Extracellular vesicle gel preparation and temperature-sensitive gel solution used for extracellular vesicle gel preparation

Abstract

The invention provides an extracellular vesicle temperature-sensitive gel preparation, which comprises an effective amount of extracellular vesicles and a gel solution for loading the extracellular vesicles, and can generate gelation at a preset temperature. The invention further provides a method for preparing the extracellular vesicle gel preparation, a method for preventing or treating diseases or symptoms responding to extracellular vesicles in a patient by using the extracellular vesicle gel preparation and / or application of the extracellular vesicle gel preparation.

Description

Field of the Invention

[0001] The field of the present invention relates to an extracellular vesicle gel preparation. Background of the Invention

[0002] Extracellular vesicles (EVs) are a rapidly growing area of ​​biomedical research and clinical translational medicine. EVs are released by virtually all mammalian cells. EVs carry a variety of biological components, such as lipids, nucleic acids, and proteins. They not only play a key role in intercellular communication but also regulate various signaling pathways in target cells. They can serve as biomarkers for disease diagnosis and possess a variety of therapeutic effects, including anti-tumor and tissue repair [1-3].

[0003] The most common method of administering EVs for disease treatment is through single high-dose or multiple low-dose injections. However, high-dose injections can result in some EVs being wasted due to excessive EV concentrations, while multiple low-dose injections complicate administration. Furthermore, EVs administered via this route tend to accumulate in the liver and spleen and have a short half-life [3,4]. To overcome these shortcomings, biomaterials, such as hydrogels, encapsulating EVs for drug delivery may be a promising strategy to address this issue.

[0004] Hydrogels are three-dimensional polymer networks composed of natural or synthetic materials, in which the liquid component is mainly water. Hydrogels have the following properties: 1) a hydrophilic porous structure, which gives them excellent water retention capacity. While absorbing water, they can maintain the integrity of the gel structure, allowing the effective ingredients in them to diffuse; 2) degradability, which causes the gel to degrade simultaneously with the release of the encapsulated effective substances; 3) a sol-gel transition, which shows a volume phase change under external stimuli such as temperature. The phase change process is conducive to drug encapsulation or good adhesion of the gel to the damaged part; 4) resistance to shear / compression forces, which can enhance the gel's adaptability to the environment and help the encapsulated environmentally sensitive substances maintain a good spatial structure [5].

[0005] Of course, the properties and applications of hydrogels are not limited to this. For example, when used in the orthopedic field, they must be given a certain strength, and when used in the cornea, they may be required to provide certain optical properties.

[0006] Currently, different hydrogels have been used to encapsulate various types of cells or EVs. EVs have been widely studied due to their advantages over cells, such as low immunogenicity and ease of uptake by various cell types [6-8]. There are two common ways to encapsulate EVs in hydrogels: 1) first preparing a porous gel carrier and then loading the EVs into it; 2) loading the EVs into the gel during the formation of the gel carrier [5]. In both methods, it should be noted that the gel monomers or other additives used should be biofriendly materials to ensure that there is no harm to the EVs during the loading process or to the patient during use.

[0007] Poloxamer (trade name: pluronic) is a non-toxic, non-irritating pharmaceutical excipient that has been widely used in oral, injectable, and topical drug formulations. Poloxamer is a copolymer of polyoxyethylene and polyoxypropylene. The United States Pharmacopoeia lists five different grades of poloxamer: poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407. Poloxamer has unique thermosensitive properties: as temperature rises, the molecules aggregate into micelles. When the molecular concentration is sufficiently high, the micelles stack in an orderly manner and gel. Poloxamer exists as a liquid at low temperatures and as a semisolid gel at high temperatures. Due to this reversible thermoresponsive behavior, poloxamer can adapt to the complex and irregular spaces of wounds, allowing bioactive drugs to more fully adhere to and cover the target area and exert their biological effects. Poloxamer has been widely used in drug delivery research and as a cell / EV encapsulation scaffold[9,10]. In addition, Poloxamer itself has mild inflammatory properties and the ability to absorb wound surface secretions, keeping the wound moist and providing a certain healing environment

[11] . In summary, Poloxamer is a good material for encapsulating EVs for delivery and can be applied to acute or chronic skin damage. In the examples, diabetic foot was used as the application scenario to screen the excipients. SUMMARY OF THE INVENTION

[0008] In a first aspect, the present application provides an extracellular vesicle gel formulation comprising an effective amount of extracellular vesicles and a gel solution loaded with extracellular vesicles, said gel solution consisting of a poloxamer, a hydroxypropyl methylcellulose (HPMC) and a physiologically acceptable aqueous solution, wherein the concentration of said poloxamer is 15-30%, the concentration of said hydroxypropyl methylcellulose is 0.25-3%, wherein said % is a weight by volume percentage of grams (g) of the related substance relative to milliliters (mL) of the aqueous solution. In a preferred embodiment, said formulation is transformed from a flow state to a gel state at human skin temperature when the gel is used in liquid form on human skin. In some specific embodiments, the time of said transformation from a flow state to a gel state is < 6.5 min, < 6 min, < 5.5 min, < 5 min, < 4.5 min, < 4 min, < 3.5 min, < 3 min, < 2.5 min, < 2 min, < 1.5 min or < 1 min.

[0009] In some specific embodiments, said poloxamer is selected from one or more of poloxamer 407, poloxamer 124, poloxamer 188, poloxamer 338. In a more specific embodiment, said poloxamer is poloxamer 407. In some specific embodiments, the concentration of said poloxamer is about 15-30%, preferably about 18-30%, more preferably about 20-25%, more preferably about 20%. In some specific embodiments, the concentration of said poloxamer is about 15%, about 16%, about 17%, about 18%, about 19%, about 21%, about 22%, about 23%, about 24%, about 25%.

[0010] In some specific embodiments, the type of said hydroxypropyl methylcellulose is 1828, 2208, 2906, 2910. In a more specific embodiment, the type of said hydroxypropyl methylcellulose is 2910. In some specific embodiments, the concentration of said hydroxypropyl methylcellulose is about 0.25-3%, preferably about 0.5-2%, more preferably about 0.5-1.5%. In some specific embodiments, the concentration of said hydroxypropyl methylcellulose is about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, more preferably about 1%.

[0011] In some specific embodiments, the human skin temperature is a temperature in the range of 24°C-37°C, 24°C-36.5°C, 24°C-36°C, 24°C-35.5°C, 24°C-35°C, 24°C-34.5°C, 24°C-34°C, 24°C-33.5°C, 24°C-33°C, 24°C-32.5°C, 24°C-32°C, 24°C-31.5°C, 24°C-31°C, 24°C-30.5°C, 24°C-30°C, 24°C-29.5°C, 24°C-29°C, 24°C-28.5°C or 24°C-28°C. In some specific embodiments, the human skin temperature is a temperature in the range of 25°C-37°C, 25°C-36.5°C, 25°C-36°C, 25°C-35.5°C, 25°C-35°C, 25°C-34.5°C, 25°C-34°C, 25°C-33.5°C, 25°C-33°C, 25°C-32.5°C, 25°C-32°C, 25°C-31.5°C, 25°C-31°C, 25°C-30.5°C, 25°C-30°C, 25°C-29.5°C, 25°C-29°C, 25°C-28.5°C or 25°C-28°C. In some specific embodiments, the human skin temperature is a temperature in the range of 26°C-37°C, 26°C-36.5°C, 26°C-36°C, 26°C-35.5°C, 26°C-35°C, 26°C-34.5°C, 26°C-34°C, 26°C-33.5°C, 26°C-33°C, 26°C-32.5°C, 26°C-32°C, 26°C-31.5°C, 26°C-31°C, 26°C-30.5°C, 26°C-30°C, 26°C-29.5°C, 26°C-29°C, 26°C-28.5°C or 26°C-28°C. In some specific embodiments, the human skin temperature is a temperature in the range of 27°C-37°C, 27°C-36.5°C, 27°C-36°C, 27°C-35.5°C, 27°C-35°C, 27°C-34.5°C, 27°C-34°C, 27°C-33.5°C, 27°C-33°C, 27°C-32.5°C, 27°C-32°C, 27°C-31.5°C, 27°C-31°C, 27°C-30.5°C, 27°C-30°C, 27°C-29.5°C, 27°C-29°C, 27°C-28.5°C or 27°C-28°C. In some specific embodiments, the human skin temperature is a temperature in the range of 28°C-37°C, 28°C-36.5°C, 28°C-36°C, 28°C-35.5°C, 28°C-35°C, 28°C-34.5°C, 28°C-34°C, 28°C-33.5°C, 28°C-33°C, 28°C-32.5°C, 28°C-32°C, 28°C-31.5°C, 28°C-31°C, 28°C-30.5°C, 28°C-30°C, 28°C-29.5°C, 28°C-29°C or 28°C-28.5°C.

