Use of mesenchymal-stem-cell-derived intracellular nanovesicle in neuroprotection

Abstract

Disclosed in the present invention is the use of a mesenchymal-stem-cell-derived intracellular nanovesicle in neuroprotection. Compared to a small extracellular vesicle with exosomes as the main component, the small intracellular nanovesicle of the present invention has a smaller particle size, a narrower particle size distribution range, and greater stability at different temperatures, and has good tissue compatibility. The small intracellular nanovesicle of the present invention can better ameliorate nerve injury or neurodegenerative diseases such as optic nerve injury, ischemic stroke and Alzheimer's disease, and has very good application and research value in the field of pharmaceuticals.

Description

Application of mesenchymal stem cell-derived intracellular nanovesicles in neuroprotection Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of mesenchymal stem cell-derived intracellular nanovesicles in neuroprotection, in particular to an application of mesenchymal stem cell-derived intracellular nanovesicles in the treatment of nerve damage and neurodegeneration. Background Art

[0002] The nervous system is the dominant system in the body that regulates physiological functions. It is primarily composed of neural tissue and is divided into two major parts: the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS includes the brain and spinal cord, whose neurons typically fail to regenerate after injury, leading to loss of neurological function. Damage to the brain nerves primarily leads to neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, and Huntington's disease), traumatic brain injury, and cerebral ischemia. Spinal cord injury often causes significant sensory and motor dysfunction in patients. The optic nerve, generally considered part of the CNS, is composed of axons from retinal ganglion cells (RGCs), which transmit visual information collected by the retina to the cerebral cortex. Glaucoma, ocular trauma, tumors, and drug poisoning can all lead to damage to the optic nerve. Once damaged, it often results in decreased vision, impaired color vision, and even vision loss. Given the non-regenerative nature of central nervous system neurons and the severe functional impairment after nerve injury, finding new neurotherapeutic strategies is of great significance for exploring the repair mechanism of central nervous system injury and promoting central nervous system regeneration.

[0003] Mesenchymal stem cells (MSCs) cell therapy has become a promising treatment for neurological diseases. This treatment method depends on the ability of mesenchymal stem cells to transdifferentiate into neural cells, as well as their self-renewal potential, pro-proliferation properties and neuroprotective effects. However, there are many bottlenecks in the application of cell therapy in neurological diseases, such as abnormal cell growth and immune rejection. Studies have found that extracellular vesicles (EVs) secreted by mesenchymal stem cells have stem cell properties and are considered to be able to replace cells to play a therapeutic role, but they also have certain defects, such as low efficiency of extracellular vesicle collection, the presence of extracellular vesicles in the extracellular matrix after secretion, and the presence of exogenous substances in the cell culture medium, which leads to a decrease in purity.

[0004] It is worth noting that there are many nanoscale vesicles in the cell, which are located between various membrane-rich organelles and are responsible for the transport and secretion of substances within the cell. These intracellular vesicles (IVs) are produced by a process called vesicle budding and can originate from various organelles, including the endoplasmic reticulum, Golgi apparatus, endosomes, and plasma membrane. They are composed of a variety of membrane-rich particles, such as constitutive secretory vesicles, synaptic vesicles, COP-coated vesicles, Golgi-derived vesicles, clathrin-coated vesicles, and transport vesicles between the endoplasmic reticulum and the Golgi apparatus. These IVs maintain the basic life activities within the cell and contain a large number of biological macromolecules. They participate in the secretion of specific proteins, hormones, and other biological molecules through exocytosis.

[0005] The inventors of the present invention have discovered that these intracellular nanovesicles can not only replace cells in their therapeutic effects but also have potential applications in neuroprotection. However, previous research has primarily focused on the signaling and regulation of intracellular nanovesicle-mediated substance transport, and there are no reports of intracellular nanovesicles being used for neuroprotection. Summary of the Invention

[0006] To overcome the deficiencies of the prior art, the present invention provides an application of intracellular nanovesicles derived from mesenchymal stem cells in neuroprotection (particularly in protection against nerve damage and neurodegeneration).

[0007] In a first aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a medicament for preventing and / or treating nervous system diseases.

[0008] Specifically, the vesicles are small intracellular nanovesicles (sIVs).

[0009] Specifically, the vesicle is double-layered horseshoe-shaped or teacup-shaped.

[0010] Specifically, the average particle size of the vesicles is 50-100 nm (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 nm), especially 65-85 nm.

[0011] Specifically, the vesicles express TMEM214 protein.

[0012] Specifically, the vesicles low-express exosome markers and high-express marker proteins of intracellular membrane-rich organelles and the Clathrin protein family.

[0013] Specifically, the vesicles are relatively stable at -80°C to 37°C (e.g., -80, -50, -20, -10, 0, 5, 25, 37°C).

[0014] Specifically, the vesicles are prepared by a method comprising an ultrasonic treatment step. More specifically, the vesicles are prepared by a method comprising ultrasonic treatment, centrifugation, and ultracentrifugation steps (performed sequentially).

[0015] In a preferred embodiment of the present invention, the vesicles are prepared by a method comprising the following steps:

[0016] (1) Dispersing mesenchymal stem cells in a suspension solvent and performing ultrasonic treatment;

[0017] (2) centrifuging the liquid obtained in step (1) once or multiple times, discarding the cell membrane and organelle debris, and taking the supernatant;

[0018] (3) subjecting the supernatant obtained in step (2) to ultracentrifugation, and collecting the precipitate as intracellular nanovesicles;

[0019] Optionally, (4) resuspending the precipitate obtained in step (3).

[0020] Specifically, the cells in step (1) are isolated cells obtained after culture, digestion, and washing (after discarding the cell culture medium, digestion, washing, etc., the possibility of obtaining extracellular vesicles by separation can be eliminated).

[0021] Specifically, the method may further include cell digestion and counting steps; in some embodiments of the present invention, the cell digestion step includes: culturing cells to 90% confluence, discarding the cell culture medium, washing the cells, adding trypsin to digest the cells, and then neutralizing and washing the cells.

[0022] Specifically, the cell density of the cells in the suspension solvent is 1-4×10 6 / mL, for example 1×10 6 / mL, 2×10 6 / mL, 3×10 6 / mL, 4×10 6 In some embodiments of the present invention, the cell density is 1×10 6 pieces / mL.

[0023] Specifically, the suspension solvent is any buffer suitable for culturing cells, such as PBS, Tris buffer, or glycine buffer. In some embodiments of the present invention, the solvent is PBS.

[0024] Specifically, the mesenchymal stem cells are derived from mammals, especially humans.

[0025] Specifically, the mesenchymal stem cells are selected from: umbilical cord mesenchymal stem cells (UC-MSC), bone marrow mesenchymal stem cells (BM-MSC), adipose mesenchymal stem cells (AD-MSC), dental pulp mesenchymal stem cells, placenta and amniotic fluid and amniotic membrane mesenchymal stem cells, especially umbilical cord mesenchymal stem cells (UC-MSC).

[0026] Specifically, the amplitude of the ultrasonic treatment in step (1) is 20%-35% (e.g., 20%, 22%, 24%, 25%, 30%, 35%), preferably 20%-25%; in some embodiments of the present invention, the amplitude of the ultrasonic treatment is 20%.

[0027] Specifically, the ultrasonic treatment time in step (1) is 15-60s (e.g., 15, 20, 25, 30, 40, 50, 60s), preferably 15s; in some embodiments of the present invention, the ultrasonic treatment time is 15s, on 2s, off 2s.

[0028] Specifically, the ultrasonic treatment time in step (1) is 10-20 s (e.g., 10, 15, 18, 20 s).

[0029] In some embodiments of the present invention, the number of centrifugation treatments in step (2) is two, and the respective parameters are:

[0030] 1000-3000 g (e.g., 1000, 1500, 2000, 2500, 3000 g), 5-20 minutes (e.g., 5, 8, 10, 12, 15, 20 minutes);

[0031] 10,000-30,000 g (e.g., 10,000, 15,000, 20,000, 25,000, 30,000 g), 20-40 minutes (e.g., 20, 25, 28, 30, 32, 35, 40 minutes).

