiator LAP standard solution). In a specific embodiment, a 3% to 10% hydrogel can be obtained by changing the volume of the initiator LAP standard solution and the mass of the methacrylic anhydride gelatin, specifically 1mL:0.05g to obtain a 5% hydrogel. In a specific embodiment, in the initiator LAP standard solution, the mass volume concentration of the LAP initiator is 0.2 to 0.3%. Specifically, for example, the preparation method of a 0.25% (w / v) initiator LAP standard solution is to mix 20 mL of PBS buffer with 0.05 g of initiator LAP to obtain a uniform solution. In order to better dissolve the initiator LAP standard solution, the initiator LAP standard solution is dissolved in a water bath at 40 to 50°C for 15 minutes, with several oscillations during the period. In a specific embodiment, the temperature of the heating and dissolving is 60 to 70°C, may be 62 to 78°C, and may be specifically 65°C. In a specific embodiment, the heating and dissolving time is 20 to 30 minutes, may be 22 to 28 minutes, and may be specifically 25 minutes. The above conditions can better obtain a uniformly dissolved methacrylic anhydride gelatin solution.

[0038] The composite hydrogel of the present invention has good biocompatibility and does not cause immune rejection. The three-dimensional network structure of the composite hydrogel can absorb and retain sufficient water, maintain a moist environment for the wound, and help accelerate cell migration and tissue regeneration. At the same time, the composite hydrogel can continuously release the engineered nanovesicles and bioactive substances derived from apoptotic mesenchymal stem cells, ensuring the stability and long-term effect of the drug efficacy, and significantly improving the therapeutic effect.

[0039] The present invention also provides the use of the engineered nanovesicles derived from apoptotic mesenchymal stem cells described in the above technical solution or the composite hydrogel described in the above technical solution in the preparation of wound healing drugs. In the present invention, the wound healing includes chronic wound healing. Chronic wounds refer to skin damage caused by external factors or intrinsic diseases, usually accompanied by long-term destruction of skin integrity and continuous loss of normal tissue. In the present invention, the chronic wound healing includes skin damage, infection or other difficult-to-heal injuries caused by chronic diseases (such as diabetes). In the present invention, the chronic wound is preferably a difficult-to-heal wound caused by diabetes, which is characterized by persistent inflammation, blocked angiogenesis and delayed tissue repair.

[0040] In the present invention, the drug preferably also includes other auxiliary components, such as immunomodulatory proteins (TGF-β, IL-10), etc. The composite hydrogel of the present invention preferably has engineered nanovesicles derived from apoptotic mesenchymal stem cells as the only active ingredient.

[0041] To further illustrate the present invention, the engineered nanovesicles, composite hydrogels, preparation methods and applications derived from apoptotic mesenchymal stem cells provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] An engineered nanovesicle derived from apoptotic mesenchymal stem cells, the preparation process is as follows:

[0044] 1. Preparation of apoptotic mesenchymal stem cells

[0045] The mesenchymal stem cells were revived and cultured in 10% FBS MSCM medium (Mesenchymal Stem Cell Medium; Catalog No: 7501; brand: science cell) until the third generation. When the fusion rate of the mesenchymal stem cells reached 70% to 95%, ultraviolet light (UVB) in the 280-320 nm band was irradiated at 100 μW / cm 2 The cells were irradiated with a power of 1 h and then placed in an incubator (37°C, 5% CO2) for another 4 h to obtain apoptotic mesenchymal stem cells.

[0046] 2. Preparation of engineered nanovesicles derived from apoptotic mesenchymal stem cells

[0047] (1) collecting the apoptotic mesenchymal stem cells obtained above and resuspending them in phosphate buffered saline (PBS) to form a single cell suspension;

[0048] (2) The single cell suspension was passed through microporous filter membranes with pore sizes of 3 μm, 0.6 μm, and 0.2 μm in sequence;

[0049] (3) Repeat step (2) three times to obtain engineered nanovesicles derived from apoptotic mesenchymal stem cells (abbreviated as AVs).

