Composition containing megakaryocyte-derived vesicle, preparation method therefor, and use thereof

Abstract

The present application relates to the technical field of biomedicine, in particular to a composition containing a megakaryocyte-derived vesicle, a preparation method therefor, and use thereof. The use is use of the composition containing the megakaryocyte-derived vesicle in the preparation of a drug for treating physiological hemostatic process disorders caused by various causes, wherein the composition is prepared by breaking down megakaryocytes into vesicles by means of extrusion. Provided in the embodiments of the present application are a composition containing a megakaryocyte-derived vesicle, a preparation method therefor, and novel use of the composition in the preparation of a drug for treating physiological hemostatic process disorders, thereby achieving a low-cost, safe, and effective platelet alternative therapy.

Description

Compositions containing megakaryocyte-derived vesicles, their preparation methods and uses

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. CN202410696612.2, filed on May 31, 2024, entitled “Composition Containing Vesicles of Megakaryocyte Derivatives and Methods for Preparation Thereof and Use Thereof,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This application relates to the field of biomedical technology, specifically to compositions comprising megakaryocyte-derived vesicles, methods for their preparation, and uses. Background Technology

[0004] Clinically, physiological hemostasis difficulties and disorders can induce more critical symptoms or even death. Under normal physiological conditions, apart from acute large-area trauma complicated by massive bleeding, physiological hemostasis difficulties and disorders are mainly caused by physiological thrombocytopenia.

[0005] Thrombocytopenia is a common clinical condition with many contributing factors, including genetic predisposition, drug-induced, tumor-induced, or infection-induced factors. Thrombocytopenia is often accompanied by a very high risk of bleeding; severe thrombocytopenia can lead to uncontrollable bleeding, endangering the patient's life.

[0006] Platelet transfusions are commonly used clinically to preventively or urgently increase platelet levels in patients. However, the clinical application of platelet products faces numerous limitations, such as limited donor availability, low yield, complex manufacturing processes, difficulties in storage and transportation, short shelf life, and high costs. Related technologies utilize methods such as in vitro differentiated platelets and in vitro fully synthetic platelet analogs (in nanoscale liposome form) to prepare platelet products or their analogs to address the clinical shortage of platelet products. However, these technologies still suffer from complex manufacturing processes, low yields, and high costs. Furthermore, in vitro fully synthetic platelet analogs further suffer from short post-injection half-life, limited functionality (some only assist platelet function without possessing hemostatic or coagulation properties), and the introduction of xenogeneic immunogens.

[0007] Therefore, there is an urgent need to develop a low-cost, safe, and effective platelet replacement therapy. Summary of the Invention

[0008] This application addresses at least one of the problems of the related technology in the following aspects.

[0009] Therefore, a first aspect of this application provides the use of a composition comprising megakaryocyte-derived vesicles in the preparation of a medicament for treating disorders of physiological hemostasis, wherein the composition is obtained by extruding the megakaryocytes into vesicles.

[0010] In some embodiments, the physiological hemostasis disorder includes thrombocytopenia, wherein the thrombocytopenia is selected from the group consisting of: hereditary thrombocytopenia and acquired thrombocytopenia.

[0011] The hereditary thrombocytopenias mentioned above include Fanconi anemia, congenital thrombocytopenic purpura with megakaryocytes, and May-Hegglin abnormality.

[0012] Acquired thrombocytopenia includes primary blood-related diseases, drugs, malignancies, infections, and ionizing radiation-induced damage to hematopoietic stem cells or bone marrow;

[0013] Optionally, the physiological hemostasis disorder includes thrombocytopenia, wherein the thrombocytopenia is selected from the group consisting of: increased destruction of physiologically normal platelets and increased consumption of physiologically normal platelets.

[0014] The increased destruction of physiologically normal platelets includes immune thrombocytopenia, systemic lupus erythematosus, and drug-induced immune thrombocytopenia.

[0015] The increased consumption of physiologically normal platelets includes thrombocytopenia caused by thrombotic diseases and thrombocytopenia caused by viral infectious diseases, wherein the thrombotic diseases include disseminated intravascular coagulation and thrombotic thrombocytopenic purpura;

[0016] Optionally, the physiological hemostasis disorder includes thrombocytopenia, wherein the thrombocytopenia is a decrease in circulating platelets caused by splenomegaly and hypersplenism due to chronic liver disease.