[0032] In one embodiment of the invention, the first centrifugation is performed at 2000 g for 10 minutes.

[0033] In one embodiment of the invention, the second centrifugation is performed at 20,000 g for 30 minutes.

[0034] Specifically, the parameters of the ultracentrifugation treatment in step (3) include 100,000-180,000 g (e.g., 100,000, 120,000, 140,000, 150,000, 160,000, 180,000 g), 50-100 minutes (e.g., 50, 60, 65, 70, 75, 80, 90, 100 minutes).

[0035] In one embodiment of the invention, the ultracentrifugation is performed at 150,000 g for 70 minutes.

[0036] Specifically, the resuspension solvent in step (4) is any buffer suitable for culturing cells, such as PBS, Tris buffer, or glycine buffer. In some embodiments of the present invention, the resuspension solvent is PBS.

[0037] Specifically, one or more of the ultrasonic treatment, centrifugation and ultracentrifugation are performed at low temperature, for example, 0-5°C; in particular, the ultrasonic treatment, centrifugation and ultracentrifugation are all performed on ice; or, the ultrasonic treatment, centrifugation and ultracentrifugation are all performed at 4°C.

[0038] In some embodiments of the present invention, the vesicles are prepared by a method comprising the following steps: taking 1×10 6 For a cell suspension with a density of 1 cell / mL, an ultrasonic probe is placed in the center of the liquid surface, and ultrasonic treatment is performed with an ultrasonic amplitude parameter range of 20%, a time parameter range of 15s, on (run) 2s, and off (pause) 2s; the liquid is then transferred to a centrifuge tube for centrifugation with centrifugation parameters of 2000g×10min and 20000g×30min, and the supernatant is collected; the supernatant is transferred to an ultracentrifuge tube for centrifugation with centrifugation parameters of 150000g×70min.

[0039] In some embodiments of the present invention, the nervous system disease is nerve damage and nerve degeneration and related diseases.

[0040] Specifically, the nerve damage can be nerve damage caused by various reasons, for example, infection (such as viruses, bacteria, fungi, parasites, etc.), trauma (such as chemical damage, photochemical damage, knife cuts, burns, firearm injuries, abrasions, squeezing, impact, etc.), compression or traction injuries (such as intervertebral disc herniation, spinal stenosis, brachial plexus injury, etc.), autoimmune mechanisms (such as myasthenia gravis, multiple sclerosis, etc.), ischemic injuries (such as stroke, cerebral thrombosis, etc.), metabolic diseases (such as diabetes, etc.), tumors, hereditary, nutritional, inflammation, drug damage, and its manifestations may include, but are not limited to, movement disorders (such as muscle weakness, paralysis or tremor, etc.), sensory disorders (such as numbness, tingling, paresthesia or loss of sensation, etc.), autonomic nervous system disorders (such as hypotension, syncope, constipation or urinary incontinence, etc.), cognitive disorders (such as amnesia, confusion or inattention, etc.), mental disorders (such as depression, anxiety, abnormal behavior or mental confusion, etc.).

[0041] Specifically, the nerve injury-related diseases include, but are not limited to, spinal cord diseases, optic nerve diseases, cerebrovascular diseases, traumatic brain injury, extrapyramidal diseases, demyelinating diseases, neurodegenerative diseases, central nervous system infectious diseases, cerebral palsy, hydrocephalus, cranial nerve diseases, nerve root diseases, plexus diseases, single nerve diseases, multiple nerve diseases, neuromuscular junction diseases, ataxia, leukodystrophy, mental retardation, familial amyloid polyneuropathy, Charcot-Marie-Tooth disease, and neurosis.

[0042] Specifically, the neurodegeneration-related diseases include, but are not limited to, motor neuron disease (such as amyotrophic lateral sclerosis, progressive spinal muscular atrophy, pyramidal tract degeneration, progressive bulbar palsy, pseudobulbar palsy, true bulbar palsy, primary lateral sclerosis, spinal bulbar muscular atrophy, lower motor neuron syndrome, upper motor neuron syndrome, flail arm syndrome, Madras motor neuron disease), Alzheimer's disease, multiple system atrophy, senile brain atrophy, Pick's disease dementia, alcoholic nervous system degeneration (such as alcoholic cerebellar degeneration, alcoholic polyneuropathy, alcoholic myopathy, central pontine myelinolysis, corpus callosum degeneration, alcoholic dementia, alcoholic mental disorder), frontotemporal dementia, subacute necrotizing encephalopathy, corticostriatal spinal degeneration, Lewy body dementia, Machado-Joseph disease.

[0043] Specifically, the spinal cord disease is selected from: spinal cord injury, myelitis, poliomyelitis, encephalomyelitis, spinal cord tumor, spinal muscular atrophy, motor neuron disease, spinal cord compression, syringomyelia, syringomyelia, vascular myelopathy (such as spinal cord hemorrhage, spinal cord ischemia, spinal cord embolism, spinal cord infarction, spinal cord necrosis, spinal cord edema, spinal artery thrombosis, posterior spinal artery syndrome, anterior spinal artery occlusive syndrome, spinal vascular malformation, spinal arteriovenous malformation), hepatic myelopathy, cervical spondylotic myelopathy, spinal meningitis, epidural abscess, spinal disease, cauda equina syndrome, HTLV-1 associated myelopathy / tropical spastic paralysis.

[0044] Specifically, the optic nerve disease is selected from: optic nerve tumors, ischemic optic neuropathy, hereditary optic neuropathy, compressive optic neuropathy, optic nerve injury, nutritional optic neuropathy, toxic optic neuropathy, optic neuritis, papilledema, optic nerve atrophy, and glaucoma.

[0045] Specifically, the cerebrovascular disease is selected from: ischemic cerebrovascular disease (such as cerebral ischemia, cerebral infarction, cerebral thrombosis, cerebral embolism, ischemic-hypoxic encephalopathy), hemorrhagic cerebrovascular disease (such as cerebral hemorrhage, subarachnoid hemorrhage, non-traumatic subdural hemorrhage, non-traumatic epidural hemorrhage), cerebral atherosclerosis, cerebral arteritis, cerebral aneurysm, cerebral arteriovenous fistula, amyloid angiopathy, intracranial venous thrombosis, intracranial blood pressure malformation (such as cerebral arteriovenous malformation, cerebral cavernous malformation, dural arteriovenous fistula, carotid cavernous sinus fistula), intracranial telangiectasia, reversible cerebral vasoconstriction syndrome, vascular dementia (such as Binswanger's disease, multiple infarct dementia, hemorrhagic dementia), hypertensive encephalopathy, moyamoya disease, red core syndrome, Weber syndrome, Benedict's syndrome, vascular leukoencephalopathy.

[0046] Specifically, the extrapyramidal disease is selected from: Hallevorden-Spatz disease, progressive supranuclear palsy, multiple system atrophy, calcification of the basal ganglia, dystonia, orofacial movement disorders, Meige syndrome, essential tremor, myoclonus, chorea, restless legs syndrome, stiff-person syndrome, and Wilson's disease.

[0047] Specifically, the demyelinating disease is selected from: multiple sclerosis (such as brainstem multiple sclerosis, spinal cord multiple sclerosis, multiple sclerosis dementia), acute disseminated encephalomyelitis, neuromyelitis optica, acute hemorrhagic leukoencephalitis, diffuse sclerosis, central pontine myelinolysis, extrapontine myelinolysis, acute transverse myelitis, subacute necrotizing myelitis, concentric sclerosis, tumor-like demyelinating lesions, white matter demyelination, demyelinating myelitis, and clinically isolated syndrome.

[0048] Specifically, the neurodegenerative disease is selected from the group consisting of epilepsy, cerebral ischemia, brain injury, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, ataxia-telangiectasia syndrome, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, multiple sclerosis, primary lateral sclerosis, spinal muscular atrophy, and spastic paraplegia.

