Exosome as well as preparation method and application thereof

Abstract

The invention relates to an exosome as well as a preparation method and application thereof. The 3D culture carrier can slowly and continuously release NO, and the released NO can stimulate stem cells, so that the expression level of VEGF, miR-126 and other cell factors released into the exosome is greatly increased. Compared with an exosome obtained by a general means, the extracted exosome (3D-NO-exo) has better effects of promoting angiogenesis, promoting cell proliferation and regulating inflammatory response. Although the 3D culture carrier contains an NO donor, the separated and extracted 3D-NO-exo does not directly contain NO, so that possible potential harm of NO to a human body under specific conditions can be avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical materials, and in particular to exosomes and a preparation method and application thereof. Background Art

[0002] Diabetes is a common chronic disease characterized by elevated blood glucose concentrations, primarily related to the abnormal biology of beta cells and their effects on insulin action. Of particular concern is that patients with diabetes are characterized by a high number of complications, of which 19% to 34% will develop refractory diabetic wounds such as diabetic foot (DFU). Diabetic foot ulcers (DFU) are chronic wounds that are difficult to heal and, if not properly managed, can lead to serious consequences, including high mortality and amputation rates. The 1-year mortality rate is 14.4%, and the total amputation rate is 19.3%. Diabetic foot ulcers are the main source of preventable morbidity in adults with diabetes. The consequences of foot ulcers include decreased functional status, infection, hospitalization, lower limb amputation, and death. The lifetime risk of foot ulcers is 19% to 34%, and this number is rising as diabetic patients live longer and their medical complexity increases. The morbidity after an ulcer occurs is high, with a 3-5 year recurrence rate of 65%, a lifetime lower limb amputation rate of 20%, and a 5-year mortality rate of 50-70%.

[0003] The healing process of diabetic wounds is affected by multiple factors, including infection and biofilm, chronic inflammation, poor angiogenesis, and reduced collagen production. These factors work together to make diabetic wounds difficult to heal. For example, persistent chronic inflammation not only delays wound healing, but may also lead to further tissue damage. At the same time, poor angiogenesis limits the supply of necessary nutrients and oxygen, further hindering the healing process. In addition, reduced collagen production affects the structural integrity and strength of the wound. Classifying ulcers according to the degree of tissue loss, ischemia, and infection helps identify the risk of limb-threatening diseases. Compared with conventional care, there are several interventions that can reduce the risk of ulcers, such as decompression shoes and treatment of pre-ulcer signs. Surgical debridement, reducing the pressure of weight-bearing on ulcers, and treating lower limb ischemia and foot infections are the main treatments. However, it often brings great pain to patients and cannot effectively and quickly heal them. Therefore, finding effective treatments is crucial to improving the quality of life of these patients, and it is urgent to solve the problem of difficult healing of diabetic wounds in future research.

[0004] Existing studies have shown that some exosomes cultured under specific conditions have shown significant efficacy in accelerating wound healing, including mechanisms such as promoting angiogenesis, cell proliferation, and regulating inflammatory responses. However, because different culture conditions have a significant impact on the yield and quality of exosomes, exploring how to obtain exosomes that have more advantages such as promoting angiogenesis, cell proliferation, and regulating inflammatory responses has become a hot topic in the current biomedical field. Summary of the invention

[0005] The purpose of the present invention is to disclose an exosome and a preparation method and application thereof, so as to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0006] The first aspect of the present invention is to provide a method for preparing exosomes.

[0007] The second aspect of the present invention is to provide exosomes prepared by the preparation method described in the first aspect of the present invention.

[0008] The third aspect of the present invention is to provide an application direction of the exosomes described in the second aspect of the present invention.