[0017] In some embodiments, the megakaryocytes are mature megakaryocytes and / or megakaryocyte progenitor cells differentiated from stem cells selected from the group consisting of: hematopoietic stem cells derived from umbilical cord blood or peripheral blood, embryonic stem cells, induced pluripotent stem cells and adipose-derived mesenchymal stem cells. Preferably, the megakaryocytes are CD41 and CD42b double-positive megakaryocytes.

[0018] Optionally, the megakaryocyte-derived vesicles have a particle size of 50 to 1000 nM, preferably 100 to 600 nM.

[0019] An embodiment of the second aspect of this application provides a method for preparing a composition comprising megakaryocyte-derived vesicles for treating disorders of physiological hemostasis, comprising:

[0020] (1) A suspension containing vesicles is prepared by extruding the megakaryocytes into vesicles;

[0021] (2) Purify the suspension containing vesicles.

[0022] The megakaryocytes therein are mature megakaryocytes and / or megakaryocyte progenitor cells derived from stem cells selected from the group consisting of: hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells and adipose-derived mesenchymal stem cells derived from umbilical cord blood or peripheral blood. Preferably, the megakaryocytes are CD41 and CD42b double-positive megakaryocytes.

[0023] In some embodiments, the method further includes pretreating the megakaryocytes before squeezing them, wherein the pretreating is treating the megakaryocytes with a protease inhibitor;

[0024] Optionally, the megakaryocyte-derived vesicles have a particle size of 50 to 1000 nM, preferably 100 to 600 nM.

[0025] In some embodiments, the protease inhibitor includes a specific inhibitor of integrin metalloproteinase 17 (ADAM17), preferably the small molecule inhibitor TAPI-1;

[0026] Optionally, the pretreatment is to treat the megakaryocytes with the protease inhibitor at a final concentration of 1 to 1000 μM;

[0027] Optionally, the pretreatment involves treating the megakaryocytes with the protease inhibitor at a final concentration of 1 to 1000 μM for 12 to 48 hours.

[0028] In some embodiments, the step of preparing the suspension containing vesicles by extruding the megakaryocytes into vesicles includes:

[0029] The vesicle-containing suspension is prepared by extruding a megakaryocyte suspension through a polymer membrane; or the vesicle-containing suspension is prepared by extruding a megakaryocyte suspension through a microfluidic chip.

[0030] In some embodiments, the step of extruding the megakaryocyte suspension through a polymer membrane to obtain the vesicle-containing suspension comprises: repeatedly extruding the megakaryocyte suspension through a polymer membrane with a pore size of 1 to 5 μm, optionally a polycarbonate membrane, at an extrusion speed of 5 to 10 seconds per extrusion, an extrusion volume of 0.5 to 2 ml per extrusion, and a number of extrusions of 5 to 20 times, to obtain the vesicle-containing suspension.

[0031] In some embodiments, the step of extruding the megakaryocyte suspension through a microfluidic chip to obtain the vesicle-containing suspension includes: passing the megakaryocyte suspension sequentially through a first microfluidic chip and a second microfluidic chip, wherein the predetermined width of the first microfluidic chip is 5 to 20 μm, and the predetermined width of the second microfluidic chip is 1 to 5 μm.

[0032] In some embodiments, the concentration of the megakaryocyte suspension is 5 × 10⁻⁶. 5 Up to 5×10 7 Cells / mL.

[0033] The embodiments of the third aspect of this application provide compositions prepared by the preparation method of any of the embodiments of the second aspect described above.

[0034] The embodiments of this application achieve the following beneficial effects:

[0035] Compositions containing megakaryocyte-derived vesicles, prepared by extruding megakaryocytes, can be used as platelet replacement or supplementary drugs in the treatment of physiological hemostasis disorders caused by thrombocytopenia (such as uncontrollable bleeding throughout the body). They possess the potential to regulate endogenous megakaryocyte platelet production in vivo, exhibit good biocompatibility, and are easy to store and transport. This application provides a method for preparing compositions containing megakaryocyte-derived vesicles and their novel use in the preparation of drugs for treating physiological hemostasis disorders, thereby achieving a cost-effective, safe, and efficient platelet replacement therapy. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 is a schematic diagram of a method for preparing a composition containing megakaryocyte-derived vesicles provided in an embodiment of this application.

