Application of mesenchymal stem cell exosome composition in scald healing

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of an exosome composition to anti-inflammation, scald healing or wound healing acceleration. Specifically, the invention provides an application of the exosome composition in preparation of medicines for preventing and / or inhibiting and / or relieving and / or improving and / or treating scalds, and the exosome composition comprises fresh mesenchymal stem cell exosomes, sialic acid, glucan and albumin. The active substances such as sialic acid, glucan and albumin are added into the exosome storage liquid to prepare the exosome composition, and the composition can be used for improving the in-vitro storage stability of the exosome preparation on one hand, and can promote the uptake of the exosome by immune cells on the other hand, so that the collection of the exosome to a wound is promoted, and the wound healing is promoted. Therefore, the curative effect of the exosome is enhanced, the effects of resisting inflammation and promoting proliferation are synergistically exerted, wound healing is optimized, and a method which is more effective than that of a traditional exosome preparation is provided.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of an exosome composition in anti-inflammation, burn wound healing, or accelerating wound healing. Background Art

[0002] As the largest organ of the human body, the skin tissue not only occupies an important position in terms of structure, but also undertakes various key physiological functions such as defending against pathogens and mechanical injuries, and sensing environmental changes. In addition, the skin participates in body temperature regulation through the dilation and constriction of blood vessels to maintain relative body temperature stability. However, skin integrity may be damaged due to various reasons such as burns, scalds, trauma, surgery, and diabetic complications. Wound healing is a complex regeneration process involving the coordinated action of multiple stages. This process is generally divided into an inflammatory phase, a proliferative phase, and a maturation phase. In the inflammatory phase, immune cells are activated to clear pathogens and necrotic tissues to prevent infection; subsequently, in the proliferative phase, cell proliferation and tissue repair become crucial, and the activities of fibroblasts and vascular endothelial cells are particularly prominent; finally, in the maturation phase, the newly formed tissue gradually gains strength and function, and collagen fibers are reorganized, and scar formation occurs.

[0003] However, the healing process of chronic wounds is often disrupted and cannot follow the typical healing cascade reaction. This may lead to the persistence of chronic inflammation, causing great pain to patients and imposing a significant socioeconomic burden. Although remarkable progress has been made in the field of wound healing in regenerative medicine, wound infection and effective healing remain urgent problems to be solved. Chronic wounds are prone to various complications, such as excessive inflammation, persistent infection, the formation of drug-resistant microbial biofilms, and the sluggish response of dermal and / or epidermal cells to reparative stimuli. These problems not only affect the wound healing rate but may also have a negative impact on the clinical treatment outcomes of patients.

[0004] Anti-inflammation is an important research direction for wound healing. However, traditional anti-inflammatory and wound healing methods have many limitations, such as drug side effects, slow healing speed, etc. In recent years, exosomes derived from mesenchymal stem cells have shown great therapeutic potential because of their advantages such as low immunogenicity, high biological activity, and biological targeting, which can significantly reduce the risk of side effects.

[0005] Exosomes are extracellular vesicles with a diameter of 30 - 150 nm secreted by living cells and having information - transmitting functions. Exosomes have a bilayer lipid membrane structure and can mediate inter - cellular signal communication by delivering the deoxyribonucleic acid (DNA), ribonucleic acid (RNA), lipids, and proteins they carry to target cells. Exosomes are widely involved in various physiological / pathological regulations of the body and can be used for the diagnosis, treatment, and prognosis evaluation of various diseases. Currently, mesenchymal stem cell exosomes and their products have been proven to have important pharmacological effects such as anti - aging (SciAdv. 2022 Oct 21; 8(42): eabq2226.), regulating immune cells (J Extracell Vesicles. 2024 Aug; 13(8): e12472.), promoting cell proliferation, improving skin condition (Carbohydr Polym. 2025 Mar 1:351:123098.), promoting wound healing (J Extracell Vesicles. 2024 Nov; 13(11): e70013.), etc. They have obtained approval from the US Food and Drug Administration (FDA) to conduct clinical trials for atopic dermatitis and cerebral infarction and have high clinical application potential. Summary of the Invention

[0006] Existing exosome preparations have defects such as short in - vivo half - life, poor targeting, and low biological activity, which limit the efficacy of exosome preparations. In the present invention, an exosome composition is prepared by adding active substances such as sialic acid, dextran, and albumin to the exosome storage solution. On the one hand, this composition can be used to improve the stability of exosome preparations during in - vitro storage. On the other hand, it can promote the uptake of exosomes by immune cells, thereby promoting the recruitment of exosomes to the wound site, enhancing the efficacy of exosomes, and synergistically playing an anti - inflammatory and proliferation - promoting role to optimize wound healing, providing a method that is more effective than traditional exosome - based treatments.