[0049] Specifically, the central nervous system infectious disease is selected from: encephalitis, meningitis (such as pachymeningitis, leptomeningitis, arachnoiditis), myelitis, encephalomyelitis, ventriculitis, cerebellitis, brainstem inflammation, intracranial abscess (such as brain abscess, epidural abscess, subdural abscess), intracranial granuloma (such as brain granuloma, epidural granuloma, subdural granuloma), neurosyphilis, brain parasitic disease, prion disease.

[0050] Specifically, the cerebral palsy is selected from: spastic cerebral palsy, dyskinetic cerebral palsy, ataxic cerebral palsy, and Worster-Drought syndrome.

[0051] Specifically, the hydrocephalus is selected from: communicating hydrocephalus, obstructive hydrocephalus, compensatory hydrocephalus, and congenital hydrocephalus.

[0052] Specifically, the cranial nerve disease is selected from: trigeminal nerve disease (such as trigeminal neuralgia), facial nerve disease (such as Bell's palsy, geniculate ganglionitis, Merrow syndrome, hemifacial spasm, hemifacial atrophy, facial neuritis), olfactory nerve disease, glossopharyngeal nerve disease, vagus nerve disease, hypoglossal nerve disease, and multiple cranial nerve diseases.

[0053] Specifically, the nerve root disease is selected from: nerve compression, nerve root sleeve cyst.

[0054] Specifically, the plexus disease is selected from: brachial plexus injury and lumbar plexus injury.

[0055] Specifically, the mononeuropathy is selected from: upper limb mononeuropathy (such as carpal tunnel syndrome, ulnar neuropathy, radial nerve damage), lower limb mononeuropathy (such as sciatic nerve damage, femoral neuropathy, tibial neuropathy, tarsal tunnel syndrome, plantar nerve damage, intercostal neuropathy).

[0056] Specifically, the polyneuropathy is selected from: idiopathic progressive neuropathy, inflammatory polyneuropathy, secondary polyneuropathy (such as diabetic polyneuropathy, infectious polyneuropathy, dystrophic polyneuropathy).

[0057] Specifically, the neuromuscular junction disease is selected from: myasthenia gravis, myasthenic syndrome, muscular dystrophy, myotonia-related diseases (such as neuromyotonia, myotonia atrophica, dystrophic myotonia, myotonia congenita, paramyotonia), mitochondrial myopathy, alcoholic myopathy, inflammatory myopathy, muscular atrophy, periodic paralysis, ocular myopathy, and myofibrillar myopathy.

[0058] Specifically, the ataxia is selected from: cerebellar ataxia (such as Marinesco-Sjogren syndrome, Ramsay-Hunt syndrome, paroxysmal ataxia, Friedreich ataxia, spinocerebellar ataxia, hereditary spastic ataxia, Kearn-Sayre syndrome, ataxia telangiectasia, CANVAS syndrome), hereditary spastic paraplegia.

[0059] Specifically, the neurosis is selected from the group consisting of hysteria, anxiety disorder, obsessive-compulsive disorder, autism, depression, panic disorder, neurasthenia, phobia, hypochondriasis, and somatoform disorder.

[0060] In some embodiments of the present invention, the nervous system disease is an optic nerve disease, for example, compressive optic neuropathy.

[0061] In some embodiments of the present invention, the nervous system disease is a cerebrovascular disease, for example, ischemic cerebrovascular disease, in particular cerebral ischemia.

[0062] In some embodiments of the present invention, the nervous system disease is a nervous system degenerative disease, in particular Alzheimer's disease or Parkinson's disease.

[0063] In some embodiments of the present invention, the neurological disease is traumatic brain injury.

[0064] In some embodiments of the present invention, the nervous system disease is a spinal cord disease, in particular, a spinal cord injury.

[0065] Specifically, the nervous system disease is selected from: nerve damage disease and neurodegenerative disease.

[0066] Specifically, the nerve injury disease is selected from: optic nerve disease, ischemic cerebrovascular disease, traumatic brain injury and spinal cord injury.

[0067] Specifically, the neurodegenerative disease is selected from: Alzheimer's disease and Parkinson's disease.

[0068] In some embodiments of the present invention, the nerve damage disease is selected from: optic nerve damage, compressive optic neuropathy, ischemic optic neuropathy, optic neuritis, optic atrophy, glaucoma, cerebral ischemia, cerebral infarction, cerebral thrombosis, cerebral embolism and hypoxic-ischemic encephalopathy.

[0069] In some embodiments of the invention, the neurodegenerative disease is Alzheimer's disease.

[0070] Specifically, the drug can be administered in any suitable manner, including but not limited to oral administration, injection (such as intravenous injection, intramuscular injection, intraperitoneal injection), subcutaneous administration, skin administration, ocular administration (such as eye drops, eye ointment, subconjunctival injection, vitreous cavity injection), and nasal administration (such as nasal administration).

[0071] In one embodiment of the present invention, the administration method is ocular administration, particularly intravitreal injection.

[0072] In one embodiment of the present invention, the administration method is injection, particularly intravenous injection or intraperitoneal injection.

[0073] In one embodiment of the present invention, the administration method is nasal administration, especially nasal administration.

[0074] Specifically, the drug can be formulated into any suitable preparation form, such as, but not limited to, cream, foam, paste, ointment, emulsion, liquid solution, eye drops, injection, powder injection, gel, spray, suspension, microemulsion, eye mask or contact lens, etc., especially injection.

[0075] The present invention also provides a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating nerve damage diseases.

[0076] The present invention also provides a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of drugs for preventing and / or treating neurodegenerative diseases.

[0077] In a second aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a medicament for preventing and / or treating optic nerve damage, compressive optic neuropathy, ischemic optic neuropathy, optic neuritis, optic atrophy or glaucoma.

[0078] Specifically, the mesenchymal stem cell-derived intracellular nanovesicles are as described in the first aspect of the present invention.

[0079] In a third aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a medicament for preventing and / or treating cerebral ischemia, cerebral infarction, cerebral thrombosis, cerebral embolism or hypoxic-ischemic encephalopathy.

[0080] Specifically, the mesenchymal stem cell-derived intracellular nanovesicles are as described in the first aspect of the present invention.

[0081] In a fourth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a medicament for preventing and / or treating Alzheimer's disease.

[0082] Specifically, the mesenchymal stem cell-derived intracellular nanovesicles are as described in the first aspect of the present invention.

[0083] In a fifth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a medicament for preventing and / or treating Parkinson's disease.

[0084] Specifically, the mesenchymal stem cell-derived intracellular nanovesicles are as described in the first aspect of the present invention.

[0085] In a sixth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a medicament for preventing and / or treating traumatic brain injury.

[0086] Specifically, the mesenchymal stem cell-derived intracellular nanovesicles are as described in the first aspect of the present invention.

[0087] In a seventh aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating spinal cord diseases.

[0088] Specifically, the mesenchymal stem cell-derived intracellular nanovesicles are as described in the first aspect of the present invention.

[0089] Specifically, the spinal cord disease is spinal cord injury.

[0090] In an eighth aspect of the present invention, a method for preventing and / or treating a nervous system disease is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0091] Specifically, the nervous system disease is as described in the first aspect of the present invention.

[0092] Specifically, the subject is a mammal, especially a human.

[0093] Specifically, the administration can be carried out in any suitable manner, including but not limited to oral administration, injection (such as intravenous injection, intramuscular injection, intraperitoneal injection, subconjunctival injection, and vitreous cavity injection), subcutaneous administration, skin administration, ocular administration (such as intraocular administration, ocular surface administration, and periocular administration, specifically such as eye drops, eye ointment, subconjunctival injection, vitreous cavity injection, vitreous implantation, retrobulbar injection, peribulbar administration, subbulbar administration, intracameral injection, and eye gel administration), and nasal administration (such as nasal administration).

