Pharmaceutical composition for treating nerve injury comprising milk exosomes as active ingredient

Abstract

The present invention provides a pharmaceutical composition for treating nerve injury, comprising milk exosomes as an active ingredient, which can rapidly treat nerve injury caused by trauma due to excellent nerve regeneration promoting effects without side effects.

Description

Pharmaceutical composition for treating nerve damage comprising milk exosomes as an active ingredient

[0001] The present invention relates to a novel use of milk exosomes, and more particularly, to a pharmaceutical composition for treating nerve damage comprising milk exosomes as an active ingredient.

[0002] Exosomes, small membrane-enclosed vesicles derived from endosomes, have attracted considerable attention over the past decade. Their importance has been increasingly recognized since their initial discovery in the late 1980s. These naturally occurring extracellular nanovesicles contain biologically active substances such as proteins, microRNA, mRNA, DNA, and other molecules, all of which are essential for facilitating communication between different cell types. Exosomes can be effectively isolated from various milk sources, including cow, porcine, yak, camel, human, and goat. Isolated exosomes retain their size and biological activity even when stored at -80°C due to a protective phospholipid bilayer. This layer effectively protects microRNA within the exosomes from degradation in the gastrointestinal tract and limits further absorption into the intestine.

[0003] In this regard, Korean Patent Publication No. 2021-0025568 discloses a composition for preventing, treating, or improving systemic sclerosis, comprising adipose-derived stem cell-derived exosomes as an active ingredient, and Korean Patent Publication No. 2022-0073565 discloses a composition for treating neurological diseases, comprising stem cell-derived exosomes pretreated with natural product extracts, and its use. However, there is still no known use for treating nerve damage using milk-derived exosomes as an active ingredient.

[0004] The present invention aims to solve various problems, including the above-described problems, and provides a pharmaceutical composition for treating nerve damage, comprising milk exosomes as an active ingredient, which can rapidly treat nerve damage caused by various trauma due to their excellent nerve regeneration promotion effect and no side effects. However, these tasks are exemplary and the scope of the present invention is not limited thereby.

[0005] According to one aspect of the present invention, a pharmaceutical composition for treating nerve damage is provided, comprising exosomes isolated from milk or goat milk as an active ingredient.

[0006] According to another aspect of the present invention, exosomes isolated from milk or goat milk are provided for use in treating nerve damage.

[0007] According to another aspect of the present invention, there is provided a method for treating nerve damage in a subject, comprising administering to the subject a therapeutically effective amount of exosomes isolated from milk or goat milk used for treating nerve damage.

[0008] The pharmaceutical composition for treating nerve damage comprising milk exosomes of the present invention as an active ingredient, as described above, exhibits excellent nerve regeneration-promoting effects on the central or peripheral nerves, thereby enabling rapid nerve damage recovery without side effects. Furthermore, it can be utilized as a therapeutic agent for treating various types of nerve damage. Of course, the scope of the present invention is not limited by these effects.

[0009] Figure 1 is a process diagram schematically showing the manufacturing process of a fibrin patch containing exosomes of the present invention.

[0010] Figure 2 is a schematic diagram schematically showing the research process of the present invention. The nerve defect repair method of 24 SD rats was equally divided into three groups (n=8 per group): (1) autologous nerve transplantation, (2) acellular nerve graft (ANG) using an exosome-fibrin patch, and (3) acellular nerve graft (ANG) using only a fibrin patch.

[0011] Figure 3 illustrates the in vivo surgical procedure using rats. (A) is a photograph showing preoperative preparation. The surgical site on the left hind leg was shaved and sterilized with 75% ethanol. (B) is a photograph confirming the exposure of the sciatic nerve. An incision was made along the femoral axis, and the thigh muscle was incised to expose the sciatic nerve. A 20 mm portion of the nerve was marked and resected. (C) is a photograph showing the nerve reconstruction. The nerve defect was treated using autologous graft (Group I), acellular nerve graft with an exosome-fibrin patch (Group II), or acellular nerve graft with a fibrin patch alone (Group III). The proximal and distal ends were sutured, and the exosome-containing fibrin patch or the fibrin patch alone was applied to the suture site and the nerve graft site. (D) is a photograph showing the suture after surgery. The skin incision was closed with 4-0 Vicryl, and postoperative care included antibiotics and analgesics to ensure adequate recovery.

[0012] Figure 4a is a transmission electron microscope (TEM) analysis image observing the size of milk-derived exosomes (M-EVs).

[0013] Figure 4b is a graph showing the size distribution and protein marker analysis of milk-derived exosomes (M-EVs). The size distribution of M-EVs and the results of Western blot analysis of TSG101, MFG-E8, CD63, and CD9 are shown. The size distribution of milk-derived exosomes (M-EVs) was analyzed by dynamic light scattering (DLS), and M-EVs exhibited a size distribution of approximately 100–150 nm, which is consistent with the typical size range of exosomes. Western blot analysis confirmed the presence of TSG101, CD63, CD9, which are representative exosome marker proteins, and MFG-E8, a major protein of milk-derived exosomes, in M-EVs. These results demonstrate the successful isolation and characterization of milk-derived exosomes.

[0014] Figure 4c is a graph showing the results of cell proliferation evaluation using a CCK-8 assay 24 hours after M-EV treatment in NIH-3T3 cells and SVEC4-10 cells.

[0015] Figure 4d is a representative image of the wound scratch migration assay in NIH-3T3 cells (0.8 mg / mL).

[0016] Figure 4e is a graph showing the results of quantitative analysis of M-EV in the wound closure area.

[0017] Figure 4f is a representative image of the tube formation assay in SVEC4-10 cells after treatment with 0.4 mg / mL M-EV for 6 hours.

[0018] Figure 4g is a graph showing the results of analyzing the number of branch points and the quantification of tube length 6 hours after M-EV treatment. All data are means ± SEM ( * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001) is indicated.

[0019] Figure 5a shows representative images of ankle position at final stance during video gait analysis at 8 and 16 weeks post-surgery in rats. The angle formed between the tibia and foot at final stance (hereafter, ankle angle) was measured as an indicator of sciatic nerve regeneration.

[0020] Figure 5b is a graph showing the quantitative analysis of ankle angles in the final stance between groups at 8 and 16 weeks after rat surgery, based on video gait analysis. Group I (autologous graft) showed a significantly higher angle than Group III (acellular nerve graft with fibrin patch alone) at both time points (P<0.05). Group II (acellular nerve graft with exosome-fibrin patch) showed a higher angle than Group III at 16 weeks (P<0.05), but no significant difference was observed between Groups I and II.

[0021] Figure 6a shows representative images of the tibialis anterior (TA) muscle harvested from the affected side (left) and the unaffected side (right) 16 weeks after surgery. The size and mass of the TA muscle on the affected side were relatively larger in both the autologous graft group (group I) and the acellular nerve graft group with exosome-fibrin patch application (group II) compared to the acellular nerve graft group with fibrin patch application alone (group III). Group III showed a noticeably smaller muscle size compared to groups I and II.