[0050] 3. Preparation of engineered nanovesicles derived from mesenchymal stem cells

[0051] (1) resuscitating mesenchymal stem cells and culturing them in 10% FBS MSCM medium until the third generation, when the fusion rate of the mesenchymal stem cells reaches 70% to 95%, collecting the mesenchymal stem cells and resuspending them in phosphate buffered saline (PBS) to form a single cell suspension;

[0052] (2) The single cell suspension was passed through microporous filter membranes with pore sizes of 3 μm, 0.6 μm, and 0.2 μm in sequence;

[0053] (3) Repeat step (2) three times to obtain engineered nanovesicles of mesenchymal stem cells (abbreviated as NVs).

[0054] 4. Identification of engineered nanovesicles derived from isolated apoptotic mesenchymal stem cells by transmission electron microscopy

[0055] (1) Take 10 μL of freshly prepared engineered nanovesicle solution derived from apoptotic mesenchymal stem cells and drop it on the clean surface of the sealing film.

[0056] (2) The copper mesh was placed on top of the engineered nanovesicle droplet derived from apoptotic mesenchymal stem cells, with the membrane surface in contact with the droplet. After suspending for 10 min, the mesh was slowly blotted dry with filter paper.

[0057] (3) Transfer the copper mesh to a drop of 3% glutaraldehyde fixative and fix for 5 min before absorbing the fixative.

[0058] (4) Transfer the copper mesh to the surface of the water droplet and wash it repeatedly for 10 times, suspending it for 2 minutes each time, and then use filter paper to absorb the water.

[0059] (5) The copper mesh was then placed on the surface of a 4% uranyl acetate droplet and suspended for 10 min before the remaining solution was absorbed.

[0060] (6) Finally, place the copper mesh in a 1% methylcellulose droplet and let it stand for 5 min before absorbing the water.

[0061] (7) The copper mesh was left at room temperature for more than 30 minutes, and after drying, it was observed under a transmission electron microscope and photographed. The observation results of the engineered nanovesicles derived from apoptotic placental mesenchymal stem cells were as follows: Figure 1 shown.

[0062] Figure 1The results showed that the engineered nanovesicles derived from the isolated apoptotic placental mesenchymal stem cells were in the shape of a pancake, slightly dark in the middle, and with a slightly brighter circle at the edge.

[0063] 5. Detection of the particle size of engineered nanovesicles derived from isolated apoptotic mesenchymal stem cells

[0064] The particle size of engineered nanovesicles derived from apoptotic mesenchymal stem cells was measured using ZetaVIEW PMX 110. 100 μL of PBS solution containing engineered nanovesicles derived from apoptotic mesenchymal stem cells was diluted to an appropriate multiple so that the final concentration became 10 7 The parameters were set as follows: particle size range 50-200nm, molecular weight range 1000-20107Da, temperature 25℃, laser 4.0mV He-Ne laser, wavelength 633nm. Each sample was analyzed three times continuously, and the NTA values ​​were recorded and analyzed at different positions. ZetaVIEW 8.04.02 software was used for data analysis.

[0065] Figure 2 Particle size distribution of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells. Figure 2 The results showed that the particle size of the engineered nanovesicles derived from apoptotic mesenchymal stem cells was about 186nm.

[0066] VI. Identification of engineered nanovesicles derived from isolated apoptotic mesenchymal stem cells by polyacrylamide gel electrophoresis

[0067] (1) 100 μL of lysis buffer was added to 50 μL of mesenchymal stem cells (MSC), mesenchymal stem cell vesicles (NVs), and engineered nanovesicles (AVs) derived from apoptotic mesenchymal stem cells, respectively, and the mixture was placed on crushed ice for 30 min.

[0068] (2) Add 20 μL of loading buffer to the appropriate amount of the mixed solution after lysis, boil it in a 100°C metal bath for 10 min, and store it at -80°C after cooling.