[0094] Specifically, the dosage depends on many factors, including the age, weight, sex, disease, severity, route and frequency of administration of the subject, and therefore can vary. In some embodiments of the present invention, the dosage can be 1 μg-60 μg vesicles / eye, for example, 1 μg vesicles / eye, 2 μg vesicles / eye, 4 μg vesicles / eye, 5 μg vesicles / eye, 6 μg vesicles / eye, 8 μg vesicles / eye, 10 μg vesicles / eye, 15 μg vesicles / eye, 20 μg vesicles / eye, 25 μg vesicles / eye, 30 μg vesicles / eye, 35 μg vesicles / eye, 40 μg vesicles / eye, 45 μg vesicles / eye, 50 μg vesicles / eye, 55 μg vesicles / eye, and 60 μg vesicles / eye.

[0095] In a ninth aspect of the present invention, a method for preventing and / or treating optic nerve damage is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof;

[0096] Alternatively, a method for preventing and / or treating compressive optic neuropathy, ischemic optic neuropathy, optic neuritis, optic atrophy or glaucoma is provided, which comprises the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0097] Specifically, the subject is a mammal, especially a human.

[0098] Specifically, the administration can be carried out by any suitable administration method, in particular ocular administration (eg, intraocular administration, ocular surface administration), such as eye drops, eye ointment, subconjunctival injection, intravitreal injection, in particular intravitreal injection.

[0099] In some embodiments of the present invention, the administration method is intravitreal injection, and the administration amount can be 1 μg-30 μg vesicles / eye.

[0100] In a tenth aspect of the present invention, a method for preventing and / or treating cerebral ischemia is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof;

[0101] Alternatively, a method for preventing and / or treating cerebral infarction, cerebral thrombosis, cerebral embolism or hypoxic-ischemic encephalopathy is provided, which comprises the step of administering intracellular nanovesicles derived from mesenchymal stem cells (as described in the first aspect of the present invention) to a subject in need thereof.

[0102] Specifically, the subject is a mammal, especially a human.

[0103] Specifically, the administration can be carried out in any suitable manner, in particular nasal administration, especially nasal cavity administration.

[0104] In an eleventh aspect of the present invention, a method for preventing and / or treating Alzheimer's disease is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0105] Specifically, the subject is a mammal, especially a human.

[0106] Specifically, the administration can be carried out in any suitable manner, in particular nasal administration, especially nasal cavity administration.

[0107] In a twelfth aspect of the present invention, a method for preventing and / or treating Parkinson's disease is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0108] Specifically, the subject is a mammal, especially a human.

[0109] Specifically, the administration can be carried out in any suitable manner, in particular nasal administration, especially nasal cavity administration.

[0110] In a thirteenth aspect of the present invention, a method for preventing and / or treating traumatic brain injury is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0111] Specifically, the subject is a mammal, especially a human.

[0112] Specifically, the administration can be carried out in any suitable manner, in particular nasal administration, especially nasal cavity administration.

[0113] In a fourteenth aspect of the present invention, a method for preventing and / or treating spinal cord injury is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0114] Specifically, the subject is a mammal, especially a human.

[0115] Specifically, the administration can be carried out by any suitable administration method, in particular, injection administration, for example, intravenous injection administration, intramuscular injection administration, intraperitoneal injection administration, in particular intravenous injection administration.

[0116] The present invention has the following excellent effects:

[0117] (1) The present invention provides a method for collecting intracellular nanovesicles derived from mesenchymal stem cells by ultrasonic cell disruption, which can avoid the tedious steps and impurities of collecting extracellular vesicles, that is, the operation of separating vesicles is simple, and the yield of the obtained vesicles is high. The obtained small intracellular nanovesicles have a smaller particle size, a narrower particle size distribution range, and are more stable at different temperatures compared to extracellular vesicles with exosomes as the main component. The obtained intracellular nanovesicles have good tissue compatibility. Compared with extracellular vesicles, the intracellular nanovesicles have a wider range and degree of distribution when injected in situ into the tissue. When used as a carrier to load drugs, they have a higher encapsulation rate and drug loading rate. For example, drugs loaded by intracellular nanovesicles in the form of intravitreal injection can be absorbed faster by the retina.

[0118] (2) The small intracellular nanovesicles prepared by the present invention have very good application and research value in nerve damage or neurodegenerative diseases (such as optic nerve damage, ischemic stroke, Alzheimer's disease, etc.). The small intracellular nanovesicles can inhibit the apoptosis of ganglion cells, slow down the reduction of GCC thickness, reduce neuronal pathological damage and the relative number of apoptotic cells, activate microglia and astrocytes, reduce inflammatory stress on neural tissue, increase the expression of β-tubulinⅢ, Occludin and ZO-1, improve the motor coordination ability and neuromuscular ability of animals, improve the learning disabilities and cognitive disorders of animals, and stimulate the spontaneous exploratory behavior of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 shows a schematic diagram of the process and steps of the method for producing intracellular nanovesicles, wherein Figure 1A shows a flow chart and Figure 1B shows the steps.

[0120] Figure 2 shows the optimization process for the isolation parameters of intracellular nanovesicles. Figures 2A and 2B show the protein yield (A) and vesicle yield (B) of sIVs obtained at different sonication times and an ultrasonic amplitude of 20%. At a sonication amplitude of 20%, the vesicle yield dropped sharply when the sonication time exceeded 10 seconds or exceeded 20 seconds. Figures 2C and 2D show the protein yield (C) and vesicle yield (D) of sIVs obtained at different sonication times and an ultrasonic amplitude of 15 seconds. At a sonication time of 15 seconds, the vesicle yield dropped sharply when the sonication amplitude exceeded 25%. Figure 2E shows transmission electron micrographs of sIVs obtained at different sonication amplitudes and an ultrasonic amplitude of 15 seconds. Scale bar: 100 nm. Figure 2F shows transmission electron micrographs of sIVs obtained at different sonication times and an ultrasonic amplitude of 20%. Scale bar: 100 nm.

[0121] Figure 3 shows transmission electron microscopy images of sEVs and sIVs from MSCs. Scale bar for wide-field images: 200 nm; scale bar for close-up images: 200 nm.

[0122] Figure 4 shows the results of nanoparticle size analysis, which shows the particle size distribution of MSCs cells and their sEVs and sIVs.

[0123] FIG5 shows the statistical analysis results of the particle sizes of MSCs cells and their sEVs and sIVs (**p<0.01, ***p<0.001 indicate significant differences among the groups).

[0124] Figure 6 shows the statistical analysis results of the number of sEVs and sIVs vesicles (A) and total protein production of MSCs cells with equal cell numbers (B) (**p < 0.01, ***p < 0.001 and ****p < 0.0001 indicate significant differences between the groups).

[0125] FIG7 shows the results of Coomassie Brilliant Blue staining, which shows the protein distribution of MSCs cells and their sEVs and sIVs.

[0126] FIG8 shows the results of Western blot, which shows the expression of exosome marker proteins (Alix, HSP70, TSG101, CD63, CD81) of MSCs cells.

[0127] Figure 9 shows transmission electron micrographs of sEVs and sIVs of MSCs at different temperatures. Scale bar: 200 nm.

[0128] Figure 10 shows the results of nanoparticle size analysis of sEVs and sIVs grown on MSCs at different temperatures (-80°C, 4°C, and 37°C). Figure 10A shows the particle size distribution, and Figure 10B shows the statistical analysis of particle size (*p < 0.05, **p < 0.01 indicate significant differences between groups).

[0129] FIG11 shows the sIVs-specific proteins in MSCs cells, arranged from high to low abundance, and displays the top 50 most highly expressed proteins of each.

