Engineered nanovesicle derived from apoptotic mesenchymal stem cell, composite hydrogel, preparation method and use

Abstract

The present invention specifically relates to the technical field of biomedical engineering, and provides an engineered nanovesicle derived from an apoptotic mesenchymal stem cell, a composite hydrogel, a preparation method and a use. Provided is a preparation method for the engineered nanovesicle derived from the apoptotic mesenchymal stem cell, comprising the following steps: performing apoptosis treatment on mesenchymal stem cells to obtain apoptotic cells; and extruding the apoptotic cells through a microporous filter membrane to obtain the engineered nanovesicle derived from the apoptotic mesenchymal stem cells. The apoptotic vesicles prepared by the preparation method have a rich yield, the preparation process is efficient and can achieve large-scale production, and the apoptotic vesicles have strong transformation potential.

Description

Engineered nanovesicles and composite hydrogels derived from apoptotic mesenchymal stem cells, their preparation methods, and applications. Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to an engineered nanovesicle, composite hydrogel, preparation method, and application of apoptotic mesenchymal stem cells. Background Technology

[0002] Traditional wound dressings, such as gauze and bandages, act as a physical barrier in wound healing, providing hemostasis and protection. However, their effectiveness is limited to isolating the wound and reducing contamination; they cannot provide a suitable moist environment or bioactive substances that promote tissue repair, resulting in slower wound healing and an inability to effectively suppress the risk of infection. Furthermore, traditional dressings lack active factors related to tissue regeneration, thus offering poor efficacy in treating chronic wounds, diabetic ulcers, and other difficult-to-heal wounds.

[0003] Cell therapy has been increasingly applied to regenerative medicine and wound repair in recent years, demonstrating certain advantages, particularly 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 rates after transplantation, difficulty in effectively integrating into damaged tissues, and potential immune rejection. Furthermore, many cell therapies require the use of non-degradable or potentially toxic carrier materials to support the transplanted cells, which not only increases the biocompatibility requirements of the materials but may also trigger side effects. Moreover, the complex and costly processes of cell expansion, isolation, and transplantation limit its widespread adoption in wound healing treatment. Technical issues

[0004] To overcome the aforementioned limitations, cell-derived vesicles (such as exosomes and microvesicles) have emerged as a promising alternative. These vesicles possess excellent biocompatibility and bioactivity, and can carry molecules that regulate wound healing, such as growth factors, anti-inflammatory factors, and microRNAs, demonstrating broad application potential in chronic wound repair. However, traditional cell-secreting vesicles have limited production volumes, and their purification and production processes are complex, failing to meet the demands of large-scale applications. Therefore, exploring drugs that effectively promote tissue regeneration and are easily mass-produced is crucial as a novel strategy for promoting chronic wound healing. Technical solutions

[0005] The purpose of this invention is to provide engineered nanovesicles, composite hydrogels, preparation methods, and applications derived from apoptotic mesenchymal stem cells. The apoptotic vesicles prepared by the method described in this invention exhibit abundant yields, high efficiency, and the potential for large-scale production, as well as significant transformational potential.

[0006] This invention provides a method for preparing engineered nanoapoptotic vesicles 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 using a microporous filter membrane to obtain engineered nanoapoptotic vesicles derived from apoptotic mesenchymal stem cells.

[0007] Preferably, the apoptosis treatment includes ultraviolet irradiation.

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

[0009] Preferably, the ultraviolet irradiation is followed by incubation; the incubation conditions are: incubation at 37°C in a 5% CO2 incubator for 4-6 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 is a microporous filter membrane with 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: engineered nanovesicles derived from apoptotic mesenchymal stem cells as described in the above technical solution and a methacrylic anhydride-modified gelatin solution; the raw materials for preparing the methacrylic anhydride-modified gelatin solution include LAP photoinitiator and methacrylic anhydride-modified gelatin.

[0014] Preferably, the number of engineered nanovesicles derived from apoptotic mesenchymal stem cells in the methacrylic anhydride-modified gelatin solution is (1~9)×10⁻⁶. 8 per mL.