[0009] The method for preparing exosomes according to the first aspect of the present invention comprises the following steps: 1) preparing an inner layer solution and a surface layer solution respectively, wherein the inner layer solution contains stem cells and a stem cell culture matrix, and the surface layer solution includes a polymer material with gelling properties and an NO donor; 2) using a biological 3D printing device to prepare a 3D culture carrier precursor in which the inner layer solution is wrapped by the surface layer solution, and gelling the surface layer solution to obtain a 3D culture carrier; 3) placing the 3D culture carrier in a culture medium to induce stem cells to secrete exosomes; 4) isolating and extracting the exosomes.

[0010] Nitric oxide (NO) is an attractive therapeutic agent due to its ability to regulate inflammation and angiogenesis in wound healing. In many chronic ulcers, especially those for diabetic patients, continuous supplementation of NO can improve the healing process, so stents that can continuously release NO have become a new therapeutic strategy to promote diabetic wound healing.

[0011] The 3D culture carrier of the present invention can slowly and continuously release NO, which can stimulate stem cells and significantly increase the expression levels of cytokines such as vascular endothelial growth factor (VEGF) and microRNA-126 (miR-126) released into exosomes. The extracted exosomes (3D-NO-exo) have better effects on promoting angiogenesis, cell proliferation and regulating inflammatory response than exosomes obtained by general means.

[0012] In a further application embodiment, the NO donor is selected from organic nitrates, nitrosoimines or nitrosothiols.

[0013] In a further application embodiment, the organic nitrate NO donor may be nitroglycerin or isosorbide dinitrate; the nitrosoimine NO donor may be S-nitroso-N-acetyl-DL-penicillamine (SNAP); and the nitrosothiol NO donor may be nitrosothiol glutathione (GSNO).

[0014] In a further application embodiment, the stem cells are umbilical cord mesenchymal stem cells (UC-MSCs) and / or adipose-derived mesenchymal stem cells (ADMSCs).

[0015] In a further application embodiment, the polymer material with gelling properties is selected from at least one of sodium alginate, gelatin, hyaluronic acid, chondroitin sulfate or polyethylene glycol. The sodium alginate mainly uses a calcium ion-containing crosslinker; the gelatin mainly uses an isocyanate crosslinker; the hyaluronic acid commonly uses 1,4-butanediol diglycidyl ether, divinyl sulfone, toluene diisocyanate, etc. as a crosslinker; the chondroitin sulfate mainly uses ethylene glycol dimethacrylate (EDGMA) as a crosslinker; the polyethylene glycol mainly uses polyethylene glycol SMCC crosslinker. The crosslinker can be added to the receiving container, and the printed product extruded by the coaxial microfilament printing device is directly injected into the receiving container, so that the surface solution is in direct contact with the crosslinker, thereby crosslinking and forming a 3D culture carrier. The outer layer of the 3D culture carrier locks the inner layer solution inside to prevent the loss of the stem cell suspension, providing a 3D culture environment for stem cells.

[0016] In a further application embodiment, the concentration of the stem cells contained in the inner layer solution is 5×10 6 / mL to 5×10 9 Pieces / mL.

[0017] In a further application embodiment, the inner layer solution also contains a NO donor with a mass concentration of 0.05%. The NO donor added to the inner layer solution can provide NO stimulation to stem cells more directly, but due to the lack of sustained release control means, it can only play an early promotion role.

[0018] In further application implementations, the polymer material with gelling properties can be modified to achieve a photo-crosslinking effect. For example, natural polymer materials modified by methacrylation: methacrylated gelatin (GelMA), methacrylated chitosan, methacrylated chondroitin sulfate, methacrylated sodium alginate, etc. A photoinitiator is added to the surface solution of the methacrylated natural polymer material, and when the coaxial microfilament printing device extrude the printed product, it is directly exposed to the irradiation environment of ultraviolet light or blue light, so that the printed product is quickly cross-linked and formed.

[0019] In a further application embodiment, the surface solution includes the polymer material at a mass concentration of 1.5-3% and the NO donor at a mass concentration of 0.01-5%. The biological function of NO is concentration-dependent, and it can only provide stimulation to stem cells within a suitable concentration range, thereby increasing the expression level of cytokines in exosomes.