[0130] Figure 12 shows the results of super-resolution microscopy and total internal reflection fluorescence structured illumination microscopy. Among them, Figure 12A shows the TIRF-SIM mode (showing the cell membrane), which shows that there are CD63-positive areas on the cell membrane surface (left picture), while almost no TMEM214-positive areas are observed (right picture). Figure 12B shows the wide field-2DSM mode (showing the whole cell), which proves that CD63-positive (left picture) and TMEM214-positive (right picture) signals are present in the whole cell. Figure 12C shows time-lapse screenshots of dynamic observation of living cells. The left picture shows CD63-labeled late endosomes, sEVs and cell membranes in the cell. The arrow at 0s shows that sEVs have just been released from the cell membrane to the outside of the cell, and gradually move away from the cell membrane from 6min to 14min. The right picture shows TMEM214, which shows that sIVs are diffusely distributed in the cell and are not released outside the cell.

[0131] FIG13 is a Venn diagram showing the total protein species in MSCs, sEVs, and sIVs.

[0132] FIG14 shows the principal component analysis results of the total proteins identified in MSCs cells, sEVs, and sIVs.

[0133] FIG15 is a heat map showing differentially expressed proteins between sEVs and sIVs of MSCs cells.

[0134] FIG16 shows a volcano plot showing the top five significantly differentially expressed proteins between sEVs and sIVs of MSCs cells.

[0135] FIG17 shows a heat map showing the differential expression of exosomal markers between sEVs and sIVs of MSCs cells.

[0136] FIG18 shows a heat map showing the differential expression of organelle markers between sEVs and sIVs of MSCs cells.

[0137] FIG19 is a heat map showing the differential expression of Clathrin family proteins between sEVs and sIVs of MSCs cells.

[0138] Figure 20 shows the cellular component enrichment analysis of sIVs-expressed proteins in MSCs, where “Summary” represents the representative pathways after project cluster analysis, and “Project” represents the pathways and specific entries of the pathways.

[0139] Figure 21 shows the biological process enrichment analysis of sIVs-expressed proteins in MSCs, where “Summary” represents the representative pathways after project cluster analysis, and “Project” represents the pathways and specific entries of the pathways.

[0140] Figure 22 shows the differences in IL-1β and IGF2 cytokine levels between sEVs and sIVs of MSCs detected by protein profiling. Figure 22A shows the differences in IL-1β cytokine levels between sEVs and sIVs of MSCs detected by protein profiling, and Figure 22B shows the differences in IGF2 cytokine levels between sEVs and sIVs of MSCs detected by protein profiling (*p < 0.05, ***p < 0.001 indicate significant differences between groups).

[0141] Figure 23 shows the differences in the levels of IGF-1, EGF, IL-10, IL-6, and TNFα cytokines carried by sEVs and sIVs of MSCs detected by ELISA. Figure 23A shows the differences in the levels of IGF-1 cytokines carried by sEVs and sIVs of MSCs detected by ELISA, Figure 23B shows the differences in the levels of EGF cytokines carried by sEVs and sIVs of MSCs detected by ELISA, Figure 23C shows the differences in the levels of IL-10 cytokines carried by sEVs and sIVs of MSCs detected by ELISA, Figure 23D shows the differences in the levels of IL-6 cytokines carried by sEVs and sIVs of MSCs detected by ELISA, and Figure 23E shows the differences in the levels of TNFα cytokines carried by sEVs and sIVs of MSCs detected by ELISA (**p < 0.01, ***p < 0.001 indicate significant differences between the groups, ns indicates no statistical difference).

[0142] FIG24 shows the relative RNA abundance in sEVs and sIVs of MSCs cells (ns indicates no statistical difference).

[0143] Figure 25 shows the percentage of small noncoding RNAs read from small RNAs in sEVs and sIVs from MSCs. miRNA: micro-RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; tRNA: transfer RNA; rRNA: ribosomal RNA.

[0144] FIG26 is a Venn diagram showing the types of miRNAs contained in sEVs and sIVs of MSCs cells.

[0145] FIG27 shows the principal component analysis results of the MSCs cell miRNA dataset.

[0146] FIG28 is a matchstick chart showing the top 10 highly abundant miRNAs in sEVs and sIVs of MSCs cells.

[0147] FIG29 is a heat map showing that there are many differentially expressed miRNAs between sEVs and sIVs of MSCs cells.

[0148] Figure 30 shows a volcano plot showing the top five differentially expressed miRNAs between sEVs and sIVs of MSCs. The abscissa represents the fold change (log2 fold difference) of miRNA expression between different samples or comparison combinations, and the ordinate represents the significance level of the expression difference.

[0149] Figure 31 shows enrichment analysis of candidate target genes of miRNAs differentially expressed in sEVs and sIVs derived from MSCs. Figure 31A shows the top 10 entries in GO analysis for Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Figure 31B shows KEGG enrichment analysis of candidate target genes of miRNAs differentially expressed in sEVs and sIVs derived from MSCs.

[0150] Figure 32 shows the types and proportions of metabolites contained in sEVs and sIVs of MSCs cells.

[0151] FIG33 shows the principal component analysis results of lipids contained in MSCs cell sEVs and sIVs.

[0152] FIG34 is a heat map showing the differential lipid types contained in MSC cell sEVs and sIVs.

[0153] Figure 35 shows a lipidome bar chart comparing the MSCs sIVs group and the sEVs group. The horizontal axis represents the relative percentage change in the content of each substance between the two groups. A relative percentage change of zero indicates that the substance is present at the same level in both groups; a positive relative percentage change indicates that the substance is present at a higher level in the sIVs group; a negative relative percentage change indicates that the substance is present at a higher level in the sEVs group. The vertical axis of the lipidome bar chart represents lipid classification information.

[0154] Figure 36 shows the results of an investigation into the ability of intracellular nanovesicles to be endocytosed by RPE cells cultured in vitro. Figures 36A and B show that DiD-labeled sEVs and sIVs were co-incubated with RPE cells for 3 h, 12 h, 24 h, and 48 h. The first row of figures shows the cytoskeleton, the second row of figures shows the vesicles, and the third row of figures shows a composite of the cytoskeleton, vesicles, and nuclei. Scale bar: 20 μm. Figure 36C shows the statistical results of DiD fluorescence intensity in the cells (*p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences between the groups).

[0155] Figure 37 shows the results of an investigation into the ability of intracellular nanovesicles to be endocytosed by HRMECs cultured in vitro. Figures 37A and B show DiD-labeled sEVs and sIVs co-incubated with HRMECs for 3 h, 12 h, 24 h, and 48 h. The first row of images shows the cytoskeleton, the second row of images shows the vesicles, and the third row of images shows a composite image of the cytoskeleton, vesicles, and nucleus. Scale bar: 20 μm. Figure 37C shows the statistical results of DiD fluorescence intensity within the cells (*p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences between the groups).

[0156] Figure 38 shows the results of an investigation into the ability of intracellular nanovesicles to be internalized by the retina. Figure 38A shows the distribution of DiD-labeled sEVs and sIVs on retinal sections 24h and 48h after subconjunctival injection. Figure 38B shows the distribution of DiD-labeled sEVs and sIVs on retinal sections 8h and 24h after intravitreal injection. DAPI staining shows the cell nucleus. Scale bar: 20μm. Figure 38C shows the statistical results of DiD fluorescence intensity in the retina of the subconjunctival injection group. Figure 38D shows the statistical results of DiD fluorescence intensity in the retina of the intravitreal injection group (*p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 indicate significant differences between the groups).

[0157] Figure 39 shows the results of retinal flat mount staining experiments after successful ONC mouse model establishment, showing the changes in retinal ganglion cell numbers at different clamping times (A) and statistical analysis results (B) (****p<0.0001 indicates significant differences between groups).

[0158] Figure 40 shows the results of a retinal flat mount staining experiment in ONC mice at different treatment concentrations. It shows the status of ONC mouse retinal ganglion cells (A) and statistical analysis results (B). L represents the low concentration (1.25 mg / ml) and H represents the high concentration (2.5 mg / ml) (*p < 0.05, indicating significant differences between groups).

[0159] Figure 41 shows the results of retinal flat mount staining experiments in ONC mice after different treatment times. It shows the status of ONC mouse retinal ganglion cells (A) and statistical analysis results (B and C) after 7 and 21 days of administration, respectively (*p < 0.05, indicating significant differences between groups).