[0007] First, the present invention provides the use of an exosome composition in the preparation of a drug for preventing and / or inhibiting and / or alleviating and / or improving and / or treating scalds, wherein the exosome composition comprises fresh mesenchymal stem cell exosomes, sialic acid, dextran, and albumin.

[0008] Preferably, the exosome composition comprises a matrix and mesenchymal stem cell exosomes; the particle concentration of the mesenchymal stem cell exosomes in the exosome composition is not less than 1×10 8 particles / mL. More preferably, the particle concentration of the mesenchymal stem cell exosomes in the exosome composition is 1×10 8~1×10 10 particles / mL。

[0009] Preferably, the matrix comprises: by mass percentage, 1-5 wt% of animal serum albumin, 1-5 wt% of dextran, 0.1-0.5 wt% of sialic acid, and 89.5-97.9 wt% of phosphate buffer without Ca 2+ , Mg 2+ .

[0010] Preferably, the dextran is dextran with a molecular weight of 20,000-100,000; preferably, the dextran is dextran 70 and / or dextran 40; more preferably, the dextran is dextran 70 with a molecular weight of 60,000-80,000.

[0011] Preferably, the animal serum albumin is serum albumin of cattle, human, horse, donkey, sheep, dog or pig, and preferably bovine serum albumin.

[0012] Preferably, the pH value of the phosphate buffer without Ca 2+ , Mg 2+ is 6.4-8.8, preferably 6.8-8.8, and more preferably 7.4.

[0013] Preferably, the method for preparing the exosome composition comprises the following steps: Step (1): Sterilize the phosphate buffer without Ca 2+ , Mg 2+ by high temperature and high pressure, and then let it stand at room temperature and cool to 45°C-50°C; Step (2): Add dextran to the phosphate buffer obtained in step (1) and vortex and stir until the dextran is completely dissolved; Step (3): When the solution obtained in step (2) cools to room temperature, add sialic acid to the solution and stir and mix until dissolved; Step (4): Add animal serum albumin to the solution obtained in step (3) and invert to dissolve to obtain an exosome diluent; Step (5): Filter the exosome diluent obtained in step (4) using a filter membrane; Step (6): Add fresh mesenchymal stem cell exosomes to the exosome diluent filtered in step (5) to obtain the exosome composition.

[0014] Preferably, the pore size of the filter membrane is 0.2-0.3 μm, and preferably 0.22 μm.

[0015] Preferably, the pH of the exosome composition is 5.0-6.5, and preferably 5.8.

[0016] Preferably, the drug comprises an effective amount of the exosome composition and a pharmaceutically acceptable carrier or excipient.

[0017] Preferably, the pharmaceutical preparation form of the drug is an injection preparation, an oral preparation, a nasal drop, a spray preparation, an ointment preparation or a patch.

[0018] Beneficial effects:

[0019] Compared with conventional mesenchymal stem cell exosome preparations, the exosome composition of the present invention is rich in fresh mesenchymal stem cell exosomes, sialic acid, dextran and albumin, can maintain the stability of nucleic acids in exosomes in vitro for a long time, and at the same time, the composition of the exosome composition is simple and clear, with high biosafety, can be directly used for intravenous injection, and can improve the uptake of mesenchymal stem cell exosomes by immune cells when used as a drug, enhance the recruitment of exosomes to the wound, and then enhance the anti-inflammatory and wound healing activities of mesenchymal stem cell exosomes, and has an excellent wound healing and / or accelerating wound healing effect. Description of the drawings

[0020] Figure 1 Flowcharts showing the extraction of fresh mesenchymal stem cell exosomes by ultra-high speed centrifugation and the preparation of the exosome composition. Wherein a is the preparation flowchart of fresh mesenchymal stem cell exosomes; b is the preparation flowchart of the exosome composition.

[0021] Figure 2 Showing the screening process and stability of the exosome composition. Among them, a is the screening process of the main components of the exosome composition; b is the effective component ratio screened by big data multi-modal language model (AI) analysis; c is that the exosome composition can effectively improve the stability of nucleic acids in exosomes during in vitro storage.

[0022] Figure 3 Showing that the exosome composition can effectively promote the wound healing of scalded mice. Among them, a is the wound healing condition after injecting the exosome composition for scald; b is the statistical chart of the wound healing condition; c is the statistical chart of the skin regeneration condition of the mouse wound.

[0023] Figure 4 a is the in vivo imaging diagram of the wound of the scalded mouse. Figure 4 b is the immunofluorescence picture at the scald wound (green: immune cells positive for CD11b, red: exosomes labeled with PKH26, blue: nuclei labeled with DAPI). Figure 4 c is the GSEA analysis result after RNA-seq detection of the wound tissue sample, and multiple pathways related to immune inflammatory responses are regulated. It shows that the exosome composition can significantly promote the aggregation of exosomes at the scald wound and inhibit the immune inflammatory response of scalded mice.