[0022] Figure 6b is a graph showing the quantitative analysis of TA muscle recovery expressed as a percentage of the contralateral side at 16 weeks after surgery, based on the analysis of the adductor tibialis muscle weight in rats. Group I (autologous transplantation) showed the highest recovery rate (77.64% ± 6.78%), followed by Group II (acellular nerve graft group with exosome-fibrin patch applied, 66.22% ± 17.66%) and Group III (acellular nerve graft group with fibrin patch applied alone, 39.86% ± 14.45%).

[0023] Figure 7a is a representative image of the hind limb of a rat 16 weeks after surgery, analyzing the ankle contracture angle. The affected side (left) and the normal side (right) of each experimental group are shown. Groups I (autologous graft) and II (acellular nerve graft group with exosome-fibrin patch applied) showed relatively symmetrical limb postures between the affected and normal sides, while Group III (acellular nerve graft group with fibrin patch applied alone) showed a notable difference between the affected and normal sides, reflecting a higher degree of ankle contracture in Group III.

[0024] Figure 7b is a graph showing the ankle contracture angles of rats, analyzed 16 weeks after surgery across the three groups. The average contracture angle was highest in Group I (112.50°±7.69°), followed by Group II (101.75°±8.66°) and Group III (89.75°±10.42°). A lower contracture angle reflects a greater degree of ankle contracture.

[0025] Figure 8 is a graph showing representative CMAP (Compound Muscle Action Potential) waveforms and quantitative results at 16 weeks after rat surgery as an electrophysiological evaluation. Group I (autologous graft) showed the highest CMAP amplitude, followed by Group II (acellular nerve graft group with exosome-fibrin patch applied) and Group III (acellular nerve graft group with fibrin patch applied alone). A significant difference in amplitude was observed between Groups I and III and Groups II and III (P = 0.003), but no significant difference was observed in latency.

[0026] Figure 9a shows representative toluidine blue-stained cross-sectional images of regenerated sciatic nerves in rats 16 weeks after surgery. The images represent the proximal (upper row) and distal (lower row) sections of each experimental group: autologous graft (Group I), acellular nerve graft group with exosome-fibrin patch application (Group II), and acellular nerve graft group with fibrin patch application alone (Group III). All images were taken at 100x magnification, and the scale bar represents 400 μm.

[0027] Figure 9b shows toluidine blue-stained cross-sectional images taken at 400x magnification, with the scale bar representing 100 μm: autologous transplantation (group I), acellular nerve graft group with exosome-fibrin patch applied (group II), and acellular nerve graft group with fibrin patch applied alone (group III).

[0028] Figure 9c is a graph showing the results of a quantitative analysis of the total number of axons in the proximal and distal segments of the regenerated sciatic nerve in rats 16 weeks after surgery. In the proximal segment, Group I showed a significantly higher number of axons than Group III (P = 0.018). In the distal segment, both Group I and Group II showed significantly higher axonal numbers than Group III (both comparisons, P = 0.037).

[0029] Figure 10a is a representative NF200 immunohistochemical staining analysis showing cross-sections of proximal and distal nerve segments in rats 16 weeks after surgery. The images represent the distribution and intensity of NF200 staining across three groups: autologous graft (Group I), acellular nerve graft with exosome-fibrin patch (Group II), and acellular nerve graft with fibrin patch alone (Group III). Scale bar = 400 μm.

[0030] Figure 10b is a graph showing the results of quantitative analysis of NF200 signal intensity in the proximal and distal segments. The average pixel intensity (mean ± SD) was highest in Group I, followed by Group II, and lowest in Group III in both segments. Statistical analysis revealed significantly higher intensities in Groups I and II compared to Group III (*P<0.05). Error bars represent the 95% confidence interval.

[0031] Figure 11a is a representative image showing S100β expression in the proximal and distal nerve segments of Group I (autologous graft), Group II (acellular nerve graft group with exosome-fibrin patch application), and Group III (acellular nerve graft group with fibrin patch application alone) at 16 weeks after rat surgery, according to immunohistochemical staining analysis. The images were taken at 100x magnification, and the scale bar represents 400 μm. Higher S100β signal intensities were observed in Group I and Group II compared to Group III, indicating enhanced nerve regeneration.

[0032] Figure 11b is a graph showing the results of a quantitative analysis of the mean pixel intensity of S100β immunolabeling in the proximal and distal sections. In the proximal section, Groups I and II showed significantly higher mean pixel intensities than Group III (P<0.05). Similarly, in the distal section, Groups I and II showed significantly higher intensities than Group III (P<0.05). Data are expressed as mean ± standard deviation, and error bars represent the 95% confidence interval.

[0033] Definition of terms:

[0034] The term "exosome" as used herein refers to nano-sized cell-derived vesicles that may be present in all biological fluids, including blood, urine, and cell culture media. Exosomes are known to be between 30 and 100 nm in size and are released from cells when multivesicular bodies fuse with the cell membrane or are released directly through the cell membrane. Exosomes are known to play important roles in various processes, such as coagulation, intercellular signaling, and metabolic waste management. In this regard, the term "milk exosome" as used herein refers to exosomes derived from mammalian milk, such as commercial milk or colostrum. Similarly, "colostrum exosome" refers to exosomes derived from milk secreted by mother cows immediately after giving birth to their young.

[0035] The term "milk" used in this document refers to cow's milk, but milk from other mammals, such as horses, sheep, goats, and camels, can also be considered functionally equivalent. However, considering availability and price, commercially available cow's milk is the most preferable. Goat's milk, produced and distributed from goats, can also be an excellent substitute. Goat's milk, in particular, is similar in composition to breast milk, making it easily digestible and nutritious, and is considered a premium milk. Horse's milk, while less common, is also used as a cow's milk substitute by nomadic peoples in Central Asia. Meanwhile, milk secreted from the end of pregnancy to the first few days after delivery is called "colostrum." Unlike regular milk, colostrum contains more nutrients and antibodies necessary for survival and growth. It also contains higher levels of antioxidants, such as lactoferrin and hemopexin, than regular milk, making it a preferred choice for health and nutritional reasons.

[0036] The term "nerve damage" as used in this document refers to damage to peripheral nerves caused by physical pressure or shock, rather than damage caused by disease.

[0037] Detailed description of the invention:

[0038] According to one aspect of the present invention, a pharmaceutical composition for treating nerve damage is provided, comprising exosomes isolated from milk or goat milk as an active ingredient.

[0039] In the above pharmaceutical composition, the milk or goat milk may be raw milk, commercially available milk, goat milk or colostrum.

[0040] In the pharmaceutical composition, the nerve damage may be damage to a peripheral nerve, and the peripheral nerve damage may be trauma, damage to a motor nerve, or damage to a sensory nerve, and the trauma may be selected from the group consisting of burns, bruises, lacerations, puncture wounds, cuts, abrasions, bedsores, and wounds.