[0160] Figure 42 shows the results of optical coherence tomography experiments, which show the structural changes of each retinal layer at the optic disc level (A) and the statistical analysis results of the thickness of the retinal ganglion cell complex (B) (*p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences between groups).

[0161] Figure 43 shows the results of the rotarod test in each group of stroke-affected mice. It shows that the time it took for mice in the PBS group to fall from the rotarod was significantly earlier and shorter than that in the MSC-sEVs and MSC-sIVs groups. The time it took for animals in the MSC-sEVs and MSC-sIVs groups to fall from the rotarod was significantly longer than that in the PBS group on days 3, 5, and 7.

[0162] Figure 44 shows the results of the forelimb grip strength test in each group of stroke mice. It shows that ischemia induced a decrease in forelimb grip strength at various time points within 7 days after ischemia, while administration of MSC-sIVs and MSC-sEVs significantly increased forelimb grip strength 3 days after ischemia.

[0163] Figure 45 shows the results of TTC tissue staining in a mouse stroke model. After TTC staining, the PBS group showed the largest white infarct area, while the MSC-sIVs and MSC-sEVs groups showed smaller white infarct areas. Significant differences were observed between the PBS group and the MSC-sEVs and MSC-sIVs groups.

[0164] Figure 46 shows the results of TUNEL assay for neuronal apoptosis in the brain tissue of each group of stroke mice. Compared with the PBS group, the MSC-sIVs and MSC-sEVs treatment groups significantly reduced the number of apoptotic neurons in the cerebral cortex of photochemical stroke mice, and the differences were statistically significant compared with the PBS group (*p < 0.05).

[0165] Figure 47 shows the staining results for IBa-1, a microglial marker, in the brain tissue of each group of stroke-affected mice. The activation of microglia in the ischemic cortex was reduced in both the MSC-sIVs and MSC-sEVs groups compared to the PBS group (*p < 0.05 indicates a significant difference between groups). Compared to MSC-sEVs, MSC-sIVs intervention significantly reduced the number of activated microglia.

[0166] Figure 48 shows the results of immunofluorescence analysis of GFAP, a marker of astrocyte activation. It shows that GFAP protein expression in the brain tissue of stroke mice in the PBS group was significantly higher than that in both the MSC-sIVs and MSC-sEVs groups. MSC-sIVs can reduce GFAP expression, and the effect is more significant than that of MSC-sEVs (*p<0.05, ***p<0.001, and ****p<0.0001 indicate significant differences between the groups).

[0167] Figure 49 shows the results of immunofluorescence staining of β-tubulin III protein in the brain tissue of stroke mice. It shows that MSC-sEVs treatment significantly increased β-tubulin III protein expression in mouse brain tissue (*p < 0.05 indicates a significant difference between groups), and MSC-sIVs can increase β-tubulin III expression more significantly than MSC-sEVs.

[0168] Figure 50 shows the results of immunofluorescence staining for CD31, occludin, and ZO-1 in the brain tissue of stroke mice. It shows that the expression of CD31, occludin, and ZO-1 in the brain tissue of mice in the PBS group was significantly reduced. Compared with the PBS group, the levels of CD31, occludin, and ZO-1 in the brain tissue of mice treated with MSC-sIVs and MSC-sEVs were significantly increased.

[0169] Figure 51 shows the results of the Morris water maze test on Alzheimer's mice. Figure 51A shows that in the navigation test (finding the platform), the escape latency of the PBS group mice from day 4 to day 6 was significantly higher than that of the normal group, while the escape latency of the 5xFAD mice treated with MSC-sIVs and MSC-sEVs on days 4-6 was significantly lower than that of the 5xFAD mice treated with PBS. In addition, the MSC-sIVs group had a significantly shorter latency to reach the platform compared with the PBS group on days 4 and 6 of navigation, and the difference was statistically significant (*p < 0.05). Figure 51B shows that during the navigation phase on day 6, we found that the PBS mice had difficulty finding the platform and swam around the edge compared with the normal, MSC-sIVs, and MSC-sEVs groups, indicating that the PBS group had more severe spatial memory impairment.

[0170] Figure 52 shows the results of the open field test on Alzheimer's mice, which shows that the activity trajectories of mice in the PBS group tended to be in the peripheral area of ​​the open field.

[0171] Figure 53 shows the results of the Y-maze test in Alzheimer's mice. It shows that the spontaneous alternation rate of mice in the PBS group was significantly reduced; compared with the PBS group, the spontaneous alternation rates of mice in the normal group, MSC-sIVs, and MSC-sEVs groups were significantly increased (*p < 0.05, **p < 0.01 indicates significant differences between groups), indicating that learning and memory dysfunction in these mice was alleviated after treatment. DETAILED DESCRIPTION

[0172] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0173] The terms "patient" or "subject" and the like are used interchangeably herein to refer to any animal or cell thereof to be treated according to the methods described herein, whether in vitro or in situ. Specifically, the aforementioned animals include mammals, e.g., rats, mice, guinea pigs, rabbits, dogs, monkeys, humans, and particularly humans.

[0174] The term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and / or halting one or more clinical symptoms of a disease after onset of the disease.

[0175] The term "prevent" or "prevent" refers to treating a disease before it occurs to avoid, minimize, or make the onset or development of the disease more difficult.

[0176] The term "intracellular vesicle" is also called intracellular nanovesicle, small intracellular vesicle, and small intracellular nanovesicle.

[0177] The term "extracellular vesicles", also known as small extracellular vesicles, are a type of vesicles with a double membrane structure that are secreted by cells into the extracellular environment.

[0178] The nerve damage in the present invention may be caused by various reasons, for example, infection (some viruses, bacteria, fungi, parasites and other pathogens can cause nerve damage), trauma (such as chemical damage, photochemical damage, knife cuts, burns, firearms, abrasions, extrusions, impacts and other nerve damage), compression or traction injuries (such as common clinical intervertebral disc herniation or spinal stenosis, resulting in compression of spinal nerves and spinal cord, brachial plexus injury and lower limb nerve damage caused by women during pregnancy and childbirth), autoimmune mechanism (such as myasthenia gravis, multiple sclerosis and other nerve damage), ischemic injury (such as stroke, cerebral thrombosis), etc. ischemic nerve damage caused by formation), metabolic diseases (such as nerve damage caused by peripheral neuropathy such as diabetes), tumors, hereditary, nutritional, inflammatory, and drug damage (such as nerve damage caused by isoniazid and metformin affecting the absorption of vitamin B12). Its manifestations may include, but are not limited to, movement disorders (such as muscle weakness, paralysis or tremor), sensory disorders (such as numbness, tingling, paresthesia or loss of sensation), autonomic nervous system disorders (such as hypotension, syncope, constipation or urinary incontinence), cognitive disorders (such as amnesia, confusion or inattention), and mental disorders (such as depression, anxiety, abnormal behavior or mental confusion).

[0179] The term "neurological injury-related diseases" is also called neurological injury diseases, which refers to diseases that affect the function of the nervous system, including but not limited to spinal cord diseases, optic nerve diseases, cerebrovascular diseases, traumatic brain injury diseases, extrapyramidal diseases (also known as movement disorders), demyelinating diseases, neurodegenerative diseases, central nervous system infectious diseases, cerebral palsy, hydrocephalus, cranial nerve diseases, nerve root diseases, plexus diseases, single nerve diseases, multiple nerve diseases, neuromuscular junction diseases, ataxia, leukodystrophy, mental retardation, familial amyloid polyneuropathy (FAP), Charcot-Marie-Tooth disease (CMT, also known as hereditary motor sensory neuropathy (HMSN)), neurosis (such as hysteria, anxiety disorder, obsessive-compulsive disorder, autism, depression, panic disorder, neurasthenia, phobia, hypochondriasis, somatoform disorder).

