Use of vesicles in the preparation of extracorporeal blood coagulants or extracorporeal hemostatic agents

Abstract

Use of inducible vesicles in the preparation of extracorporeal blood coagulation promoters or extracorporeal hemostatic agents. The inducible vesicles are obtained by adding an apoptosis inducer and contacting them with stem cells or somatic cells. Expression of tissue factor is an important factor for the inducible vesicles to achieve extracorporeal blood coagulation promoters or extracorporeal hemostatic agents.

Description

[Technical Field]

[0001] The present disclosure is in the field of biomedicine and relates to the use of vesicles in the preparation of extracorporeal blood coagulants or extracorporeal hemostatic agents. [Background technology]

[0002] More than 30% of deaths worldwide are due to uncontrolled bleeding from surgical procedures, traffic accidents, or war wounds. Therefore, rapid and effective hemostasis is crucial to reduce the mortality and morbidity of acute wound-related injuries. Conventional hemostatic materials, including hemostatic gauze, hemostatic bandages, gelatin sponges, and hemostatic powder, do not provide ideal hemostatic effects. Therefore, the development of rapid, highly effective, and safe hemostatic agents is the primary goal of current hemostatic materials.

[0003] Extracellular vesicles (EVs) are nano-sized particles with a membrane structure that contain proteins, nucleic acids, and various cytokines secreted by cells. Extracellular vesicles are generally divided into three types: exosomes, microvesicles, and apoptotic vesicles. Currently, research in this field has focused primarily on exosomes. Exosomes are extracellular vesicles with diameters of approximately 30–150 nm and contain components such as RNA, lipids, and proteins. Research has shown that exosomes play an important role in maintaining internal stability and promoting tissue repair. Currently, mesenchymal stem cells (MSCs) are considered to be the cells with the strongest exosome-producing capacity. Numerous studies have shown that exosomes derived from MSCs have broad application prospects and play an important role in the repair and regeneration of various tissue injuries. However, there are still many challenges facing exosome-based extracellular vesicle therapy. For example, the exosome extraction and purification process is complex and time-consuming, has high requirements for equipment and reagents, and produces low physiological exosome yields, all of which limit the clinical translation and application of exosome therapy. Summary of the Invention

[0004] In some embodiments, the present disclosure provides for the use of inducible vesicles in the preparation of an extracorporeal procoagulant or extracorporeal hemostatic agent.

[0005] In some embodiments, the present disclosure provides a method for treating extracorporeal blood procoagulation or extracorporeal hemostasis, comprising administering inducible vesicles to a patient or subject.

[0006] In some embodiments, the present disclosure provides inducible vesicles for use in extracorporeal blood procoagulation or extracorporeal hemostasis.

[0007] In some embodiments, the inducible vesicles provide inducible vesicles that are positive for tissue factor expression.

[0008] Through research, the inventors have discovered that, unlike the treatment of hemophilia with inducible vesicles, the expression of tissue factor (TF) is an important factor in treating extracorporeal bleeding, and vesicles lacking TF expression cannot effectively achieve extracorporeal hemostasis.

[0009] In some embodiments, the present disclosure further provides a method for screening in vitro blood coagulation promoting agents or in vitro hemostatic agents, detecting the expression of tissue factor in inducible vesicles as candidate drugs to be screened, and screening inducible vesicles that are positive for tissue factor expression.

[0010] In some embodiments, the present disclosure further provides a method for quality inspection, quality control, or quality evaluation of an extracorporeal blood coagulation promoting drug or extracorporeal hemostatic drug, the method comprising: inspecting inducible vesicles; using tissue factor expression parameters as evaluation indicators; and determining that the quality of the drug is acceptable when the tissue factor expression parameters reach a predetermined level.

[0011] In some embodiments, the tissue factor expression parameter is the tissue factor expression level or the tissue factor expression positivity rate.

[0012] In some embodiments, the tissue factor expression positivity rate is the percentage of induced vesicles that are positive for tissue factor expression to the total number of induced vesicles.

[0013] In some embodiments, the present disclosure provides for the use of an inducible vesicle product with increased tissue factor expression positivity in the preparation of an extracorporeal procoagulant or extracorporeal hemostatic agent.

[0014] In some embodiments, the present disclosure provides a method for treating extracorporeal blood coagulation or extracorporeal hemostasis, comprising administering to a patient or subject inducible vesicles having an increased positive tissue factor expression rate.

[0015] In some embodiments, the present disclosure provides inducible vesicles with increased tissue factor expression positivity for use in extracorporeal blood procoagulation or extracorporeal hemostasis.

[0016] In some embodiments, the induced vesicle product with an improved tissue factor expression positivity rate has a percentage of induced vesicles that are positive for tissue factor expression of the total induced vesicles that is above a predetermined level.

[0017] In some embodiments, the predetermined level is a tissue factor expression positivity rate of ≧30%.

[0018] In some embodiments, the predetermined level is a tissue factor expression positivity rate of ≧31%.

[0019] In some embodiments, the predetermined level is a tissue factor expression positivity rate of ≧32%.

[0020] In some embodiments, the predetermined level is a tissue factor expression positivity rate of ≧35%.

[0021] In some embodiments, the predetermined level is a tissue factor expression positivity rate of ≧40%.

[0022] In some embodiments, the predetermined level is a tissue factor expression positivity rate of ≧45%.

[0023] In some embodiments, the predetermined level is a tissue factor expression positivity rate of ≧50%.

[0024] In some embodiments, the predetermined level is a tissue factor expression positivity rate of ≧55%.

[0025] In some embodiments, the predetermined level is a tissue factor expression positivity rate of ≧60%.

[0026] In some embodiments, the present disclosure provides a composition comprising an inducible vesicle and tissue factor.

[0027] In some embodiments, the composition further comprises a blood coagulant or hemostatic agent.

[0028] In some embodiments, the procoagulant or hemostatic agent is one or more selected from prothrombin, thrombin, fibrinogen, fibrin, adhesion proteins, clotting factors, collagen, gelatin, vasopressin, plasminogen activator inhibitors, platelet activators, and synthetic peptides with hemostatic activity.

[0029] In some embodiments, the present disclosure provides for the use of the composition in the preparation of an extracorporeal blood procoagulant or extracorporeal hemostatic agent.

[0030] In some embodiments, the present disclosure provides a method of treating extracorporeal blood procoagulation or extracorporeal hemostasis, comprising administering the composition to a patient or subject.

[0031] In some embodiments, the present disclosure provides compositions for use in extracorporeal blood procoagulation or extracorporeal hemostasis.

[0032] In some embodiments, the present disclosure provides a method for preparing an inducible vesicle, the method comprising contacting stem cells or somatic cells with an apoptosis-inducing agent, thereby causing the stem cells or somatic cells to produce the inducible vesicle, and contacting the stem cells or somatic cells with the apoptosis-inducing agent for about 3-16 hours.

[0033] In some embodiments, the contact time with the apoptosis-inducing agent is about 3-15 hours.

[0034] In some embodiments, the contact time with the apoptosis-inducing agent is about 4-14 hours.

[0035] In some embodiments, the contact time with the apoptosis-inducing agent is about 6-14 hours.

[0036] In some embodiments, the contact time with the apoptosis-inducing agent is about 8-14 hours.

[0037] In some embodiments, the contact time with the apoptosis-inducing agent is about 10-14 hours.

[0038] In some embodiments, the contact time with the apoptosis-inducing agent is about 11-13 hours.

[0039] In some embodiments, the contact time with the apoptosis-inducing agent is about 12 hours.