[0015] The present invention also provides the application of engineered nanovesicles derived from apoptotic mesenchymal stem cells as described in the above-mentioned technical solutions or the composite hydrogels as described in the above-mentioned technical solutions in the preparation of wound healing drugs.

[0016] This invention provides a method for preparing engineered nanovesicles derived from apoptotic mesenchymal stem cells. The engineered nanovesicles prepared by this method exhibit abundant yield, high efficiency, and the potential for large-scale production, as well as significant transformational potential. The engineered nanovesicles described in this invention can promote the proliferation and migration of various cell types, accelerate angiogenesis, and regulate macrophage phenotype, thereby promoting wound healing. Experimental results show that the engineered nanovesicles derived from apoptotic mesenchymal stem cells prepared by this method can significantly promote the proliferation and migration of vascular endothelial cells, while simultaneously regulating the polarization state of macrophages, effectively improving the inflammatory microenvironment of chronic diabetic wounds. The engineered nanovesicles derived from apoptotic mesenchymal stem cells demonstrate excellent immunomodulatory and tissue repair potential, providing a novel and highly promising clinical solution for the treatment of chronic wounds. Beneficial effects

[0017] The composite hydrogel provided by this invention, as a novel wound dressing, not only possesses good biocompatibility and extremely low cytotoxicity, but also can simulate a moist wound environment, providing more suitable conditions to promote cell migration and tissue regeneration. It enables the sustained release of engineered nanovesicles derived from apoptotic mesenchymal stem cells, modulates the inflammatory environment, and effectively promotes angiogenesis. The use of engineered nanovesicles derived from apoptotic mesenchymal stem cells loaded onto the composite hydrogel provides a more efficient and controllable solution for the treatment of chronic wounds. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

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

[0021] Figure 3 shows the polyacrylamide gel electrophoresis results of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 1 of this invention.

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

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

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

[0025] Figure 7 shows the detection results of HaCaT cell proliferation using engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 3 of the present invention.

[0026] Figure 8 shows the detection results of HaCaT cell proliferation using engineered nanovesicles derived from apoptotic bone marrow mesenchymal stem cells provided in Example 3 of the present invention.

[0027] Figure 9 shows the uptake experiment results of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 3 of the present invention.

[0028] Figure 10 shows the HaCaT cell migration promotion effect of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 3 of the present invention.

[0029] Figure 11 is a quantitative diagram of 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] Figure 12 is a flow cytometry result of engineered nanovesicles reprogramming macrophage phenotypes derived from apoptotic placental mesenchymal stem cells provided in Example 3 of the present invention.

[0031] Figure 13 shows the Transwell results of HUVEC migration induced by engineered nanovesicles derived from apoptotic placental mesenchymal stem cells provided in Example 3 of the present invention. Embodiments of the present invention

[0032] This invention provides a method for preparing engineered nanovesicles (hereinafter referred to as apoptotic vesicles) 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 using a microporous filter membrane to obtain engineered nanovesicles derived from apoptotic mesenchymal stem cells.

[0033] This invention involves apoptosis treatment of mesenchymal stem cells to obtain apoptotic cells. In specific embodiments, the mesenchymal stem cells include placental mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose-derived mesenchymal stem cells. In specific embodiments, the apoptosis treatment includes ultraviolet (UV) irradiation. In specific embodiments, the UV irradiation is performed when the mesenchymal stem cell fusion rate is 70-95%, or more specifically 85-90%. In specific embodiments, before the apoptosis treatment, the mesenchymal stem cells can be cultured in MSCM medium containing 10% FBS. This invention does not have specific limitations on the source of the FBS, MSCM medium, and stem cell growth additives; commercially available products in the art can be used. In specific embodiments, the MSCM medium is a dedicated culture medium containing mesenchymal stem cell growth additives (the brand of the culture medium is Science Cell). In specific embodiments, the UV irradiation is in situ irradiation of the mesenchymal stem cells. In specific embodiments, the wavelength of the UV irradiation is 280-320 nm (UVB), and the power of the UV irradiation is 100 μW / cm². 2 UVB is more damaging to DNA than UVA, easily causing direct cell damage and resulting in more apoptotic cells. The UV irradiation time is 15-60 minutes, specifically 60 minutes. Appropriate UV irradiation can induce a certain degree of DNA damage in cells, thereby triggering programmed cell death. Too short an irradiation time results in insufficient apoptosis, while too long an irradiation time leads to excessive apoptosis and may cause cell necrosis. In a specific embodiment, the UV irradiation is followed by incubation; the incubation conditions are: 4-6 hours at 37°C in a 5% CO2 incubator, specifically 4 hours. UV irradiation can induce apoptosis in large quantities, improving efficiency, and the resulting engineered nanovesicles are pure. The engineered nanovesicles derived from apoptotic mesenchymal stem cells of this invention are abundant, and more apoptotic vesicles can be obtained through engineered extrusion, increasing the yield of apoptotic vesicles.