[0038] Figure 2 shows a schematic diagram of the microchannel structure (Figure 2A) and a topographical diagram of the microfluidic chip (Figure 2B).

[0039] Figure 3 shows the detection results of vesicle particle size distribution in the composition containing megakaryocyte-derived vesicles prepared in Example 2 of this application in Example 3 of this application. Figure 3A shows the vesicle particle size distribution results in the composition prepared by polymer membrane extrusion method, and Figure 3B shows the vesicle particle size distribution results in the composition prepared by microchannel extrusion method.

[0040] Figure 4 shows the results of characterizing the morphology of megakaryocyte-derived vesicles in the composition prepared in Example 2 of this application by transmission electron microscopy in Example 3 of this application.

[0041] Figure 5 shows the results of flow cytometry characterization of platelet hemostasis and coagulation-related active proteins derived from megakaryocytes on the surface of vesicles in the composition prepared in Example 2 of this application, in Example 3 of this application.

[0042] Figure 6 shows the in vitro agglutination results of the megakaryocyte-derived vesicle composition prepared in Example 2 of this application in Example 3 of this application, wherein Figure 6A shows the in vitro agglutination results of the composition prepared by polymer membrane extrusion method, and Figure 6B shows the in vitro agglutination results of the composition prepared by microchannel extrusion method.

[0043] Figure 7 shows the in vivo functional evaluation results of the composition containing megakaryocyte-derived vesicles in Example 4 of this application. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0045] This application is based on the inventor's following understanding:

[0046] Platelets are enucleated, biconcave, flattened disc-shaped granules produced in the body by mature megakaryocytes using the shear force of blood. In this form, they are in an inactive state. During physiological hemostasis and coagulation, platelets first adhere to the damaged blood vessel wall via the glycoprotein GPIb-IX-V complex on their surface. They release their own thrombin, ADP, and other activating factors and vasoconstrictive substances, mediating their own activation. The increased levels of active glycoproteins GP IIb / IIIa increase platelet aggregation at the bleeding site, thereby accelerating the hemostasis process. The negatively charged surface of platelets can also adsorb various coagulation factors, promoting the occurrence of the coagulation cascade at the bleeding site and accelerating the coagulation process.

[0047] Thrombocytopenia is a common clinical problem with many potential causes, including genetics, medications, tumors, and infections. Immune factors, coagulation disorders, or viruses can increase platelet destruction or consumption; abnormal liver and spleen function can lead to abnormal platelet distribution. Thrombocytopenia is often accompanied by bleeding symptoms, and severe cases can cause uncontrollable bleeding, endangering the patient's health. Clinically, platelet transfusions are commonly used to prophylactically or urgently increase a patient's platelet level. However, the development of platelet products faces many limitations, such as a very limited pool of donors, a complex platelet separation process, difficulties in preservation and transportation, short shelf life, susceptibility to bacterial infection, and high costs. Current platelet product production cannot meet the total clinical transfusion demand, making in vitro platelet production or platelet replacement therapy an urgent need.

[0048] In related technologies, platelet products or their analogues are prepared through in vitro platelet differentiation and in vitro totalized platelet analogues (in nanoscale liposome form) to meet the clinical shortage of platelet products. However, these technologies still suffer from problems such as complex preparation processes, low yields, and high costs. Furthermore, in vitro totalized platelet analogues have further drawbacks, including short post-injection half-life, limited functionality (some only assist platelet function without possessing hemostatic and coagulation effects), and the introduction of xenogeneic immunogens. Therefore, there is an urgent need to develop a low-cost, safe, and effective platelet replacement therapy.

[0049] In response, the inventors conducted extensive research and proposed a new use of compositions containing megakaryocyte-derived vesicles in the preparation of medicaments for treating physiological hemostasis disorders, as well as a method for preparing such compositions, providing a low-cost, safe, and effective platelet replacement therapy.

[0050] The first aspect of this application provides the use of a composition comprising megakaryocyte-derived vesicles in the preparation of a medicament for treating disorders of physiological hemostasis, wherein the composition is obtained by extruding the megakaryocytes into vesicles.

[0051] Physiological hemostasis: Under normal circumstances, bleeding caused by damage to small blood vessels will stop spontaneously within a few minutes. This phenomenon is called physiological hemostasis. The physiological hemostasis process includes three processes: vasoconstriction, platelet thrombus formation, and blood coagulation. These three processes occur sequentially and overlap, and are closely related to each other, with platelets playing a crucial role.