[0040] In some embodiments, the method further comprises: (1) culturing mesenchymal stem cells in a medium; (2) collecting the culture supernatant from step (1); (3) isolating the vesicles from the culture supernatant in step (2); Includes.

[0041] In some embodiments, the step (1) of culturing mesenchymal stem cells comprises: (4) isolating mesenchymal stem cells from the tissue; (5) adding a medium to culture mesenchymal stem cells and contacting the mesenchymal stem cells with an apoptosis-inducing agent in the medium; Includes.

[0042] In some embodiments, the apoptosis-inducing agent comprises a combination of one or more of staurosporine, ultraviolet radiation, starvation, or heat stress.

[0043] In some embodiments, the apoptosis-inducing agent is staurosporine.

[0044] In some embodiments, the concentration of staurosporine is about 200 nM-1000 nM.

[0045] In some embodiments, the concentration of staurosporine is about 250 nM-750 nM.

[0046] In some embodiments, the concentration of staurosporine is about 400 nM-600 nM.

[0047] In some embodiments, the concentration of staurosporine is about 450 nM-550 nM.

[0048] In some embodiments, the concentration of staurosporine is about 500 nM.

[0049] In some embodiments, the present disclosure provides the above methods for use with induced vesicles that are positive for tissue factor expression.

[0050] In some embodiments, the present disclosure provides the above method for use in obtaining an inducible vesicle product with an increased rate of positive tissue factor expression.

[0051] In some embodiments, the present disclosure provides an inducible vesicle prepared by the above method.

[0052] In some embodiments, the present disclosure provides the use of inducible vesicles prepared by the above method in the preparation of an extracorporeal blood coagulant or extracorporeal hemostatic agent.

[0053] In some embodiments, the present disclosure provides a method for treating extracorporeal blood procoagulation or extracorporeal hemostasis, comprising administering to a patient or subject the induced vesicles prepared by the above-described methods.

[0054] In some embodiments, the present disclosure provides inducible vesicles prepared by the above methods for use in extracorporeal blood procoagulation or extracorporeal hemostasis.

[0055] In some embodiments, the induced vesicles are vesicles that are produced by inducing apoptosis by adding an external factor when stem cells are normally viable.

[0056] In some embodiments, the stem cells are mesenchymal stem cells.

[0057] In some embodiments, the source of the mesenchymal stem cells includes bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon, or peripheral blood.

[0058] In some embodiments, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0059] In some embodiments, the mesenchymal stem cells are derived from a mammal.

[0060] In some embodiments, the mammal is selected from a primate or a murine.

[0061] In some embodiments, the primate is a human.

[0062] In some embodiments, the drug is selected from a lyophilized powder injection, an injection, an aerosol inhalant, an oral formulation, or a topical formulation.

[0063] In some embodiments, the medicament is a topical formulation.

[0064] In some embodiments, the topical formulation is selected from a tincture, a poultice, a paste, a powder, an ointment, a salve, a cream, a lotion, a rub, a spray, an aerosol, a film, a patch, a gel, a solution, a sponge, or a gauze.

[0065] In some embodiments, the gel comprises an aqueous gel and an oily gel.

[0066] In some embodiments, the topical formulation is a sponge formulation.

[0067] In some embodiments, the medicament further comprises a pharmaceutically acceptable pharmaceutical carrier.

[0068] In some embodiments, the pharmaceutical carrier comprises one or more of a diluent, excipient, filler, binder, disintegrant, surfactant, and lubricant. [Brief explanation of the drawings]

[0069] [Figure 1] 1 shows the identification of UC-MSCs cell surface molecules by flow cytometry. [Figure 2] The effects of PS inclusion, TF inclusion, and TF knockdown on the procoagulant effect of IEVs are shown in Figure 2A. Figure 2B shows the procoagulant effect of IEVs after antibody-mediated blockade of PS and TF, respectively, and after siRNA knockdown of TF (****: P<0.0001). [Figure 3]Figure 3 shows scanning electron microscopy detection of the promotion of clot stabilization by IEVs. Figure 3A shows a scanning electron microscopy image of the outer surface of a clot. Figure 3B shows a proteomic comparison of IEVs and exocrine glands. Figure 3C shows detection of siRNA knockdown efficiency by Western blot. Figure 3D shows scanning electron microscopy images of the inner and outer surfaces of a clot. [Figure 4] The whole blood clotting times of different numbers of IEVs are shown (*: P<0.05, **: P<0.01, ***: P<0.001). [Figure 5] The expression profiles of TF and PS in IEVs obtained at different time points from umbilical cord mesenchymal stem cells induced with different concentrations of STS, as detected by flow cytometry. [Figure 6] Figure 6A shows a comparison of the whole blood clotting times of IEVs. Figure 6B shows a comparison of the whole blood clotting times of IEVs stored at -80°C, 4°C, and 25°C for 28 days (****: P<0.0001, ns: not significant). [Figure 7] The effect of the IEVs-gelatin sponge complex on a rat femoral artery bleeding model is shown in Figure 7A. Figure 7B shows an SD rat femoral artery bleeding model. Figure 7C shows the 10-s free bleeding volume of the superficial femoral artery and vein in each group. Figure 7D shows the 10-min compression bleeding volume of each group (*: P<0.05, **: P<0.01, ns: no significant difference). [Figure 8] The effect of the IEVs-gelatin sponge complex on a New Zealand rabbit femoral artery bleeding model is shown in Figure 8A. Figure 8B shows the femoral artery of a New Zealand rabbit. Figure 8C shows the 10-second free bleeding volume of the femoral artery in each group. Figure 8D shows the 10-minute compression bleeding volume of each group (*: P<0.05, ns: not significant). [Figure 9] 1 shows a comparison of mouse wound healing results. [Figure 10] 1 shows a comparison of mouse wound healing rates. [Figure 11]The whole blood clotting time of IEVs derived from different stem cells (*: P<0.05, ****: P<0.0001, ns: not significant) is shown. [Figure 12] The IEVs-gelatin sponge complex promotes oral fascial wound healing in rats and has a hemostatic effect on post-tooth extraction bleeding in rats. Figure 12A shows images of oral wounds at different time points in each group of rats. Figure 12B shows the area of ​​oral fascial wounds at different time points in each group of rats. Figure 12C shows the time to hemostasis and the amount of blood loss after post-tooth extraction bleeding in each group of rats (*: P<0.05, **: P<0.01, ns: not significant). [Figure 13] The hemostatic effect of the IEVs-gelatin sponge complex on bleeding after human mandibular wisdom tooth extraction is shown in Figure 13A. Figure 13B shows the time to hemostasis after human mandibular wisdom tooth extraction (*: P<0.05). [Figure 14] Fabrication and characterization of IEVs hydrogel sponge are shown. Figure 14A shows the fabrication process of the IEVs hydrogel sponge. Figure 14B shows a digital photograph of the IEVs hydrogel sponge. Figure 14C shows the swelling property of the IEVs hydrogel sponge. Figure 14D shows the adhesion ability of the IEVs hydrogel sponge to the heart, liver, spleen, lung, kidney, and skin tissues of mice. Figure 14E shows the blood coagulation index (BCI) measurement. Figure 14F shows the in vitro IEVs release curve of the IEVs hydrogel sponge. Figure 14G shows a scanning electron microscope image. [Figure 15] The hemostatic effect of IEVs hydrogel sponge on rat liver bleeding is shown. Figure 15A shows the surgical procedure for a rat liver bleeding model. Figure 15B shows the time to hemostasis for rats in each group. Figure 15C shows the amount of blood loss for rats in each group. Figure 15D shows HE-stained images of rat livers in each hemostatic material group. Figure 15E shows immunofluorescence images of CD3 cells in the livers of rats in each hemostatic material group. [Figure 16]The effect of IEVs hydrogel sponge in a rat femoral artery bleeding model is shown. Figure 16A shows the surgical procedure for the rat femoral artery bleeding model. Figure 16B shows the time to hemostasis in each group of rats. Figure 16C shows the amount of blood loss in each group of rats in the femoral artery bleeding model (*: P<0.05, **: P<0.01, ***: P<0.001, ns: no significant difference). [Figure 17] The effect of the IEVs hydrogel sponge in a femoral artery hemorrhage model in New Zealand rabbits is shown. Figure 17A shows the surgical procedure for the femoral artery hemorrhage model in New Zealand rabbits. Figure 17B shows the time to hemostasis in each group of New Zealand rabbits. Figure 17C shows the amount of blood loss in each group of New Zealand rabbits (*: P<0.05, **: P<0.01, ***: P<0.001, ns: no significant difference). DETAILED DESCRIPTION OF THE INVENTION