[0034] After obtaining apoptotic cells, this invention uses a microporous membrane to extrude the apoptotic cells, obtaining engineered nano-apoptotic vesicles derived from apoptotic mesenchymal stem cells. In a specific embodiment, the apoptotic cells are resuspended in PBS buffer to obtain a single-cell suspension, which is then extruded using a microporous membrane to obtain engineered nanovesicles. In a specific embodiment, the microporous membrane is a microporous membrane with pore sizes of 3 μm, 0.6 μm, and 0.2 μm. In a specific embodiment, when extruding the single-cell suspension using the microporous membrane, the single-cell suspension can be filtered and extruded sequentially through microporous membranes with pore sizes of 3 μm, 0.6 μm, and 0.2 μm. In a specific embodiment, the extrusion is performed 2 to 4 times, and more specifically, 3 times.

[0035] This invention also provides engineered nanovesicles derived from apoptotic mesenchymal stem cells prepared by the method described in the above technical solution. These engineered nanovesicles contain bioactive molecules such as miRNA, growth factors, and proteins. Apoptotic vesicles play an important role in tissue repair and immune regulation, capable of transmitting signaling molecules, precisely targeting lesion sites, regulating the behavior of surrounding cells, promoting the transformation of M1 (pro-inflammatory) macrophages into M2 (anti-inflammatory) macrophages, and promoting wound healing. This invention, by constructing engineered nanovesicles derived from apoptotic mesenchymal stem cells to replace autocrine vesicles, not only obtains the biological advantages derived from stem cells, especially in immune regulation, wound repair, and targeted therapy, but also achieves a more stable and efficient preparation process.

[0036] This invention also provides a composite hydrogel comprising the following raw materials: engineered nanovesicles derived from apoptotic mesenchymal stem cells as described in the above technical solution and a methacrylic anhydride-modified gelatin solution; the raw materials for preparing the methacrylic anhydride-modified gelatin solution include a LAP photoinitiator and methacrylic anhydride-modified gelatin. In a specific embodiment, the number of engineered nanovesicles derived from apoptotic mesenchymal stem cells in the methacrylic anhydride-modified gelatin is (1~9)×10⁻⁶. 8 Cells / mL, specifically 8 × 10 8 per mL.

[0037] The preparation method of the composite hydrogel of the present invention includes the following steps: mixing engineered nanovesicles derived from apoptotic mesenchymal stem cells with a methacrylic anhydride-modified gelatin solution, and irradiating with a light source to gelatinize, thereby obtaining a composite hydrogel. In a specific embodiment, the irradiation wavelength of the light source is 405 nm. In a specific embodiment, the irradiation time is 10-40 s, which can be 20-30 s. In the present invention, the preparation method of the methacrylic anhydride-modified gelatin solution includes: mixing LAP photoinitiator and methacrylic anhydride-modified gelatin (brand: EFL), and heating to dissolve. In a specific embodiment, the LAP photoinitiator is mixed with the methacrylic anhydride-modified gelatin in the form of a solution (initiator LAP standard solution). In a specific embodiment, by changing the volume of the initiator LAP standard solution and the mass of the methacrylic anhydride-modified gelatin, a 3%-10% hydrogel can be obtained, specifically 1 mL: 0.05 g to obtain a 5% hydrogel. In a specific embodiment, the mass-volume concentration of the LAP initiator in the initiator LAP standard solution is 0.2-0.3%. Specifically, a 0.25% (w / v) initiator LAP standard solution is prepared by mixing 20 mL of PBS buffer with 0.05 g of initiator LAP until homogeneous. To better dissolve the initiator LAP standard solution, it is dissolved in a water bath at 40-50°C for 15 min, with several agitations during the process. In a specific embodiment, the dissolving temperature is 60-70°C, specifically 62-78°C, and more specifically 65°C. In a specific embodiment, the dissolving time is 20-30 min, specifically 22-28 min, and more specifically 25 min. These conditions ensure a more homogeneous and well-dissolved methacrylic anhydride-modified gelatin solution.