[0012] In some specific embodiments, the extracellular vesicles are exosomes. In some specific embodiments, the exosomes are enriched in one or more miRNAs selected from miR-24, miR-21, miR-146, miR-150, and Let-7a. In some specific embodiments, the exosomes are enriched in one or more proteins selected from thrombospondin-1, heat shock protein 70 (heat shock cognate 71kDa protein), pyruvate kinase PKM (pyruvate kinase PKM), heat shock protein 90 (heat shock protein HSP 90-alpha), peroxiredoxin-1, and peroxiredoxin-2.

[0013] In some specific embodiments, the content of extracellular vesicles is 1×10 8 Particles / ml-1×10 11 particles

[0014] / ml, 1×10 9 Particles / ml-1×10 11 Particles / ml, 1×10 10 Particles / ml-1×10 11 Particles / ml, 1×10 8 Particles / ml-1×10 10 Particles / ml, 1×10 9 Particles / ml-1×10 10 particles / ml, or 1×10 8 Particles / ml-1×10 9 Particles / ml.

[0015] In particular, in a second aspect, a method for obtaining mesenchymal stem cell exosomes suitable for the purpose of the present invention is provided, comprising the steps of:

[0016] (a) providing mesenchymal stem cells;

[0017] (b) pre-culturing the mesenchymal stem cells using a first culture medium under appropriate culture conditions to obtain pre-cultured mesenchymal stem cells having a confluency of 60-90%;

[0018] Wherein, the first culture medium is αMEM culture medium containing platelet lysate;

[0019] (c) incubating the cultured mesenchymal stem cells with a second culture medium for 24 to 48 hours to obtain an incubation culture medium containing the cultured mesenchymal stem cells and exosomes produced by the cultured stem cells;

[0020] Wherein, the second culture fluid is an ultrafiltrate obtained by ultracentrifuging, filtering and ultrafiltration of an αMEM culture medium containing platelet lysate; and

[0021] (d) isolating exosomes from the incubation culture medium obtained in the previous step.

[0022] In another embodiment, the stem cells are mesenchymal stem cells of passage 2 (P2) to passage 6 (P6) in vitro, preferably mesenchymal stem cells of passage 4 (P4) in vitro.

[0023] In another embodiment, the mesenchymal stem cells are derived from mammals, preferably humans.

[0024] In another embodiment, the mesenchymal stem cells are derived from human tissues, such as adipose tissue, bone marrow, or placenta.

[0025] In another embodiment, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0026] In another embodiment, in step (b), the stem cell confluency is 60-90%, more preferably 70-90%, and most preferably 75-80%.

[0027] In another embodiment, the concentration of the platelet lysate in the first culture medium is 2% to 15%, more preferably 5% to 10%, and most preferably 5%, wherein the percentages are volume percentages.

[0028] In another embodiment, the concentration of the platelet lysate in the second culture medium is 2% to 15%, more preferably 5% to 10%, and most preferably 5%, wherein the percentages are volume percentages.

[0029] In another embodiment, the speed of the ultracentrifugation is 100,000 to 120,000 g, more preferably 100,000 g; most preferably 120,000 g.

[0030] In another embodiment, the ultracentrifugation time is 6 to 24 hours.

[0031] In another embodiment, the filtration is performed using a filter with a pore size of 0.22 μm to 0.45 μm, more preferably 0.22 μm.

[0032] In another embodiment, the ultrafiltration is performed using an ultrafiltration tube with a pore size of 100 to 300 KD, more preferably 300 KD.

[0033] In another embodiment, in step 1, the second culture solution is prepared using the following method:

[0034] (5) providing an αMEM medium containing 2% to 15% platelet lysate;

[0035] (ii) ultracentrifuging the αMEM medium containing 2% to 15% platelet lysate for 6 to 24 hours to obtain a supernatant; wherein the ultracentrifugation speed is 100,000 to 120,000 g, more preferably 100,000 g, and most preferably 120,000 g;

[0036] (iii) filtering the supernatant after centrifugation using a filter with a pore size of 0.22 μm to 0.45 μm (more preferably 0.22 μm) to obtain a filtered filtrate;

[0037] (iv) ultrafiltration is performed on the filtrate after filtration using an ultrafiltration tube with a pore size of 100 to 300 kD (more preferably 300 kD), thereby obtaining an ultrafiltrate, that is, obtaining the second culture fluid.

[0038] In another embodiment, the second culture medium has the following characteristics:

[0039] (1) Contains 2% to 15% platelet lysate;

[0040] (2) contains almost no exosomes in platelet lysates;

[0041] (3) Maintaining optimal proliferation capacity of mesenchymal stem cells;

[0042] (4) maintaining the optimal stem cell properties of mesenchymal stem cells; and

[0043] (5) Promote mesenchymal stem cells to secrete more exosomes.

[0044] In another embodiment, the “almost no exosomes in platelet lysate” means that after centrifugation or ultrafiltration of the second culture medium, the total amount of exosomes in the platelet lysate is reduced by 60%, more preferably by 70%, and most preferably by 80% compared to the first culture medium.

[0045] In another embodiment, in step (c), the incubation time is 24-72 hours, more preferably 24-48 hours, most preferably 48 hours.

[0046] In another embodiment, in step (d), comprising:

[0047] (d1) separating the incubated culture solution to obtain a cell-free supernatant, wherein the supernatant contains the extracellular vesicles;

[0048] (d2) incubating the supernatant of step (d1) with a polyethylene glycol (PEG) solution overnight to obtain a mixture; (d3) separating the mixture of step (d2) to obtain a precipitate, wherein the precipitate contains the extracellular vesicles.

[0049] In another embodiment, in step (d1), the incubated culture solution is centrifuged to obtain a cell-free supernatant.

[0050] In another embodiment, the centrifugation condition is 3000-10000g for 10-40 minutes.

[0051] In another embodiment, in step (d2), the polyethylene glycol (PEG) is PEG4000-PEG8000, preferably PEG6000.

[0052] In another embodiment, in step (d2), the final concentration of polyethylene glycol (PEG) in the polyethylene glycol (PEG) solution is 8%-20%.

[0053] In another embodiment, in step (d3), the mixture is centrifuged to obtain a precipitate, wherein the precipitate contains the extracellular vesicles.

[0054] In another embodiment, the centrifugation condition is 3000-15000g for 40-70 minutes.

[0055] In another embodiment, the method further comprises step (e): detecting the particle concentration, marker expression level of the extracellular vesicles obtained in step (d); detecting the cell proliferation, cell viability, cell surface marker of the cells obtained in step (c).

[0056] In another embodiment, the detection of the marker expression level comprises detecting the protein expression level of the marker.

[0057] In another embodiment, the marker comprises an extracellular vesicle-specific marker, a cell-specific marker, or a combination thereof.

[0058] In another embodiment, the extracellular vesicle-specific marker is CD81.

[0059] In another embodiment, the cell-specific marker is selected from the group consisting of CD73, CD90, CD105, CD34, CD45, HLA DR, or a combination thereof.

[0060] Therefore, in a third aspect, the present invention provides cellular exosomes suitable for the purpose of the present invention, wherein the cellular exosomes are obtained by the method described in the previous aspect of the present invention.

[0061] In another embodiment, the exosomes are derived from human cells.

[0062] In another embodiment, the human cells are selected from induced stem cells and mesenchymal stem cells.

[0063] In another embodiment, compared with the exosomes obtained by incubation and culture in the "control culture medium", the exosomes obtained by the method according to the first aspect of the present invention have one or more characteristics selected from the following group:

[0064] (1) High exosome production;

[0065] (2) The expression of exosome-specific markers is high.

[0066] In another embodiment, compared to stem cells incubated and cultured with a "control culture medium", the stem cells incubated and cultured by the method of claim 1 have one or more characteristics selected from the following group:

[0067] (1) The cells have strong proliferation ability and good activity;

[0068] (2) The cell-specific markers meet the standards for mesenchymal stem cell-specific markers.

[0069] In another embodiment, the exosome-specific marker is CD81.

[0070] In another embodiment, the cell-specific marker is selected from CD73, CD90, CD105, CD34, CD45, HLA DR, or a combination thereof.

[0071] In another embodiment, the standards for the mesenchymal stem cell-specific markers are: CD73+ / CD90+ / CD105+>95%; CD34+ / CD45+ / HLA DR+<2%.

[0072] In another embodiment, the "control culture medium" is selected from the following group: αMEM basal medium, αMEM+5% platelet lysate, B solution, B-100KD, or serum-free medium.

[0073] In another embodiment, the B solution is the supernatant obtained by ultracentrifuging an αMEM culture medium containing 5% platelet lysate at 100,000 to 120,000 g for 6 to 24 hours.

[0074] In another embodiment, the B-100KD is an αMEM culture medium containing 5% platelet lysate, which is ultracentrifuged at 100,000 to 120,000 g for 6 to 24 hours to obtain the supernatant, which is then filtered through a filter with a pore size of 0.22 μm, and the filtrate is then ultrafiltered through an ultrafiltration tube with a pore size of 100KD to obtain the ultrafiltrate.

[0075] In another embodiment, the serum-free medium is KnockOut Serum Replacement produced by Thermo Corporation.

[0076] In another embodiment, the platelet lysate is EliteGro, a product of EliteCell.