[0024] Figure 5It is shown that the exosome composition can effectively promote the uptake of exosomes by human epidermal cells and promote cell proliferation and migration. Among them, a is a fluorescence microscopic image of the uptake of exosomes in the exosome composition by human epidermal cells (red: exosomes delivered to cells labeled with PKH26, blue: cell nuclei labeled with DAPI); b is the cell migration of human epidermal cells after scratching the human epidermal cells treated with the exosome composition. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with the accompanying drawings and the following implementation manners. It should be understood that the accompanying drawings and the following implementation manners are only used to illustrate the present invention, rather than limiting the present invention. It should be noted that the following description of the present invention is illustrative by way of example, and the scope of protection required by the present invention is not limited by this. For example, in the following description, some functions and advantages other than the object of the present invention may be more obvious.

[0026] The term "mesenchymal stem cell" used herein refers to a type of adult stem cell with multi-directional differentiation potential, mainly derived from tissues such as bone marrow, adipose tissue, umbilical cord blood, and placenta.

[0027] The term "exosome" used herein refers to a nanoscale vesicle with a membrane structure secreted or released from cells into the extracellular space, and is also referred to as an extracellular vesicle or microvesicle.

[0028] The term "mesenchymal stem cell exosome" is intended to include all exosomes isolated from, for example, the conditioned medium of human umbilical cord mesenchymal stem cells, blood, human umbilical cord tissue, or biological products equivalent thereto.

[0029] The term "dextran" used herein refers to a homopolysaccharide composed of glucose as a monosaccharide, and the glucose units are linked by glycosidic bonds.

[0030] The term "effective amount" means an amount by which mesenchymal stem cell exosomes can: (i) treat a specific disease, disorder, or condition, (ii) attenuate, improve, or eliminate one or more symptoms of a specific disease, disorder, or condition, or (iii) prevent or delay the onset of one or more symptoms of a specific disease, disorder, or condition described herein.

[0031] As used herein, the term "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for use in the human body and must have sufficient purity and sufficiently low toxicity. "Compatibility" herein means that the components in the exosome composition can be intermingled with the active ingredient without significantly reducing the efficacy of the active ingredient. Pharmaceutically acceptable carriers or excipients include, but are not limited to, cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween, etc.), wetting agents (such as sodium dodecyl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0032] As used herein, the term "wound" refers to a condition in which a part or all of the body is damaged, and is intended to cover pathological conditions in which the tissues (such as skin, muscle, nerve tissue, bone, soft tissue, internal organs or vascular tissue) that constitute the internal or external surface of the body are damaged or destroyed. By way of example and not limitation of the present invention, some examples of wounds include abrasions, lacerations, punctures, incisions, avulsions, pressure ulcers, tissue damage caused by radiation, penetrating wounds, gunshot wounds, burns, frostbite, surgical wounds, sutured wounds after plastic surgery, wounds caused by chemicals, etc., and can include any damage to any part of an individual.

[0033] The present invention provides the use of an exosome composition in promoting wound healing. The exosome composition contains exosomes derived from fresh mesenchymal stem cells, sialic acid, dextran and albumin as active ingredients. This exosome composition aims to enhance the therapeutic effect of exosomes in wound healing. In addition, the exosome composition provided by the present invention has certain advantages in maintaining the in vitro stability of exosomes and can maintain the content of nucleic acids in exosomes for a long time.

[0034] The exosome composition includes a matrix and mesenchymal stem cell exosomes. In some embodiments, the components of the matrix include: by mass percentage, 1-5 wt% of bovine serum albumin, 1-5 wt% of dextran (dextran 70), 0.1-0.5 wt% of sialic acid, and phosphate buffer solution (PBS) without Ca 2+ 、Mg 2+ 89.5-97.9 wt%. In some embodiments, the sum of the mass percentages of the components of the matrix is 100%.

[0035] The mesenchymal stem cell exosomes can be mesenchymal stem cell exosomes of mammals. By way of example and not limitation of the present invention, the mesenchymal stem cell exosomes are human umbilical cord mesenchymal stem cells, which have the advantages of wide sources and applications, strong biological activity, and great research potential. Mesenchymal stem cells from other mammals such as dogs, cats, rodents, horses, cows, monkeys, and pigs, as well as from other tissues such as adipose, bone marrow, and placenta, also fall within the scope of the exosome composition of the present invention.

[0036] The preparation method of mesenchymal stem cell exosomes is a conventional technique in the art. By way of example and not limitation of the present invention, the ultra-high-speed centrifugation method is the "gold standard" for exosome extraction methods and is applicable to the extraction and purification of most exosomes. It should be understood that other extraction methods for exosomes such as differential centrifugation, density gradient centrifugation, kit methods, size exclusion chromatography, and precipitation methods are also applicable to the extraction of exosomes in the present invention.