[0070] The technical solutions of the present disclosure are further described below through specific examples. The specific examples do not limit the scope of protection of the present disclosure. Any non-essential modifications and adjustments made by those skilled in the art based on the present disclosure still fall within the scope of protection of the present disclosure.

[0071] In the examples of the present disclosure, IEVs is an abbreviation for inducible vesicles. Inducible extracellular vesicles refer to subcellular products produced by apoptosis induced or induced in progenitor cells (e.g., stem cells) during normal cell survival. Typically, such subcellular products have a membrane structure, express apoptosis markers, and partially contain genetic material, DNA. The inventors have discovered that inducible vesicles are distinct from cells and conventional extracellular vesicles (e.g., exosomes). In some embodiments, the cells in a normal cell state are, for example, non-apoptotic cells, non-senescent cells, cells not arrested in growth due to senescence, cells not resuscitated after freezing, cells not undergoing abnormal growth due to malignant transformation, or undamaged cells. In some embodiments, the cells in a normal cell state are harvested from cells that have reached 80-100% confluence during cell culture. In some embodiments, the cells in a normal cell state are harvested from cells in the logarithmic growth phase. In some embodiments, the normally viable cells are derived from primary and subsequent cultures of cells derived from human or mouse tissue. In some embodiments, the normally viable cells are derived from established cell lines or cell strains. In some embodiments, the progenitor cells are derived from primary cells. In some embodiments, the inducible vesicles may be derived from umbilical cord mesenchymal stem cells (UC-MSCs) at passages P3-P8.

[0072] The STS in this disclosure is staurosporine.

[0073] TF in the present disclosure is Tissue Factor.

[0074] As used herein, the term "tissue factor expression parameter" refers to the expression level of tissue factor and the positive rate of tissue factor expression.

[0075] As used herein, the term "positive tissue factor expression rate" refers to the percentage of induced vesicles that are positive for tissue factor expression among all induced vesicles.

[0076] "Comprising," "including," or "containing" means that combinations (e.g., media) and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" means excluding other elements of any material significance to the intended combination. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the disclosure. "Consisting of" means excluding trace elements and substantial method steps of other components. All embodiments defined by each of these transitional terms are within the scope of the present disclosure. [Example]

[0077] Example 1: Isolation and culture of umbilical cord mesenchymal stem cells (UC-MSCs) First, umbilical cords (obtained from the Department of Obstetrics and Gynecology, Guangdong Provincial People's Hospital) were washed with phosphate-buffered saline (PBS) containing penicillin and streptomycin to remove blood. The washed cords were cut into 3-4 cm lengths in a culture dish, and the umbilical vessels were removed. The cords were then cut into small pieces and digested with a solution containing 2 mg / mL type I collagenase and 4 mg / mL dispase II in phosphate-buffered saline (PBS) at 37°C for 1 hour. The digested tissue was seeded into a 100 mm culture dish containing complete medium. After incubation at 37°C under 5% carbon dioxide for 72 hours, the culture was washed twice with PBS to remove nonadherent cells. After replacing the medium with fresh complete medium, confluent primary UC-MSC colonies were observed 7-10 days later. Characteristic markers of UC-MSCs were detected using a flow cytometer. The results show that the resulting UC-MSCs express CD29, CD44, and CD90 (≥95%), but not CD34 or CD45 (≤5%) (Figure 1). The UC-MSCs were digested and subcultured by incubation with trypsin at 37°C. The complete medium was then replaced every 3 days, and the cells were passaged after the culture dish was filled. The components of the complete medium are listed in Table 1.

[0078] [Table 1]

[0079] Example 2: Obtaining Inducible Egg Vesicles (IEVs) from UC-MSCs The UC-MSCs may be, but are not limited to, passages 3 to 8. In this example, the UC-MSCs specifically used were passage 7.

[0080] Using UC-MSCs at passage 7 (80-90% confluence) cultured as described in Example 1, we washed them with PBS, added them to alpha medium containing 500 nM staurosporine (STS), and incubated them at 37°C for 12 hours. The supernatant was then collected and subjected to the following procedures: centrifuging at 800 g for 10 minutes at 4°C, collecting the supernatant, centrifuging at 2000 g for 10 minutes at 4°C, collecting the supernatant, centrifuging at 16000 g for 30 minutes at 4°C, discarding the supernatant, and centrifugation at 16000 g for 30 minutes at 4°C. The resulting precipitate was IEVs. The precipitate was resuspended in 500 μl of PBS and centrifuged at 16000 g for 30 minutes at 4°C. The supernatant was discarded, and the washed IEVs were obtained.

[0081] Example 3: Role of TF and PS in the process of promoting blood coagulation by IEVs We performed plasma coagulation experiments using IEVs treated with antibody blocking and siRNA knockdown techniques to investigate the mechanism by which IEVs promote coagulation.

[0082] (1) Plasma clotting experiment: Add 12.5 μL of 1 × 10 cells to a flow cytometry tube. 8 IEVs were added (and a control group (12.5 μL of PBS was added to the flow cytometry tube) was simultaneously prepared), and the tube was preheated at 37°C for 10 min. 40 μL of mouse plasma was added and mixed uniformly. 7 μL of 0.2 M CaCl2 solution was added, and timing was started immediately after. The tube was rocked up and down once every 15 s, and the time until the plasma clot could no longer be rocked was defined as the clotting time.

[0083] (2) Antibody blocking of TF and PS: IEVs obtained in Example 2 were resuspended in PBS, and the number of IEVs was measured using a nanoparticle tracking analyzer (Particle Metrix, Germany). 8 IEVs were incubated with 1 μg / mL TF-blocking antibody (neutralized TF, R&D Systems, FAB3178P, USA), 1 μg / mL TFPI (abmart, PU299161, China), or 1 μg / mL Annexin V (Sino Biological Inc., 10039-01, China) at 4°C for 30 minutes. After antibody blocking, IEVs were directly used in plasma coagulation experiments. The results showed that PS blocking did not affect the procoagulant function of IEVs, whereas treatment with TF-neutralizing antibody and tissue factor pathway inhibitor (TFPI) significantly reduced the procoagulant effect of IEVs (Figure 2A).