[0041] In the pharmaceutical composition described above, the exosomes can promote the regeneration of nerve cells themselves or enhance the proliferation or function of Schwann cells surrounding the axons of nerve cells. It has been confirmed that the exosomes can contribute to the recovery of nerve damage by increasing the number of nerve cell axons. However, the mechanism of this effect may involve the promotion of nerve cell regeneration or the enhancement of the proliferation and function of Schwann cells surrounding the nerve cell axons.

[0042] In the above pharmaceutical composition, the exosome may have a size of 50 to 250 nm.

[0043] The pharmaceutical composition may be administered in a dosage form selected from the group consisting of microinjection into neural tissue, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and intranasal administration. More specifically, the pharmaceutical composition of the present invention may be formulated in the form of a transdermal patch, including, but not limited to, microneedles.

[0044] According to another aspect of the present invention, exosomes isolated from milk or goat milk are provided for use in treating nerve damage.

[0045] According to another aspect of the present invention, there is provided a use of exosomes isolated from milk or goat milk for the manufacture of a medicament for preventing and treating nerve damage.

[0046] According to another aspect of the present invention, there is provided a method for treating nerve damage in a subject, comprising administering to the subject a therapeutically effective amount of exosomes isolated from milk or goat milk used for treating nerve damage.

[0047] According to another aspect of the present invention, a method for treating nerve damage in a subject is provided, comprising administering to the subject a therapeutically effective amount of exosomes isolated from milk or goat milk.

[0048] In the present invention, the term "pharmaceutical composition" means a composition manufactured for the purpose of preventing or treating nerve damage, and may be formulated and used in various forms according to conventional methods. For example, it may be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and may be formulated and used in the form of external skin preparations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas, suppositories, and sterile injection solutions.

[0049] The pharmaceutical composition of the present invention can be prepared in any dosage form commonly manufactured in the art (e.g., see [Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), and the form of the preparation is not particularly limited, but is preferably an external preparation. The external preparation of the present invention can include conventional forms of external preparations such as sheets, liquid applications, sprays, lotions, creams, patches, powders, penetrating pads, sprays, gels including hydrogels, pastes, liniments, ointments, aerosols, powders, suspensions, and transdermal absorption agents. These dosage forms are described in the document [Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), which is a prescription book generally known to all pharmaceutical chemistry.

[0050] The pharmaceutical composition of the present invention may vary depending on the type of the patient's affected area, application site, number of treatments, treatment time, formulation, patient's condition, type of adjuvant, etc. The dosage is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The above daily dose may be administered once a day, or divided into 2 to 3 times a day at appropriate intervals, or intermittently at intervals of several days.

[0051] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration are contemplated, including, for example, oral, parenteral, injection, needle-free device, inhalation spray, topical, rectally, nasally, buccally, vaginally, or via implanted reservoirs. Oral administration, injection, needle-free device administration, or transdermal administration are preferred. The term "parenteral" as used herein includes transdermal administration and subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0052] The intrathecal injection is a method of injecting drugs or biopharmaceuticals through a needle into the spinal canal and subarachnoid space. The spinal canal is formed by the continuous connection of vertebral foramina located in each vertebra. The spinal cord, meninges, blood vessels, and some peripheral nerves are located within the spinal canal and protect them. In addition, because the intrathecal injection is injected into the subarachnoid space, the test substance reaches the cerebrospinal fluid (CSF) between the arachnoid and pia mater. In mice, the injection is made between L5 and L6 of the lumbar vertebrae. The lumbar vertebrae are six vertebrae extending downward from the thoracic vertebrae. They are the largest vertebrae because they support body weight and have greater mobility than other vertebrae. The sciatic nerve is the largest single nerve that branches off from the lumbosacral plexus, which is formed by the union of the lumbar and sacral nerves. The dorsal root ganglia (DRG) are the areas where the cell bodies of axons of the posterior roots of the spine are gathered. The dorsal root is the portion of the spinal nerve that enters the spinal cord from the back and contains only sensory fibers. The cell bodies of these fibers are distributed only on the outside of the nerve. The ventral root is the portion of the spinal nerve located ventrally and contains motor fibers that originate in the spinal cord. Intrathecal injections are also called subarachnoid space injections because they are injected between the L5 and L6 lumbar vertebrae. This is because the spinal cord ends at the L2 lumbar vertebrae in mice, and L4 and L5 are areas where only cerebrospinal fluid exists without a spinal cord. The spinal cord is protected by the spine and lumbar vertebrae, and sensory and motor neurons are gathered within the spinal cord.

[0053] The pharmaceutical composition of the present invention can be applied to a nerve injury site by covering the affected area with a fibrin scaffold. The fibrin scaffold may be a biodegradable polymer scaffold, and the biodegradable polymer may include PLGA {poly(lactic-co-glycolic) acid), PGA {poly(glycolic acid)}, PLA {poly(lactic acid)}, collagen, fibrin, hyaluronic acid, or decellularized ECM (extracellular matrix). In addition to the biodegradable polymer scaffold, a biodegradable polymer hydrogel may also be used as a drug delivery vehicle.

[0054] The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. In addition, solid or liquid formulation additives may be used in the manufacture of the pharmaceutical composition. The formulation additives may be either organic or inorganic. Examples of excipients include lactose, sucrose, sucrose, glucose, cornstarch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, dextrin, and pectin. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any colorant that is generally approved for addition to pharmaceuticals can be used. These tablets and granules can be appropriately coated with sugar, gelatin, or other agents as needed. In addition, preservatives, antioxidants, and the like can be added as needed. In addition, when the pharmaceutical composition is a drug, it can additionally contain one or more selected from fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, or preservatives. Meanwhile, the formulation of the pharmaceutical composition of the present invention may be in a desirable form depending on the method of use, and in particular, it is preferable to formulate it by adopting a method known in the art so as to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal.Examples of specific dosage forms include: PLASTERS, GRANULES, LOTIONS, LINIMENTS, LEMONADES, POWDERS, SYRUPS, LIQUIDS AND SOLUTIONS, AEROSOLS, EXTRACTS, ELIXIRS, FLUIDEXTRACTS, EMULSIONS, SUSPENSIONS, DECOCTIONS, INFUSIONS, TABLETS, SUPPOSITORIES, INJECTIONS, SPIRITS, CATAPLSMA, CAPSULES, TROCHES, TINCTURES, PASTES, PILLS, SOFT Or it may be any one selected from hard gelatin capsules. The pharmaceutical composition of the present invention may further include additional ingredients commonly used, such as stabilizers, solubilizers, and flavoring agents, and carriers.

[0055] In the present invention, the term "administration" refers to introducing the pharmaceutical composition of the present invention into a subject by any appropriate method, and the administration route may be administered through various routes, such as oral or parenteral, as long as it can reach the target tissue. The pharmaceutical composition may be appropriately administered to a subject according to a conventional method, administration route, and dosage used in the art according to the purpose or need. Examples of the administration route include oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and parenteral injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, an appropriate dosage and frequency of administration may be selected according to a method known in the art, and the actual amount of the pharmaceutical composition of the present invention to be administered and the frequency of administration may be appropriately determined by various factors, such as the type of symptom to be treated, administration route, sex, health condition, diet, age and weight of the subject, and severity of the disease.