[0069] (3) After the protein electrophoresis gel was prepared, 5 μL of mesenchymal stem cells (MSCs), mesenchymal stem cell vesicles (NVs), and engineered nanovesicles (AVs) derived from apoptotic mesenchymal stem cells were added to the gel lanes and 5 μL of Protein Maker was added to the adjacent lanes. The compression gel was run at 80 V and the separation gel was run at 120 V.

[0070] (4) Remove the gel from the lane where the sample is located, add Coomassie Brilliant Blue, protect from light, shake on a shaker to develop the color, wash off the excess dye with pure water, observe and take photos.

[0071] Figure 3 Figure 2 is the result of polyacrylamide gel electrophoresis of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells. Figure 3 As shown, placental mesenchymal stem cells, placental mesenchymal stem cell vesicles, and engineered nanovesicles derived from apoptotic placental mesenchymal stem cells have the same protein expression.

[0072] Based on the electron microscopy results, average particle size and protein expression, the product finally prepared in this example meets the characteristics of engineered nanovesicles, indicating that the engineered nanovesicles finally isolated in this example are indeed derived from apoptotic placental mesenchymal stem cells.

[0073] Example 2

[0074] 1. This embodiment provides a composite hydrogel, and the preparation method of the composite hydrogel is as follows:

[0075] (1) Preparation of methacrylic anhydride gelatin solution

[0076] S1. Take 20 mL of PBS and add it into a brown bottle containing 0.05 g of initiator LAP to prepare a 0.25% (w / v) initiator LAP standard solution.

[0077] S2. Heat the initiator LAP standard solution in a 45°C water bath for 15 minutes, shaking several times during the process.

[0078] S3. Take 0.25 g of methacrylic anhydride gelatin and put it into a brown centrifuge tube. Add 5 mL of initiator LAP standard solution, heat and dissolve it in a 65°C water bath in the dark for 25 minutes. Shake it several times during the process. Immediately sterilize it through a 0.22 μm sterile syringe filter (to prevent low-temperature gelation) to obtain a methacrylic anhydride gelatin solution.

[0079] (2) Preparation of composite hydrogel

[0080] According to the preparation method of Example 1, engineered nanovesicles derived from apoptotic placental mesenchymal stem cells were prepared, and 8×10 8 The mixture was added into 1 mL of methacrylic anhydride gelatin solution and mixed evenly, and irradiated with a 405 nm light source for 30 s to achieve gelation, thereby obtaining a composite hydrogel (abbreviated as Gel-AVs).

[0081] The structure of methacrylic anhydride gelatin hydrogel was observed by scanning electron microscopy. Figure 4 shown.

[0082] Figure 4 The results showed that the gel surface of the composite hydrogel presented a network pore structure.

[0083] 2. Release efficiency of engineered nanovesicle composite hydrogels loaded with apoptotic placental mesenchymal stem cells

[0084] The prepared Gel-AVs were added to a Transwell chamber (membrane pore size 8 μm, suitable for 24-well plates) filled with PBS. At a specific time, the liquid in the lower chamber was collected and supplemented with fresh PBS. The concentration of AVs released in the liquid was detected using a BCA protein reagent test kit. Release rate (%) = detection concentration / total concentration × 100%.

[0085] Figure 5 This is a graph showing the rate of apoptotic vesicle release from composite hydrogels. Figure 5 The results showed that the composite hydrogel could continuously release apoptotic vesicles, and the maximum release amount was basically reached on the fourth day.

[0086] 3. Engineered nanovesicle composite hydrogels loaded with apoptotic placental mesenchymal stem cells for in vivo diabetic wound healing

[0087] (1) Diabetic mouse model

[0088] C57BL / 6J mice, 7 weeks old, weighing about 20 g, male, were selected; modeling method: 1% streptozotocin (prepared and used immediately) was administered at 150 mg / kg for two consecutive days (fasting for 12 hours before administration), and blood sugar was tested after one week (blood was collected by tail tip puncture, fasting). If the blood sugar value was greater than 16.6 mM for three consecutive days, the model was successfully established.