[0038] The composite hydrogel of this invention exhibits excellent biocompatibility and does not induce immune rejection. Furthermore, its three-dimensional network structure absorbs and retains sufficient moisture, maintaining a moist environment in the wound and facilitating accelerated cell migration and tissue regeneration. Simultaneously, the composite hydrogel continuously releases engineered nanovesicles and bioactive substances derived from apoptotic mesenchymal stem cells, ensuring stable and prolonged drug efficacy and significantly improving therapeutic outcomes.

[0039] This invention also provides the application of engineered nanovesicles derived from apoptotic mesenchymal stem cells or the composite hydrogels described in the above-mentioned technical solutions in the preparation of wound healing drugs. In this invention, wound healing includes chronic wound healing. Chronic wounds refer to skin damage caused by external factors or internal diseases, usually accompanied by long-term disruption of skin integrity and continuous loss of normal tissue. In this invention, chronic wound healing includes skin lesions, infections, or other difficult-to-heal injuries caused by chronic diseases (such as diabetes). In this invention, the chronic wound is preferably a difficult-to-heal wound caused by diabetes, characterized by persistent inflammation, inhibited angiogenesis, and delayed tissue repair.

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

[0041] To further illustrate the present invention, the engineered nanovesicles, composite hydrogels, preparation methods, and applications of apoptotic mesenchymal stem cells provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions 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 was prepared as follows:

[0044] I. Preparation of Apoptotic Mesenchymal Stem Cells

[0045] 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 mesenchymal stem cell confluence rate reached 70%~95%. Then, ultraviolet (UVB) at a wavelength of 280~320nm was applied at 100μW / cm². 2 Irradiate with high power for 1 hour, then incubate in an incubator (37℃, 5% CO2) for another 4 hours to obtain apoptotic mesenchymal stem cells.

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

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

[0048] (2) Pass the single-cell suspension through microporous 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 (abbreviated as AVs) derived from apoptotic mesenchymal stem cells.

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

[0051] (1) Resuscitate mesenchymal stem cells and culture them in 10% FBS MSCM medium until the third generation. When the fusion rate of mesenchymal stem cells reaches 70%~95%, collect the mesenchymal stem cells and resuspend them in phosphate-buffered saline (PBS) to form a single-cell suspension.

[0052] (2) Pass the single-cell suspension through microporous 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] IV. Identification of engineered nanovesicles derived from isolated apoptotic mesenchymal stem cells using transmission electron microscopy.

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

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

[0057] (3) Transfer the copper mesh onto the droplet of 3% glutaraldehyde fixative, fix for 5 minutes and then blot dry the fixative.

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

[0059] (5) The copper mesh was then suspended on the surface of a 4% acetic acid dicationic uranium droplet for 10 minutes before the remaining solution was absorbed.

[0060] (6) Finally, place the copper mesh in a 1% methylcellulose solution droplet, let it stand for 5 minutes, and then absorb the moisture.

[0061] (7) The copper mesh was left to stand at room temperature for more than 30 minutes, dried, and then observed and photographed by transmission electron microscopy. The observation results of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells are shown in Figure 1.

[0062] Figure 1 shows that the engineered nanovesicles derived from apoptotic placental mesenchymal stem cells were disc-shaped, slightly dark in the center and with a slightly brighter edge.