[0077] In another aspect, the present invention provides a method for preparing a gel formulation for carrying extracellular vesicles, the method comprising the steps of:

[0078] (a) adding a physiologically acceptable aqueous solution to a beaker, (b) weighing poloxamer and HPMC, adding them to the beaker in the previous step under stirring, and continuing stirring for at least 10 minutes, preferably at least 20 minutes, and (c) refrigerating the solution in a refrigerator at 2-8°C overnight (>8 hours, preferably >12 hours).

[0079] Furthermore, the present invention provides a method for preparing a gel preparation containing extracellular vesicles, the method comprising the following steps:

[0080] (a) adding a physiologically acceptable aqueous solution to a beaker, (b) weighing poloxamer and HPMC, adding them to the beaker in the previous step under stirring, and continuing stirring for at least 10 minutes, preferably at least 20 minutes, (c) refrigerating the solution in a refrigerator at 2-8°C overnight (>8 hours, preferably >12 hours), and (d) adding the extracellular vesicle suspension to the solution.

[0081] In some specific embodiments, the physiologically acceptable aqueous solution is an isotonic saline solution. In some specific embodiments, the isotonic saline solution is sodium chloride injection.

[0082] In some specific embodiments, the stirring conditions in step (b) are carried out by an electric stirrer. In some specific embodiments, the stirring conditions in step (b) include a stirring speed of 200 to 500 rpm.

[0083] In some specific embodiments, the poloxamer is selected from one or more of poloxamer 407, poloxamer 124, poloxamer 188, and poloxamer 338. In a more specific embodiment, the poloxamer is poloxamer 407. In some specific embodiments, the final concentration of the poloxamer is 18-30%, preferably 20-25%, and more preferably 20%, wherein the % is the weight volume percentage of grams (g) of the relevant substance relative to milliliters (mL) of the aqueous solution.

[0084] In some specific embodiments, the type of hydroxypropyl methylcellulose is 1828, 2208, 2906, or 2910. In a more specific embodiment, the type of hydroxypropyl methylcellulose is 2910. In some specific embodiments, the final concentration of hydroxypropyl methylcellulose is 0.25-3%, preferably 0.5-2%, and more preferably 1%, wherein the % is the weight volume percentage of grams (g) of the relevant substance relative to milliliters (mL) of the aqueous solution.

[0085] In some specific embodiments, the aqueous solution is weighed at 100 parts by volume. In some specific embodiments, the poloxamer is weighed at 18-30 parts by weight, preferably 20-25 parts by weight, and more preferably 20 parts by weight. In some specific embodiments, the hydroxypropyl methylcellulose is weighed at 0.25-3 parts by weight, preferably 0.5-2 parts by weight, and more preferably 0.5 parts by weight. In the above embodiments, 1 part by weight:1 part by volume corresponds to a ratio of 1 gram:1 milliliter.

[0086] In some specific embodiments, the extracellular vesicles are exosomes.

[0087] In some specific embodiments, the extracellular vesicles are derived from human or other mammalian mesenchymal stem cells, preferably human mesenchymal stem cells. In some more specific embodiments, the mesenchymal stem cells are derived from human tissue, such as fat, bone marrow, or placenta.

[0088] In some specific embodiments, the final content of the extracellular vesicles is 1×10 8 Particles / ml-1×10 11 Particles / ml.

[0089] In some specific embodiments, the extracellular vesicle suspension is prepared according to the method of the second aspect of the present invention.

[0090] In another aspect, the present invention provides a method for preventing or treating a disease or condition responsive to extracellular vesicles in a patient, the method comprising administering to the patient an effective amount of an extracellular vesicle gel formulation of the present invention. In some specific embodiments, the disease or condition responsive to extracellular vesicles is a skin or mucosal lesion. In some specific embodiments, the skin or mucosal lesion is a diabetic skin lesion, such as diabetic foot, such as a partial or full-thickness injury, wound, and / or ulcer involving the skin of the foot.

[0091] Accordingly, the present invention provides use of the extracellular vesicle gel preparation of the present invention in the preparation of a medicament for treating a disease or condition responsive to extracellular vesicles.

[0092] The extracellular vesicle gel preparation of the present invention has a suitable gelation temperature and gelation time, and has suitable rheological properties. When applied to the human body, such as skin lesions, the extracellular vesicle gel preparation of the present invention can quickly phase-transform to form a gel, increase the adhesion and retention time of the preparation at the application site, form a physical barrier to cover and protect the wound surface, and locally control and prolong the release process and action process of the active ingredient. Therefore, the extracellular vesicle gel preparation of the present invention can serve as a more optimal route of administration for extracellular vesicles (especially the extracellular vesicles of the present invention). BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 Shown are A: uptake of exosomes by HFF-1 cells 48 h after administration of exosome solution and exosome gel; B: relative quantification of fluorescence labeled with exosomes.

[0094] Figure 2 Shown are the histopathological examinations of the wounds of mice in each group on day 14. Panel A is the HE staining result; Panel B is the Masson staining result.

[0095] Figure 3 Shown are the histopathological scores of mouse wounds at day 14 (n=9, Mean±SD). Detailed Description of the Invention

[0096] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used with respect to a particular stated value or range of values, the term "about" as used herein will be used with respect to the value with which it is associated with a fluctuation of ±10%. For example, as used herein, the expression "about 100" includes 90 and 110 and all values ​​therebetween (e.g., 90.5, 95, 101, 105, 109.95, etc.). For ranges, the term "about" is used for each number of the range so defined.

[0097] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0098] As used herein, the term "weight percent by volume" or "% w / v" refers to the percentage weight of a single component relative to the total volume of a mixture containing that component. For example, 500 mg of a component in a total volume of 8 mL is 6.25% w / v, and 500 mg of a component in a total volume of 5 mL is 10% w / v. Alternatively, the equivalent is expressed as, for example, mg / mL.

[0099] An "isolated" material has been altered by the hand of man from its natural state. If an "isolated" component or substance occurs in nature, it has been altered or removed from its original environment, or both. For example, the extracellular vesicles referred to herein, when present in a suspension, composition, or formulation, are substantially isolated unless otherwise indicated.

[0100] As referred to herein, a formulation refers to a combination of at least one active ingredient (e.g., extracellular vesicles) and at least one inactive ingredient, which, when combined with the active ingredient and / or one or more additional inactive ingredients, directly or indirectly renders the formulation more suitable for therapeutic administration to a human or non-human animal.

[0101] The term "extracellular vesicle (EV)" refers to any type of vesicle that can be obtained from any form of cell, such as exosomes (e.g., any vesicle derived from the endolysosomal pathway, microvesicles, any vesicle shed from the plasma membrane of a cell), apoptotic bodies (e.g., obtainable from apoptotic cells), microparticles (which can be derived from, for example, platelets), exosomes (which can be derived from, for example, neutrophils and monocytes in serum), etc., with the main types being exosomes, microvesicles, and apoptotic bodies. The size of EVs can vary considerably, and the size distribution can be quite dispersed even between EVs of the same origin, but EVs typically have a hydrodynamic size of the nanometer size, i.e., less than 2000 nm, in liquid / suspension, and most EVs have a size of 30-200 nm, representing the average and / or median size of the vesicle population. EVs can be derived from any cell type in vivo, in vitro, and ex vivo. EV has a double lipid membrane structure, and its surface is rich in four transmembrane proteins such as CD63, CD81, CD9, etc., and contains cytokines, growth factors, signaling lipids, mRNA, microRNA and siRNA substances derived from mother cells. In addition, the term also covers extracellular vesicle mimics, which are cell membrane-based vesicles obtained by, for example, membrane extrusion, ultrasonic treatment or other techniques. It is obvious to those skilled in the art that the technical solutions of the present invention generally involve a population of EVs, wherein EVs can be present in various concentration ranges and concentrations, such as 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17EV particles / unit volume (e.g., mL). In some embodiments of the present invention, the EVs in the preparation may be of multiple types. The EVs of the present invention can carry various types of payloads (e.g., drugs), and / or may contain specific molecules such as mRNA, shRNA, miRNA, proteins, peptides, etc., for example, genetically engineered to contain specific molecules such as mRNA, shRNA, miRNA, proteins (e.g., antibodies), peptides, etc. The genetic engineering modification may also be intended to enhance its activity as a delivery vehicle for exogenous drug payloads. As endogenously produced vesicles, EVs have various advantages such as non-toxicity, low immunogenicity, and higher stability. The use of EVs instead of cell therapy may reduce the risks of whole-cell therapy, such as uncontrolled cell division or cytokine storm.

[0102] The term exosome refers to a subtype of EVs derived from endosomes, mostly ranging in diameter from 50 to 150 nm. They are a major component of parasite secretions from various cell types, including mesenchymal stem cells (MSCs). As carriers of rich cargos, they function primarily through the continuous transport of microRNAs (miRNAs) and proteins. Over 150 miRNAs and 850 unique proteins have been identified in MSC-derived exosomes, which modulate various activities of target cells through diverse pathways. MicroRNAs contained in MSC exosomes participate in physiological processes such as immune regulation and tissue repair. For example, miR-24, miR-21, and miR-146 can inhibit inflammation, while miR-150 and Let-7a can promote tissue repair and angiogenesis, playing a crucial role in skin damage repair. These three proteins play a crucial role in stimulating angiogenesis and may underlie the tissue repair effects of exosomes.