[0089] (2) Diabetic full-thickness excisional wound model

[0090] Modeling method: After depilation, skin tissue (including epidermis and dermis) with a diameter of 1 cm was excised from the middle of the mouse's back.

[0091] Dosing strategy: The drug was administered from the day after modeling, divided into three doses, with an interval of 2 to 3 days between each dose.

[0092] Drug administration groups: control (normal mice, the rest of the groups are diabetic mice); PBS (no drug administration); GelMA; AVs; Gel-AVs.

[0093] Dosage: The dosing volume for each group was 100 μL, 100 mg for the AVs group and 100 mg for the Gel-AVs group.

[0094] Wound tissues were collected for immunofluorescence on the 7th and 14th days after administration. The tissues on the 7th day were stained for CD86\CD206 proteins, and the tissues on the 14th day were stained for α-SMA / CD31.

[0095] Figure 6This is an immunofluorescence image of tissue sections showing that the composite hydrogel is used to promote diabetic wound healing in vivo. Figure 6 The results showed that the CD86 fluorescence of the Gel-AVs group was weaker than that of the PBS group, and the CD206 fluorescence was stronger than that of the PBS group, the GelMA group, and the AVs group. It can be seen that the Gel-AVs group contained more M2 macrophages, which regulated the inflammatory environment in the wound. At the same time, the α-SMA / CD31 fluorescence was stronger than that of the PBS group, the GelMA group, and the AVs group, indicating that there were more blood vessels formed in the wound tissue of the Gel-AVs group. Based on various indicators, the therapeutic effect of the composite hydrogel Gel-AVs was significant.

[0096] The product finally prepared in this example has ideal hydrogel characteristics, indicating that a composite hydrogel is finally prepared in this example and has excellent therapeutic effect on promoting wound healing.

[0097] Example 3

[0098] Cytological experiments: In the following experiments, engineered nanovesicles (AVs) derived from apoptotic mesenchymal stem cells were prepared according to the preparation method provided in Example 1.

[0099] 1. Cell proliferation detection experiment

[0100] Stably cultured HaCaT cells were seeded in 96-well plates with 100 μL per well at a density of 6 × 10 4 Cells / mL. Observe the cell adhesion, update the culture medium, and add 100 μL of culture medium containing different concentrations (10 μg / mL, 30 μg / mL, 50 μg / mL, 70 μg / mL, 100 μg / mL, 120 μg / mL, 150 μg / mL) of AVs (AVs here are apoptotic vesicles from placental mesenchymal stem cells) to each well as the experimental group, and set up 5 replicates for each concentration. At the same time, add 100 μL of culture medium to treat HaCaT cells as the control group (corresponding to Figure 7 0 in the experiment, and the wells without cells added with 100 μL of culture medium were used as blank control groups, and 5 duplicate wells were set up respectively. After incubation in a constant temperature incubator for 24 hours, the old culture medium was discarded and 100 μL of serum-free culture medium containing 10% CCK-8 working solution was added, and then the wells were moved into the incubator for incubation for 1 to 4 hours. The absorbance of each well at 405 nm was detected by an ELISA instrument to detect cell activity, record and calculate the cell proliferation rate. The formula for calculating the cell proliferation rate is: (absorbance value of the experimental group - absorbance value of the blank control) / absorbance value of the control group - absorbance value of the blank control) × 100%.

[0101] The results of the test on the proliferation of HaCaT cells by engineered nanovesicles derived from apoptotic placental mesenchymal stem cells are as follows Figure 7As shown, when the concentration of AVs is higher than 10 μg / mL, it shows a significant effect on promoting the proliferation of HaCaT cells. Among them, when the concentration of AVs is 50-70 μg / mL, the effect of promoting the proliferation of HaCaT cells is the most obvious, and the highest proliferation rate is 199.4%.