[0063] V. 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 a ZetaVIEW PMX 110. 100 μL of PBS solution containing engineered nanovesicles derived from apoptotic mesenchymal stem cells was diluted appropriately to achieve a final concentration of 10 μL / mL. 7 Approximately 100 vesicles were selected. The parameters were set as follows: particle size range 50–200 nm, molecular weight range 1000–20107 Da, temperature 25°C, 4.0 mV He-Ne laser, wavelength 633 nm. Each sample was analyzed three times consecutively, and the NTA values ​​were recorded and analyzed at different locations. ZetaVIEW 8.04.02 software was used for data analysis.

[0065] Figure 2 shows the particle size distribution of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells. The results in Figure 2 indicate that the particle size of the engineered nanovesicles derived from apoptotic mesenchymal stem cells is approximately 186 nm.

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

[0067] (1) Add 100 μL of lysis buffer 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 place them on crushed ice for 30 min for lysis.

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

[0069] (3) After the protein electrophoresis gel is prepared, take 5 μL of mesenchymal stem cells (MSC), mesenchymal stem cell vesicles (NVs) and engineered nanovesicles (AVs) derived from apoptotic mesenchymal stem cells and add them to the gel lanes. Add 5 μL of protein Maker to the adjacent lanes. Run the compression gel at 80V and the separation gel at 120V.

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

[0071] Figure 3 shows the polyacrylamide gel electrophoresis results of engineered nanovesicles derived from apoptotic placental mesenchymal stem cells. As shown in Figure 3, 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 combined results of electron microscopy, average particle size, and protein expression, the final product prepared in this embodiment conforms to the characteristics of engineered nanovesicles, indicating that the final isolated product in this embodiment is indeed an engineered nanovesicle derived from apoptotic placental mesenchymal stem cells.

[0073] Example 2

[0074] I. 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 to a brown bottle containing 0.05 g of initiator LAP to prepare a 0.25% (w / v) initiator LAP standard solution.

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

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

[0079] (2) Preparation of composite hydrogel

[0080] Following the preparation method of Example 1, engineered nanovesicles derived from apoptotic placental mesenchymal stem cells were prepared, and 8 × 10⁸ nanovesicles were taken. 8 Add one part to 1 mL of methacrylic anhydride gelatin solution and mix well. Irradiate with a 405 nm light source for 30 s to gel, and obtain composite hydrogel (abbreviated as Gel-AVs).

[0081] The structure of the methacrylic anhydride gelatin hydrogel was observed using a scanning electron microscope, and the results are shown in Figure 4.

[0082] The results in Figure 4 show that the surface of the composite hydrogel exhibits a network-like porous structure.

[0083] II. 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 (8 μm pore size, compatible with 24-well plates) containing PBS. At specific time intervals, the liquid in the lower chamber was collected and fresh PBS was added. The concentration of released AVs in the liquid was detected using a BCA protein assay kit. Release rate (%) = (detected concentration / total concentration) × 100%.

[0085] Figure 5 shows the release rate of apoptotic vesicles from the composite hydrogel. The results in Figure 5 indicate that the composite hydrogel can continuously release apoptotic vesicles, reaching its maximum release rate on the fourth day.

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

[0087] (1) Establishment of diabetic mouse model

[0088] Select C57BL / 6J mice, 7 weeks old, weighing approximately 20g, male; Modeling method: 1% streptozotocin (prepared and used immediately), administered at 150mg / kg for two consecutive days (fasting for 12 hours before administration), blood glucose was measured one week later (blood was collected by tail tip puncture, fasting), and the model was considered successful if the blood glucose value was greater than 16.6mM for three consecutive days.

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

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

[0091] Dosing strategy: Administer the medication starting the day after model establishment, in three divided doses, with each dose spaced 2-3 days apart.

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

[0093] Dosage: 100 μL per group, 100 mg for the AVs group and 100 mg of AVs for the Gel-AVs group.