[0052] In this application, the megakaryocyte-derived vesicles are prepared by artificially and actively extruding megakaryocytes derived from various stem cell differentiations into a large number of micron- or nano-sized vesicles.

[0053] In some embodiments, the physiological hemostasis disorder includes thrombocytopenia, wherein the thrombocytopenia is selected from the group consisting of: hereditary thrombocytopenia and acquired thrombocytopenia.

[0054] The hereditary thrombocytopenias mentioned above include Fanconi anemia, congenital thrombocytopenia with malformed megakaryocytes, and May-Hegglin abnormality. The acquired thrombocytopenias mentioned above include blood-related primary diseases, drugs, malignancies, infections, and ionizing radiation-induced damage to hematopoietic stem cells or bone marrow.

[0055] In some embodiments, the physiological hemostasis disorder includes thrombocytopenia, wherein the thrombocytopenia is selected from the group consisting of: increased destruction of normal platelets and increased consumption of normal platelets.

[0056] The increased destruction of physiologically normal platelets includes immune thrombocytopenic purpura, systemic lupus erythematosus, and drug-induced immune thrombocytopenic purpura. The increased consumption of physiologically normal platelets includes thrombocytopenia caused by thrombotic diseases and thrombocytopenia caused by viral infectious diseases. The thrombotic diseases include disseminated intravascular coagulation and thrombotic thrombocytopenic purpura.

[0057] In some embodiments, the physiological hemostasis disorder includes thrombocytopenia, wherein the thrombocytopenia is a decrease in circulating platelets caused by splenomegaly and hypersplenism due to chronic liver disease.

[0058] The composition provided in this application can be used for severe bleeding symptoms associated with thrombocytopenia and acute large-area trauma, etc.

[0059] In some embodiments, the megakaryocytes are mature megakaryocytes and / or megakaryocyte progenitor cells differentiated from stem cells selected from the group consisting of: hematopoietic stem cells derived from umbilical cord blood or peripheral blood, embryonic stem cells, induced pluripotent stem cells, and adipose-derived mesenchymal stem cells.

[0060] In some embodiments, the megakaryocytes are CD41 and CD42b double-positive megakaryocytes.

[0061] In some embodiments, the megakaryocyte-derived vesicles have a particle size of 50 to 1000 nM (e.g., 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM).

[0062] In some embodiments, the megakaryocyte-derived vesicles have a particle size of 100 to 600 nM (e.g., 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM).

[0063] In this application, megakaryocytes derived from various stem cell differentiations are decomposed using an artificial active squeezing technique to obtain a composition containing a large number of vesicles with appropriate particle size distribution, which has the ability to promote physiological hemostasis.

[0064] A second aspect of this application provides a method for preparing a composition comprising megakaryocyte-derived vesicles for treating disorders of physiological hemostasis, comprising:

[0065] (1) A suspension containing vesicles is prepared by extruding the megakaryocytes into vesicles;

[0066] (2) Purify the suspension containing vesicles.

[0067] The megakaryocytes mentioned therein are mature megakaryocytes and / or megakaryocyte progenitor cells derived from stem cells selected from the following groups: hematopoietic stem cells derived from umbilical cord blood or peripheral blood, embryonic stem cells, induced pluripotent stem cells, and adipose-derived mesenchymal stem cells.

[0068] The megakaryocyte-derived vesicles in the composition prepared by the method provided in this application have the ability to promote coagulation due to the presence of platelet factor III on their surface, and have the potential to regulate the production of platelets by endogenous megakaryocytes in vivo due to the presence of effective nucleic acids or proteins from megakaryocytes. They have good biocompatibility and are easy to preserve and transport. Thus, the embodiments of this application realize a low-cost and safe and effective platelet replacement therapy through the above-described methods and uses.

[0069] In some embodiments, the megakaryocytes are CD41 and CD42b double-positive megakaryocytes.

[0070] In some embodiments, the megakaryocyte-derived vesicles have a particle size of 50 to 1000 nM (e.g., 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM).

[0071] In some embodiments, the megakaryocyte-derived vesicles have a particle size of 100 to 600 nM (e.g., 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM).