[0102] Stably cultured HaCaT cells were seeded in 96-well plates with 100 μL per well at a density of 6 × 10 4 Cells / mL. Observe the cell adhesion, update the culture medium, add 100 μL of culture medium containing different concentrations (10 μg / mL, 30 μg / mL, 50 μg / mL, 70 μg / mL, 100 μg / mL, 120 μg / mL, 150 μg / mL) of AVs (AVs here are apoptotic vesicles from bone marrow mesenchymal stem cells) to each well as the experimental group, and set up 5 replicate wells for each concentration. At the same time, add 100 μL of culture medium to treat HaCaT cells as the control group (corresponding to Figure 8 0 in the experiment, and the wells without cells added with 100 μL of culture medium were used as blank control groups, and 5 duplicate wells were set up respectively. After incubation in a constant temperature incubator for 24 hours, the old culture medium was discarded and 100 μL of serum-free culture medium containing 10% CCK-8 working solution was added, and then the wells were moved into the incubator for incubation for 1 to 4 hours. The absorbance of each well at 405 nm was detected by an ELISA instrument to detect cell activity, record and calculate the cell proliferation rate. The formula for calculating the cell proliferation rate is: (absorbance value of the experimental group - absorbance value of the blank control) / absorbance value of the control group - absorbance value of the blank control) × 100%.

[0103] The results of the test on the proliferation of HaCaT cells by engineered nanovesicles derived from apoptotic bone marrow mesenchymal stem cells are as follows Figure 8 As shown, when the concentration of AVs is higher than 10 μg / mL, it exhibits a proliferation promoting effect on HaCaT cells. Among them, when the concentration of AVs is 50 μg / mL, the proliferation promoting effect on HaCaT cells is the most obvious, with the highest proliferation rate of 145.5%.

[0104] contrast Figure 7 and Figure 8 The apoptotic vesicles of placental mesenchymal stem cells and bone marrow mesenchymal stem cells can both promote cell proliferation, and at the same concentration, the ability of apoptotic vesicles of placental mesenchymal stem cells to promote cell proliferation is better than that of apoptotic vesicles of bone marrow mesenchymal stem cells.

[0105] 2. Cellular Uptake Experiment

[0106] After resuspending the RAW264.7 cells in good growth condition, they were divided into two groups and laid in the confocal dish, namely the control group (blank control group) and AVs (AVs here are apoptotic vesicles from placental mesenchymal stem cells, with a concentration of 50μm / mL), with a cell density of 20w / mL, 1mL per well, and placed in the incubator for 24h. Prepare the DIO working solution, take 1uL of DIO and staining enhancer, add 500uL AVs (50μg / mL) solution, vortex and then add 500uL of cell culture medium, incubate in the dark for 15min. Remove the culture medium in the confocal dish, add the prepared incubation solution to the confocal dish, and incubate for 4h. Prepare Lyso-TrackerRed working solution: take 1μl Lyso-TrackerRed and add it to 15ml warm cell culture medium, mix well to get Lyso-Tracker Red working solution (Lyso-TrackerRed working solution needs to be pre-incubated at 37℃ before use). Remove the culture medium in the confocal dish and wash twice with PBS. Add the prepared Lyso-TrackerRed staining solution pre-incubated at 37°C and incubate with the cells at 37°C for 40 minutes. Then remove the Lyso-Tracker Red staining solution, wash twice with PBS, and add paraformaldehyde to fix for 30 minutes. Nuclear staining: remove the fixative, wash twice with PBS, then add a small amount (500uL) of DAPI staining solution to cover the sample and leave at room temperature for 3 to 5 minutes. Aspirate the DAPI staining solution, wash 2 to 3 times with PBS, 3 to 5 minutes each time, and then use it for confocal photography.