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

[0095] Figure 6 shows the immunofluorescence of tissue sections from which the composite hydrogel was used to promote in vivo wound healing in diabetic patients. The results in Figure 6 indicate that the CD86 fluorescence in the Gel-AVs group was weaker than that in the PBS group, while the CD206 fluorescence was stronger than that in the PBS, GelMA, and AVs groups. This suggests that the Gel-AVs group contained more M2 macrophages, thus modulating the inflammatory environment in the wound. Simultaneously, the α-SMA / CD31 fluorescence was stronger than that in the PBS, GelMA, and AVs groups, indicating greater angiogenesis in the wound tissue of the Gel-AVs group. Overall, the composite hydrogel Gel-AVs showed significant therapeutic efficacy.

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

[0097] Example 3

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

[0099] I. Cell proliferation detection experiment

[0100] Stable HaCaT cells were seeded into 96-well plates at a density of 100 μL per well, at a density of 6 × 10⁶ cells / well. 4 Cells / mL. Cell adhesion was observed, and the culture medium was replaced. 100 μL of 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 apoptotic vesicles (AVs here are apoptotic vesicles derived from placental mesenchymal stem cells) was added to each well as the experimental group, with 5 replicates for each concentration. Simultaneously, HaCaT cells treated with 100 μL of medium served as the control group (corresponding to 0 in Figure 7), and wells containing 100 μL of cell-free medium served as the blank control group, with 5 replicates for each. After incubation in a constant temperature incubator for 24 h, the old culture medium was discarded, and 100 μL of serum-free medium containing 10% CCK-8 working solution was added. The cells were then transferred to the incubator and incubated for 1–4 h. The absorbance at 405 nm was measured using a microplate reader to detect cell viability, record the data, and calculate the cell proliferation rate. The formula for calculating cell proliferation rate is: (absorbance value of experimental group - absorbance value of blank control) / absorbance value of control group - absorbance value of blank control) × 100%.

[0101] Figure 7 shows the results of the assay for HaCaT cell proliferation using engineered nanovesicles derived from apoptotic placental mesenchymal stem cells. When the concentration of AVs was higher than 10 μg / mL, it significantly promoted the proliferation of HaCaT cells. The most significant effect was observed when the AVs concentration was 50–70 μg / mL, with the highest proliferation rate of 199.4%.

[0102] Stable HaCaT cells were seeded into 96-well plates at a density of 100 μL per well, at a density of 6 × 10⁶ cells / well. 4 Cells / mL. Cell adhesion was observed, and the culture medium was replaced. 100 μL of 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 apoptotic vesicles (AVs here are derived from bone marrow mesenchymal stem cells) was added to each well as the experimental group, with 5 replicates for each concentration. Simultaneously, HaCaT cells treated with 100 μL of medium served as the control group (corresponding to 0 in Figure 8), and wells containing 100 μL of cell-free medium served as the blank control group, with 5 replicates for each group. After incubation in a constant temperature incubator for 24 h, the old culture medium was discarded, and 100 μL of serum-free medium containing 10% CCK-8 working solution was added. The cells were then transferred to the incubator and incubated for 1–4 h. The absorbance at 405 nm was measured using a microplate reader to detect cell viability, record the data, and calculate the cell proliferation rate. The formula for calculating cell proliferation rate is: (absorbance value of experimental group - absorbance value of blank control) / absorbance value of control group - absorbance value of blank control) × 100%.

[0103] Figure 8 shows the results of the assay for HaCaT cell proliferation using engineered nanovesicles derived from apoptotic bone marrow mesenchymal stem cells. When the concentration of AVs was higher than 10 μg / mL, it promoted the proliferation of HaCaT cells. The most significant effect was observed when the AVs concentration was 50 μg / mL, with the highest proliferation rate of 145.5%.

[0104] Comparing Figures 7 and 8, both apoptotic vesicles of placental mesenchymal stem cells and bone marrow mesenchymal stem cells can promote cell proliferation. Moreover, at the same concentration, the apoptotic vesicles of placental mesenchymal stem cells have a better ability to promote cell proliferation than the apoptotic vesicles of bone marrow mesenchymal stem cells.