[0072] The composition prepared in this application has an appropriate vesicle particle size distribution, mainly in the range of 100 to 600 μm. This not only significantly promotes the adhesion and aggregation of platelets with abnormally low concentrations below physiological levels in blood vessels at the site of injury, but also reduces the probability of hemostatic thrombus formation without the ability to change sexual function, thereby avoiding leakage caused by the hemostatic thrombus.

[0073] In some embodiments, the method further includes pretreating the megakaryocytes before squeezing them, wherein the pretreating is treating the megakaryocytes with a protease inhibitor.

[0074] In some embodiments, the protease inhibitor includes a specific inhibitor of integrin metalloproteinase 17 (ADAM17), preferably the small molecule inhibitor TAPI-1.

[0075] In some embodiments, the pretreatment is to treat the megakaryocytes with the protease inhibitor at a final concentration of 1 to 1000 μM (e.g., 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM).

[0076] In some embodiments, the pretreatment is to treat the megakaryocytes with the protease inhibitor at a final concentration of 1 to 1000 μM (e.g., 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM) for 12 to 48 hours (e.g., 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours).

[0077] In some embodiments, the step of preparing the suspension containing vesicles by extruding the megakaryocytes into vesicles includes:

[0078] The vesicle-containing suspension is prepared by squeezing a megakaryocyte suspension through a polymer membrane; or

[0079] The vesicle-containing suspension is prepared by squeezing a megakaryocyte suspension through a microfluidic chip.

[0080] In some embodiments, the process of extruding the megakaryocyte suspension through a polymer membrane to obtain the vesicle-containing suspension includes: an extrusion speed of 5 to 10 seconds per stroke (e.g., 6 seconds per stroke, 7 seconds per stroke, 8 seconds per stroke, 9 seconds per stroke), and an extraction rate of 0.5 to 2 ml per stroke (e.g., 0.6 ml per stroke, 0.7 ml per stroke, 0.8 ml per stroke, 0.9 ml per stroke, 1.0 ml per stroke, 1.1 ml per stroke, 1.2 ml per stroke, 1.3 ml per stroke, 1.4 ml per stroke, 1.5 ml per stroke). The megakaryocyte suspension is repeatedly squeezed through a polymer membrane with a pore size of 1 to 5 μm (e.g., 1.6 mL / time, 1.7 mL / time, 1.8 mL / time, 1.9 mL / time) and 5 to 20 times (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 times) to obtain the vesicle-containing suspension.

[0081] In some embodiments, the step of extruding the megakaryocyte suspension through a microfluidic chip to obtain the vesicle-containing suspension includes: passing the megakaryocyte suspension sequentially through a first microfluidic chip and a second microfluidic chip, wherein the predetermined width of the first microfluidic chip is 5 to 20 μm (e.g., 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm), and the predetermined width of the second microfluidic chip is 1 to 5 μm (e.g., 2 μm, 3 μm, 4 μm).

[0082] In some embodiments, the concentration of the megakaryocyte suspension is 5 × 10⁻⁶. 5 Up to 5×10 7 Cells / mL (e.g., 6 × 10⁶) 5 10 cells / mL, 7×10 5 cells / mL, 8×10 5 cells / mL, 9×10 5 cells / mL, 1×10 6 cells / mL, 2×10 6 cells / mL, 3×10 6 cells / mL, 4×10 6 cells / mL, 5×10 6 cells / mL, 6×10 6 10 cells / mL, 7×10 6 cells / mL, 8×10 6cells / mL, 9×10 6 cells / mL, 1×10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, 4×10 7 (cells / mL).

[0083] The third aspect of this application provides a composition prepared by the preparation method of any of the second aspects described above.

[0084] In this application, the term "comprising" is an open-ended expression, meaning it includes the content specified in this invention, but does not exclude other aspects.

[0085] The following embodiments are used to further illustrate the advantages and features of this method, and are not intended to limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0086] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Unless otherwise specified, all quantitative analysis experiments in the following examples were performed in triplicate, and the results were averaged.

[0087] Example 1: Preparation of a composition containing megakaryocyte-derived vesicles

[0088] Figure 1 shows a schematic diagram of vesicle preparation. First, mononuclear cells were isolated from human umbilical cord blood. The mononuclear cells were resuspended in megakaryocyte differentiation medium and added to cell culture flasks in appropriate quantities. The cells were then incubated statically in a 5% CO2, 37°C incubator for 14 consecutive days. The differentiated cells were collected on day 14, and cell counting was performed. The positivity rate of megakaryocyte-related markers (CD41 / CD42b) in the cells was detected by flow cytometry to determine the number of megakaryocytes.