[0107] The results of the uptake experiment of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells are as follows Fig. 9 As shown, the green fluorescence and the red fluorescence mostly overlap, indicating that AVs are engulfed into lysosomes and AVs can be well taken up by RAW264.7 cells.

[0108] 3. Cell scratch assay

[0109] Place the Culture-Insert tightly in a 6-well plate and prepare a 3×10 5HaCaT cell suspension with a concentration of 1 cell / mL was added to each well of Culture-Insert 2 at a standard of 70 μL per well, and care was taken to avoid touching the insert and causing it to move. Subsequently, the insert was moved into a constant temperature incubator together with the 6-well plate and incubated at 37°C and 5% CO2 for 24 hours. The cell density was checked under a microscope, and the ideal confluence was achieved, and the Culture-Insert was gently removed with sterile tweezers. Subsequently, it was washed with PBS solution to remove cell debris and non-attached cells. Finally, 5% fetal bovine serum culture medium with protein concentrations of 30 μg / mL, 50 μg / mL, and 70 μg / mL AVs (AVs here are apoptotic vesicles from placental mesenchymal stem cells) was added, respectively, and placed in a constant temperature incubator. At the same time, the same volume of 5% fetal bovine serum culture medium was added as a control group, and each treatment group had 3 replicates. At different time points, the changes in the scratches were observed and photographed using an inverted microscope, and the scratch healing rate was calculated according to the following formula:

[0110] Scratch healing rate (%) = (initial scratch area - final scratch area) / initial scratch area × 100%. The initial scratch area is the scratch area set before the test, and the final scratch area is the scratch area after treatment in different treatment groups. The results are shown in Fig.10 and Fig.11 .

[0111] Fig.10 This is a diagram of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells promoting HaCaT cell migration; Fig.11 The figure is a quantitative diagram of the migration area of ​​HaCaT cells promoted by engineered nanovesicles derived from apoptotic placental mesenchymal stem cells. Fig.10 and Fig.11 As shown, compared with the blank control group (control), the cell scratch healing in each group with AVs added was significantly accelerated, showing a significant migration promotion effect.

[0112] 4. Regulation of Macrophage Reprogramming Experiment

[0113] First, RAW264.7 cells with good growth status were resuspended and plated in a 12-well plate, 1 mL per well, at a density of 5×10 5 / mL. At the same time, lipopolysaccharide (LPS) solution and IFN-γ solution were added to each well to make the final concentrations of 500ng / mL and 4ng / mL, respectively. After 24h of induction, 0μg / mL, 50μg / mL, and 70μg / mL of AVs (AVs here are apoptotic vesicles from placental mesenchymal stem cells) were added to each group and co-incubated with cells. After 48h of culture, the cells were collected and resuspended with PBS and centrifuged. Resuspended with 100μL PBS buffer, 1μL of CD11b, CD86, and CD206 flow antibodies were added, and stained for 30min. Then centrifuged to remove excess antibodies and measured by flow cytometry. Among them, CD86 is a protein highly expressed after M1 macrophage polarization; CD206 is generally considered to be a marker for M2 macrophages.

[0114] Flow cytometry results of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells reprogramming macrophage phenotypes Fig.12 As shown in the figure, after AVs were added, the expression of M1 marker CD86 in the cells was significantly reduced, while the expression of M2 marker CD206 was significantly increased. This shows that AVs successfully reversed the phenotype of some M1 macrophages.