[0105] II. Cell Uptake Experiment

[0106] After resuspending healthy RAW264.7 cells, they were divided into two groups and seeded in confocal microplates: a control group (blank control group) and AVs (AVs are apoptotic vesicles derived from placental mesenchymal stem cells, concentration 50 μm / mL). The cell density was 20 w / mL, 1 mL per well, and incubated for 24 h. DIO working solution was prepared by adding 1 μL each of DIO and staining enhancer to 500 μL of AVs (50 μg / mL) solution, vortexing, and then adding 500 μL of cell culture medium. The mixture was incubated in the dark for 15 min. The culture medium in the confocal microplates was removed, and the prepared incubation solution was added to the confocal microplates for co-incubation for 4 h. Lyso-Tracker Red working solution was prepared by adding 1 µL of Lyso-Tracker Red to 15 mL of warm cell culture medium and mixing well. (Lyso-Tracker Red working solution needs to be preheated to 37°C before use.) Remove the culture medium from the confocal microscopy dish and wash twice with PBS. Add the prepared Lyso-Tracker Red staining working solution pre-incubated at 37°C and incubate with the cells at 37°C for 40 min. Then remove the Lyso-Tracker Red staining working solution, wash twice with PBS, and fix with paraformaldehyde for 30 min. Nuclear staining: Remove the fixative, wash twice with PBS, then add a small amount (500 μL) of DAPI staining solution to cover the sample and incubate at room temperature for 3-5 min. Aspirate the DAPI staining solution, wash 2-3 times with PBS for 3-5 min each time, and then use for confocal imaging.

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

[0108] III. Cell Scratch Test

[0109] The Culture-Insert was tightly attached to the 6-well plate, and samples were prepared with a density of 3×10⁻⁶. 5HaCaT cell suspension was added at a rate of 70 μL per well to each well of a Culture-Insert 2 plate, taking care to avoid moving the insert. The insert and the plate were then transferred to an incubator at 37°C and 5% CO2 for 24 hours. Cell density was checked under a microscope; once ideal confluence was achieved, the Culture-Insert was gently removed with sterile forceps. The plates were then washed with PBS to remove cell debris and non-attached cells. Finally, culture medium containing 5% fetal bovine serum (FBS) was added at protein concentrations of 30 μg / mL, 50 μg / mL, and 70 μg / mL, respectively. The plates were incubated simultaneously, with a control group receiving the same volume of FBS. Each treatment group had three replicates. At different time points, scratch wound changes were observed and photographed using an inverted microscope, and the scratch healing rate was calculated using the following formula:

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

[0111] Figure 10 shows the migration of HaCaT cells promoted by engineered nanovesicles derived from apoptotic placental mesenchymal stem cells; Figure 11 shows the quantitative map of the migration area of ​​HaCaT cells promoted by engineered nanovesicles derived from apoptotic placental mesenchymal stem cells. As shown in Figures 10 and 11, compared with the blank control group, the scratch healing of cells in each group with added AVs was significantly accelerated, showing a significant migration-promoting effect.

[0112] IV. Macrophage Reprogramming Experiment

[0113] First, resuspend healthy RAW264.7 cells and seed them into 12-well plates at a density of 5 × 10⁻⁶ mL per well. 5Cells were cultured at concentrations of 500 ng / mL and 4 ng / mL, respectively. Simultaneously, lipopolysaccharide (LPS) and IFN-γ solution were added to each well. After 24 h of induction, 0 μg / mL, 50 μg / mL, and 70 μg / mL of apoptotic vesicles (AVs derived from placental mesenchymal stem cells) were added to each group for co-incubation. After 48 h of culture, cells were collected, resuspended in PBS, and centrifuged. The cells were then resuspended in 100 μL of PBS buffer, and 1 μL of flow cytometry antibodies for CD11b, CD86, and CD206 were added for staining for 30 min. Excess antibodies were removed by centrifugation, and the cells were then analyzed by flow cytometry. CD86 is a protein highly expressed after polarization of M1 macrophages; CD206 is generally considered a marker of M2 macrophages.

[0114] Figure 12 shows the flow cytometry results of reprogramming macrophage phenotypes using engineered nanovesicles derived from apoptotic placental mesenchymal stem cells. After the addition of AVs, the expression of the M1 marker CD86 in the cells was significantly reduced, while the expression of the M2 marker CD206 was significantly increased. This indicates that AVs successfully reversed the phenotype of some M1 macrophages.