[0020] The exosomes 3D-NO-exo described in the second aspect of the present invention are prepared by the above-mentioned preparation method.

[0021] Although the 3D culture carrier provided by the present invention contains NO donor, the separated and extracted 3D-NO-exo does not directly contain NO, which can avoid the potential harm of NO to the human body under specific conditions.

[0022] The application described in the third aspect of the present invention refers to the use of the 3D-NO-exo for preparing a drug, which has the effect of inhibiting chronic inflammation and / or promoting wound repair.

[0023] The beneficial effect of the present invention is that the loaded stem cells and NO donors are combined, and the 3D culture carrier constructed by biological 3D printing technology is used to produce exosomes. While obtaining a higher output, the stem cells can also release more cytokines into the exosomes 3D-NO-exo, so that the 3D-NO-exo can achieve the effects of promoting angiogenesis, promoting cell proliferation and regulating inflammatory response far exceeding conventional exosomes. The 3D-NO-exo can be used for rapid repair of diabetic wounds to help patients relieve pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a bar graph of the relative expression of various biomolecules in different exosomes; Figure 2 These are experimental photos of the effects of different exosomes on the migration of HUVECs; Figure 3 It is a line graph showing the effects of different exosomes on the proliferation capacity of HUVEC; Figure 4 These are experimental photos of the effects of different exosomes on the migration of HACAT; Figure 5 It is a line graph showing the effects of different exosomes on the value-added capacity of HACAT; Figure 6 is the effect of different exosomes on HUVEC in the Transwell cell migration assay; Figure 7 is the effect of different exosomes on the tube-forming ability of HUVECs; Figure 8 is the effect of different exosomes on wound healing in nude mice; Fig. 9 This is the HE staining result of different exosomes on wound healing in nude mice. DETAILED DESCRIPTION

[0025] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention.

[0026] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0027] Example 1: Preparation of 3D-NO-exo via coaxial filamentous carriers.

[0028] (1) Cultivation of ADMSCs.

[0029] ADMSCs (Sciencell TM , Research Laboratories, Inc.) were placed in a 150 mm cell culture dish, 25-30 mL of MSC culture medium (7521, Sciencell, USA) was added, and cultured at 37°C and 5% CO2 to obtain ADMSCs suspension.

[0030] (2) Preparation and cultivation of three-dimensional culture carriers.

[0031] The prepared ADMSCs suspension was used as the inner fluid, and a solution containing 1.5-3% sodium alginate and 0.5-5% SNAP was used as the surface fluid. The solution was extruded into a 3-4% CaCl2 solution through a coaxial microfilament printing device to complete gelation, forming a 3D culture carrier with an outer gel encapsulating the inner cell suspension. The inner core was formed by mixing ADMSCs or UC-MSCs with 0.5% SNAP to form a core flow. The 3D culture carrier was rinsed three times with a 0.9% sodium chloride solution and transferred to a new culture dish. 25-30 mL of DMEM culture medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) was added and incubated at 37°C and 5% CO2.

[0032] (3) Isolation of exosomes.

[0033] The culture supernatant can be directly collected after every 24 or 48 hours of incubation.

[0034] The DMEM medium collected for culturing the 3D culture carrier was subjected to sequential centrifugation and filtration, first at 300×g for 10 minutes to precipitate intact cells, and then at 2000×g for 30 minutes to precipitate dead cells. The partially purified medium from which cells and cell debris were removed was then centrifuged at 10,000×g for 30 minutes. The supernatant was filtered through a 0.22 μm filter to remove vesicles or protein aggregates. Next, the supernatant was centrifuged at 100,000×g for 70 to 80 minutes. The resulting precipitate was washed with PBS, transferred to a new tube, and centrifuged at 100,000×g for 70 minutes. The 3D-NO-exo was obtained.