[0089] ADAM17 can mediate the cleavage of the enzymatically degradable region of CD42b on the cell membrane under mechanical stimulation, thereby causing the loss of CD42b and reducing its adhesive function for vesicles. Therefore, in order to reduce the loss of CD42b on the cell surface during preparation, megakaryocytes are pretreated before vesicle preparation. The pretreatment reagent is TAPI-1, a specific small molecule inhibitor of integrin metalloproteinase 17 (ADAM17).

[0090] Differentiated cells were pretreated with medium supplemented with 10 μM TAPI-1 for 12 h. After incubation, cells were collected and counted according to the ratio of CD41+ and CD42b+ double-positive cells. The medium was removed by centrifugation at 400g for 5 min at room temperature, and the cells were washed once with PBS. 5 Megakaryotic cells were resuspended at a concentration of cells / ml.

[0091] Then, a suspension containing vesicles is prepared by squeezing the megakaryocyte suspension using either method one or method two.

[0092] Method 1: Preparation of cell vesicles using polymer membrane extrusion: Take a polycarbonate membrane with a pore size of 5μm and a diameter of 19mm, immerse it completely in PBS, and clean the liposome extruder accessories; assemble the extruder accessories and polycarbonate membrane, use one side of the extruder syringe to draw about 1ml of well-mixed cell suspension, assemble it into the extrusion module, and assemble the empty syringe on the other side into the extrusion module as well. Use both syringes to push the cell suspension back and forth evenly, about 10 seconds per squeeze, for a total of 11 squeezes;

[0093] After squeezing, the vesicle-containing suspension obtained by squeezing is collected in the syringe on the other side.

[0094] Method 2: Preparation of cell vesicles using microchannel extrusion: Schematic diagram of the microchannel structure (Figure 2A) and morphology of the microfluidic chip (Figure 2B). As shown in Figure 2, the microchannel in Figure 2A is made of polydimethylsiloxane, which is tightly bonded to a glass slide to obtain the microfluidic chip shown in Figure 2B. A syringe is connected to the prepared microfluidic chip using a polytetrafluoroethylene (PTFE) conduit with an outer diameter of 0.9 mm and an inner diameter of 0.5 mm. The microfluidic channel is first rinsed with 75% alcohol, then rinsed with PBS, and finally the microfluidic channel is degassed. 1 mL of the mixed cell suspension is drawn up using a 1 mL sterile syringe, and the syringe is loaded onto a microinjection pump. After setting the syringe size to 1 mL (inner diameter 4.69 mm), the injection is performed at a linear velocity of 50 μL / min, and the suspension containing vesicles is collected from the outlet.

[0095] The stepwise centrifugation purification steps of the suspension containing vesicles are as follows: First, the squeezed suspension is centrifuged at 400g for 5min to remove unsqueezed cells and larger particles; the supernatant is collected by centrifugation at 14000g for 10min to collect vesicles, the supernatant containing most of the cell contents is discarded, and the precipitate is resuspended with an appropriate amount of PBS to obtain a composition containing vesicles derived from megakaryocytes.

[0096] Example 2: Removal of free DNA from compositions containing megakaryocyte-derived vesicles

[0097] The composition prepared in Example 1 contained excessive megakaryocyte DNA, which adversely affected the in vivo biosafety of the vesicles. Therefore, the free DNA in the composition should be removed. An appropriate amount of the composition prepared in Example 1 was taken, and 0.5 mg / ml of DNase I was added. The mixture was incubated in a 37°C water bath for 1 hour. After incubation, the vesicles were collected by centrifugation and resuspended in an equal volume of PBS. An equal volume of physiological saline was used as a control group. The DNA content was detected using a Qubit fluorometer (Invitrogen). The results showed that compared to the amount of DNA in the physiological saline control group, the DNase I group degraded approximately 89.3% of the residual DNA.