[0115] V. Transwell Chemotaxis Experiment

[0116] HUVEC cells in good growth condition were resuspended in serum-free medium to achieve a cell density of 4 × 10⁶ cells / year. 6 Cells were seeded in 24-well plates with 8 μm pores. 200 μL of the cell suspension was added to the upper chamber, and 600 μL of 10% FBS medium containing 0 μg / mL, 50 μg / mL, and 70 μg / mL AVs (AVs are apoptotic vesicles derived from placental mesenchymal stem cells) and 50 μg / mL NVs were added to the lower chamber, respectively. Three replicates were prepared for each group. After culturing the cell-filled Transwell chambers in an incubator for 48 h, the cells in the upper chamber were wiped off with a cotton ball. The chambers were carefully removed with forceps, the medium in the upper chamber was aspirated, and the Matrigel and cells in the upper chamber were gently wiped off with a cotton swab. 600 μL of 4% paraformaldehyde was added to a new 24-well plate, and the chambers were placed in the plate and fixed at room temperature for 20 min. The chambers were then removed, the fixative in the upper chamber was aspirated, and the cells were transferred to wells pre-filled with approximately 600 μL of crystal violet and stained at room temperature for 10 min. After staining, the chambers are rinsed with pure water 3-6 times and then photographed under a microscope.

[0117] The Transwell results of HUVEC migration induced by engineered nanovesicles derived from apoptotic placental mesenchymal stem cells are shown in Figure 13. Compared with the control group, more cells in the NVs group penetrated the membrane, while more cells in the AVs group penetrated the membrane than in the NVs group. This indicates that AVs can induce cell migration and that HUVEC cells can exhibit chemotaxis towards AVs.

[0118] In summary, the engineered nanovesicles derived from apoptotic mesenchymal stem cells used in the composite hydrogel of this invention exhibit high preparation efficiency. Furthermore, AVS (Apoptotic Mesenchymal Stem Cells) are readily taken up by cells, demonstrating excellent effects in promoting cell proliferation and migration, and regulating inflammatory responses. Moreover, the composite hydrogel exhibits extremely low cytotoxicity, enabling sustained release of engineered nanovesicles derived from apoptotic mesenchymal stem cells, which can regulate the inflammatory environment and effectively promote angiogenesis and tissue regeneration. Simultaneously, the microporous membrane extrusion method of this invention achieves high yield, approximately 7 × 10⁻⁶. 6 Each cell can collect 800 μg of engineered apoptotic nanovesicles while ensuring the proper function of the apoptotic vesicles, and has greater potential for transformation.

[0119] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for the preparation of engineered nanometric apoptotic vesicles of mesenchymal stem cell-derived apoptosis, characterized by, Includes the following steps: Mesenchymal stem cells were subjected to apoptosis treatment to obtain apoptotic cells; The apoptotic cells were extruded using a microporous filter membrane to obtain engineered nanoapoptotic vesicles derived from apoptotic mesenchymal stem cells.

2. The production method according to claim 1, characterized by, The mesenchymal stem cells include placental mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose-derived mesenchymal stem cells.

3. The preparation method according to claim 1, characterized in that, The apoptosis 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 time of the ultraviolet irradiation is 15-60 min.

5. The preparation method according to claim 3, characterized in that, The ultraviolet irradiation process also includes incubation; the incubation conditions are: incubation at 37°C in a 5% CO2 incubator for 4-6 hours.

6. The preparation method according to claim 1, characterized in that, The microporous filter membrane is a microporous filter membrane 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 materials include the following: engineered nanovesicles derived from apoptotic mesenchymal stem cells as described in claim 7 and a methacrylic anhydride-modified gelatin solution; the preparation materials for the methacrylic anhydride-modified gelatin solution include LAP photoinitiator and methacrylic anhydride-modified gelatin.

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

10. The use of engineered nanovesicles derived from apoptotic mesenchymal stem cells as described in claim 7 or the composite hydrogel as described in claim 8 or 9 in the preparation of wound healing drugs.

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