[0056] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art. Administration may be administered once a day or divided into several doses.

[0057] The effective dose of the pharmaceutical composition may be adjusted according to various factors including the type of disease, severity of the disease, types and contents of the active ingredient and other ingredients contained in the composition, type of formulation, and the patient's age, weight, general health, sex, and diet, administration time, administration route, and excretion rate of the composition, treatment period, and concurrently used drugs. For example, in the case of adults, a dose of 10 to 4000 mg / day may be administered once or several times a day at regular intervals, depending on the judgment of a doctor or pharmacist.

[0058] Meanwhile, the above composition may also be applied as a quasi-drug composition containing milk exosomes as an active ingredient. The quasi-drug composition may be used together with other quasi-drugs or quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredients may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). The quasi-drug composition may be, but is not limited to, a disinfectant, shower foam, gargle, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment, or filter filler.

[0059] Exosomes are vesicles that are tens to hundreds of nanometers in size and are made of a double phospholipid membrane that has the same structure as the cell membrane. Inside, they contain proteins and nucleic acids (mRNA, miRNA, etc.), which are called exosome cargoes. These exosome cargoes include a wide range of signaling factors, and these signaling factors are known to be cell type-specific and regulated differently depending on the environment of the secreting cell. Exosomes are intercellular signaling mediators secreted by cells, and various cell signals transmitted through them are known to regulate cell behaviors, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Exosomes are known to contain specific genetic material and bioactive factors depending on the nature and state of the cell from which they are derived. Because exosomes are fundamentally derived from cells, they are biocompatible, unlike other nanoparticles, and can be loaded or labeled with drugs or biologically active ingredients inside or on their surface. Therefore, attempts are continuously being made to use them as drug delivery vehicles or raw materials for cosmetics and pharmaceuticals.

[0060] Bovine colostrum is the first milk produced by cows immediately after birth. Compared to mature milk, colostrum contains higher protein concentrations of extracellular vesicles (EVs) and is richer in miRNAs, particularly those involved in immune, developmental, and tissue repair. While colostrum-derived exosomes have not yet been reported to participate in neural regeneration, previous studies have shown that they can contribute to wound healing by increasing microRNA RNA-21 (miR-21). miR-21 creates a favorable environment for neural repair by reducing inflammation and oxidative stress, inhibits apoptosis, and regulates key signaling pathways essential for cell survival, growth, and differentiation. Furthermore, miR-21 can promote axonal regeneration by targeting genes such as SPRY2. It has been reported to be positively correlated with GAP43 and DNMT3A10, proteins associated with axonal growth, neural development, and plasticity. Based on these results, the inventors hypothesized that bovine colostrum-derived exosomes can contribute to peripheral nerve regeneration by regulating the expression of microRNA-21, and in the present invention, the potential of nerve regeneration was confirmed using bovine colostrum-derived exosomes (BCE) through an animal model.

[0061] Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, but can be implemented in various different forms. The following examples are provided to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention.

[0062] Example 1: Isolation of exosomes

[0063] The present inventors have designed a new exosome isolation method by modifying a previously reported method for isolating exosomes from milk (Yamauchi et al., Drug Dev. Ind. Pharm. 45(3): 359-364, 2019) (Patent No. 10-2523065). The method reported by Yamauchi et al. used a process of removing fat and cells by centrifuging raw milk at 2,000×g at 4℃ for 20 minutes (first centrifugation), diluting it with an equal volume of distilled water (distilled water dilution), adjusting the pH to 4.6 by adding 6 N HCl to remove casein by isoelectric precipitation (isoelectric precipitation), centrifuging it at 5,000×g at room temperature for 20 minutes (second centrifugation), and sequentially filtering it using 1.0, 0.45, and 0.2 μm filters (sequential filtration). The present inventors changed the first centrifugation process in the above method to performing it at 5,000×g at 4℃ for 30 minutes and filtering it using a 40 μm filter mesh (strainer, Falcon) before diluting it with distilled water. ® , Corning, USA) was introduced to further remove fat and cells, and a single filtration process using only a 0.2 μm filter was used without sequential filtration after secondary centrifugation.

[0064] Example 2: Dynamic Light Scattering Analysis

[0065] The size distribution of EVs was measured using dynamic light scattering (DLS) (Zetasizer Nano; Malvern Instruments, UK). EV samples were diluted in phosphate-buffered saline (PBS) and analyzed at a 173° backscattering angle in disposable cuvettes (Kartell Labware, Italy). Data were processed using the software provided with the instrument, and the particle size distribution was expressed as a percentage of the number of particles in the sample.

[0066] Example 3: Transmission electron microscopy (TEM) analysis

[0067] The exosomes isolated in Example 1 were photographed using a transmission electron microscope (Tecnal F20 TEM, FEI Company, USA).

[0068] Example 4: Immunoblotting

[0069] Western blot analysis was performed to confirm the presence of extracellular vesicle (EV)-specific markers. EV protein samples were prepared by lysing EVs in RIPA buffer (Thermo Fisher Scientific, USA) containing a cocktail of protease and phosphatase inhibitors (Cell Signaling Technology, USA) to prevent protein degradation. The protein concentration in the lysate was quantified using the bicinchoninic acid (BCA) assay, a colorimetric method for measuring protein concentration. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to properly separate proteins by molecular weight. Proteins were then transferred to nitrocellulose membranes (Bio-Rad, USA) using the Turbo transfer system, a fast and efficient membrane transfer method. After transfer, the membranes were blocked in 5% skim milk prepared in Tris-buffered saline (TBS-T) containing 0.1% Tween 20 for 1 h at room temperature. Blocking was performed to minimize nonspecific binding of antibodies. The membranes were then incubated overnight at 4°C with primary antibodies targeting EV-specific markers. The following primary antibodies were used for detection: TSG101 (Abcam, UK, ab83, dilution 1:1000), a well-known EV marker associated with the ESCRT pathway; CD63 (Invitrogen, USA, MA1-19301, dilution 1:2000), a tetraspanin commonly found on EV membranes; CD9 (Invitrogen, USA, MA1-19301, dilution 1:2000), another tetraspanin frequently used to identify EVs; MFG-E8 (R&D Systems, USA, AF2805, dilution 1:1000), a marker frequently associated with milk-derived EVs.

[0070] After incubation with primary antibody, the membrane was thoroughly washed to remove unbound antibody. It was then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody that specifically binds to the primary antibody for 1 hour at room temperature. After additional washing steps to remove excess secondary antibody, protein bands were visualized using chemiluminescence detection using the iBright imaging system (Invitrogen, USA).