[0180] The term "neurodegeneration-related diseases" is also called neurodegenerative diseases or nervous system degenerative diseases. It is a group of diseases with unknown causes that chronically progressively damage the central nervous system and peripheral nervous system and other tissues, including but not limited to motor neuron disease, Alzheimer's disease, multiple system atrophy (MSA), senile cerebral atrophy, Pick's disease dementia, alcoholic nervous system degeneration, frontotemporal dementia, subacute necrotizing encephalopathy (also known as Leigh syndrome), corticostriatal degeneration (CJD, also known as Creutzfeldt-Jakob disease), dementia with Lewy bodies (DLB), Machado-Joseph disease (MJD, also known as Azores disease).

[0181] The term "spinal cord disease" refers to diseases caused by spinal cord injury or damage, including but not limited to spinal cord injury, myelitis, poliomyelitis, encephalomyelitis, spinal cord tumors, spinal muscular atrophy (SMA), motor neuron disease, spinal cord compression, syringomyelia, syringomyelia, vascular myelopathy (such as spinal cord hemorrhage, spinal cord ischemia, spinal cord embolism, spinal cord infarction, spinal cord necrosis, spinal cord edema, spinal artery thrombosis, posterior spinal artery syndrome, anterior spinal artery occlusive syndrome, spinal vascular malformation, spinal arteriovenous malformation (AVMs)), hepatic myelopathy, cervical spondylotic myelopathy, spinal arachnoiditis, epidural abscess, spondylosis, cauda equina syndrome, HTLV-1 associated myelopathy / tropical spastic paralysis (HAM / TSP).

[0182] The term "optic nerve disease" refers to diseases of the optic nerve. Any interruption of optic nerve function due to any cause (vascular, compressive, toxic, or developmental abnormalities) results in an optic nerve disease, characterized by visual impairment. These diseases include, but are not limited to, optic nerve tumors, ischemic optic neuropathy, hereditary optic neuropathy, compressive optic neuropathy, optic nerve injury (also known as traumatic optic neuritis, traumatic optic neuropathy, traumatic optic nerve injury, TON), nutritional optic neuropathy, toxic optic neuropathy, optic neuritis, papilledema, optic atrophy, and glaucoma.

[0183] The term "cerebrovascular disease" refers to a group of diseases that occur in the blood vessels of the brain and cause brain tissue damage due to impaired intracranial blood circulation, including but not limited to ischemic cerebrovascular disease (such as cerebral ischemia, cerebral infarction (also known as ischemic stroke), cerebral thrombosis, cerebral embolism, ischemic-hypoxic encephalopathy), hemorrhagic cerebrovascular disease (such as cerebral hemorrhage, subarachnoid hemorrhage, non-traumatic subdural hemorrhage, non-traumatic epidural hemorrhage), cerebral atherosclerosis, cerebral arteritis, cerebral aneurysm, cerebral arteriovenous fistula, amyloid angiopathy, intracranial venous thrombosis, intracranial blood pressure malformation (such as cerebral arteriovenous malformation, cerebral cavernous malformation, dural arteriovenous fistula, carotid cavernous sinus fistula), intracranial telangiectasia, reversible cerebral vasoconstriction syndrome, vascular dementia (such as Binswanger's disease, multi-infarct dementia, hemorrhagic dementia), hypertensive encephalopathy, moyamoya disease, red core syndrome, Weber syndrome, Benedict's syndrome, and vascular leukoencephalopathy.

[0184] The term "extrapyramidal disease" refers to a group of unique movement disorders caused by extrapyramidal pathological changes mainly in the basal ganglia of the brain, mainly manifested by dysfunction of voluntary movement regulation, muscle strength, sensation and cerebellum are not affected, including but not limited to Hallevorden-Spatz disease (HSD, also known as pallidum-nigral degeneration), progressive supranuclear palsy, multiple system atrophy, calcification of the nucleus basalis (Fahr's disease), dystonia, orofacial movement disorders, Meige syndrome, essential tremor, myoclonus, chorea, restless legs syndrome, stiff-person syndrome, and hepatolenticular degeneration.

[0185] The term "demyelinating disease" is an autoimmune disease characterized by multifocal and inflammatory demyelination of the central nervous system, including but not limited to multiple sclerosis (such as brainstem multiple sclerosis, spinal cord multiple sclerosis, multiple sclerosis dementia), acute disseminated encephalomyelitis (ADEM), neuromyelitis optica, acute hemorrhagic leukoencephalitis (AHLE), diffuse sclerosis, central pontine myelinolysis, extrapontine myelinolysis, acute transverse myelitis, subacute necrotizing myelitis (FAS), concentric sclerosis (Balo disease), tumor-like demyelinating lesions (TDLs), white matter demyelination, demyelinating myelitis, and clinically isolated syndrome.

[0186] The term "neurodegenerative disease" is caused by the loss of neurons and / or their myelin sheaths, which worsens over time and causes functional impairment, including but not limited to epilepsy, cerebral ischemia (CI), brain injury (BI), Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), ataxia-telangiectasia syndrome, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, multiple sclerosis (MS), primary lateral sclerosis (PLS), spinal muscular atrophy, and spastic paraplegia.

[0187] The term "infectious diseases of the central nervous system" is divided into diseases caused by viruses, bacteria, fungi, rickettsia, spirochetes, parasites, etc., including but not limited to encephalitis, meningitis (such as pachymeningitis, leptomeningitis, arachnoiditis), myelitis, encephalomyelitis, ventriculitis, cerebellitis, brainstemitis, intracranial abscess (such as brain abscess, epidural abscess, subdural abscess), intracranial granuloma (such as brain granuloma, epidural granuloma, subdural granuloma), neurosyphilis, brain parasitic disease, and prion disease.

[0188] The term "cerebral palsy", also known as cerebral palsy or cerebral palsy, is a syndrome caused by non-progressive brain damage and developmental defects from conception to infancy, mainly manifested by movement disorders and abnormal postures, including but not limited to spastic cerebral palsy, dyskinetic cerebral palsy, ataxic cerebral palsy, and Worster-Drought syndrome.

[0189] The term "hydrocephalus" refers to a buildup of fluid in cavities deep within the brain that causes pressure on the brain and functional problems. This includes, but is not limited to, communicating hydrocephalus, obstructive hydrocephalus, compensated hydrocephalus, and congenital hydrocephalus.

[0190] The term "cranial nerve disease" refers to diseases caused by damage or dysfunction of the areas of the brain that control cranial nerves, nerve fibers in the brain that connect to cranial nerve centers, or cranial nerves, including but not limited to trigeminal nerve diseases (such as trigeminal neuralgia), facial nerve diseases (such as Bell's palsy, geniculate ganglionitis (also known as Hunter syndrome, Ramsay-Hunt syndrome), Merrow syndrome, hemifacial spasm, hemifacial atrophy, facial neuritis), olfactory nerve diseases, glossopharyngeal nerve diseases, vagus nerve diseases, hypoglossal nerve diseases, and multiple cranial nerve diseases.

[0191] The term "radiculopathy" refers to a class of acute or chronic conditions caused by long-term compression of nerve roots by lesions in or adjacent to the spine, including but not limited to nerve compression and nerve root sleeve cysts.

[0192] The term "plexus disease" refers to diseases of the brachial or lumbosacral plexuses that cause mixed motor and sensory impairments, occurring in the upper or lower extremities, respectively, including but not limited to brachial plexus injury and lumbosacral plexus injury.

[0193] The term "mononeuropathy" refers to damage to a single peripheral nerve, including but not limited to upper extremity mononeuropathies (e.g., carpal tunnel syndrome, ulnar neuropathy, radial nerve damage) and lower extremity mononeuropathies (e.g., sciatic nerve damage, femoral neuropathy, tibial neuropathy, tarsal tunnel syndrome, plantar nerve damage, intercostal neuropathies).

[0194] The term "polyneuropathy" refers to a group of diseases caused by diffuse peripheral nerve disorders whose symptoms are not limited to a single nerve distribution area or a single limb and are often bilaterally symmetrical, including but not limited to idiopathic progressive neuropathies, inflammatory polyneuropathy, and secondary polyneuropathy (such as diabetic polyneuropathy, infectious polyneuropathy, and nutritional polyneuropathy).