[0037] N-acetylneuraminic acid, also known as sialic acid, is a class of monosaccharide derivatives containing nine carbon atoms. Its core structure is a pyranose-structured keto acid with a negative charge. Sialic acid is widely distributed at the ends of the oligosaccharide chains of glycoproteins or glycolipids on the surface of eukaryotic cells and is an important component of glycoproteins and glycolipids on cell membranes, having important physiological functions such as regulating immune inflammatory responses (J Biomed Sci. 2015 Oct 24; 22: 96.), improving muscle generation (Brain. 2014 Oct; 137(Pt 10): 2670 - 2679), and regulating neural development (Crit Rev Food Sci Nutr. 2023; 63(29): 9875 - 9894). Currently, there is no mature sialic acid preparation for anti-inflammatory or wound healing, and it is urgent to explore and develop.

[0038] Sialic acid has strong acidity. When the content is too high (>0.5%), it will produce cytotoxicity, and when the content is too low (<0.05%), it cannot provide an acidic environment for the exosome composition, reducing the exosome activity. For example, the proportion of sialic acid used in the exosome composition is 0.25 wt%.

[0039] Dextran 70 and dextran 40 are active ingredients that have been approved for marketing by the State Administration for Market Regulation. In some embodiments of the present invention, dextran 70 exhibits stronger stability than dextran 40. The molecular weight of the dextran can be 20,000 - 100,000. When the molecular weight is too high (>100,000), it may lead to difficult dissolution and a non-uniform solution, and when the molecular weight is too low (<20,000), it may lead to difficulty in forming a colloidal protective film and unable to provide physical protection for exosomes. Preferably, the dextran used is dextran 70 with a molecular weight of 60,000 - 80,000.

[0040] The dextran used in the specific implementation is dextran 70 with an average molecular weight of 60,000 to 80,000, and its molecular formula is shown as follows:

[0041] The PBS without Ca 2+ and Mg 2+ has a pH value of 6.4 to 8.8, preferably 6.8 to 8.8, and more preferably 7.4.

[0042] The albumin used can be serum albumin from any one of animals such as cows, humans, horses, donkeys, sheep, dogs, or pigs, and bovine serum albumin is preferred. Bovine serum albumin can undergo the Maillard reaction with dextran to form a more stable compound. In addition, bovine serum albumin has a wide source and low cost, and has been approved for use as a pharmaceutical excipient in clinical applications. In contrast, human serum albumin, horse serum albumin, and donkey serum albumin do not have the above advantages.

[0043] The preparation method of the exosome composition is described below.

[0044] The PBS without Ca 2+ and Mg 2+ is sterilized by high temperature and high pressure and then left to stand at room temperature until it cools to 45°C to 50°C. The PBS without Ca 2 + and Mg 2+ needs to be sterilized by high temperature and high pressure. The sterilization temperature can be 95 to 130°C, and the sterilization time can be 5 to 45 minutes. For example, the sterilization conditions are 121°C for 20 minutes. Unsterilized PBS may contain potential bacteria and microorganisms, posing a risk of contamination.

[0045] Preferably, the PBS solution needs to be left to stand at room temperature until it cools to 45°C to 50°C. The purpose of this step is to facilitate the rapid dissolution of dextran (such as dextran 70). Dextran 70 is a high molecular weight polymer, and its dissolution rate is slow at room temperature. Warm water at 50°C can significantly increase the dissolution rate of dextran 70. Too high a temperature may cause the degradation of dextran 70, while too low a temperature may result in a slow dissolution rate of dextran 70.

[0046] Add dextran powder in proportion and vortex and stir until completely dissolved.

[0047] When the above solution cools to room temperature, add sialic acid powder to the solution and stir evenly to dissolve.

[0048] Add (bovine serum) albumin powder and dissolve it by inversion (avoiding violent vortexing) to obtain the exosome diluent. When dissolving (bovine serum) albumin, inversion mixing should be used and violent vortexing should be avoided as much as possible. Violent vortexing or mixing will cause bubbles that are difficult to remove to form in the (bovine serum) albumin solution, which will affect subsequent operations.

[0049] Filter the above-mentioned exosome diluent through a filter membrane. The exosome diluent needs to be filtered through a filter membrane. Preferably, the pore size of the filter membrane can be 0.2 - 0.3 μm, preferably 0.22 μm. A filter membrane with a pore size of 0.2 - 0.3 μm can effectively remove insoluble impurities and contaminants such as bacteria and fungi in the preparation. If the pore size is too large, small particle impurities cannot be removed, and if the pore size is too small, the filter membrane will become blocked.

[0050] Add freshly prepared mesenchymal stem cell exosomes to the obtained exosome diluent to obtain the exosome composition.