[0103] "Diameter" and "median diameter" refer to the intensity-volume diameter corresponding to the extracellular vesicle particle preparation / composition, which can be measured using conventional determination methods in the art.

[0104] In another embodiment of the present invention, the vesicles contain different types of microRNAs (miRNAs), small RNAs (sRNAs), non-coding DNA fragments, transfer RNAs (t-RNAs), soluble cytokines, growth factors, and other proteins with specific functions.

[0105] In another embodiment of the present invention, the cells used to produce the extracellular vesicles include cells from the following sources:

[0106] (a) Cells directly isolated and purified from human tissues;

[0107] (b) cells obtained by direct isolation and purification from human tissues, followed by proliferation of these cells through minimal manipulation in a GMP laboratory;

[0108] (c) Cells obtained in a GMP laboratory after specific gene modification, specific gene editing, specific gene transduction, or introduction of specific microRNAs;

[0109] (d) Cells obtained by pretreatment under special culture conditions in a GMP laboratory.

[0110] In another embodiment of the present invention, the cells used to produce the extracellular vesicles include primary cells and passaged cells with a passage number of 1-10.

[0111] In another embodiment of the present invention, the cells used to produce the extracellular vesicles include genetically unmanipulated cells and genetically manipulated cells.

[0112] In another embodiment of the present invention, the gene manipulation includes gene editing, gene introduction, gene knock-down, gene knock-out, or a combination thereof.

[0113] In another embodiment of the present invention, the cells used to produce the extracellular vesicles are pretreated cells.

[0114] In another embodiment of the present invention, the somatic cells are adipose-derived mesenchymal stem (progenitor) cells, placenta-amniotic-derived mesenchymal stem cells, or a combination thereof.

[0115] In another embodiment of the present invention, the adipose-derived mesenchymal stem (progenitor) cells are obtained in the following manner: for healthy male or female volunteers who have undergone ethical review and filing, signed informed consent, and passed strict laboratory examinations and meet the inclusion criteria for the establishment of an adipose stem (progenitor) cell product bank, fat is removed by abdominal wall skin puncture and liposuction, or abdominal wall surgery, and the fat is stored in a cell preservation solution and transported at low temperature to a qualified GMP laboratory. After separation, purification, and proliferation treatment, the adipose-derived mesenchymal progenitor cells, i.e., working bank cells (intermediate products), are obtained.

[0116] In another embodiment of the present invention, the human adipose tissue-derived mesenchymal stem (progenitor) cells are produced in a GMP production laboratory according to the intermediate product production process, meet the negative standards of various pathogen detection, meet the specific surface markers such as CD73 positive, CD90 positive, CD105 positive cell percentage of approximately 98%, CD34 positive / CD45 positive and HLA-DR positive cell percentage of less than 2%, and meet the antibiotic residue and serum and serum substitute residue detection standards, and are P1-P6 adipose mesenchymal stem (progenitor) cells.

[0117] In another embodiment of the present invention, the adipose-derived mesenchymal stem (progenitor) cells are P3-P4 adipose-derived mesenchymal stem (progenitor) cells.

[0118] In another embodiment of the present invention, the somatic cells are mesenchymal stem (progenitor) cells derived from adipose tissue stored under deep cryogenic conditions of -196°C to -80°C (more preferably -196°C to -135°C) for 0-36 months (more preferably 0-24 months or 0.5-24 months).

[0119] The platelet lysate of the present invention is obtained by directly lysing human platelet cells through repeated freeze-thaw cycles, allowing them to release large amounts of activated growth factors. Therefore, human platelet lysate is a xeno-free, animal serum-free cell culture medium supplement derived from human platelets. It contains all the growth factors and proteins necessary for cell growth.

[0120] The present invention provides a method for producing stem cell exosomes in vitro, specifically comprising the steps of:

[0121] (1) Culture P2 to P6 adipose-derived mesenchymal stem cells in αMEM medium containing 5% platelet lysate at a cell seeding density of 10,000 to 15,000 cells / cm 2 , the cells reached a confluence of 60% to 90%.

[0122] (2) Prepare B-300K solution, which is an αMEM culture medium containing 5% platelet lysate. After ultracentrifugation at 100,000-120,000 g for 6-24 hours, obtain the supernatant, and filter the supernatant through a filter with a pore size of 0.22-0.45 μm. The filtrate is then ultrafiltered through an ultrafiltration tube with a pore size of 100-300 KD to harvest the resulting ultrafiltrate.

[0123] (3) After incubating the cells in step (1) with B-300K solution for 24 to 48 hours, the conditioned medium was collected.

[0124] (4) The conditioned medium obtained in step (3) was subjected to differential centrifugation at 3000-10000 g to obtain the supernatant.

[0125] (5) The supernatant obtained in step (4) is incubated overnight with PEG4000-8000 containing a final concentration of 8%-20%, and then subjected to differential centrifugation at 3000-150000 g to obtain a precipitate, which is the human stem cell-derived exosomes.

[0126] In another embodiment, the mesenchymal stem cells are human tissue-derived mesenchymal stem cells, and the cells are selected from the P2, P3, P4, P5, and P6 generations of primary cells that have been subcultured. In another more preferred embodiment, the mesenchymal stem cells are adipose-derived mesenchymal stem cells derived from human adipose tissue. In another more preferred embodiment, the mesenchymal cells are selected from the P4 generation.

[0127] In another embodiment, the seeding density of the mesenchymal stem cells is 10,000 cells / cm 2 .

[0128] In another embodiment, the platelet lysate is used at a final concentration of 2%, 5% or 10%.

[0129] In another embodiment, the platelet lysate is used at a final concentration of most preferably 5%, less preferably 10%.

[0130] In another embodiment, the confluence of the mesenchymal stem cells reaches any value in the range of 70% to 90%.

[0131] In another embodiment, the mesenchymal stem cells have a confluence of 80% and less preferably 70%.

[0132] In another embodiment, the B-300K solution is an αMEM medium containing platelet lysate, and the optimal concentration of the platelet lysate is 5%, and the more preferred concentration is 10%.

[0133] In another embodiment, the B-300K solution is prepared as follows: αMEM culture medium containing 5% platelet lysate is ultracentrifuged at 120,000 g for 6 hours to obtain a supernatant, the supernatant is filtered through a filter with a pore size of 0.22 μm to obtain a filtrate, and the filtrate is ultrafiltered through an ultrafiltration tube with a pore size of 300KD to obtain an ultrafiltrate.

[0134] In another embodiment, in step (4), the conditioned medium obtained in step (3) is centrifuged at 3000 g for 15 minutes.

[0135] In another embodiment, in step (4), the conditioned medium obtained in step (3) is centrifuged at 10,000 g for 30 minutes.

[0136] In another embodiment, in step (5), the supernatant obtained in step (4) is incubated with liquid C overnight, and the liquid C contains PEG4000-8000 with a final concentration of 8% to 20%.

[0137] In another embodiment, the solution C contains PEG6000 with a final concentration of 12%.

[0138] In another embodiment, in step (5), the supernatant obtained in step (4) is incubated with solution C overnight and then subjected to differential centrifugation at 3000-15,000 g.

[0139] In another embodiment, in step (5), the supernatant obtained in step (4) is incubated with solution C overnight and then centrifuged at 3000 g for 1 hour.

[0140] In another embodiment, in step (5), the supernatant obtained in step (4) is incubated with solution C overnight and then centrifuged at 120,000 g for 70 minutes.

[0141] In another embodiment, the method further comprises the step of: (6) detecting cell activity and protein expression levels of detection markers.

[0142] In another embodiment, in step (6), the monitoring includes detecting human cell-derived exosome-specific protein markers: CD9 and CD81 using a western blot method. The markers are positively correlated with the expression level of the exosome-specific proteins.

[0143] In another embodiment, in step (6), the monitoring comprises detecting the concentration of human cell exosome particles using the NTA method.

[0144] The methods screened by the present invention have significant effects on increasing the production and specific markers of cell exosomes, and have no effect on the activity and specific markers of cells.

[0145] Extracellular vesicle gel preparation

[0146] The extracellular vesicle gel formulation described herein advantageously has thermosensitive properties, rapidly transitioning from a free-flowing state to a gel state at human physiological temperature (preferably, skin temperature).