[0035] All centrifugations were performed at 4°C using a benchtop ultracentrifuge.

[0036] Example 2: Preparation of 3D-NO-exo via core-shell microsphere carriers.

[0037] (1) Culture of UC-MSCs.

[0038] UC-MSCs (Sciencell TM , Research Laboratories, Inc.) were placed in a 150 mm cell culture dish, 25-30 mL of MSC culture medium (7521, Sciencell, USA) was added, and cultured at 37°C and 5% CO2 to obtain UC-MSCs suspension.

[0039] (2) The prepared UC-MSCs suspension was sucked into a 1 mL syringe as the inner fluid, and a solution containing 1.5-3% methacryloyl gelatin and 0.5-5% Diazeniumdiolate (NONOates) was sucked into a 1 mL syringe as the surface fluid (containing 0.3% LAP photoinitiator). Corn oil was sucked into a 50 mL syringe. The three syringes were loaded onto the corresponding core, shell, and oil phase bins of the droplet 3D printing device to print the spherical 3D culture carrier, and the printed product was cross-linked by UV curing.

[0040] (3) The method for isolating exosomes is the same as that in Example 1.

[0041] Example 3: Comparative experiment of existing exosomes and 3D-NO-exo.

[0042] (1) Preparation of 2D-exo.

[0043] About 1.5×10 6 ADMSCs (Sciencell TM , Research Laboratories) were inoculated into a 150 mm culture dish and cultured to 60-80% confluence, rinsed two to three times with PBS buffer (Gibco, USA), 5 minutes each time, and then the culture medium was replaced with MSC conditioned medium (NC0103, Yocon, China). Then, the exosomes were separated and purified according to the exosome separation method provided in Example 1 to obtain planar cultured exosomes 2D-exo.

[0044] (2) Preparation of 3D-exo.

[0045] According to the operation process provided in Example 1, only SNAP as the NO donor is discarded, thereby obtaining exosomes 3D-exo in which the stem cells are not in contact with NO during the culture process.

[0046] The 3D-NO-exo extracted in Example 1 and the 2D-exo and 3D-exo prepared above were tested and analyzed. Figure 1 As shown, the contents of ACAT2, CD31, and VEGF in the 3D-NO-exo group were significantly different from those in the 2D-exo group and the 3D-exo group; the content of Col1a1 in the 3D-NO-exo group was similar to that in the 3D-exo group, but significantly higher than that in the 2D-exo group.

[0047] Example 4: Cell scratch migration assay and CCK-8 assay.

[0048] (3) Human immortalized keratinocytes (HACAT) and human umbilical vein endothelial cells (HUVEC) were used as the experimental objects to investigate the effects of 2D-exo, 3D-exo and 3D-NO-exo on cell migration ability.

[0049] The density is 5×10 5 HACAT and HUVEC were inoculated in six-well culture plates. They were cultured in a humidified incubator at 37°C and 5% CO2. When the cell confluence reached 90%, they were washed three times with PBS and the culture medium was replaced with fresh FBS-free culture medium. A sterile 200 μL pipette tip was then used to scrape the confluent cell monolayer. No exosomes were added to the blank control group (Control group). 5×10 8 Cells were treated with exosomes at 1000 particles / mL. Images were collected at 0, 12, 24, and 48 hours after exosome addition. The migration area was measured and quantified.

[0050] CCK-8 experiments were performed on each group simultaneously.

[0051] The results are as follows Figures 2 to 5 shown.

[0052] The effects of different exosomes on the migration and proliferation ability of HUVECs. Figure 2 In the middle (A), it can be observed that: there is no significant change between 24 hours and 0 hours in the Control group; a certain degree of scratch narrowing can be observed in the 2D-exo group at 24 hours compared with 0 hours; obvious scratch narrowing can be observed in the 3D-exo group at 24 hours compared with 0 hours; and significant scratch narrowing can be observed in the 3D-NO-exo group at 24 hours compared with 0 hours. By comparing the 24-hour scratch narrowing distance, the following can be obtained: Figure 2 In the column shown in the middle (B), the migration rate of the 3D-NO-exo group is as high as 60%, which is much higher than that of other groups.