[0071] Example 5: Cell culture

[0072] NIH-3T3 (mouse fibroblast cell line; ATCC, USA) and SVEC4-10 (mouse endothelial cell line; ATCC, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% antibiotic-antimycotic solution (Gibco, USA). Cells were maintained in a humidified incubator at 37°C with 5% CO₂. For consistency and experimental reliability, SVEC4-10 cells were used only up to passage 7.

[0073] Example 6: Cell proliferation assay

[0074] The effects of milk-derived extracellular vesicles (Milk-EVs) on cell proliferation were evaluated using Cell Counting Kit 8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). NIH-3T3 fibroblasts and SVEC4-10 endothelial cells were seeded at a density of 6 × 10³ cells per well in 96-well plates and allowed to attach in complete medium for 12 h. After incubation, the medium was removed, and the cells were washed with Dulbecco's phosphate-buffered saline (DPBS). The cells were then treated with various concentrations of Milk-EVs (0, 0.1, 0.4, 0.8, and 1.2 mg / mL) in serum-free medium for 24 h. After 24 h of treatment, 20 μL of CCK-8 solution (10% of the total medium volume) was added to each well, and the plates were incubated at 37°C for an additional 2 h. Absorbance was measured at 450 nm using a SpectraMax 34 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell proliferation rates were calculated and expressed as a percentage of the untreated control. All experiments were performed in triplicate, and the results are expressed as the mean ± SEM.

[0075] Example 7: Wound Scratch Migration Analysis

[0076] To evaluate the effect of milk-derived extracellular vesicles (M-EVs) on cell migration, a wound scratch assay was performed using NIH-3T3 fibroblasts. NIH-3T3 cells were seeded in a 6-well plate at a density of 2 × 10 per well. 5 Cells were seeded at a density of 100 μg / mL and cultured overnight at 37°C in a 5% CO₂ incubator to form a confluent monolayer. Once the monolayer was formed, a straight scratch was made using a sterile scratcher (SPLScar; SPL Life Sciences, Korea), and the cells were carefully washed with phosphate-buffered saline (PBS) to remove debris. M-EVs were cultured in serum-free medium at a concentration of 100 μg / mL (5 × 107 The cells were added to the wells at a concentration of 10 particles / mL, and the cells were cultured for 24 h. Images of the wound area were taken at 0 h and 24 h using a CK40 culture microscope (Olympus, Japan). The degree of wound closure was quantified by measuring the closed wound area using ImageJ software (NIH).

[0077] Example 8: Tube formation assay

[0078] The angiogenic effect of M-EVs was evaluated using a tube formation assay using SVEC4-10 endothelial cells. Matrigel (50 μL; Corning Matrigel Matrix, Corning, USA) was added to each well of a 96-well plate and polymerized at 37°C for 30 min. Then, SVEC4-10 cells were seeded on the solidified Matrigel at a density of 2 × 10⁴ cells per well and treated with 100 μg / mL (5 × 10 7 Cells were treated with M-EV at a concentration of 10 particles / mL. After culturing the cells for 8 h, images of tube-like structures were captured using a CK40 culture microscope (Olympus, Japan). Tube formation was analyzed by quantifying the total tube length and number of branch points using ImageJ software (NIH).

[0079] Example 9: Characterization of exosomes

[0080] The size distribution of exosomes was measured using dynamic light scattering (DLS) spectrometry (Zetasizer Nano, Malvern Instruments, UK). Exosome samples were diluted in phosphate-buffered saline (PBS), placed in disposable cuvettes (Kartell Labware, Italy), and analyzed under a 173° backscatter angle. Data were processed using the software provided by the instrument, and the particle size distribution within the sample was expressed as a particle number ratio (%). Western blot analysis was also performed to confirm the presence of exosome (EV)-specific markers. EV protein samples were prepared by lysing with RIPA buffer (Thermo Fisher Scientific, USA) and a protein / phosphatase inhibitor mixture (Cell Signaling Technology, USA). Protein concentration was quantified using the bicinchoninic acid (BCA) assay, a colorimetric assay for measuring protein concentration. The prepared proteins were separated by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) to ensure good resolution of the separated proteins according to molecular weight. The proteins were then transferred to nitrocellulose membranes using a Turbo Transfer System (Bio-Rad, USA). The transferred membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBS-T) containing 5% skim milk for 1 h at room temperature to minimize nonspecific antibody binding. The membranes were then reacted with primary antibodies targeting EV-specific markers overnight at 4°C.The primary antibodies used were as follows: TSG101 (Abcam, UK, ab83, dilution 1:1000), CD63 (Invitrogen, USA, MA1-19301, dilution 1:2000), CD9 (Invitrogen, USA, MA1-19301, dilution 1:2000), MFG-E8 (R&D Systems, USA, AF2805, dilution 1:1000). After the primary antibody reaction, the membrane was thoroughly washed to remove unbound antibodies and then reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. After additional washing to remove excess secondary antibodies, protein bands were visualized using chemiluminescence detection with an iBright imaging system (Invitrogen, USA).

[0081] Example 10: Preparation of a fibrin patch containing exosomes

[0082] Exosomes extracted from colostrum were provided in a stable lyophilized form at room temperature, prepared as follows. Exosomes were initially dissolved in PBS buffer at a concentration of 20 μg / mL (1 × 10 7 The EVs were suspended at a concentration of 10 particles / mL. A total of 1 mg of EV was placed in a 1.5 mL tube, completely frozen in liquid nitrogen, and then dehydrated in a freeze dryer (Ilshin Biobase, Korea) with the lid open at -85°C and 58 mTorr or less for 2 days. The lyophilized form maintained the specific exosome properties described previously. The carrier used for the exosomes was a commercially available fibrin sealant (Tisseel, Baxter, Deerfield, Illinois, USA). The lyophilized exosomes were mixed with Tisseel to prepare a fibrin patch containing exosomes (Kim H, et al., Adv. Healthc. Mater. 11(6):e2102027, 2022).

[0083] Specifically, lyophilized exosomes were dissolved in 1 ml of phosphate buffered saline (PBS) and 5×10 8 An exosome solution with a concentration of 100% was prepared. The exosome mixture solution (2 × 10 8 400 μl of a dog vesicle and 600 μl of a fibrin-dissolving inhibitor solution were added to the silane protein concentrate to form fibrinogen. Then, 1000 μl of calcium chloride solution was added to prepare a thrombin solution. Finally, the two reagents were combined to obtain an exosome concentration of 20% (1 × 10 8 Fibrin patches containing exosomes (100 vesicles / ml) were produced (Fig. 1).

[0084] Example 11: Animal Model and Experimental Design

[0085] All experimental protocols were approved by the Institutional Animal Care and Use Committees (IACUC) of Seoul Asan Medical Center and Ulsan University College of Medicine (Approval No. 2023-30-298). A total of 32 7-week-old Sprague-Dawley rats (weighing 250–300 g) were purchased from JA BIO (Suwon, Gyeonggi-do). Eight rats were used for sciatic nerve harvesting to generate acellular nerves, and the remaining 24 rats were used for in vivo experiments. All animal care and handling procedures were performed in strict compliance with the IACUC guidelines of the Asan Institute for Life Sciences.