[0147] In some specific embodiments, the temperature of transition from the flow state to the gel state is a temperature in the range of 20°C-31°C, 20°C-30°C, 21°C-31°C, 21°C-30°C, 22°C-31°C, 22°C-30°C, 23°C-31°C, 23°C-30°C, 24°C-31°C, 24°C-30°C, 25°C-31°C, 25°C-30°C, 26°C-31°C or 26°C-30°C. In some specific embodiments, the temperature of transition from the flow state to the gel state is a temperature in the range of 25.1°C-31.0°C or 25.1°C-30.9°C. In some specific embodiments, the temperature of transition from the flow state to the gel state is a temperature in the range of 25.1°C-31.0°C or 25.1°C-30.9°C. In some specific embodiments, the temperature at which the fluid state is transformed into the gel state is 20.0°C, 20.1°C, 20.2°C, 20.3°C, 20.4°C, 20.5°C, 20.6°C, 20.7°C, 20.8°C, 20.9°C, 21.0°C, 21.1°C, 21.2°C, 21.3°C, 21.4°C, 21.5°C, 21.6°C, 21. 7℃, 21.8℃, 21.9℃, 22.0℃, 22.1℃, 22.2℃, 22.3℃, 22.4℃, 22.5℃, 22.6℃, 22.7℃, 22.8℃, 22.9℃, 23.0℃, 23.1℃, 23.2℃, 23.3℃, ​​23.4℃, 23.5℃, 23.6℃, ​​23.7℃, 23.8℃, 23.9℃. 24.0℃、24.1℃、24.2℃、24.3℃、24.4℃、24.5℃、24.6℃、24.7℃、24.8℃、24.9℃、25.0℃、25.1℃、25.2℃、25.3℃、25.4℃、25.5℃、25.6℃、25.7℃、25.8℃、25.9℃、26.0℃、26.1℃、26.2℃、26.3℃、26.4℃、26.5℃、26.6℃、26.7℃、26.8℃、26.9℃、27.0℃、27.1℃、27.2℃、27.3℃、27.4℃、27 .5° C., 27.6° C., 27.7° C., 27.8° C., 27.9° C., 28.0° C., 28.1° C., 28.2° C., 28.3° C., 28.4° C., 28.5° C., 28.6° C., 28.7° C., 28.8° C., 28.9° C., 29.0° C., 29.1° C., 29.2° C., 29.3° C., 29.4° C., 29.5° C., 29.6° C., 29.7° C., 29.8° C., 29.9° C., 30.0° C., 30.1° C., 30.2° C., 30.3° C., 30.4° C., 30.5° C., 30.6° C., 30.7° C., 30.8° C., 30.9° C. or about 31° C. In some more preferred embodiments, the temperature of transition from the flow state to the gel state is a temperature between 24.0° C. and 30.0° C.In some most preferred embodiments, the temperature of transition from the fluid state to the gel state is between 25.0°C and 27.0°C.

[0148] The extracellular vesicle gel formulation described herein advantageously promotes the extracellular vesicles loaded therein to exert their effects as active ingredients, for example, preferably, to exert more effective and sustained effects in a sustained-release manner.

[0149] The extracellular vesicle gel formulations described herein advantageously promote stable preservation of the extracellular vesicles loaded therein as active ingredients.

[0150] In some embodiments, the aqueous composition of the present invention can be stored stably at 2-8°C (e.g., 4°C) for at least 24 hours. In some embodiments, the aqueous composition of the present invention is stable at any temperature between -90°C and -70°C (e.g., -80°C), for example, at any temperature, for example, for at least 3 months, preferably at least 12 months, more preferably at least 24 months. The stability is, for example, manifested in that the concentration of extracellular vesicle particles in the dissolved state is greater than 70%, greater than 80%, greater than 85% or greater than 90%, preferably greater than 95%, more preferably greater than 98%. The stability is, for example, manifested in that the particle size of the extracellular vesicle particles in the dissolved state changes by less than 30%, less than 20%, less than 15% or less than 10%, preferably less than 5%, more preferably less than 3%, more preferably less than 2%, more preferably less than 1%, compared to the concentration at the time of preparation.

[0151] In some embodiments, the extracellular vesicle gel preparation is highly dispersible in a free-flowing state, that is, it is colorless and transparent, and has no flocs or precipitates visible to the naked eye.

[0152] Methods and uses

[0153] The extracellular vesicles and thermosensitive gel preparations containing extracellular vesicles of the present invention can be formulated to be suitable for direct administration. Therefore, in some specific embodiments, the gel preparation is a pharmaceutical composition. Thus, the present invention also provides the use of the gel preparation for the preparation of a medicament.

[0154] In some embodiments, the medicament is used to treat a disease that would benefit from the administration of extracellular vesicles. In some embodiments, the disease is a dermatological condition or lesion, for example, inflammation and / or damage to the skin or mucous membranes. In some embodiments, the dermatological condition or lesion is selected from the group consisting of: acute skin lesions; chronic and acute skin diseases; diabetic foot ulcers; wounds caused by chemical burns or physical factors (e.g., burns, mechanical action, local compression, etc.); skin or tissue damage caused by one or more situations such as injury, disease or surgical procedures, viral infections, bacterial infections, fungal infections, autoimmune reactions, hair follicle diseases, disorders or conditions, wrinkles and loss of firmness. In some embodiments, the dermatological condition or lesion is a diabetic foot ulcer, a leg ulcer, a pressure ulcer / sore, and 1st and 2nd degree partial thickness burns, surgical wounds, traumatic wounds, periodontal disease, anal fissures and hemorrhoids, and an artificial opening caused by ostomy. In some embodiments, the dermatological condition or lesion involves part of the skin and its related tissues and structures (e.g., blood vessels, nerves). In some embodiments, the dermatological condition or lesion involves the full thickness of the skin and its associated tissues and structures (eg, blood vessels, nerves).

[0155] In some embodiments, the disease is diabetic skin lesions, such as diabetic foot skin lesions, wounds, and / or ulcers.

[0156] Diabetic foot is a complication of diabetes. Diabetes is a chronic disease in which patients are unable to control elevated blood sugar levels due to insufficient insulin secretion and / or insulin resistance. Abnormally elevated blood sugar levels and the resulting damage to tissues and organs (particularly peripheral nerves and blood vessels) can lead to a variety of complications, one of the most common of which is diabetic foot. Due to neuropathy and local ischemia, patients are unable to promptly and correctly perceive or ignore minor injuries to their feet, resulting in the gradual aggravation of these minor injuries. Furthermore, due to high blood sugar and neurovascular disease, tissue regeneration is difficult, leading to the formation of difficult-to-heal ulcers and / or increased susceptibility to infection, further exacerbating the damage. Wounds can progress from tiny localized lesions to larger ulcers, gangrene, penetration of the full-thickness skin, and even invasion of the bones and bone marrow. Ultimately, severe diabetic foot requires amputation. The amputation rate among patients with diabetes is 15 times that of the general population.

[0157] The thermosensitive gel preparation of extracellular vesicles of the present invention physically protects the wound surface, isolates pathogens, and locally controls and prolongs the release and action process of exosomes, continuously regulates the local immune state, and promotes tissue regeneration, thereby effectively treating various skin and mucous membrane damage. It is particularly suitable for breaking the vicious cycle of diabetic foot and similar diseases and helping damage recovery.

[0158] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples can be conventional methods in the art, for example, with reference to "Molecular Cloning Laboratory Manual" (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the supplier. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial channels unless otherwise specified. Example

[0159] Example 1: Blank Gel Prescription Screening - Excipient Types

[0160] Based on the existing technical literature records, the inventor's previous experience and other information, a blank gel formula containing excipients of different types and concentrations was preferably formulated. Screening was carried out based on the blank gel state, gelation temperature, gelation time, viscoelastic curve and other aspects to further optimize the excipient composition and concentration in the formula.

[0161] Table 1 Experimental instrument and equipment information

[0162]

[0163]

[0164] Table 2 Experimental material information

[0165]

[0166] 1.1 State of blank gel solution

[0167] For each formulation, use a graduated cylinder to measure a certain volume of sodium chloride injection into a beaker. Weigh the predetermined amounts of gel excipients: poloxamer 407, poloxamer 188, sodium alginate, carbomer 974P, carbomer 980, and hypromellose. Add the solid excipients to the beaker containing the sodium chloride injection while stirring (preferably with magnetic stirring) at 500-700 rpm. After all excipients are added, continue stirring for 20 minutes. Refrigerate the mixture in a refrigerator at 2-8°C overnight (>12 hours). Observe and record the state of the blank gel solution after overnight refrigeration.

[0168] Table 3 Status of blank gel solution (refrigerated at 2-8°C overnight)

[0169]

[0170]

[0171] Note: As can be seen in the table, 23013102 and 23020606 are blank gel formulation of the same composition but different preparation batches.

[0172] 1.2 Determination of gelation temperature

[0173] The formulations 23013101, 23013102, 23013103, 23013104, 23020604, 23020605 which passed the state test in 1.1 were investigated. 6 mL of each blank gel solution was taken into a 10 mL size transparent vial, and placed in a water bath, with the liquid surface 2 cm below the water surface, the water bath was heated from 15 ℃ to 50 ℃ at a rate of 1 ℃ / min. During the heating process, the temperature of the blank gel solution was monitored in real time with a thermometer, and the state of the gel solution was observed, with an angle of 60° between the horizontal plane and the liquid surface, and the liquid was observed once every 10-15 s. The gelation temperature was recorded when the liquid did not flow within 10 s. The thermometer was accurate to 0.1 ℃. The test was repeated three times, and the average value was calculated.