[0051] As an example, the present invention also provides a method for preparing the above-mentioned exosome composition. The preparation method includes the following steps: (1) Sterilize PBS without Ca 2+ and Mg 2+ by autoclaving at high temperature and high pressure, and then let it stand at room temperature and cool to 45°C - 50°C; (2) Add dextran (dextran 70) powder at a ratio of 2 wt% and vortex and stir until completely dissolved; (3) When the above solution cools to room temperature, add 0.25 wt% of sialic acid powder to the solution and stir evenly to dissolve; (4) Add 2 wt% of bovine serum albumin powder and dissolve it by inversion (avoiding violent vortexing) to obtain the exosome diluent; (5) Filter the above-mentioned exosome diluent through a 0.22 μm filter membrane; (6) Add an appropriate amount of mesenchymal stem cell exosome freeze-dried powder or exosome precipitate obtained by centrifugation to the obtained exosome diluent to obtain the exosome composition.

[0052] The present invention also discloses the use of a pharmaceutical composition in the prevention and / or treatment of inflammation and promoting wound healing. The pharmaceutical composition contains an effective amount of the exosome composition and a pharmaceutically acceptable carrier or excipient.

[0053] The drug or its exosome composition can be made into injection preparations, oral preparations, nasal drops, spray preparations, ointment preparations or patches, etc. according to the common methods of drug preparation. In addition, the exosome composition can also be used in combination with other drugs for preventing and / or treating inflammation and promoting wound healing.

[0054] The present invention also discloses a method for using an exosome composition for treating inflammation and promoting wound healing. That is, the present invention provides a method for using the above exosome composition to prevent, inhibit, alleviate, improve or treat inflammatory diseases and promote wound healing.

[0055] The method includes administering a therapeutically effective amount of the exosome composition to a mammal, or applying the exosome composition to the skin, an inflammatory region or an injured region. The mammal can be at least one mammal selected from the following: human, dog, cat, rodent, horse, cow, monkey and pig.

[0056] By way of example and not limitation of the present invention, the exosome composition according to an embodiment of the present invention can be administered or treated by injection, microneedle method, application or a combination thereof. For example, the exosome composition can be an injectable preparation, an infusion preparation, a spray preparation, a liquid preparation or a patch preparation.

[0057] Preferably, the treatment mode is: intravenous injection of the exosome composition (the content of mesenchymal stem cell exosomes ≥ 2×10 9 particles / mL) rich in an effective amount of mesenchymal stem cell exosomes dissolved in the exosome diluent in the above preparation step once every three days for 12 days.

[0058] Preferably, the exosome diluent is a mixed solution prepared by the above steps and containing, by mass percentage, 2 wt% of bovine serum albumin, 2 wt% of dextran (dextran 70), 0.25 wt% of sialic acid, and 95.75 wt% of PBS without Ca 2+ and Mg 2+ .

[0059] Preferably, the effective amount of mesenchymal stem cell exosomes should be not less than 2×10 9 particles / mL. If the concentration is too low, the effective concentration may not be achieved, and if the concentration is too high, a strong immune rejection reaction may occur.

[0060] It should be understood that the content of mesenchymal stem cells in the drug exosome composition according to an embodiment in the preparation can be appropriately adjusted and selected according to the specific condition of the injury and different application forms, etc.

[0061] The exosome composition can be made into an injection preparation, an oral preparation, a nasal drop preparation, a spray preparation or a microneedle preparation, etc. according to the conventional methods of drug preparation in the art.

[0062] In summary, the inventors have found through research an exosome composition with mesenchymal stem cell exosomes, sialic acid, dextran and albumin as active ingredients, which can maintain the nucleic acid stability of exosomes during long-term in vitro storage and can be used simultaneously for treating inflammation, promoting wound healing or accelerating wound healing.

[0063] Compared with ordinary exosome preparations, the exosome composition of the present invention has clear components and reasonable ratios, can be directly used for intravenous injection, and at the same time promotes the uptake of exosomes by immune cells and the recruitment of exosomes to wounds, thereby improving / maintaining the activity of exosomes and enhancing the efficacy of exosomes in improving burn healing. In addition, the exosome composition of the present invention has low cost and simple preparation, and the selected components have all been approved by the National Market Regulatory Administration or the FDA for application in human trials, indicating that the exosome composition has extremely high clinical application prospects, thus completing the present invention.

[0064] The following further lists examples to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are also only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description in this article, rather than being limited to the specific values in the following examples. All relevant operations comply with ethical operation standards and procedures.