[0086] Example 12: Decellularization protocol

[0087] The present inventors collected peripheral nerve tissue from eight 7-week-old Sprague-Dawley rats (weighing 250–300 g) purchased from JA BIO (Suwon, Gyeonggi-do). A total of 16 acellular nerves were obtained from the sciatic nerves obtained from both lower extremities of the rats through a decellularization process. The decellularization process utilized the previously reported supercritical CO2 technique (Choi SJ, et al., Sci. Rep. 14(1):23696. 2024). The decellularization process removes cells and cellular material from nerve tissue while preserving the extracellular matrix. This process removes substances such as DNA and RNA that can induce immune responses, while preserving the mechanical and physical properties of the tissue. Therefore, decellularized acellular nerves can be used as artificial nerves. While these acellular nerves can serve as structural support, they are difficult to induce nerve connections and functional recovery, and they do not directly support angiogenesis, which is essential for nerve tissue regeneration and survival. In this experiment, histological and immunohistochemical examinations and functional evaluations were performed on autologous nerves, acellular nerves, and exosomes applied to acellular nerves to determine the effect of exosomes on the regeneration and functional recovery of acellular nerves.

[0088] Specifically, rats were anesthetized, and both hind legs were shaved and sterilized with 75% ethanol. An incision was made along the lateral aspect of the femur to expose the sciatic nerve, and the skin and fascia were incised. A portion of the sciatic nerve longer than 2 cm was carefully resected to ensure sufficient length for subsequent processing. After careful removal of surrounding fat and muscle tissue, the harvested nerve was washed in sterile 1× phosphate-buffered saline (PBS, Cat# L0615-500, BioWest, USA) for 30 min. Supercritical CO2 extraction was performed using equipment consisting of a 0.3 L cylindrical vessel (DOF Inc., Hwaseong, Gyeonggi-do). The nerve tissue was immersed in 1× PBS containing 1% antibiotics overnight on a rocker set at 30 rpm. The supercritical CO2 process was performed in a controlled environment using ethanol (30–70%, Cat# 1.00983.5000, Sigma Aldrich, USA) as a cosolvent. The CO2 flow rate was controlled by a gas flow meter, and a pressure of 200–300 bar was applied for 3 h. After treatment, the nerves were washed twice with 1× PBS for 15 min each under the same rocking conditions (Fig. 2).

[0089] Example 13: In vivo surgical procedure

[0090] Twenty-four male Sprague-Dawley rats were randomly assigned to three experimental groups. In the autograft group (group I, n=8), the resected sciatic nerve segment underwent nerve suturing along the original direction of the proximal and distal transection. Group II (n=8) received an exosome-fibrin patch to promote nerve regeneration, while group III (n=8) received a fibrin patch alone without exosomes applied to the nerve graft site. All rats were anesthetized using 3% to 4% isoflurane administered via a nasal mask connected to an inhalation anesthesia device. The surgical site on the left hind limb was shaved and sterilized with 75% ethanol. An incision was made along the femoral axis, and the femoral muscle was incised to expose the sciatic nerve. The sciatic nerve was transected near the obturator muscle in the mid-thigh, and a 20 mm segment of the nerve was resected. In Group I, the resected nerve segment was resutured in the same direction as the original nerve course after proximal and distal amputations. In Groups II and III, the prepared decellularized nerve was cut into 20 mm lengths and sutured to the nerve defect site. To ensure coverage, fibrin containing exosomes (Group II) or fibrin alone (Group III) was applied along the entire length of the graft, including the proximal and distal junctional sites. The skin incision was closed with 4-0 Vicryl (Ethicon, Somerville, NY, USA), and the animals were administered antibiotics and analgesics postoperatively and monitored for signs of infection, wound healing complications, or significant weight loss (Figure 3).

[0091] Example 14: Video Walking Ankle Angle Analysis

[0092] We assessed sciatic nerve recovery by measuring ankle angles during the toe-off phase of the rats, as this phase is closely related to functional nerve regeneration. Measurements were performed 16 weeks post-surgery using a custom-made walking apparatus consisting of an acrylic platform measuring 100 cm in length, 10 cm in width, and 20 cm in height. A black chamber was attached to one end to encourage forward movement. A DSLR video camera (Nikon D810, Japan) was positioned laterally to capture sagittal plane movements. Before recording, the rats were trained to walk along the track to familiarize themselves with the apparatus. The hind limbs were shaved, and anatomical landmarks, including the lower third of the tibia, knee joint, ankle joint, and fifth metatarsal head, were marked with black ink to consistently identify movement segments. Multiple video recordings were performed to ensure reproducibility, and the walking environment was maintained undisturbed. The toe-off phase was defined as the moment of maximal plantar flexion of the experimental ankle joint. Ankle angle was determined by measuring the angle formed between a line connecting the knee and ankle joint and a line connecting the ankle joint and the head of the fifth metatarsal. The angle was expressed in degrees and analyzed using ImageJ software (version 1.54; National Institutes of Health, Bethesda, Maryland, USA). This approach provided a quantitative assessment of functional recovery in the experimental group.

[0093] Example 15: Electrophysiology

[0094] Compound muscle action potentials (CMAPs) were recorded 16 weeks after surgery to assess functional recovery of the sciatic nerve and tibialis anterior in all experimental rats (N=24). The rats were anesthetized using 3%–4% isoflurane administered via a nasal mask connected to an inhalation anesthesia device. Stainless steel microelectrodes capable of supporting a sampling rate of up to 48 kHz per channel were placed in the tibialis anterior. The sciatic nerve on the operated side was surgically exposed, and electrical stimulation was delivered proximal to the nerve using a tandem hook-shaped electrode (9013S0022, Natus Ultrapro S100, USA). The acquired signals were amplified by a multichannel amplifier with a frequency range of 1–10,000 Hz (SKU#9031E0172, Natus Ultrapro S100, USA) and digitized at a rate of 2 kHz using the Natus HL7 Solutions system integrated with Natus Elite software (Natus Ultrapro S100, USA). Negative deflection (depolarization) was represented by an upward-facing waveform, and the average waveform was calculated from 10 consecutive traces. CMAP amplitude was determined as the difference between the peak voltage and the baseline. Duration was defined as the time interval between the two points where the CMAP waveform crossed the baseline during the rising and falling phases. Latency was measured as the time between the stimulus artifact and the point where the CMAP waveform crossed the baseline during the rising phase.

[0095] Example 16: Tibialis anterior muscle assessment

[0096] Sixteen weeks after surgery, the adductor tibialis muscle was excised from the surrounding tissue and weighed using an electronic scale. The weight of the tibialis anterior muscle in each group was photographed and recorded. To compare the groups, the ratio of the adductor tibialis muscle weight in the surgical area was calculated and compared with the same muscle weight in the non-surgical area.