[0084] (3) TF knockdown: TF expression in UC-MSC-derived IEVs was eliminated using small interfering RNA (siRNA) technology. The IEVs were transfected with Lipofectamine according to the manufacturer's instructions. TM UC-MSCs were transfected with RNAiMAX transfection reagent (Invitrogen, USA), siRNA negative control (si-NC), and siRNA-TF (si-TF) (Santa Cruz Biotechnology, USA). Transfection efficiency was measured 48 hours after transfection by Western blotting. Plasma coagulation experiments were performed using IEVs extracted from UC-MSCs 72 hours after siRNA transfection. The IEV extraction steps and extraction conditions were the same as in Example 2, and the blood coagulation experiments were performed according to step (1). The results show that the procoagulant effect of IEVs with TF knockdown was significantly reduced compared to the control group, and this was statistically significant (Figure 2B).

[0085] As can be seen from the above experimental results, IEVs promote rapid blood coagulation mainly through TF. This differs from the expression and mechanism of IEV-based hemophilia treatment disclosed in CN114306238A. CN114306238A also discloses that the mechanism of IEV-based treatment of hemophilia mice is unrelated to TF and PS. This indicates that the mechanism of the blood coagulation promotion therapy disclosed here differs from the mechanism of hemophilia treatment.

[0086] Example 4: Enhancement of clot stabilization by IEVs detected by scanning electron microscopy Using the IEVs obtained in Example 2, 50 μL of 1 × 10 10 IEVs were placed in the bottom of a 48-well plate (a PBS control group was also set up; 50 μL of PBS was placed in the bottom of the 48-well plate) and preheated at 37°C for 10 min. 0.5 mL of citrate-anticoagulated rat whole blood was added to each well and mixed evenly. 50 μL of 0.2 mol / L CaCl2 was then added and incubated at 37°C for 1 h. The samples were then fixed with 2 mL of 2.5% (v / v) glutaraldehyde at 4°C for 4–6 h. The samples were then dehydrated in 50%, 70%, 85%, and 100% ethanol for 10 min, dried, and gold-plated. Finally, the samples were observed using a scanning electron microscope.

[0087] Electron micrographs show that clots from the PBS group had loosely intertwined surface fibers and relatively large pore sizes, whereas clots from the IEVs group had finer, denser surface fibers and smaller pore sizes (Figure 3A). Proteomic quantitative analysis of IEVs and exosomes was performed using protein DIA quantification technology. Results showed that IEVs contained significantly more intergrin α5 than exosomes derived from allogeneic MSCs (Figure 3B). Since intergrin α5 is an important substance that binds to fibrin, we obtained IEVs with low intergrin α5 expression by knocking down intergrin α5 using siRNA in P5-passage UC-MSCs (Figure 3C). This enabled us to determine whether IEVs stabilize fibrin through intergrin α5. Electron micrographs showed that IEVs with low intergrin α5 expression had looser fibrillar arrangements and larger pore sizes at the inner and outer surfaces of clots compared with IEVs with normal intergrin α5 expression (Figure 3D). These results suggest that IEVs can promote clot stabilization by making fibrin in the clot more compact through intergrin α5.

[0088] Here, in the proteomic quantitative analysis, the MSCs from which exosomes and IEVs were extracted were the same cell line.

[0089] Exosome isolation and extraction method: Using UC-MSCs at passage 7 (80-90% confluence) cultured as described in Example 1, cells were washed with PBS, serum-free medium (α-MEM medium) was added, and the cells were incubated at 37°C for 48 hours. The cell supernatant was collected for exosome isolation and extraction. The extraction steps included centrifugation at 800g for 10 minutes, supernatant collection, centrifugation at 2000g for 10 minutes, supernatant collection, centrifugation at 16000g for 30 minutes, supernatant collection, centrifugation at 120000g for 90 minutes, removal of the supernatant, resuspension of the pellet in sterile PBS, and further centrifugation at 120000g for 90 minutes, removal of the supernatant, collection of the bottom exosomes, and resuspension in sterile PBS.

[0090] Example 5: Comparison of the blood coagulation promotion time of IEVs derived from different numbers of UC-MSCs Using the IEVs obtained in Example 2, 1 × 10 9 , 4×10 8 , 2 × 10 8 , 1×10 8 A whole blood coagulation experiment was performed using 10 IEVs. The procedure for the whole blood coagulation experiment was as follows: IEVs resuspended in 500 μL of PBS were added to a flow cytometry tube (blank control: 500 μL of PBS was added to the flow cytometry tube), preheated at 37°C for 10 minutes, and 1 mL of rat sodium citrate-anticoagulated whole blood was added. After uniform mixing, 100 μL of 0.1 M CaCl2 solution was added. Time was immediately started, and the tube was rocked up and down once every 10 seconds. The blood coagulation time was determined as the time until the blood clot could no longer be rocked. The experimental results showed that the blood coagulation time increased with a decrease in the number of IEVs, and this was statistically significant, indicating that the blood coagulation promotion process of IEVs is dose-dependent (Figure 4).

[0091] Example 6: Obtaining UC-MSC-derived inducible vesicles (IEVs) under different conditions P7 passage UC-MSCs cultured as described in Example 1 at 80-90% confluence were washed with PBS and added to alpha medium containing 250 nM, 500 nM, or 750 nM staurosporine (STS). The supernatants were collected at four time points: 4 h, 8 h, 12 h, and 16 h. The supernatants collected at each time point were then subjected to the following procedures: centrifuged at 800 g for 10 minutes at 4°C, collected, centrifuged at 2000 g for 10 minutes at 4°C, collected, and centrifuged at 16000 g for 30 minutes at 4°C. The supernatant was discarded, and the resulting precipitate was IEVs. The precipitate was resuspended in 500 μl of PBS and centrifuged at 16000 g for 30 minutes at 4°C. The supernatant was discarded, and the washed IEVs were obtained.

[0092] Example 7: TF and PS expression status in inducible vesicles (IEVs) derived from UC-MSCs IEVs obtained by inducing different concentrations of STS at different time points in Example 6 were used. IEVs were resuspended in PBS and the number of IEVs was measured using a nanoparticle tracking analyzer (Particle Metrix, Germany). 1 × 10 IEVs were collected in 98 μL of FACS buffer. 10 The IEVs were resuspended, and 2 μL of PE-conjugated anti-human TF (BioLegend, USA) or APC-conjugated Annexin V (BioLegend, USA) was added, followed by incubation at 4°C for 30 minutes. TM The percentage of positive staining was analyzed using software (NovoCyte, USA). As can be seen from the results, the expression of tissue factor (TF) and phosphatidylserine (PS) was highest in IEVs induced with 500 nM STS for 12 h (Figure 5).

[0093] Example 8: Preparation of freeze-dried inducible vesicles (IEVs) derived from UC-MSCs The IEVs obtained in Example 2 were resuspended in 100 μL of PBS and mixed to homogenize. The lyophilization solution consisted of 4 mL of 0.65X PBS, 100 mM trehalose, and 5% polyvinylpyrrolidone 40 (PVP40). The lyophilized IEVs were placed in a -80°C refrigerator overnight. The next day, the lyophilized samples were removed from the -80°C refrigerator. The EP tubes containing the lyophilized IEVs were quickly opened, wrapped in plastic, and placed in a -80°C freeze dryer (Shanghai Shengwei Electronics Technology FD-A18N-80) for 30 hours to obtain lyophilized vesicles.

[0094] Example 9: Comparison of the blood coagulation promotion time of fresh IEVs and freeze-dried IEVs after leaving them at room temperature for 28 days Whole blood coagulation experiment: Fresh IEVs group (1 × 10 9 IEVs in 500 μL of PBS), lyophilized IEVs (1 × 10 9The subjects were divided into five groups: fresh PBS group (500 μL of PBS), freeze-dried PBS group (freeze-dried PBS, 500 μL of PBS), and freeze-dried liquid group (500 μL of freeze-dried liquid), and a whole blood coagulation experiment was performed according to the procedure in Example 5.