[0174] Table 4 Determination results of water bath gelation temperature

[0175]

[0176] Chronic skin damage such as diabetic foot, the foot temperature range is 30.25±3.1 ℃

[12] , therefore the determination value of the results was set to ≤30 ℃.

[0177] 1.3 Determination of gelation time

[0178] This time only the formulations which passed the tests in 1.1 and 1.2, 23013101, 23013102, 23020604, 23020605 were investigated. The excipients in them only contained poloxamer 407 and hypromellose.

[0179] 2 mL of each blank gel solution was taken into a 10 mL size transparent vial, and placed in a 27 ℃ water bath, and the timing was started immediately, with the liquid surface 2 cm below the water surface, and the state of the gel solution was observed in real time. The liquid was observed once every 10-15 s, with an angle of 60° between the horizontal plane and the liquid surface, and the gelation time was recorded when the liquid did not flow within 10 s. The test was repeated three times, and the average value was calculated. The gelation time should be ≤5 min.

[0180] Table 5 Determination results of water bath gelation time

[0181]

[0182] Note: N / A means not detected.

[0183] With the exception of sample 23013101, which contained only poloxamer 407 at a concentration of ≤18%, the remaining formulations all achieved gel times of less than 5 minutes, meeting the requirements. The differences between the results for 23013102 and 23020606 were due to errors arising from external conditions in different batches, but both were significantly less than 5 minutes, and therefore the conclusions are considered reliable.

[0184] 1.4 Linear viscoelastic curve detection

[0185] This test only examined the following batches: 23020604, 23020605, and 23020606. Blank gel solutions based on these batches were clear and transparent, with gelation temperatures below 27°C and gelation times ≤5 minutes.

[0186] The linear viscoelastic curve of the gel solution was measured using a cone-plate (C35 2° / Ti rotor). Approximately 1 mL of the gel sample solution was added to the rotational rheometer test plate. The linear viscoelastic curve was measured under strains of 0.00001% to 2.00%, a frequency of 1 Hz, and a temperature of 27°C. Each sample was measured at least three times in parallel.

[0187] Table 6 Summary of linear viscoelastic curve test results of different batches of blank gel solutions

[0188]

[0189] It can be seen from the results of linear viscoelastic curve measurement that the yield value τ of the blank gel solutions with batch numbers 23020606, 23020604, and 23020605 at 27°C tends to gradually increase, that is, with the increase of the amount of HPMC, the gel strength of the blank gel solution gradually increases.

[0190] Therefore, the subsequent excipient investigation was the combination of poloxamer 407 and hypromellose.

[0191] Example 2: Blank Gel Prescription Screening - Excipient Concentration

[0192] Table 7 Experimental instrument and equipment information

[0193]

[0194]

[0195] Table 8 Experimental material information

[0196]

[0197] In the determination of Example 1 above, preliminary screening revealed that the required excipients were poloxamer 407 and hypromellose, used in combination. This Example further screened for the optimal concentration / content of the two in the formulation to obtain a thermosensitive gel formulation with optimal performance.

[0198] 2.1 Gelation temperature detection

[0199] The experimental conditions of this determination are the same as those of Example 1.2. The test results are shown in the table below.

[0200] Table 9 Water bath gelation temperature measurement results

[0201]

[0202] 2.2 Gel time test

[0203] The experimental conditions of this determination are the same as those of Example 1.3. The test results are shown in the table below.

[0204] Table 10 Gel time measurement results

[0205]

[0206]

[0207] At a 20% concentration of poloxamer 407, the gelation time of the blank gel solution was ≤2 minutes, meeting clinical requirements. Compared to an 18% concentration of poloxamer 407, a 20% concentration of poloxamer 407 exhibited a significantly shorter gelation time and slightly lower gelation temperature. Therefore, in subsequent formulations, the poloxamer 407 concentration was tentatively set at 20%.

[0208] 2.3 Linear viscoelastic curve detection

[0209] The experimental conditions of this test were the same as those of Example 1.4, and two formulations with a 20% concentration of Poloxamer 407 were tested. The test results are shown in the table below.

[0210] Table 11 Summary of linear viscoelastic curve test results of blank gel solutions from batches 23021301-23021302

[0211] Conclusion: The results of linear viscoelastic curve determination show that at 27°C, the elastic modulus G' and yield value τ of the two batches of blank gel solutions are similar, and there is no significant difference in their gelation temperatures, indicating that the addition of 0.5% and 1% HPMC has little effect on the gel properties of the blank gel solution.

[0212] Example 3: Obtaining Mesenchymal Stem Cell Exosomes (MSC-Exos)

[0213] 3.1 Preparation of MSC-Exos

[0214] Experimental Materials

[0215] (1) Human adipose-derived mesenchymal stem cells

[0216] Fat from volunteer donors was harvested and enzymatically digested under sterile conditions. The cell pellet was collected and cultured in αMEM containing 5% EliteGro®. The cells were then incubated at 37°C in a 5% CO2 incubator to serve as P1. When the cells reached approximately 90% confluency, they were digested and passaged using 0.25% trypsin. After four passages, they were cryopreserved as P4 cell banks and stored in liquid nitrogen for future use.

[0217] (2) Culture medium

[0218] αMEM + 5% EliteGro: αMEM medium contains non-essential amino acids, sodium pyruvate, lipoic acid, vitamin B12, biotin and ascorbic acid, ribonucleosides, deoxyribonucleosides, and does not contain proteins, lipids, or growth factors; 5% platelet lysate (EliteGro) is further added.

[0219] Solution B: αMEM+5% EliteGro medium, obtained by ultracentrifugation at 100,000-120,000 g for 6-24 hours.

[0220] B-300KD (B-300K solution): B solution is further filtered through a filter with a pore size of 0.22 μm, and the filtrate is then ultrafiltered through an ultrafiltration tube with a pore size of 300KD to obtain the ultrafiltrate.

[0221] Experimental methods

[0222] The preparation method of MSC-Exos includes the following steps:

[0223] (a) Provide P4 human adipose-derived mesenchymal stem cells at a rate of 1×10 4 pieces / cm 2 vaccination;

[0224] (b) pre-culturing the stem cells using αMEM+5% EliteGro medium to obtain pre-cultured stem cells with a confluency of 60-90%;

[0225] (c) discarding the culture medium, then adding solution B or B-300K solution, and incubating the cultured stem cells for 24 to 48 hours to obtain an incubation culture solution containing the incubated stem cells and exosomes produced by the incubated stem cells;

[0226] (d) Isolating exosomes from the incubation medium obtained in step (c): collecting the cell culture supernatant, centrifuging at 3000g for 15 minutes, aspirating the supernatant, and removing the precipitate; centrifuging at 10,000g for 0.5 hours, aspirating the supernatant, and removing cell debris. The centrifuged culture medium is supplemented with 24% PEG 6000 and incubated at 4°C overnight; centrifuging at 3200g for 1 hour at 4°C, discarding the supernatant, and resuspending the precipitate in PBS; centrifuging at 120,000g for 70 minutes, discarding the supernatant, and finally, resuspending the precipitate at the bottom of the centrifuge tube with PBS or 0.9% saline, wherein the precipitate contains the exosomes, thereby obtaining an exosome suspension (stock solution).

[0227] 3.2 Characterization of MSC-Exos Content

[0228] According to the method of Example 3.1, mesenchymal stem cell exosomes were prepared in multiple batches for subsequent experiments.

[0229] From three batches of exosome stock solution, 500 μl was taken from each aliquot and RNA was extracted using the Qiagen miRNeasy miniKit kit. The aliquots were sealed and placed in sealed bags and transported on dry ice to Shanghai Meiji Biotechnology Co., Ltd. for quality inspection, library construction, and sequencing.

[0230] The microRNAs contained in the MSC exosomes prepared in Example 3.1 are involved in physiological processes such as immune regulation and tissue damage repair. For example, miR-24, miR-21, and miR-146 can inhibit inflammation, while miR-150 and Let-7a can promote tissue repair and angiogenesis, playing an important role in skin damage repair.

[0231] Table 12 List of microRNAs contained in three batches

[0232]

[0233]

[0234]

[0235]

[0236] In addition, three replicates of 300 μl were taken from the same three batches of exosome stock solution and sent to Shanghai Meiji Biotechnology Co., Ltd. for TMT mass spectrometry analysis to obtain the protein expression of the three batches of products.

[0237] According to the data of ExoCarta, more than 900 proteins have been collected from MSC exosomes. The exosomes produced in Example 3.1 have 81 coincident proteins with the database for characterizing the exosome properties of the product. Among them, Thrombospondin-1, Heat shock cognate 71 kDa protein, Pyruvate kinase PKM, Heat shock protein HSP 90-alpha, Peroxiredoxin-1 and Peroxiredoxin-2 are involved in cell adhesion, cell migration, tissue repair, oxidative stress and other processes, and can play a role in skin repair.

[0238] Table 13 List of exosome-related proteins contained in three batches of preparations

[0239]

[0240]

[0241]

[0242] Example 4: Preparation of gel preparation containing mesenchymal stem cell exosomes (MSC-Exos)

[0243] From the blank gel formula determined according to the screening of Examples 1-2 and the MSC-Exos prepared in Example 3, a gel preparation containing exosomes was prepared.