[0098] Example 3: In vitro characterization of megakaryocyte-derived vesicles

[0099] (1) Physical property characterization of megakaryocyte-derived vesicles: The vesicles in the composition obtained in Example 2 were diluted appropriately, and then the particle size distribution and the number of vesicles in the 0-1 μm size range were characterized using a nanoparticle tracking analyzer. The particle size distribution results are shown in Figure 3. The number of vesicles in the 0-1 μm size range is approximately 2000-5000 times the number of raw megakaryocytes. The vesicle potential was characterized using a Malvern nanoparticle potentiometer. A small amount of vesicle fluid was diluted to 1 ml with physiological saline, and its Zeta potential value was detected. The result was approximately -13 mV. The morphology of the vesicles was characterized using transmission electron microscopy (TEM). The results are shown in Figure 4. The prepared vesicles have a typical vesicle-like "cup-shaped" or "disc-shaped" structure. Flow cytometry was used to characterize the levels of hemostatic markers (CD41 and CD42b) and coagulation-related factor PF3 on the vesicle surface. The results are shown in Figure 5. The proportion of CD41+ vesicles was 65.42%, the proportion of CD42b+ vesicles was 24.16%, and the proportion of CD41+ & CD42b+ dual-positive vesicles was 17.76%. This proportion was related to the ratio of CD41 and CD42b on the megakaryocyte surface. The proportion of PF3 single-positive vesicles was 85.23%.

[0100] (2) In vitro platelet-like hemostatic function characterization of compositions containing megakaryocyte-derived vesicles—CD41-mediated agglutination assay: 1.0 × 10⁻⁶ oz. 6 CD41 and CD42b double-positive megakaryocytes were washed once with PBS and resuspended to a concentration of 5.0 × 10⁶ cells. 5 / ml concentration; vesicles were prepared according to the extrusion and purification conditions in Example 1, and the extruded particles were finally resuspended in 500 μl of physiological saline; according to the experimental groups in Table 1 below, physiological saline, vesicles, human fibrinogen (5 μg / μl), and ADP (200 μM) were respectively activated at room temperature for 20 min, and thrombin (0.2 U / μl) was added to each tube, and then incubated in a 37°C water bath for 1 h.

[0101] Table 1

[0102] The vesicle aggregation state of each tube (tube 1 corresponds to Figure 6A, tube 2 corresponds to Figure 6B) is shown in Figure 6.

[0103] Example 4: In vivo functional evaluation of a composition containing megakaryocyte-derived vesicles

[0104] Fifteen 8-week-old female NCG mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were irradiated with a dose of 2.0 Gy to construct thrombocytopenia. The mice's autologous platelet levels were lowest on day 7 post-irradiation, therefore this was selected as the experimental site for injecting megakaryocyte-derived vesicles. The experimental groups are shown in Table 2 below. The preparation method provided in this application allows megakaryocytes to produce approximately 2000-5000 times the number of vesicles, i.e., approximately 5 × 10⁻⁶ vesicles. 5 One megakaryocyte can produce approximately 10 9 There are 10 CD41 and CD42b dual-positive vesicles, therefore the setting contains 10 9 A combination of platelets (platelet group) was used as a positive control.

[0105] Table 2

[0106] The megakaryocyte differentiation and compression method is as described in Examples 1 and 2, and the composition containing megakaryocyte-derived vesicles is prepared by injecting 200 μl of the volume resuspension membrane per mouse using the compression method.

[0107] Irradiated mice were divided into three groups based on their platelet levels on Day 6 after irradiation. Ear tags were recorded, and each group underwent tail vein injection. Hemostasis was evaluated 0.5 hours after injection using the needle puncture method. Specifically, a 23G lancet was inserted vertically into the tail artery at a distance of 15 mm from the beginning of the tail. Timing was started immediately after the needle was removed, and any spilled blood was absorbed near the wound with laboratory paper until bleeding ceased, marking the endpoint of hemostasis.

[0108] The hemostasis time results of the above experiment are shown in Figure 7. The results show that megakaryocyte compression of vesicles has a significant hemostatic effect, indicating that vesicles derived from megakaryocytes have platelet-like hemostatic function.

[0109] The above embodiments are preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the essential principles of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

[0110] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0111] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of a composition comprising megakaryocyte-derived vesicles in the preparation of a medicament for treating disorders of physiological hemostasis, wherein the composition is prepared by extruding the megakaryocytes into vesicles.