[0097] Example 17: Measurement of ankle contracture angle

[0098] Ankle contracture angles were assessed 16 weeks postoperatively according to a previously established protocol. Measurements were performed during maximum ankle plantar flexion, focusing on the angle formed between the anterior border of the tibia and the dorsal surface of the foot. To provide a baseline, contracture angles were also measured on the contralateral, non-implanted side. Results were analyzed to assess the degree of recovery of neurological function and muscle performance in the implanted group. All measurements were captured under standardized conditions with a full-frame DSLR camera (Nikon D810, Japan), and angles were quantified using ImageJ software.

[0099] Example 18: Toluidine blue staining analysis

[0100] Transplanted peripheral nerves were harvested and divided into proximal and distal halves for staining and analysis. Each half was stained with toluidine blue, and the number of myelin fibers was counted in all treated rats. The specimens were fixed overnight in 4% paraformaldehyde at 4°C and washed with tap water for 8 hours. Dehydration was performed using graded ethanol solutions (50%, 70%, 90%, and 100%) at room temperature for 1 hour each, followed by overnight treatment in 100% xylene at room temperature. The specimens were then embedded in paraffin, sectioned into 4-μm-thick slices using a scalpel and microtome, and stained with 1% toluidine blue. Stained images of the proximal and distal halves were captured at 100x and 400x magnification using a light microscope (EVOS FL Auto, Life Technologies). The total number of axons in each nerve fiber was calculated in each section using ImageJ (National Institutes of Health) software.

[0101] Example 19: Immunohistochemical analysis

[0102] Transplanted nerves were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 μm-thick slices. After deparaffinization and rehydration with ethanol and xylene, antigen retrieval was performed in sodium citrate buffer (pH 6.0) using a microwave. The sections were cooled to room temperature, washed in TBS-T, and treated with 3% hydrogen peroxide to block endogenous peroxidase activity. A hydrophobic pen was used to create a barrier around the samples for staining. For immunohistochemical evaluation, the sections were incubated overnight at 4°C with primary antibodies against S100β (1:100 dilution, Sigma) and NF200 (1:50 dilution, Sigma). After washing, sections were treated with diaminobenzidine (DAB) solution for signal generation, counterstained with Mayer's hematoxylin for 1 minute, and washed with tap water for 3 minutes before mounting with coverslips. DAB signals were analyzed using ImageJ software with a color deconvolution plugin. To ensure consistency across all samples, intensity thresholds were determined based on the staining patterns observed in the positive control. The mean pixel intensity was calculated within five randomly selected regions of interest per section to provide representative data. Positive controls were obtained from healthy sciatic nerve sections to verify the specificity of S100β and NF200 staining. Intensity data were expressed as arbitrary units (AU) of intensity.

[0103] Example 20: Statistical Analysis

[0104] Statistical analysis was performed using SPSS version 26 (IBM, USA). Appropriate statistical methods were selected based on the data distribution. For nonparametric data, the Kruskal-Wallis test was used, followed by pairwise comparisons using the Bonferroni correction. For normally distributed data, a one-way analysis of variance (ANOVA) was performed, followed by the Bonferroni post hoc test for multiple comparisons. Results are expressed as the mean ± standard deviation (mean ± SD). A P value less than 0.05 was considered statistically significant.

[0105] Experimental Example 1: Exosome Characterization

[0106] To characterize exosomes in detail, we utilized dynamic light scattering (DLS). DLS is a non-invasive and well-established technique for measuring the size distribution of nanoparticles in suspension. It calculates particle size by analyzing changes in the intensity of scattered light based on the Brownian motion of the particles. Furthermore, we imaged milk-derived exosomes using a transmission electron microscope. As shown in Figures 4a and 4b, milk-derived exosomes were confirmed to be nanosized particles with an average particle diameter of 68.05 nm and a particle size distribution ranging from 30 to 100 nm. This is consistent with the previously reported characteristics of milk-derived exosomes. Furthermore, to verify the successful extraction of EVs, we performed Western blot analysis to detect specific marker proteins, including TSG101, CD63, and CD9, which are associated with luminal vesicles and EV biosynthesis. Western blot is a method for detecting the presence and expression level of specific proteins in protein samples, detecting target proteins through antigen-antibody reactions. These proteins are widely used as markers of exosomes and play a crucial role in confirming the presence of exosomes. Furthermore, milk fat globule EGF factor 8 (MFG-E8), a major protein in milk-derived exosomes, was also clearly identified. MFG-E8 is a protein abundant in milk-derived exosomes and is closely related to exosome function. These results clearly demonstrate the effective isolation and characterization of exosomes.

[0107] Experimental Example 2: Activation of cell proliferation by milk EVs

[0108] To evaluate the effects of milk-derived extracellular vesicles (M-EVs) on cellular functions related to wound healing, a series of in vitro assays focusing on cell proliferation, migration, and angiogenesis were performed. The effects of milk-derived extracellular vesicles (M-EVs) on cellular functions were assessed through a series of in vitro experiments. Proliferation of NIH-3T3 fibroblasts and SVEC4-10 endothelial cells was assessed using the Cell Counting Kit-8 (CCK-8) assay. As a result, treatment with M-EVs enhanced cell proliferation in a dose-dependent manner in both cell types. In particular, proliferation was significantly increased at concentrations of 0.8 mg / mL and 1.2 mg / mL, indicating that higher concentrations of M-EVs effectively promoted cell proliferation (Fig. 4c).

[0109] Experimental Example 3: Analysis of wound scratch movement and tube formation

[0110] To evaluate the effect of milk-derived extracellular vesicles (M-EVs) on fibroblast migration, a wound-wounding assay was performed using NIH-3T3 cells. Results showed that cell migration was significantly enhanced in the M-EV-treated group. Specifically, treatment with colostrum-derived EVs (Colos EVs) resulted in approximately a fourfold increase in wound closure compared to the control group (Figures 4d and 4e). Furthermore, the angiogenic potential of M-EVs was evaluated through a tube formation assay performed on endothelial cells, focusing on the number of branch points and total tube length. As a result, we observed that M-EVs promoted in vitro angiogenesis compared to the control group (Figures 4f and 4g).

[0111] Experimental Example 2: Video Walking Angle Analysis

[0112] We evaluated sciatic nerve regeneration by measuring the ankle angle in the final posture at 8 and 16 weeks after surgery in rats. As a result, at 8 weeks, the mean ankle angle of Group I (autologous graft) was 54.25°±4.07°, the mean ankle angle of Group II (acellular nerve graft with exosome-fibrin patch) was 52.38°±4.07°, and the mean ankle angle of Group III (acellular nerve graft with fibrin patch) was 43.38°±4.07°. A significant difference was found between Groups I and III (P= 0.043), but no significant difference was found between Groups I and II or between Groups II and III. Additionally, at week 16, the mean ankle angles were 84.25°±5.14° in Group I, 78.13°±5.14° in Group II, and 62.00°±5.14° in Group III. Group I showed a significantly higher angle than Group III (P= 0.001), and Group II also showed a significantly higher angle than Group III (P= 0.015). However, no significant differences were observed between Groups I and II at this time point (Figures 5a and 5b).