[0065] Unless otherwise specified, for the technologies or conditions not specified in the present invention, the conventional technical conditions in the art are followed or the product instructions are followed. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0066] Example 1: Extraction method of fresh mesenchymal stem cell exosomes

[0067] Figure 1 Figure a shows the flow chart of extracting fresh mesenchymal stem cell exosomes by ultra-high speed centrifugation. Human umbilical cord mesenchymal stem cells in the logarithmic growth phase were selected for culture and exosomes were extracted. The human umbilical cord mesenchymal stem cells were purchased from Shanghai Fuheng Biotechnology Co., Ltd., and the serum-free low-glucose DMEM medium was purchased from Suzhou Kemel Biotechnology Co., Ltd. The specific method is as follows: When the cell confluence reaches 80-90%, add serum-free low-glucose DMEM medium and continue to culture for 48 h, and then collect the conditioned medium; then, use ultra-high speed centrifugation or ultrafiltration to separate and extract exosomes: Take an appropriate amount of cell culture medium and centrifuge at a centrifugal force of 300 g for 10 min to remove cells, and then centrifuge at a centrifugal force of 3000 g for 20 min to further remove cell debris and impurities; the obtained supernatant is centrifuged at 10000 g for 60 min, and then the supernatant is aspirated and discarded. The exosomes are contained in the precipitate at the bottom of the tube; add an appropriate amount of precooled PBS without Ca 2+ and Mg 2+ to resuspend the exosomes, and centrifuge at 11000 g for 90 min to wash the exosomes. The obtained precipitate is mesenchymal stem cell exosomes.

[0068] Example 2: Screening Process of Exosome Composition

[0069] Figure 2 a of shows the screening method of the exosome composition. Based on the publicly available database The HumanMetabolome Database (HMDB), small molecule metabolites widely present in various human body fluids, including cerebrospinal fluid, blood, urine, milk, saliva, and feces, were selected as a benchmark, and further screened for compounds that can be synthesized and metabolized endogenously in the human body. In addition, it was further clarified that the content of the above compounds in the human body can be accurately measured, and it was ensured that there were no significant differences in the content of the compounds among different genders, different ages, pregnant or not, and different disease states (such as diabetes, hypertension, Alzheimer's disease, preeclampsia, and kidney diseases, etc.).

[0070] Figure 2 b of shows the screening and proportioning strategy of the main components of the exosome composition: Based on the Chinese Pharmacopoeia and the publicly available databases Pubmed and Chemicalbook, a reasonable component ratio was analyzed with the help of a big data multi-modal language model (AI).

[0071] Promoting angiogenesis and epidermal regeneration are important strategies for accelerating wound healing, and cell proliferation and toxicity detection are recognized methods for detecting the above indicators in wound healing. Referring to the method described by Yang et al. (Biomaterials. 2024 Jul:308:122558.), the effects of exosome compositions with different formulations on the cell viability of human epidermal cells (HaCaT) and human microvascular endothelial cells (HCMEC / D3) were detected by CCK8 reagent (purchased from Shanghai Beyotime Biotechnology Co., Ltd.), so as to reflect the role of exosome compositions with different formulations in wound healing.

[0072] Table 1 Effects of Exosome Composition on Cell Viability

[0073] The exosome composition in Table 1 contains 2×10 9 particles / mL of fresh mesenchymal stem cell exosomes in addition to the matrix. It can be found that the exosome composition formulation with the simultaneous addition of sialic acid, albumin, and dextran 70 has the best effect on enhancing cell viability and is suitable for the development of wound healing drugs. Among them, when the content of sialic acid, albumin, and carbohydrate (dextran) meets the requirement of 0.1wt% - 8wt%, it is suitable for the preparation of the exosome composition. Preferably, the matrix of the exosome composition includes: by mass percentage, animal serum albumin 1 - 5wt%, dextran 1 - 5wt%, sialic acid 0.1 - 0.5wt%, without Ca 2+ 、Mg2+ 89.5 - 97.9 wt% of phosphate buffer solution; and the exosome composition contains 2×10 9 particles / mL of fresh mesenchymal stem cell exosomes.

[0074] Example 3: Preparation process of exosome composition

[0075] Figure 1 b shows the preparation process of the exosome composition. The preparation method of the exosome composition includes the following steps: (1) Sterilize PBS without Ca 2+ and Mg 2+ by high temperature and high pressure, and let it stand at room temperature until cooled to 45℃ - 50℃; (2) Add dextran (dextran 70) powder at a ratio of 2 wt% and vortex and stir until completely dissolved; (3) When the above solution is cooled to room temperature, add sialic acid powder at a ratio of 0.25 wt% and stir evenly to dissolve; (4) Add bovine serum albumin powder at a ratio of 2 wt% and invert to dissolve (avoid violent vortex) to obtain an exosome diluent; (5) Filter the above exosome diluent with a 0.22 μm filter membrane; (6) Add an appropriate amount of mesenchymal stem cell exosome lyophilized powder or exosome precipitate obtained by centrifugation to the obtained exosome diluent to obtain the exosome composition.