2. The use according to claim 1, characterized in that, The physiological hemostasis disorders include thrombocytopenia, wherein the thrombocytopenia is selected from the group consisting of: hereditary thrombocytopenia and acquired thrombocytopenia, wherein the hereditary thrombocytopenia includes Fanconi anemia, congenital thrombocytopenia with malformations and megakaryocytes and May-Hegglin abnormality, and wherein the acquired thrombocytopenia includes hematologic diseases, drugs, malignancies, infections and ionizing radiation that damage hematopoietic stem cells or bone marrow; Optionally, the physiological hemostasis disorder includes thrombocytopenia, wherein the thrombocytopenia is selected from the group consisting of: increased destruction of normal platelets and increased consumption of normal platelets, wherein the increased destruction of normal platelets includes immune thrombocytopenia, systemic lupus erythematosus and drug-induced immune thrombocytopenia, wherein the increased consumption of normal platelets includes thrombocytopenia caused by thrombotic diseases and thrombocytopenia caused by viral infectious diseases, wherein the thrombotic diseases include disseminated intravascular coagulation and thrombotic thrombocytopenic purpura; Optionally, the physiological hemostasis disorder includes thrombocytopenia, wherein the thrombocytopenia is a decrease in circulating platelets caused by splenomegaly and hypersplenism due to chronic liver disease.

3. The use according to claim 1 or 2, characterized in that, The megakaryocytes are mature megakaryocytes and / or megakaryocyte progenitor cells differentiated from stem cells selected from the following groups: hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells and adipose-derived mesenchymal stem cells derived from umbilical cord blood or peripheral blood. Preferably, the megakaryocytes are CD41 and CD42b double-positive megakaryocytes. Optionally, the megakaryocyte-derived vesicles have a particle size of 50 to 1000 nM, preferably 100 to 600 nM.

4. A method for preparing a composition containing megakaryocyte-derived vesicles for treating disorders of physiological hemostasis, comprising: (1) A suspension containing vesicles is prepared by extruding the megakaryocytes into vesicles; (2) Purify the suspension containing vesicles. The megakaryocytes therein are mature megakaryocytes and / or megakaryocyte progenitor cells derived from stem cells selected from the group consisting of: hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells and adipose-derived mesenchymal stem cells derived from umbilical cord blood or peripheral blood. Preferably, the megakaryocytes are CD41 and CD42b double-positive megakaryocytes.

5. The preparation method according to claim 4, characterized in that, Before squeezing the megakaryocytes, the method further includes pretreating the megakaryocytes, wherein the pretreating is treating the megakaryocytes with a protease inhibitor; Optionally, the megakaryocyte-derived vesicles have a particle size of 50 to 1000 nM, preferably 100 to 600 nM.

6. The preparation method according to claim 5, characterized in that, The protease inhibitor includes a specific inhibitor of integrin metalloproteinase 17, preferably the small molecule inhibitor TAPI-1. Optionally, the pretreatment is to treat the megakaryocytes with the protease inhibitor at a final concentration of 1 to 1000 μM; Optionally, the pretreatment involves treating the megakaryocytes with the protease inhibitor at a final concentration of 1 to 1000 μM for 12 to 48 hours.

7. The preparation method according to claim 4, characterized in that, The process of preparing the suspension containing vesicles by extruding the megakaryocytes into vesicles includes: The vesicle-containing suspension is prepared by squeezing a megakaryocyte suspension through a polymer membrane; or The vesicle-containing suspension is prepared by squeezing a megakaryocyte suspension through a microfluidic chip.

8. The preparation method according to claim 7, characterized in that, The method of obtaining the vesicle-containing suspension by extruding a megakaryocyte suspension through a polymer membrane includes: repeatedly extruding the megakaryocyte suspension at an extrusion speed of 5 to 10 seconds per extrusion, an extrusion volume of 0.5 to 2 ml per extrusion, and 5 to 20 extrusions to pass it through a polymer membrane with a pore size of 1 to 5 μm, optionally a polycarbonate membrane, to obtain the vesicle-containing suspension; or The process of extruding a megakaryocyte suspension through a microfluidic chip to obtain the vesicle-containing suspension includes: The megakaryocyte suspension is passed sequentially through a first microfluidic chip and a second microfluidic chip, wherein the predetermined width of the first microfluidic chip is 5 to 20 μm, and the predetermined width of the second microfluidic chip is 1 to 5 μm.

9. The preparation method according to claim 7 or 8, characterized in that, The concentration of the megakaryocyte suspension was 5 × 10⁻⁶. 5 Up to 5×10 7 Cells / mL.

10. The composition prepared by any one of claims 4 to 9.

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