[0244] Preparation of temperature-sensitive blank gel solution

[0245] According to the prescription table, a certain volume of sodium chloride injection was measured into a beaker with a measuring cylinder, and the prescribed amount of poloxamer 407 and HPMC2910 was weighed. Under the action of the stirring paddle of the electric stirrer, poloxamer 407 and HPMC2910 were added to the beaker containing sodium chloride injection while stirring, and the stirring speed was 200-500 rpm. After all the excipients were added, stirring was continued for 20 min, and then the mixture was placed in a 2-8°C refrigerator for cold storage overnight (>12 h).

[0246] The prepared temperature-sensitive blank gel solution was divided into flow phase glass bottles, covered with bottle caps, and subjected to moist heat sterilization using a sterilization pot. The sterilization conditions were 121°C for 15 min. After sterilization, the state of the temperature-sensitive blank gel was observed, and then it was placed in a 2-8°C refrigerator for cold storage overnight (>12 h).

[0247] Preparation of gel solution containing exosomes

[0248] The exosome stock solution was further diluted with a carrier liquid (1×PBS or sodium chloride injection) to prepare an exosome dilution solution. The exosome concentration in the dilution solution was 4×10 9 particles / mL (for stability determination below) or 3.68×10 10 Particles / mL (for in vitro release assay below).

[0249] Prepare the exosome gel solution by adding the sterilized thermosensitive blank gel solution to the exosome diluent at a volume ratio of 1:4. Maintain the water bath temperature below 15°C and mix thoroughly. Aliquot into 3 mL vials. Remove five vials from each batch of exosome gel solution and immediately measure the gelation temperature, gelation time, and linear viscoelastic curve. Store any remaining exosome gel at -80°C for subsequent use (e.g., stability testing).

[0250] Table 14 Preparation Prescription Concentration

[0251]

[0252] Example 5: Identification of different formulations of gel preparations containing mesenchymal stem cell exosomes (MSC-Exos)

[0253] Table 15 Experimental instruments

[0254]

[0255] 5.1 Storage stability determination

[0256] 5.1.1 Stability of physical properties during low-temperature storage

[0257] The exosome gel preparations with batch numbers 23031702010101 and 23031704010101 were taken out of the -80°C refrigerator after 1, 2, and 3 months of storage, respectively. After complete melting at room temperature, the gel solution was gently shaken to mix evenly, and the gelation temperature and linear viscoelastic curve of the gel solution were measured.

[0258] Table 16 Information of samples to be tested

[0259]

[0260] Table 17 Stability data of exosome gel frozen storage for 3 months (gelation temperature)

[0261]

[0262] During the 3-month storage of exosome gel at -80℃, the gelation temperature of batch 23031702010101 samples had no significant difference compared with that before frozen storage; the gelation temperature of batch 23031704010101 samples had only a slight increase compared with that before frozen storage.

[0263] Table 18 Stability data of exosome gel frozen storage for 3 months (rheological data)

[0264]

[0265]

[0266] Conclusion: During the 3-month storage at -80°C, the RSDs of G' and τ of batches 23031702010101 and 23031704010101 were ≤15%, indicating that the rheological properties of the samples stored at -80°C for 3 months were relatively stable.

[0267] In summary, in the tested formulation, poloxamer 407 was 20% and hypromellose was 0.5-2.0%. After storage at -80°C for 3 months, the RSDs of the gelling temperature, elastic modulus (G'), and yield stress (τ) were all ≤15%, indicating that the physical properties of the gel formulation remained stable under low-temperature freezing conditions.

[0268] 5.1.2 Stability of active ingredients during storage / freeze-thaw at different temperatures

[0269] Exosome gel with batch number AMEXO202211001-004 and exosome injection with batch number AMEXO202202002-003 were stored at different temperatures and / or repeatedly frozen and thawed. The particle number, particle size, and content of the active ingredient (exosomes) were then measured to assess the loss of the active ingredient after storage / freeze-thaw.

[0270] Table 19 Stability of active ingredients in exosome injection

[0271]

[0272]

[0273] Table 20 Stability of active ingredients in exosome gel

[0274]

[0275] Conclusion: After repeated freeze-thaw cycles, the content and median particle size of exosomes in the gel were basically the same as before repeated freeze-thaw cycles, indicating that freeze-thaw cycles had no significant effect on the water bath gelation temperature and gelation time of the exosome gel.

[0276] 5.2 In vitro release assay

[0277] Study the effect of different concentrations of excipients on drug release rate through in vitro release experiments

[0278] Table 21 Experimental instrument information

[0279] name model serial number Manufacturer Drug Transdermal Diffusion Tester Mixdrive 6 MI-A-10-02 Hanson

[0280] Table 22 Sample information

[0281]

[0282] The specific operations are:

[0283] (1) According to the manufacturer's instructions, preheat the machine to 32°C and cut the membrane to the appropriate size (about 1.7 cm 2 ) and placed on a diffusion cell filled with receiving solution (PBS). The test sample was aspirated with a 1 mL microsyringe and briefly allowed to gel at room temperature before being slowly added to the sample loading tank. A magnetic stirrer was set at 300 rpm. Samples were taken at 2, 3, 4, 5, and 6 hours, with a 0.9 mL sample volume and a 0.9 mL refill volume. The reservoir was a 1 mL brown injection bottle. Three parallel groups (designated -1, -2, and -3) were set up for the exosome gels. No blank gel was set up for parallel experiment. The entire experiment was conducted in the dark.

[0284] (2) After collecting the samples at each sampling time point, they were placed in a refrigerator (2-8°C). After all the samples were collected, nanoflow cytometry was performed together. The sampling time for nanoflow cytometry was 120 seconds, and the transmittance for 6 hours was calculated.

[0285] Table 23 In vitro release results

[0286]

[0287]

[0288] In summary, based on the 6-hour permeability results, the concentration of 0.5-1.0% HPMC is significantly better than that of 2%, and the concentration of 0.5% is slightly better than that of 1.0%.

[0289] Example 6: Cellular Uptake of Gel Formulations Containing Mesenchymal Stem Cell Exosomes (MSC-Exos)

[0290] Through cell uptake experiments, it was verified whether the exosomes in the preferred gel formulation of the present invention were easily taken up by cells (for example, compared with the injection formulation).

[0291] Exosome solution: contains 1×10 10 particles / mL of 0.9% sodium chloride injection;

[0292] Exosome gel: containing 1 x 10 10 particles / mL of gel formulation, wherein the content of poloxamer 407 in the preparation is 20%, and the content of hypromellose in the preparation is 0.5%.

[0293] Method of operation:

[0294] HFF-1 cells were plated into the lower chamber of a 24-well plate and placed in a 37°C, 5% CO2 incubator for continued culture until complete adhesion, then exosome preparations were added. After 24 / 48 h, the culture medium was aspirated and washed once with PBS. 1 mL of PBS solution with a final concentration of 1 μg / mL Hoechst 33342 was added, and the cells were incubated at room temperature in the dark for 10 min, then washed once with PBS buffer and added with 1 mL of PBS. The cell uptake was observed using a fluorescence microscope. The green fluorescence was calculated using ImageJ to calculate the average fluorescence intensity.

[0295] The results are shown in Figure 1 (A) Compared with the control group, the gel adjuvant preparation can help the cells to better uptake Exos, and the Exos in the gel are more stable. The results prove the previous assumption of the inventors that the sustained-release effect of the gel preparation is more conducive to improving the utilization rate of exosomes than the one-time administration of injection. And this example also proves again that the gel preparation is more conducive to the preservation of active substances than the injection preparation.

[0296] Example 7: Effect of gel preparation containing mesenchymal stem cell exosomes (MSC-Exos) on wound healing of db / db mice

[0297] Through in vivo animal model experiments, it is verified whether the exosomes in the preferred gel preparation of the application have the effect of promoting repair of chronic skin damage (diabetic foot wounds).

[0298] Spontaneous type II diabetic model mice (db / db mice) with blood glucose values higher than 16.7 mmol / L were used to construct a diabetic wound model. At the same time, normal mice with blood glucose values lower than 16.7 mmol / L were used as normal wound model controls.

[0299] After the model was established, the exosome gel was administered by smearing the wound once a day for 14 consecutive days.

[0300] The composition of the treatment group preparation is: exosomes 1.32 x 10 10 particles / ml; 20% poloxamer 407; 0.5% hypromellose; total volume 113 μl.

[0301] The pathological results showed that compared with the normal group, the re-epithelialization and angiogenesis of the model group mice were significantly reduced, the collagen fibers were more disordered, and the inflammatory cell infiltration had no significant difference Figure 2 and Figure 3 ). Compared with the model group, the re-epithelialization of the treatment group mice was significantly improved, the inflammatory cell infiltration at the wound site was significantly reduced, and the collagen fibers were more orderly, similar to the normal group. In the histopathological sections, the epidermis and dermis structure of the treatment group was clear, and a small amount of hair follicle formation was observed in the dermis layer Figure 2 and Figure 3 ).