[0095] Here, fresh IEVs are the IEVs obtained in Example 2, freeze-dried IEVs are the freeze-dried IEVs obtained in Example 8, and freeze-dried PBS was prepared according to the procedure of Example 8 except that IEVs were not added.

[0096] As can be seen from the results, the blood clotting time of the freeze-dried IEVs group was significantly shorter than that of the PBS group, and there was no statistically significant difference in the blood clotting promoting effect between freeze-dried IEVs and fresh IEVs (Figure 6A). Freeze-dried IEVs were stored at -80°C, 4°C, and 25°C for 28 days. The freeze-dried IEVs stored at room temperature for 28 days still maintained a good blood clotting effect, with no statistically significant difference compared to the blood clotting effect of freeze-dried IEVs stored at -80°C and 4°C (Figure 6B). These results suggest that the inducible vesicles prepared by the freeze-drying process maintain their rapid blood clotting effect, eliminating the problems of transportation, handling, and storage for future use.

[0097] Example 10: Hemostatic effect of freeze-dried IEVs-gelatin sponge complex on femoral artery bleeding in rats 1. Preparation of Lyophilized IEVs-gelatin Sponge Complexes The IEVs obtained in Example 2 were resuspended in 100 μL of PBS, then 400 μL of lyophilization solution was added and mixed uniformly (the lyophilization solution composition was the same as in Example 8). 500 μL of this mixture was placed into a 20 mm x 20 mm gelatin sponge (Xiang'en, China). After the gelatin sponge completely absorbed the mixture, the gelatin sponge was placed in a -80°C refrigerator and left overnight. The next day, it was lyophilized for 30 hours in a -80°C freeze dryer to obtain a freeze-dried IEV-gelatin sponge complex. Preparation of PBS-gelatin sponge complex: The other procedures are the same as those for the freeze-dried IEVs-gelatin sponge complex, except that IEVs are not added.

[0098] 2. Construction of a rat hemorrhage model and grouping SD rats weighing 250g ± 20g were randomly assigned to the IEVs-gelatin sponge complex group, the PBS-gelatin sponge complex group, and the blank control group (five rats per group). During the experimental procedure, the hair was shaved and the superficial femoral artery and vein were isolated. The surgical site was then wiped with gauze to prevent exudate from affecting the experimental results. The superficial femoral artery and vein were clamped with an arterial clamp and severed with scissors. The arterial clamp was released and the blood vessel was allowed to bleed freely for 10 seconds. After 10 seconds of pressure application with one pre-weighed gauze and a 200g weight, equal weights of the IEVs-gelatin sponge complex and the PBS-gelatin sponge complex were placed at the bleeding point. For the blank control group, the gelatin sponge was not placed, but instead covered with three pre-weighed gauze pieces and pressure application with a 200g weight for 10 minutes. Blood loss was calculated using the following formula: Amount of blood loss = Weight of gauze after hemostasis - Weight of original gauze The amount of free bleeding was calculated using the formula: There was no statistically significant difference in the amount of free bleeding within 10 s between the groups, indicating that the degree of bleeding model construction was consistent (Figure 7C). On the other hand, the amount of bleeding after 10 min of compression using the IEVs-gelatin sponge complex was significantly less than the amount of bleeding after 10 min of compression using the PBS-gelatin sponge complex and the blank control group, which was statistically significant, demonstrating the effectiveness of the freeze-dried IEVs-gelatin sponge complex (Figure 7D).

[0099] Example 11: Hemostatic effect of freeze-dried IEVs-gelatin sponge complex on femoral artery bleeding in New Zealand rabbits The preparation of the freeze-dried IEVs-gelatin sponge complex and the PBS-gelatin sponge complex was the same as in Example 10.

[0100] New Zealand rabbits weighing 2 kg ± 200 g were randomly assigned to either the IEVs-gelatin sponge complex group or the PBS-gelatin sponge complex group, with five rabbits per group based on their weight. During the experimental procedure, the femoral artery was shaved and isolated, and the surgical site was wiped with gauze to prevent exudate from affecting the experimental results. The femoral artery was clamped with an arterial clamp, and a 0.5 mm diameter hole was created in the femoral artery using a 1 ml needle. The arterial clamp was then released to allow free bleeding from the blood vessel for 10 s. After 10 s of compression with one pre-weighed gauze and a 200 g weight, two IEVs-gelatin sponges or PBS-gelatin sponges were placed on the bleeding point, covered with five pre-weighed gauze pieces, and compressed with a 200 g weight. Compression was stopped after 3, 5, 7, 9, 11, 13, and 15 minutes, and hemostasis was assessed. Blood loss was calculated using the following formula: Amount of blood loss = Weight of gauze after hemostasis - Weight of original gauze The amount of free bleeding after 10 s was calculated using the formula: There was no statistically significant difference between the two groups, indicating that the degree of bleeding model construction was consistent (Figure 8C). On the other hand, the amount of bleeding after compression using the IEVs-gelatin sponge complex group was significantly less than that of the PBS-gelatin sponge complex group, which was statistically significant, demonstrating the effectiveness of the freeze-dried IEVs-gelatin sponge complex (Figure 8D).

[0101] Example 12: Promoting effect of freeze-dried IEVs-gelatin sponge complex on wound healing in mice The preparation of the freeze-dried IEVs-gelatin sponge complex and the PBS-gelatin sponge complex was the same as in Example 10.

[0102] Ten-week-old C57 mice were randomly assigned to groups based on their weight. A 1cm x 1cm full-thickness wound was created on the back of each mouse. The freeze-dried IEVs / gelatin sponge complex or freeze-dried PBS / gelatin sponge complex was applied to the wound and secured with tape. On day 7, the freeze-dried IEVs / gelatin sponge complex or freeze-dried PBS / gelatin sponge complex was removed, and the wound was photographed (Figure 9). The wound healing rate was calculated (Figure 10). The results show that the freeze-dried IEVs / gelatin sponge complex significantly improved wound healing.

[0103] Example 13: Comparison of the blood coagulation promoting time of IEVs derived from different stem cells (1) Collection of IEVs derived from different stem cells: P8-P10 human bone marrow mesenchymal stem cells (Sciencell, USA), human adipose mesenchymal stem cells (Procell, China), and human umbilical cord mesenchymal stem cells (Guangdong Provincial People's Hospital, China) were placed in alpha medium containing 500 nM staurosporine (STS), incubated at 37°C, and induced for 12 hours. After incubation, the supernatant was collected and centrifuged at 800 g for 10 minutes at 4°C. The supernatant was then collected and centrifuged at 2000 g for 10 minutes at 4°C. The supernatant was then centrifuged at 16000 g for 30 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in 500 μL of PBS and centrifuged at 16000 g for 30 minutes at 4°C. The supernatant was discarded to obtain IEVs derived from different cells.

[0104] (2) Whole blood coagulation experiment: 1 × 10 cells resuspended in 500 μL of PBS were placed in a flow cytometry tube. 9 IEVs were added (blank control: 500 μL of PBS was added to a flow cytometry tube) and preheated at 37°C for 10 minutes. 1 mL of rat sodium citrate-anticoagulated whole blood was added and mixed evenly. 100 μL of 0.1 M CaCl2 solution was added, and the blood clotting time was measured immediately. The blood was shaken up and down every 10 seconds. The time until the blood clot could no longer be shaken was recorded as the blood clotting time. The experimental results showed that IEVs derived from umbilical cord mesenchymal stem cells (UC-MSCs) had the fastest blood clotting time (Figure 11).