[0076] The exosome diluent described in this example can be directly used for the dissolution, resuspension or dilution of exosomes. The obtained exosome composition can be directly used for the detection of protein and nucleic acid concentrations.

[0077] Figure 2 c shows that the exosome composition can effectively improve the stability of nucleic acids contained in the exosomes stored therein. After being stored at -20℃ for one year, the total nucleic acids in the exosome composition were extracted using a nucleic acid extraction and purification kit (purchased from Hangzhou Bioer Technology Co., Ltd.), and the nucleic acid concentration in the extract was detected using a NanoDrop 2000c. The results showed that the nucleic acid content in the exosome composition was significantly higher than that of exosomes stored only in PBS (compared with exosomes dissolved in PBS: ****P<0.0001, n = 3, data are expressed as mean ± SEM). It is proved that the exosome composition helps to maintain the stability of the exosomes therein and avoid the degradation of nucleic acids.

[0078] Example 4: Exosome composition promotes wound healing in scalded mice.

[0079] This experiment was approved by the Animal Experiment Ethics Committee of Shanghai Jiao Tong University School of Medicine, and all operations were carried out in accordance with the animal experiment operation guidelines.

[0080] Male C57BL / 6J mice at 6 - 8 weeks of age were purchased from Suzhou Kevins Biological Model Animal Research Co., Ltd. The mice were randomly divided into a control group (intravenous injection of PBS without Ca 2+ and Mg 2+ , 200 μL); an exosome diluent group (intravenous injection of exosome diluent without exosomes, 200 μL); an exosome group (intravenous injection of exosomes at 2×10 9 particles / mL, dissolved in PBS without Ca 2+ and Mg 2+ , 200 μL); and an exosome composition group (intravenous injection of exosome composition, 200 μL, with the effective concentration of exosomes being 2×10 9 particles / mL), with 6 mice in each group.

[0081] A second - degree scald mouse model was constructed according to the method described by Yan et al. (Theranostics. 2020 Aug 8;10(22):9970 - 9983.). Briefly, after depilating both sides of the mouse abdomen, under isoflurane anesthesia, the opening at one end of a cylindrical plastic tube with a diameter of 1 cm was attached to the abdominal skin of the mouse. Then, boiling water was poured into the tube to a height of 2 cm and removed after 15 s. Immediately after scalding, intervention treatment was carried out according to the grouping, once every three days for 12 days. The wounds were recorded using a Leica camera. After 3, 6, 9, and 12 days of treatment, the healing conditions of each group were recorded by taking pictures and analyzed using ImageJ software.

[0082] Figure 3 It was shown that the exosome composition could effectively promote the wound healing of scalded mice. Among them, a shows the wound healing condition after injecting the exosome composition into the scald; b is the statistical chart of the wound healing condition; c is the statistical chart of the skin regeneration of the mouse wound.

[0083] From Figure 3 it can be seen that the exosome composition could significantly promote the wound healing and skin regeneration of scalded mice (compared with exosomes dissolved in PBS in the exosome composition group: *P < 0.05, ****P < 0.0001, n = 6, data are expressed as mean ± SEM).

[0084] Example 5: The exosome composition promotes the uptake of exosomes by immune cells, promotes cell proliferation and cell migration, and inhibits the immune inflammatory response of scald.

[0085] On the 12th day, the mice were sacrificed by cervical dislocation, and fresh scalded tissues were taken for RNA - seq and immunofluorescence detection and analysis. The experimental procedures were carried out according to the conventional operations in the field of research. The reagents used were purchased from Abcam Biotechnology Company, USA.

[0086] Figure 4a shows the in vivo imaging results at the wound site of scalded mice. Figure 4 b shows the immunofluorescence images at the scald wound site (green: immune cells positive for CD11b, red: exosomes labeled with PKH26, blue: nuclei labeled with DAPI). Figure 4 c shows the GSEA analysis results after RNA-seq detection of wound tissue samples. It shows that the exosome composition can significantly inhibit the immune inflammatory response in scalded mice.

[0087] From Figure 4 a, it can be seen that compared with mesenchymal stem cell exosomes dissolved in PBS, the exosome composition of the present invention can significantly promote the recruitment of exosomes to the wound site, thereby enhancing the efficacy of exosomes; from Figure 4 b, it can be seen that compared with the control group, compared with ordinary mesenchymal stem cell exosomes, the exosome composition of the present invention can significantly promote the recruitment of exosomes at the wound site, inhibit the infiltration of inflammatory cells at the wound, and relieve the inflammatory response (*P < 0.05, n = 3, data are expressed as mean ± SEM when comparing the exosome composition group with exosomes dissolved in PBS). From Figure 4 c, it can be seen that the exosome composition of the present invention can significantly inhibit the inflammatory response-related pathways and simultaneously up-regulate the wound healing-related pathways.