[0053] Figure 3 The line graph of the CCK-8 experiment shown validates the results of the scratch assay, confirming that 3D-NO-exo can effectively promote the proliferation capacity of HUVECs.

[0054] Figure 4 The effects of different exosomes on the migration and proliferation ability of HACAT. Figure 4In the middle (A), it can be observed that: there was no significant change between 48 hours, 24 hours and 0 hours in the Control group; compared with the situation at 0 hours, a certain degree of scratch narrowing can be observed in the 2D-exo group at 24 hours, and there is also a certain degree of scratch narrowing at 48 hours; the scratch narrowing in the 3D-exo group at 24 hours is similar to that in the 2D-exo group at 48 hours, and the scratch narrowing in the 3D-exo group at 48 hours is more obvious; compared with the situation at 0 hours, a significant scratch narrowing can be observed in the 3D-NO-exo group at 24 hours, and the degree of narrowing is similar to the photo of the 3D-exo group at 48 hours. The photo of the 3D-NO-exo group at 48 hours shows that cells on both sides of some areas have contacted each other, and the scratches in most areas are already very narrow. By comparing the scratch narrowing distance at 48 hours through the software, the following can be obtained: Figure 4 In the column shown in the middle (B), the migration rate of the 3D-NO-exo group is as high as 60%, which is much higher than that of other groups.

[0055] Figure 5 The line graph of the CCK-8 experiment shown validates the results of the scratch assay, confirming that 3D-NO-exo can effectively promote the proliferation ability of HACAT.

[0056] Example 5, Transwell cell migration assay.

[0057] HUVECs were cultured in DMEM medium containing 10% fetal bovine serum (FBS) in an environment of 5% CO2 and a temperature of 37°C. 4 HUVECs were seeded into the upper chamber of a Transwell plate (Corning, 3422), and 600 μL of fresh culture medium (Control group) or exosome-enriched culture supernatant containing 10% FBS (2D-exo group, 3D-exo group, and 3D-NO-exo group) were added to the lower chamber for 12 hours. Subsequently, the migrated cells that had passed through the basolateral membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cell invasion was observed and recorded using an optical microscope (Mshot, China).

[0058] Observation results such as Figure 6 As shown in (A), no obvious migration was observed in the Control group, and the migration of the 2D-exo group, 3D-exo group, and 3D-NO-exo group increased in turn. The stained cells in the photo were quantitatively analyzed using image recognition software. The results are shown in Figure 6 As shown in (B), the migration amount of the 3D-NO-exo group was significantly increased compared with the other groups.

[0059] Example 6, cell tube formation experiment.

[0060] Tube formation assay was used to evaluate the angiogenic properties of various exosomes. 50 μL of Matrigel containing 50% DMEM was pipetted into a 96-well plate on ice and then placed in a 37°C incubator for 30 minutes to gel. HUVEC were cultured in FBS-free medium and seeded on the surface of Matrigel after starvation for 12 hours. HUVEC (2×10 4 In the DMEM medium containing 1% FBS, the corresponding exosomes were added to the control group, 2D-exo group, 3D-exo group and 3D-NO-exo group, 5×10 8 The particles were treated and the treated cells were seeded on the surface of a 96-well Matrigel plate (Corning, USA). After 2 hours of incubation, images were taken under a microscope and analyzed using image recognition software. The number of grids, main segments, total branch length, and total number of nodes were compared. The analysis results are shown in Figure 7 As shown, it was confirmed that 3D-NO-exo can effectively promote HUVEC tube formation.

[0061] Example 7, animal experiment on chronic diabetic wounds.