[0105] Example 14: Promoting effect of freeze-dried IEVs-gelatin sponge complex on oral mucosal wound healing in rats The preparation of the freeze-dried IEVs-gelatin sponge complex and the PBS-gelatin sponge complex was the same as in Example 10.

[0106] SD rats weighing 250g ± 20g were randomly assigned to groups of 12 rats per group based on their body weight. The rats' bilateral first molars were extracted, and the extraction sockets were filled with freeze-dried IEVs / gelatin sponge composites or freeze-dried PBS / gelatin sponge composites. The gelatin sponge materials were then fixed in place with tissue adhesive. Three rats per group were euthanized on days 5, 10, and 15. Photographs of the oral wounds were taken (Figure 12A) to observe the wound area of ​​the rat oral mucosa at different time points. Results showed that treatment with the freeze-dried IEVs / gelatin sponge composite on days 5 and 15 significantly promoted wound healing in rat oral mucosa (Figure 12B).

[0107] Example 15: Hemostatic effect of freeze-dried IEVs-gelatin sponge complex on post-tooth extraction bleeding in rats The preparation of the freeze-dried IEVs-gelatin sponge complex and the PBS-gelatin sponge complex was the same as in Example 10.

[0108] Two-month-old SD rats were divided into groups of four rats based on their body weight. The rats had their first molars extracted from both sides, and the lyophilized IEVs-gelatin sponge complex or the lyophilized PBS-gelatin sponge complex was placed in the extraction socket. The time to hemostasis was observed and recorded. The results showed that the lyophilized IEVs-gelatin sponge complex significantly reduced the time to hemostasis and blood loss after tooth extraction in rats (Figure 12C).

[0109] Example 16: Hemostatic effect of freeze-dried IEVs-gelatin sponge complex on bleeding after mandibular wisdom teeth in humans The preparation of the freeze-dried IEVs-gelatin sponge complex and the PBS-gelatin sponge complex was the same as in Example 10.

[0110] The study involved 19 patients with symmetrical mandibles. Under local anesthesia, both mandibular third molars were extracted. One freeze-dried IEVs-gelatin sponge complex or one freeze-dried PBS-gelatin sponge complex was placed in each alveolar cavity, followed by hemostasis, suturing, and follow-up observation (Fig. 13A). In the experimental group, 4 × 10 freeze-dried IEVs-gelatin sponge complexes were placed in each cavity. 10The study type was a controlled experiment.

[0111] Patient inclusion criteria: 1) Patients aged 18-45 weeks (inclusive), regardless of gender; 2) Patients requiring extraction of bilaterally impacted third molars in the mandible; 3) Patients who signed informed consent.

[0112] Patient exclusion criteria: 1) The adjacent second molar in the patient's mouth was affected by pulp damage or inadequate root canal treatment, or had a fractured tooth, or had grade 3 looseness, and comprehensive clinical and imaging evaluations did not suggest a promising prognosis. 2) The adjacent second molar in the patient's mouth had a metal crown or a large amalgam restoration that interfered with imaging evaluation. 3) The patient had an uncontrolled pathological process in the mouth (acute progressive disease in the mouth). 4) Patients with malnutrition (serum albumin concentration <2 g / dl). 5) Patients diagnosed with other systemic diseases, infectious diseases, or genetic diseases, or patients in the acute inflammatory phase or acute exacerbation of a chronic disease. 6) Patients who were pregnant, breastfeeding, or planning to become pregnant. 7) The investigators determined that the patient was not suitable for this study.

[0113] As can be seen from the results, the freeze-dried IEVs-gelatin sponge complex can significantly reduce the time to hemostasis after human mandibular wisdom tooth extraction (Figure 13B).

[0114] Example 17: Preparation and characterization of IEVs hydrogel sponge 1. Preparation of oxidized hyaluronic acid modified with phenylboronic acid (PBA-HA) One gram of hyaluronic acid sodium salt (source leaf organism) was dissolved in 100 ml of deionized water with stirring (200 rpm), followed by mixing with 3-aminophenylboronic acid (Macklin, China) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (Macklin, China) in a 1:1:1 molar ratio. The mixture was stirred at room temperature for 24 hours (200 rpm) and then dialyzed against deionized water for 3 days to remove unreacted materials. The dialyzed solution was freeze-dried using a -50°C freeze dryer (LABCONCO, USA), yielding a white, sponge-like solid of phenylboronic acid-modified hyaluronic acid. One gram of phenylboronic acid-modified hyaluronic acid was dissolved in 100 ml of deionized water and then added dropwise to 10 ml of 0.25 M sodium periodate (Macklin, China). After 24 hours of reaction at room temperature in the dark, 1 mL of ethylene glycol (Macklin, China) was added to terminate the reaction, and the resulting phenylboronic acid-modified oxidized hyaluronic acid was then transferred to a dialysis membrane (MWCO 3500 Da) to purify it. After dialysis in DI water for 3 days, the purified material was finally lyophilized in a -50°C freeze dryer and stored at 4°C.

[0115] 2. Synthesis of IEVs Hydrogel Sponge 60 mg of oxidized hyaluronic acid modified with phenylboronic acid was dissolved in 500 μL of PBS and left at room temperature for 1 hour. 40 mg of polyvinyl alcohol (Aladdin, China) was dissolved in 500 μL of PBS and left at 95°C for 1 hour. 50 μL of IEVs (4 × 10 10 IEVs (IEVs obtained in Example 2) or 50 μL of PBS was added to 200 μL of lyoprotectant (200 μL, containing 200 mM trehalose and 10% PVP40). Finally, 125 μL of phenylboronic acid-modified oxidized hyaluronic acid solution and 125 μL of polyvinyl alcohol solution were added to prepare IEVs hydrogels or PBS hydrogels. The hydrogels were frozen overnight at -80°C and then lyophilized for 30 hours in a -80°C freeze dryer to obtain IEVs hydrogel sponges or PBS hydrogel sponges (Figure 14A).

[0116] 3.Water absorption and adhesion test A 60 mg IEVs hydrogel sponge was compressed and injected into deionized water to prepare a hydrated IEVs hydrogel. After 10 minutes, excess water was removed, and the IEVs hydrogel was placed on a glass slide (CITOTEST, China). Mouse heart, liver, spleen, lung, kidney, or skin (6 mm x 6 mm) was then placed on the hydrated IEVs hydrogel. The slide was then inverted to observe the adhesion ability of the IEVs hydrogel.

[0117] The results show that the IEVs hydrogel sponge and the PBS hydrogel sponge are compressible and injectable (Figure 14B). When the hydrogel sponge was placed in water, it rapidly absorbed water and formed a new hydrogel (Figure 14C). The sponge was then able to firmly adhere to the mouse heart, liver, spleen, lung, kidney, and skin tissues (Figure 14D).

[0118] 4.Blood coagulation index (BCI) 30 μL of rat anticoagulated blood and CaCl2 (0.2 mol L -1 7 μL, Sigma, C5670) was slowly dropped onto the surface of the sample or plate (negative control) of an equivalent volume, and incubated at 37°C for 5 minutes. 5 mL of PBS was carefully added so as not to disturb the clot. The sample was then gently rocked (37°C, 120 rpm, 5 minutes). The absorbance of the solution at 540 nm was recorded using a microplate reader (BioTek Synergy H1, USA). Gelatin sponge Surgifoam® and collagen sponge Avitene TM was served as a positive control.