[0113] Experimental Example 3: Analysis of the weight of the tibialis anterior muscle

[0114] The recovery of TA muscle weight, expressed as a percentage of the contralateral side, was 77.64% ± 6.78% in Group I, 66.22% ± 17.66% in Group II, and 39.86% ± 14.45% in Group III. Statistical analysis showed that Group I had a significantly higher recovery rate than Group III (P = 0.000). Similarly, Group II had a significantly higher recovery rate than Group III (P = 0.003). However, no significant difference was observed between Groups I and II (P = 0.334) (Figs. 6a and 6b).

[0115] Experimental Example 4: Ankle construction angle

[0116] To assess functional recovery, we evaluated the ankle contracture angle at 16 weeks after surgery in rats. The mean angle was 112.50°±7.69° in Group I (autograft), 101.75°±8.66° in Group II, and 89.75°±10.42° in Group III. Statistical analysis revealed significant differences between the groups. Group I showed a significantly larger angle than Group III (P<0.001), and Group II showed a larger angle than Group III (P=0.043) (Figs. 7a and 7b).

[0117] Experimental Example 5: Electrophysiological Analysis

[0118] CMAP amplitudes differed significantly between groups (P= 0.003). Group I (autologous graft) showed the highest mean amplitude of 14.26±3.54 mV, reflecting excellent nerve regeneration. Group II (exosome-fibrin with acellular nerve graft group) showed a mean amplitude of 12.66±4.05 mV, which was significantly higher than the mean amplitude of 8.21±1.29 mV in Group III (fibrin with acellular nerve graft group) (P= 0.034) (Fig. 8). No significant difference was observed between Groups I and II (P= 0.981). However, CMAP latencies did not differ statistically between groups (P= 0.257). Group I had the shortest latency, with an average of 1.78±0.25 ms, indicating efficient nerve conduction. Groups II and III showed mean latencies of 2.02±0.40 ms and 2.03±0.34 ms, respectively. Although the difference was not statistically significant, the trend suggested better functional recovery in the autologous transplant group. The electrophysiological evaluation results are summarized in Table 1.

[0119] Electrophysiological evaluation of autografts a ANG using Exosome-Tisseel b ANG using Tisseel cP value Latency (mean ± SD, ms) 1.78 ± 0.25 2.02 ± 0.40 2.03 ± 0.34 0.257 CMAP amplitude (mean ± SD, mV) 14.26 ± 3.54 12.66 ± 4.05 8.21 ± 1.29 0.003*

[0120] *Post-analysis results showed significant differences between a and c, and between b and c. ANG (Acellular Nerve Graft), CMAP (Compound Muscle Action Potential).

[0121] Experimental Example 6: Toluidine blue staining and immunohistochemical staining analysis

[0122] 6-1: Toluidine blue staining

[0123] Sixteen weeks after surgery, toluidine blue staining was performed to evaluate sciatic nerve axonal regeneration in each group. The mean myelomeningeal axon counts in the autologous transplantation group (group I), the exosome-fibrin group with allograft group (group II), and the fibrin group with allograft group (group III) were 5021.0±679.6, 4269.1±1012.7, and 3460.4±1282.4 (mean±standard deviation), respectively. Group I showed a significantly higher axon count than Group III in the proximal section (p=0.018). However, no significant differences were observed between Groups I and II or between Groups II and III. In the distal section, the corresponding values ​​were 4451.8±707.4, 3675.1±646.3, and 2607.8±952.8, respectively, indicating that group II had a significantly higher axonal number than group III (p= 0.037). Similarly, group I also had a significantly higher axonal number than group III (p= 0.037), but no significant difference was found between groups I and II (Figs. 9a, 9b, and 9c).

[0124] 6-2: NF200 immunohistochemical staining analysis

[0125] The mean pixel intensity of NF200 immunolabeling was assessed in both the proximal and distal regions. In the proximal region, the intensities were 7.76E-03±1.09E-03, 6.63E-03±1.30E-03, and 4.84E-03±6.58E-04 for groups I, II, and III, respectively. Group I showed a significantly higher intensity than group III (P= 0.000), and group II also showed a significantly higher intensity than group III (P= 0.008). In the distal section, the intensity was 7.13E-03 ± 8.78E-04, 5.95E-03 ± 1.28E-03, and 4.19E-03 ± 7.91E-04 for groups I, II, and III, respectively, with group I showing significantly higher intensity than group III (P = 0.000) and group II higher than group III (P = 0.007) (Figs. 10a and 10b).

[0126] 6-3: Immunohistochemical staining S100β analysis

[0127] The mean pixel intensities of S100β immunolabeling followed the same pattern, with group I showing the highest values ​​in both sections, followed by groups II and III. In the proximal sections, the intensities were 4.21E-03±4.31E-04, 3.65E-03±8.00E-04, and 2.71E-03±4.15E-04 for groups I, II, and III, respectively, showing significant differences between groups I and III (P = 0.000) and between groups II and III (P = 0.011). In the distal section, the intensities were 3.99E-03 ± 8.18E-04, 3.47E-03 ± 6.36E-04, and 2.55E-03 ± 4.78E-04 for groups I, II, and III, respectively, with group I showing significantly higher intensities than group III (P = 0.001), and group II also showing significantly higher intensities than group III (P = 0.033) (Figs. 11a and 11b). Higher S100β signal intensities were observed in groups I and II compared to group III, confirming enhanced nerve regeneration.

[0128] While the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.

Claims

1. A pharmaceutical composition for treating nerve damage, comprising exosomes isolated from milk or goat milk as an active ingredient.

2. In paragraph 1, The composition wherein the milk or goat milk is raw milk, commercial milk, goat milk or colostrum.

3. In paragraph 1, The above nerve damage is damage to the peripheral nerves, the composition.

4. In paragraph 3, The composition of the above peripheral nerve damage is trauma, damage to the motor nerve or damage to the sensory nerve.

5. In paragraph 4, A composition wherein the above trauma is selected from the group consisting of burns, contusions, lacerations, puncture wounds, abrasions, abrasions, bedsores, and wounds.

6. In paragraph 1, The above exosomes are a composition that promotes the regeneration of the nerve cells themselves or enhances the proliferation or function of Schwann cells surrounding the axons of the nerve cells.

7. In paragraph 1, A composition wherein the exosomes have a size of 50 to 250 nm.

8. In paragraph 1, The composition has a dosage form selected from the group consisting of microinjection into neural tissue, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, microneedles, transdermal patch, and intranasal administration.

9. Exosomes isolated from milk or goat milk used for treating nerve damage.

10. A method for treating nerve damage in a subject, comprising administering to the subject an exosome isolated from milk or goat milk used for treating nerve damage in a therapeutically effective amount.

Images