[0088] Example 6: The exosome composition promotes epithelial regeneration.

[0089] Human epidermal cells (HaCaT) were seeded in 12-well plates and divided into a control group and an exosome composition group. When the cell confluence reached about 80%, 500 μL of PKH26-labeled mesenchymal stem cell exosomes (1×10 8 particles / mL, dissolved in Ca 2+ -, Mg 2+ -free PBS) and 500 μL of exosome composition rich in an equal amount of PKH26-labeled mesenchymal stem cell exosomes (1×10 8 particles / mL) were added respectively. After adding 500 μL of complete medium to each group, the cells were further incubated statically for 24 h, and then immunofluorescence analysis was performed. The cell proliferation experiment and the cell scratch experiment were both carried out according to the conventional operations in this field. The HaCaT cell line was purchased from Dalian Meilun Biotechnology Co., Ltd., and the PKH26 exosome fluorescence labeling kit was purchased from Thermo Fisher Scientific.

[0090] Figure 5It is shown that the exosome composition can effectively promote the uptake of exosomes by human epidermal cells and promote cell proliferation and migration. Among them, a is the fluorescence microscopic image of the uptake of exosomes in the exosome composition by HaCaT (red: exosomes delivered to cells labeled with PKH26, blue: nuclei labeled with DAPI); b shows the cell migration of HaCaT after scratching with the exosome composition.

[0091] It can be seen from Figure 5 a that the exosome composition can promote the proliferation of HaCaT cells and their uptake of exosomes (compared with the exosomes dissolved in PBS in the exosome composition group: **P < 0.01, n = 5, data are expressed as mean ± SEM). It can be seen from Figure 5 b that the exosome composition can promote the cell migration rate of HaCaT (compared with the exosomes dissolved in PBS in the exosome composition group: ***P < 0.001, n = 3, data are expressed as mean ± SEM).

Claims

1. Use of an exosome composition in the preparation of a drug for preventing and / or inhibiting and / or alleviating and / or improving and / or treating burns, characterized in that: The exosome composition comprises fresh mesenchymal stem cell exosomes, sialic acid, dextran and albumin.

2. The use according to claim 1, characterized in that: The exosome composition comprises matrix and mesenchymal stem cell exosomes; the concentration of the mesenchymal stem cell exosomes in the exosome composition is not less than 1×10 8 particles / mL; the matrix comprises: in terms of mass percentage, animal serum albumin 1-5wt%, dextran 1-5wt%, sialic acid 0.1-0.5wt%, and no Ca 2+ Mg 2+ 89.5~97.9wt% of phosphate buffer.

3. The use according to claim 1 or 2, characterized in that: The dextran is dextran with a molecular weight of 20,000 to 100,000; preferably, the dextran is dextran 70 and / or dextran 40; more preferably, the dextran is dextran 70 with a molecular weight of 60,000 to 80,000.

4. The use according to any one of claims 1 to 3, characterized in that The animal serum albumin is serum albumin from cattle, humans, horses, donkeys, sheep, dogs or pigs, preferably bovine serum albumin.

5. The use according to any one of claims 1 to 4, characterized in that The Ca-free 2+ Mg 2+ The pH value of the phosphate buffer is 6.4 to 8.8, preferably 6.8 to 8.8, and more preferably 7.

4.

6. The use according to any one of claims 1 to 5, characterized in that The method for preparing the exosome composition comprises the following steps: Step (1): 2+ Mg 2+ The phosphate buffer is sterilized by high temperature and high pressure and then cooled to 45℃~50℃ at room temperature; Step (2): adding dextran to the phosphate buffer obtained in step (1) and vortexing until the dextran is completely dissolved; Step (3): When the solution obtained in step (2) is cooled to room temperature, sialic acid is added to the solution and stirred to mix and dissolve; Step (4): adding animal serum albumin to the solution obtained in step (3) and inverting and dissolving to obtain an exosome dilution solution; Step (5): filtering the exosome dilution obtained in step (4) using a filter membrane; Step (6): Add fresh mesenchymal stem cell exosomes to the exosome dilution filtered in step (5) to obtain the exosome composition.

7. The use according to claim 6, characterized in that: The pore size of the filter membrane is 0.2-0.3 μm, preferably 0.22 μm.

8. The use according to any one of claims 1 to 7, characterized in that The medicine comprises an effective amount of an exosome composition and a pharmaceutically acceptable carrier or excipient.

9. The use according to any one of claims 1 to 8, characterized in that The pH of the exosome composition is 5.0-6.5, preferably 5.

8.

10. The use according to any one of claims 1 to 9, characterized in that The drug is in the form of an injection preparation, an oral preparation, nasal drops, a spray preparation, an ointment preparation or a patch.

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