[0119] The blood coagulation index (BCI) of different materials is determined by the following formula: BCI (%) = (OD sample / OD blank control) × 100% where OD sample is the absorbance at 540 nm of the sample solution and OD blank control is the absorbance at 540 nm of the negative control solution.

[0120] As can be seen from the experimental results, the blank control, the PBS hydrogel sponge, the commercially available hemostatic products gelatin sponge Surgifoam® and collagen sponge Avitene TM In comparison, the blood coagulation index of the IEVs hydrogel sponge group was the lowest, indicating that the IEVs hydrogel sponge has extremely excellent in vitro hemostatic ability (Figure 14E).

[0121] 5. In vitro IEV release curve of IEVs hydrogel sponge (IEVs-HS) IEVs were labeled with the PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, USA) and used to synthesize PKH26-labeled IEVs hydrogel sponges. Sixty milligrams of IEVs hydrogel sponges were immersed in 1 mL of PBS at 37°C. The supernatants were collected at predetermined time points (1 min, 3 min, 5 min, 7 min, 10 min, 1 h, 3 h, 7 h, 10 h, and 24 h), and the fluorescence intensity of the supernatants was measured at 570 nm using a microplate reader.

[0122] As can be seen from the results, the IEVs hydrogel sponge rapidly and completely released IEVs within 1 h (Figure 14F). The rapid release mode indicated that IEVs could be rapidly released to prevent local bleeding, and continuously released within 1 h to prevent secondary bleeding.

[0123] 6. Scanning Electron Microscopy IEVs hydrogel sponge and PBS hydrogel sponge were made conductive by spraying gold using a sputtering coating machine (MC1000, Hitachi, Japan), and observed using a scanning electron microscope (SEM, Quanta200, Thermo Fisher, USA).

[0124] Scanning electron microscopy revealed that IEVs were present on the surface of the hydrogel sponge (Figure 14G), which ensures that IEVs are in direct contact with blood and can participate in hemostasis most quickly.

[0125] Example 18: Hemostatic effect of IEVs hydrogel sponge on liver hemorrhage in rats The preparation of IEVs hydrogel sponge and PBS hydrogel sponge was the same as in Example 17.

[0126] SD rats weighing 250g ± 20g were randomly assigned to groups of four rats according to their body weight to receive one of the following treatments: IEVs hydrogel sponge group, PBS hydrogel sponge group, blank control group, gelatin sponge Surgifoam® group, and collagen sponge Avitene® group. TM The experimental procedures were as follows: After anesthesia, the abdominal hair was shaved, and the largest lobe of the left liver was isolated. The surgical site was wiped with gauze to prevent exudate from affecting the experimental results. The central liver tissue was removed using a drill, and a wound 8 mm in diameter and 5 mm deep was created. Equal amounts of IEVs hydrogel sponge, PBS hydrogel sponge, gelatin sponge Surgifoam®, and collagen sponge Avitene® were placed in the wound. TM The blank control group had no material placed on it, and a filter paper was placed under the liver beforehand for blood absorption. The amount of blood loss was calculated using the formula: Blood loss = weight of filter paper after hemostasis - original weight of filter paper It was calculated by

[0127] As can be seen from the results, the blank control, the PBS hydrogel sponge, the commercially available hemostatic products gelatin sponge Surgifoam® and collagen sponge Avitene TM Compared with the other groups, the IEVs hydrogel sponge group had the fastest hemostasis time and the least blood loss, indicating that the IEVs hydrogel sponge has excellent in vivo hemostasis ability (Figures 15A, B, C).

[0128] Example 19: Safety of IEVs hydrogel sponge after liver hemorrhage surgery in rats After the liver hemorrhage surgery in Example 18, the rats were euthanized, and their livers were removed and paraffin sections and frozen sections were prepared. The paraffin sections were stained with hematoxylin and eosin, and the frozen sections were stained with CD3 and mounted with DAPI (Abcam, ab104139).

[0129] The experimental results showed that 4 weeks after surgery, the IEVs hydrogel sponge and other hemostatic materials were not completely degraded in the injured liver (Figure 15D), indicating that a longer time is required for the materials to completely degrade in the liver. Furthermore, large areas of necrotic liver tissue were observed in the liver of the untreated group (blank control group), suggesting that the group without any treatment experienced the most bleeding. Furthermore, the IEVs hydrogel sponge and the untreated group showed the least inflammatory cell infiltration, indicating that the IEVs hydrogel sponge has good biocompatibility in the body (Figures 15D and 15E). In contrast, after treatment with the gelatin sponge Surgifoam®, a large amount of inflammatory cell infiltration appeared at the site of the injured liver. Therefore, the IEVs hydrogel sponge has excellent biocompatibility and is expected to have good prospects for clinical application.

[0130] Example 20: Hemostatic effect of IEVs hydrogel sponge on femoral artery bleeding in rats The preparation of IEVs hydrogel sponge and PBS hydrogel sponge was the same as in Example 17.

[0131] SD rats weighing 250g ± 20g were randomly assigned to groups of four rats according to their body weight to receive one of the following treatments: IEVs hydrogel sponge group, PBS hydrogel sponge group, blank control group, gelatin sponge Surgifoam® group, and collagen sponge Avitene® group. TMThe experimental procedures were as follows: After anesthesia, the animals were shaved, the femoral artery was isolated, and the surgical site was wiped with gauze to prevent exudate from affecting the experimental results. The femoral artery was clamped with an arterial clamp, and a hole (diameter: 0.5 mm) was made in the femoral artery using a 1 ml needle. The arterial clamp was released to allow the blood vessel to bleed freely for 10 s. After that, the animals were further compressed with three pre-weighed gauze sheets and a 200 g weight for 10 s, and then an equal amount of IEVs hydrogel sponge, PBS hydrogel sponge, gelatin sponge Surgifoam®, and collagen sponge Avitene® was applied. TM The device was placed on the bleeding point, covered with five pre-weighed gauze pads, and compressed with a 200g weight. The time to hemostasis was observed every minute. The amount of blood loss was calculated using the formula: Amount of blood loss = Weight of gauze after hemostasis - Weight of original gauze It was calculated by

[0132] As can be seen from the results, the blank control, the PBS hydrogel sponge, the gelatin sponge Surgifoam® and the collagen sponge Avitene TM Compared with the control group, the IEVs hydrogel sponge group had the fastest hemostasis time and the lowest blood loss, which was statistically significant (Figures 16A, B, C), indicating that the IEVs hydrogel sponge has excellent in vivo hemostatic ability.

[0133] Example 21: Hemostatic effect of IEVs hydrogel sponge on femoral artery bleeding in New Zealand rabbits The preparation of IEVs hydrogel sponge and PBS hydrogel sponge was the same as in Example 17.