[0115] 5. Transwell Chemotaxis Assay

[0116] HUVEC cells that are growing well were resuspended in serum-free medium to a cell density of 4 × 10 6 / mL. Cells were inoculated in a chamber adapted to a 24-well plate with a membrane pore size of 8μm. 200μL of the above cell suspension was added to the upper chamber, and 600μL of 10% FBS medium containing 0μg / mL, 50μg / mL, and 70μg / mL AVs (AVs here are apoptotic vesicles from placental mesenchymal stem cells) and 10% FBS medium containing 50μg / mL NVs were added to the lower chamber. Three replicate wells were set up for each group. After the Transwell chamber containing cells was placed in an incubator and cultured for 48h, the cells in the upper chamber were wiped off with a cotton ball. Carefully remove the chamber with tweezers, dry the culture medium in the upper chamber, and gently wipe Matrigel and the cells in the upper chamber with a cotton swab. Take a new 24-well plate and add 600μL of 4% paraformaldehyde, put the chamber in and fix it at room temperature for 20min. Then take out the chamber, dry the fixative in the upper chamber, move it to the well where about 600μL crystal violet was added in advance, and stain it at room temperature for 10min. The stained chamber was rinsed with pure water 3 to 6 times and photographed under a microscope.

[0117] Transwell results of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells inducing HUVEC migration Fig.13As shown, more cells in the NVs group penetrated the membrane compared with the control group, while more cells in the AVs group penetrated the membrane compared with the NVs group, indicating that AVs can induce cell migration and HUVEC cells can produce chemotaxis to AVs.

[0118] It can be seen that the engineered nanovesicles derived from apoptotic mesenchymal stem cells used in the composite hydrogel of the present invention have high preparation efficiency. Avs can be well taken up by cells, and at the same time, they show excellent effects in promoting cell proliferation and migration, regulating inflammatory response resistance, etc. In addition, the composite hydrogel has extremely low cytotoxicity, can achieve the continuous release of engineered nanovesicles derived from apoptotic mesenchymal stem cells, can regulate the inflammatory environment, and well promote angiogenesis and tissue regeneration. At the same time, the method of extruding the microporous filter membrane of the present invention has a large output, about 7×10 6 800 μg of engineered apoptotic nanovesicles can be collected from each cell, while ensuring the proper functions of the apoptotic vesicles and having greater potential for transformation.

[0119] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing engineered nano apoptotic vesicles derived from apoptotic mesenchymal stem cells, characterized in that: The following steps are involved: Performing apoptosis treatment on mesenchymal stem cells to obtain apoptotic cells; The apoptotic cells are squeezed out by using a microporous filter membrane to obtain engineered nano apoptotic vesicles derived from apoptotic mesenchymal stem cells.

2. The preparation method according to claim 1, characterized in that: The mesenchymal stem cells include placental mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.

3. The preparation method according to claim 1, characterized in that: The apoptotic treatment includes ultraviolet irradiation.

4. The preparation method according to claim 3, characterized in that: The wavelength of the ultraviolet irradiation is 280-320 nm; the power is 100 μW / cm 2 ; The ultraviolet irradiation time is 15 to 60 minutes.

5. The preparation method according to claim 3, characterized in that: After the ultraviolet irradiation, the method further includes incubation; the incubation conditions are: incubation in a 37° C., 5% CO 2 incubator for 4 to 6 hours.

6. The preparation method according to claim 1, characterized in that: The microporous filter membranes are microporous filter membranes with pore sizes of 3 μm, 0.6 μm and 0.2 μm.

7. Engineered nanovesicles derived from apoptotic mesenchymal stem cells prepared by the preparation method according to any one of claims 1 to 6.

8. A composite hydrogel, characterized in that: The preparation method comprises the following raw materials: the engineered nanovesicles derived from apoptotic mesenchymal stem cells as described in claim 7 and a methacrylic anhydride gelatin solution; the raw materials for preparing the methacrylic anhydride gelatin solution comprise a LAP photoinitiator and methacrylic anhydride gelatin.

9. The composite hydrogel according to claim 8, characterized in that The number of engineered nanovesicles derived from apoptotic mesenchymal stem cells in the methacrylic anhydride gelatin solution is (1-9)×10 8 Pieces / mL.

10. Use of the engineered nanovesicle derived from apoptotic mesenchymal stem cells according to claim 7 or the composite hydrogel according to claim 8 or 9 in the preparation of wound healing drugs.

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