[0062] (1) Construction of diabetic chronic wound mouse model C57BL / 6 mice (after 12 hours of fasting) were used to establish a diabetes model. Streptozotocin (STZ, 50 mg / kg) dissolved in sodium citrate buffer (0.1 mol / L, pH 4.5) was injected intraperitoneally for 5 consecutive times. One week after the injection, the fasting blood glucose level of the mice was tested for 2 consecutive days. If the fasting blood glucose level was >16.7 mmol / L, they were selected as diabetic mice.

[0063] After shaving the back of the selected diabetic mice, 0.1 mg / kg buprenorphine was subcutaneously injected for analgesia before surgery, and 2% isoflurane anesthesia was used under a sterile surgical environment. The wound was prepared 2 cm below the scapula in the middle of the back, and a circular mark (2.54 cm²) was pressed perpendicularly to the skin with a 18 mm diameter trephine. The full-thickness skin (up to the deep fascia) was cut along the mark to form a circular full-thickness skin defect wound. The wounds were randomly divided into 5 groups: Control group (blank control), HA group (positive control), 2D-exo group, 3D-exo group, and 3D-NO-exo group.

[0064] (2) Wound healing analysis: The wound area was observed and measured on the 3rd, 7th and 14th days. Figure 8 As shown in (A).

[0065] The calculation formula for the percentage of wound area is: Wound area (%) = residual area / original area × 100%.

[0066] The measured wound area was analyzed using image recognition software. The analysis results are shown in Figure 8 In the middle (B), the healing effect of mice in the 3D-NO-exo group was significantly better than that in other groups.

[0067] After 14 days, the tissues around the wound were collected for H&E staining. Fig. 9 As shown, the skin structure of mice in the 3D-NO-exo group was more complete and the connective tissue was more regular, while the other groups showed irregular epidermal hyperplasia and hyperkeratosis.

[0068] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

Claims

1. A method for preparing exosomes, characterized in that: Includes steps: 1) preparing an inner layer solution and a surface layer solution respectively, wherein the inner layer solution contains stem cells and a stem cell culture matrix, and the surface layer solution includes a polymer material with gelling properties and an NO donor; 2) using a biological 3D printing device to prepare a 3D culture carrier precursor in which the inner layer solution is wrapped by the surface layer solution, and gelling the surface layer solution to obtain a 3D culture carrier; 3) placing the 3D culture carrier in a culture medium to induce stem cells to secrete exosomes; 4) isolating and extracting the exosomes.

2. The method for preparing exosomes according to claim 1, characterized in that: The NO donor is selected from organic nitrates, nitrosoimines or nitrosothiols.

3. The method for preparing exosomes according to claim 2, characterized in that: The NO donor is selected from nitroglycerin, isosorbide dinitrate, S-nitroso-N-acetyl-DL-penicillamine or nitrosothiol glutathione.

4. The method for preparing exosomes according to claim 1, characterized in that: The stem cells are umbilical cord mesenchymal stem cells and / or adipose mesenchymal stem cells.

5. The method for preparing exosomes according to claim 4, characterized in that: The concentration of the stem cells contained in the inner solution is 5×10 6 / mL to 5×10 9 Pieces / mL.

6. The method for preparing exosomes according to claim 5, characterized in that: The inner layer solution also contains a NO donor with a mass concentration of 0.05%.

7. The method for preparing exosomes according to claim 1, characterized in that: The polymer material is selected from at least one of sodium alginate, gelatin, hyaluronic acid, chondroitin sulfate or polyethylene glycol.

8. The method for preparing exosomes according to claim 7, characterized in that: The surface solution includes the polymer material with a mass concentration of 1.5-3% and the NO donor with a mass concentration of 0.01-5%.

9. An exosome, characterized in that Prepared by the exosome preparation method according to any one of claims 1 to 8.

10. Use of the exosomes according to claim 9 in the preparation of a drug, wherein the drug has the efficacy of inhibiting chronic inflammation and / or promoting wound repair.

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