[0134] New Zealand rabbits weighing 2 kg ± 200 g were randomly assigned to groups of four rabbits according to their body weight to receive one of the following treatments: IEVs hydrogel sponge group, PBS hydrogel sponge group, blank control group, gelatin sponge Surgifoam® group, and collagen sponge Avitene® group. TMThe experimental procedures were as follows: After anesthesia, the animals were shaved, the femoral artery was isolated, and the surgical site was wiped with gauze to prevent exudate from affecting the experimental results. The femoral artery was clamped with an arterial clamp, and a hole (diameter: 0.5 mm) was made in the femoral artery using a 1 ml needle. The arterial clamp was released to allow free bleeding from the blood vessel for 10 s. After further compression with three pre-weighed gauze sheets and a 200 g weight for 10 s, the animals were further injected with equal amounts of IEVs hydrogel sponge, PBS hydrogel sponge, gelatin sponge Surgifoam® group, and collagen sponge Avitene® group. TM The group was placed at the bleeding point, covered with five pre-weighed gauze pads, and pressed with a 200g weight. The time to hemostasis was observed every minute. The amount of blood loss was calculated using the formula: Amount of blood loss = Weight of gauze after hemostasis - Weight of original gauze It was calculated by

[0135] As can be seen from the results, the blank control, the PBS hydrogel sponge, the gelatin sponge Surgifoam® and the collagen sponge Avitene TM Compared with the control group, the IEVs hydrogel sponge group had the fastest hemostasis time and the lowest blood loss, which was statistically significant (Figures 17A, B, C), indicating that the IEVs hydrogel sponge has excellent in vivo hemostatic ability.

Claims

1. Use of inducible vesicles in the preparation of an extracorporeal blood coagulant or extracorporeal hemostatic agent.

2. The use according to claim 1, wherein the induced vesicles are induced vesicles that are positive for tissue factor expression.

3. 1. A method for screening an extracorporeal blood coagulant or extracorporeal hemostatic agent, comprising: A method for screening inducible vesicles as candidate drugs by detecting the expression of tissue factor in the inducible vesicles and screening for inducible vesicles that are positive for tissue factor expression.

4. A method for quality inspection, quality control, or quality evaluation of an extracorporeal blood coagulant or extracorporeal hemostatic agent, comprising: The method uses inducible vesicles as the test object, tissue factor expression parameters as the evaluation index, and when the tissue factor expression parameters reach a predetermined level, the quality of the drug is judged to be acceptable.

5. the tissue factor expression parameter is a tissue factor expression level or a tissue factor expression positive rate; The method according to claim 4, wherein the tissue factor expression positivity rate is the proportion of induced vesicles that are positive for tissue factor expression to the total number of induced vesicles.

6. Use of an inducible vesicle product with an improved tissue factor expression positivity rate in the preparation of an extracorporeal blood coagulant or extracorporeal hemostatic agent.

7. An induced vesicle product with an improved tissue factor expression positivity rate is one in which the ratio of induced vesicles that are tissue factor expression positive to the total induced vesicles is at or above a predetermined level, Preferably, the predetermined level is a tissue factor expression positive rate of ≥ 30%, Preferably, the predetermined level is a tissue factor expression positive rate of ≧31%, Preferably, the predetermined level is a tissue factor expression positive rate of ≧32%, Preferably, the predetermined level is a tissue factor expression positive rate of ≥ 35%, Preferably, the predetermined level is a tissue factor expression positive rate of ≧40%, Preferably, the predetermined level is a tissue factor expression positive rate of ≧45%, Preferably, the predetermined level is a tissue factor expression positive rate of ≧50%, Preferably, the predetermined level is a tissue factor expression positive rate of ≧55%, The use according to claim 6, wherein the predetermined level is preferably a tissue factor expression positivity rate of ≧60%.

8. A composition comprising an inducible vesicle and tissue factor, Preferably, the composition further comprises a blood coagulation promoting agent or a hemostatic agent; Preferably, the composition, wherein the blood coagulation promoter or hemostatic agent is one or more selected from prothrombin, thrombin, fibrinogen, fibrin, adhesion proteins, coagulation factors, collagen, gelatin, vasopressin, plasminogen activator inhibitors, platelet activators, and synthetic peptides having hemostatic activity.

9. 10. Use of the composition of claim 8 in the preparation of an extracorporeal blood coagulant or extracorporeal hemostatic agent.

10. adding an apoptosis-inducing agent and contacting the stem cells or somatic cells with the apoptosis-inducing agent, thereby causing the stem cells or somatic cells to produce the inducing vesicles, and the contact time with the apoptosis-inducing agent is 3 to 16 hours; Preferably, the contact time with the apoptosis-inducing agent is about 3-15 hours; Preferably, the contact time with the apoptosis-inducing agent is about 4-14 hours; Preferably, the contact time with the apoptosis-inducing agent is about 6-14 hours; Preferably, the contact time with the apoptosis-inducing agent is about 8-14 hours; Preferably, the contact time with the apoptosis-inducing agent is about 10-14 hours; Preferably, the contact time with the apoptosis-inducing agent is about 11-13 hours; Preferably, the contact time with the apoptosis-inducing agent is about 12 hours; Preferably, the method comprises: (1) culturing mesenchymal stem cells in a medium; (2) collecting the culture supernatant in step (1); (3) separating the vesicles from the culture supernatant in step (2); Including, Preferably, the step (1) of culturing mesenchymal stem cells comprises: (4) isolating mesenchymal stem cells from the tissue; (5) adding a medium to culture mesenchymal stem cells, and contacting the mesenchymal stem cells with an apoptosis-inducing agent in the medium; Including, More preferably, the apoptosis inducer comprises one or a combination of several of staurosporine, ultraviolet irradiation, starvation, and heat stress; More preferably, the apoptosis inducer is staurosporine; Preferably, the concentration of staurosporine is about 200 nM-1000 nM; Preferably, the concentration of staurosporine is about 250 nM-750 nM; Preferably, the concentration of staurosporine is about 400 nM-600 nM; Preferably, the concentration of staurosporine is about 450 nM-550 nM; Preferably, the concentration of staurosporine is about 500 nM.

11. The method according to claim 10, which is used to obtain induced vesicles that are positive for tissue factor expression, or to obtain an induced vesicle product with an increased rate of positive tissue factor expression.

12. Inducible vesicles obtainable by the method of claim 10.

13. Use of the inducible vesicles of claim 12 in the preparation of an extracorporeal blood coagulant or extracorporeal hemostatic agent.

14. The induced vesicles are vesicles that are produced by inducing apoptosis by adding an external factor when stem cells are normally alive, Preferably, the stem cells are mesenchymal stem cells; Preferably, the origin of the mesenchymal stem cells includes bone marrow, dental pulp, urine, oral cavity, fat, placenta, umbilical cord, periosteum, tendon, or peripheral blood; Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells; Preferably, the mesenchymal stem cells are derived from a mammal, Preferably, the mammal is selected from a primate or a murine; Preferably, the primate is a human. The use according to claim 1 or 2, the method according to any one of claims 3 to 5, the use according to claim 6 or 7, the composition according to claim 8, the use according to claim 9, the method according to claim 10, the induced vesicle or induced vesicle product according to claim 11, the induced vesicle according to claim 12, or the use according to claim 13.

15. the drug is selected from a lyophilized powder injection, an injection, an aerosol inhalant, an oral formulation, or a topical formulation; Preferably, the drug is a topical formulation, Preferably, the topical preparation is selected from the group consisting of tinctures, poultices, pastes, powders, ointments, salves, creams, lotions, rubs, sprays, aerosols, films, patches, gels, solutions, sponges, and gauzes; Preferably, the gel comprises an aqueous gel and an oily gel, Preferably, the topical preparation is a sponge formulation, or Preferably, the medicament further comprises a pharmaceutically acceptable pharmaceutical carrier; More preferably, the pharmaceutical carrier comprises one or more of a diluent, excipient, filler, binder, disintegrant, surfactant and lubricant. The use according to claim 1 or 2, the method according to any one of claims 3 to 5, the use according to claim 6 or 7, the composition according to claim 8, the use according to claim 9, the method according to claim 10, the induced vesicle or induced vesicle product according to claim 11, the induced vesicle according to claim 12, or the use according to claim 13.

Images