[0302] In summary, through histopathological evaluation, it was found that the mesenchymal stem cell exosome gel preparation could exert the effects of its active ingredients, promoting skin epithelial cell neogenesis at diabetic wounds, reducing dermal / subcutaneous inflammatory cell infiltration, and helping faster wound healing.

[0303] References

[0304] (1) Feng, J., et al., Plant-Derived Vesicle-Like Nanoparticles as Promising Biotherapeutic Tools:

[0305] Present and Future. Adv Mater, 2023. 35(24): p. e2207826.

[0306] (2) Testa, A., E. Venturelli, and M.F. Brizzi, Extracellular Vesicles: New Tools for Early Diagnosis of Breast and Genitourinary Cancers. Int J Mol Sci, 2021. 22(16).

[0307] (3) Li, X., et al., Exosomes from adipose-derived stem cells overexpressing Nrf2 accelerate cutaneous wound healing by promoting vascularization in a diabetic foot ulcer rat model.

[0308] Exp Mol Med, 2018. 50(4): p. 1-14.

[0309] (4) Wan, Z., et al., Mononuclear phagocyte system blockade improves therapeutic exosome delivery to the myocardium. Theranostics, 2020. 10(1): p. 218-230.

[0310] (5) Khayambashi, P., et al., Hydrogel Encapsulation of Mesenchymal Stem Cells and Their Derived Exosomes for Tissue Engineering. Int J Mol Sci, 2021. 22(2).

[0311] (6) Hunt, N.C. and L.M. Grover, Cell encapsulation using biopolymer gels for regenerative medicine. Biotechnol Lett, 2010. 32(6): p. 733-42.

[0312] (7) Akbari, A., et al., Free and hydrogel encapsulated exosome-based therapies in regenerative medicine. Life Sci, 2020. 249: p. 117447.

[0313] (8) Nicodemus, G.D. and S.J. Bryant, Cell encapsulation in biodegradable hydrogels for tissue engineering applications. Tissue Eng Part B Rev, 2008. 14(2): p. 149-65.

[0314] (9) Kaisang, L., et al., Adipose-derived stem cells seeded in Pluronic F-127 hydrogel promotes diabetic wound healing. J Surg Res, 2017. 217: p. 63-74.

[0315] (10)Yang,J.,et al.,Umbilical Cord-Derived Mesenchymal Stem Cell-Derived ExosomesCombined Pluronic F127 Hydrogel Promote Chronic DiabeticWound Healing and CompleteSkin Regeneration.Int J Nanomedicine,2020.15:p.5911-5926.

[0316] (11)Kant,V.,et al.,Topical pluronic F-127 gel application enhancescutaneous wound healingin rats.Acta Histochem,2014.116(1):p.5-13

[0317] (12)van Netten JJ,et al.,Diagnostic values for skin temperatureassessment to detect diabetes-

[0318] related foot complications.Diabetes Technol Ther.2014 Nov;16(11):714-21.

Claims

1. An extracellular vesicle gel preparation comprising an effective amount of extracellular vesicles and a gel solution loaded with the extracellular vesicles, wherein the gel solution consists of poloxamer, hydroxypropyl methylcellulose, and a physiologically acceptable aqueous solution, wherein the concentration of the poloxamer is 15-30%, and the concentration of the hydroxypropyl methylcellulose is 0.25-3%. The % is the weight volume percentage of grams (g) of the relevant substance relative to milliliters (mL) of the aqueous solution.

2. The extracellular vesicle gel preparation according to claim 1, wherein when the preparation is applied to human skin in liquid form, it changes from a fluid state to a gel state at the human skin temperature, preferably the skin temperature is higher than 24°C, more preferably 25°C-37°C, more preferably 25°C-35°C, for example, 25°C-27°C, 27°C-33°C.

3. The extracellular vesicle gel preparation according to claim 2, wherein the time for the transition is no more than 5 minutes, preferably no more than 3 minutes, more preferably no more than 2.5 minutes, and most preferably no more than 2 minutes. The extracellular vesicle gel preparation of claim 1 , wherein the poloxamer is poloxamer 407.

5. The extracellular vesicle gel preparation according to claim 1, wherein the type of hydroxypropyl methylcellulose is one or more selected from 1828, 2208, 2906, and 2910. The extracellular vesicle gel preparation according to claim 1 , wherein the concentration of the poloxamer is 18-30%, preferably 20-25%, more preferably 20%.

7. The extracellular vesicle gel preparation according to claim 1, wherein the concentration of hydroxypropyl methylcellulose is 0.5-2%, preferably 0.5-1.5%, more preferably 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%.

8. The extracellular vesicle gel preparation according to claim 1, wherein the extracellular vesicles are derived from mesenchymal stem cells, for example, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, and the like.

9. The extracellular vesicle gel preparation according to claim 8, wherein the extracellular vesicles are exosomes.

10. The extracellular vesicle gel preparation according to claim 8, wherein the extracellular vesicles are exosomes and are rich in: (a) one or more miRNAs selected from miR-24, miR-21, miR-146, miR-150 and Let-7a; and / or (b) selected from Thrombospondin-1, Heat shock protein 70 (Heat shock protein 71kDa protein), pyruvate kinase PKM, heat shock protein 90 (Heat shock protein HSP 90-alpha), peroxidase 1 (Peroxiredoxin-1) and peroxidase 2 (Peroxiredoxin-2) or more proteins.

11. The extracellular vesicle gel preparation according to any one of claims 1 to 10, wherein the extracellular vesicles are prepared according to the following steps: (a) providing a stem cell; (b) pre-culturing the stem cells using a first culture medium under appropriate culture conditions to obtain pre-cultured stem cells having a confluency of 60-90%; in, The first culture medium is a culture medium containing platelet lysate; (c) incubating the cultured stem cells with a second culture medium for 24 to 48 hours to obtain an incubation culture medium containing the cultured stem cells and exosomes produced by the cultured stem cells; Wherein, the second culture fluid is an ultrafiltrate obtained by ultracentrifuging, filtering and ultrafiltration of a culture medium containing platelet lysate; and (d) isolating exosomes from the incubation culture medium obtained in the previous step. 12 . The extracellular vesicle gel preparation according to claim 11 , wherein the first culture medium in step (b) is an αMEM medium containing platelet lysate.

13. The extracellular vesicle gel preparation according to claim 11, wherein the second culture medium in step (c) is an ultrafiltrate obtained by ultracentrifuging, filtering and ultrafiltration of an αMEM culture medium containing platelet lysate. The extracellular vesicle gel preparation according to claim 11 , wherein the stem cells are mesenchymal stem cells, preferably adipose-derived mesenchymal stem cells, more preferably P4 adipose-derived mesenchymal stem cells.

15. The extracellular vesicle gel preparation according to claim 1, wherein the content of the extracellular vesicles is 1×10 8 Particles / ml-1×10 11 Particles / ml.

16. A method for preparing a gel preparation for carrying extracellular vesicles, the method comprising the following steps: (a) Add a physiologically acceptable aqueous solution to a beaker. (b) weighing poloxamer and HPMC, adding them to the beaker in the previous step under stirring, and continuing stirring for at least 10 minutes, preferably at least 20 minutes, and (c) Refrigerate the solution in a refrigerator at 2-8°C (preferably for >8 h, more preferably for >12 h).

17. A method for preparing a gel formulation containing extracellular vesicles, comprising the steps of: (a) Add a physiologically acceptable aqueous solution to a beaker. (b) Weigh poloxamer and HPMC, add them to the beaker in the previous step under stirring, and continue stirring for at least 10 minutes, preferably at least 20 minutes. (c) placing the solution in a refrigerator at 2-8°C overnight (>8h, preferably >12h), and (d) Add the extracellular vesicle suspension to the solution.

18. The method of claim 16 or 17, wherein the poloxamer is poloxamer 407.

19. The method of claim 16 or 17, wherein the type of hydroxypropyl methylcellulose is selected from 1828, One or more of 2208, 2906, and 2910.

20. The method according to claim 16 or 17, wherein the aqueous solution is taken in an amount of 18-30 parts by weight, preferably 20-25 parts by weight, more preferably 20 parts by weight, and the poloxamer is weighed in an amount of 0.25-3 parts by weight, preferably 0.5-2 parts by weight, more preferably 0.5 parts by weight, based on 100 parts by volume of the aqueous solution, wherein 1 part by weight:1 part by volume corresponds to a ratio of 1 g:1 ml.

21. Use of the extracellular vesicle gel preparation according to any one of claims 1 to 15 in the preparation of a medicament for treating a disease or condition responsive to extracellular vesicles.

22. The method of claim 21, wherein the disease or disorder is a skin or mucosal lesion.

23. The use according to claim 21, wherein the disease or disorder is diabetic skin lesions, such as diabetic foot skin lesions, wounds and / or ulcers.

24. A pharmaceutical composition comprising the extracellular vesicle gel preparation according to any one of claims 1 to 15, or the extracellular vesicle gel preparation produced according to the method according to any one of claims 17 to 20.

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