[0195] The term "neuromuscular junction disease" refers to a group of diseases with dysfunctional transmission at the neuromuscular junction, including but not limited to myasthenia gravis, myasthenic syndrome, muscular dystrophy, myotonia-related diseases (such as neuromyotonia, myotonia atrophica, dystrophic myotonia, myotonia congenita, paramyotonia), mitochondrial myopathy, alcoholic myopathy, inflammatory myopathy, muscular atrophy, periodic paralysis (also known as periodic paralysis), ocular myopathy, and myofibrillar myopathy.

[0196] The term "ataxia" refers to a disorder of coordinated movement in the presence of normal muscle strength, including, but not limited to, cerebellar ataxias (e.g., Marinesco-Sjogren syndrome, Ramsay-Hunt syndrome (also known as Hunter syndrome), episodic ataxia (EA), Friedreich's ataxia (FA), spinocerebellar ataxia (SCA), hereditary spastic ataxia (also known as Marie's ataxia), Kearn-Sayre syndrome (KSS), ataxia telangiectasia (also known as Louis Bar syndrome), CANVAS syndrome), hereditary spastic paraplegia (HSP, also known as Strumpell-Lorrain disease).

[0197] The term "neurosis" is also called neurosis, which is a group of mental disorders characterized by decreased mental activity, worry, tension, anxiety, depression, fear, compulsion, hypochondriasis, dissociative symptoms, conversion symptoms or neurasthenia, including but not limited to hysteria, anxiety disorder, obsessive-compulsive disorder, autism, depression, panic disorder, neurasthenia, phobia, hypochondriasis, and somatoform disorder.

[0198] The term "motor neuron disease" refers to a group of chronic progressive degenerative diseases that mainly damage the anterior horn of the spinal cord, the motor nuclei of the pontine nerves and the pyramidal tracts, including but not limited to amyotrophic lateral sclerosis (ALS), progressive spinal muscular atrophy (PMA), pyramidal tract degeneration, progressive bulbar palsy (PBP), pseudobulbar palsy, true bulbar palsy, primary lateral sclerosis (PLS), spinal bulbar muscular atrophy (SBMA, also known as Kennedy's disease), lower motor neuron syndrome, upper motor neuron syndrome, flail arm syndrome, and Madras motor neuron disease.

[0199] Each experiment in the examples of the present invention was repeated three times or more, and statistical analysis was performed using GraphPad Prism 90 software. Data are presented as mean ± standard error. Comparisons between two or more groups were performed using one-way analysis of variance, and differences between groups were tested using the Bonferroni post hoc test and the nonparametric Kruskal-Wallis test with Dunn's multiple comparison test. A p < 0.05 was considered statistically significant.

[0200] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.

[0201] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0202] Example 1: Extraction and culture of mesenchymal stem cells (MSCs)

[0203] Human umbilical cords were provided by Beijing Beilai Biotechnology Co., Ltd., China. Umbilical cords were obtained from uncomplicated normal pregnancies after cesarean section and immediately placed in saline containing penicillin (100 U / mL) and streptomycin (100 mg / mL) before being transported to the laboratory within 4 hours. After removing residual blood and blood vessels, the cords were cut into 1-3 mm pieces and digested with 0.1% type II collagenase for 1 hour at 37°C. The suspension was then filtered through a 100-mesh sieve to remove undigested tissue. The supernatant from the filtration was centrifuged and washed three times with PBS. The cell pellet was resuspended in Dulbecco's modified Eagle's medium / Nutrient Mixture F12 complete medium. The medium contained 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 mg / mL streptomycin. The cells were seeded in T175 culture flasks and cultured at 37°C in a 5% CO2 incubator. The medium was changed every 3 days. When the cell confluence reached 80%, they were passaged at a subculture ratio of 1:2, and P3 to P5 cells were used for experiments.

[0204] At the same time, the above cell culture can refer to the Chinese patent application with publication number CN118207158A (publication date: 2024.06.18).

[0205] Example 2: Preparation of cell-derived nanovesicles

[0206] The process of producing intracellular nanovesicles is shown in Figure 1.

[0207] 1. Cell digestion and counting

[0208] When cells grow to 90% confluence, remove the supernatant and wash the cells twice with PBS. Then, trypsinize the cells and neutralize and wash the cells three times with PBS. Then, count the cells and adjust the cell number to 1×10 with PBS. 6 pieces / mL.

[0209] 2. Ultrasonic treatment of cells

[0210] Take 1×10 6 Add 2 mL of cell suspension at a density of 1 cell / mL to the bottom of a 50 mL centrifuge tube. Place the ultrasound probe in the center of the liquid surface. Set the ultrasound amplitude to 20% and the duration to 15 seconds, with a 2-second on cycle and a 2-second off cycle. Place the centrifuge tube on ice. Transfer the suspension to a 2 mL centrifuge tube and centrifuge.

[0211] 3. Collection of Nanovesicles in Small Cells

[0212] Centrifugation parameters were 2000g for 10 minutes and 20,000g for 30 minutes. The supernatant was then collected and transferred to an ultracentrifuge tube and centrifuged at 150,000g for 70 minutes. All operations were performed on ice. The resulting pellet was resuspended in PBS to obtain small intracellular nanovesicles (sIVs).

[0213] 4. Collection of small extracellular vesicles

[0214] FBS is a necessary condition for culturing cells in vitro, but it contains a large number of bovine extracellular vesicles, which will also be present in complete cell culture medium containing FBS. In order to remove bovine extracellular vesicles, FBS was centrifuged at 110,000g at 4°C overnight (about 12 hours). When the cell fusion reached 60%, the cells were cultured for 48 hours in complete culture medium containing 10% exosome-removed FBS. Then, the supernatant was collected and the extracellular vesicles were separated by ultracentrifugation at 4°C. The specific steps include 300g×10min, 2000g×10min, 10,000g×30min and 110,000g×70min twice. The obtained precipitate was resuspended in PBS, which was small extracellular vesicles (sEVs) mainly composed of exosomes.

[0215] Example 3: Optimization of isolation parameters for intracellular nanovesicles

[0216] Referring to the steps of Example 2, the optimization process of the separation parameters of intracellular nanovesicles is shown in FIG2 .

[0217] 1. Nanoparticle size analyzer detects the difference in the yield of sIVs

[0218] PBS-resuspended sIVs were diluted to 1 ml with PBS and analyzed using Nanoparticle Tracking Analysis (NTA) software (NTA 3.3 Dev Build 3.3.104). The temperature was set to 25°C, the laser was set to Blue 488, the flow rate was set to 50, and the mode was set to automatic detection. Three injections and three analyses were performed, and the peak value average was used as the Mode particle size result. The camera mode was sCMOS, the laser type was Blue 488, and the viscosity was 0.9 cP.

[0219] 2. Transmission electron microscopy observation of the morphology of sIVs in different cells

[0220] Take the centrifuged sIVs, resuspend them in 200μL PBS solution and mix them evenly. Take 10μL of sIVs solution and mix it with 4% PFA in a volume ratio of 1:1, drop it on a clean plastic film to form a droplet, then put the front of the electron microscope carbon grid on the droplet and leave it for 20 minutes. Then negatively stain it with 10μL phosphotungstic acid for 90 seconds, bake the carbon grid dry, and observe it using a HitacW-7500 transmission electron microscope.

[0221] Figures 2A and 2B show the protein and vesicle yields of sIVs obtained according to this embodiment at an ultrasonic amplitude of 20% for exposure times of 5s, 10s, 15s, 20s, 25s, 30s, and 60s, respectively. The results show that the number of vesicles and protein yield decreased significantly when the exposure time was less than 10 seconds or greater than 20 seconds. Figures 2C and 2D show the protein and vesicle yields of sIVs obtained according to this embodiment at an ultrasonic exposure time of 15s for exposure times of 20%, 25%, 30%, 35%, and 40%, respectively. The results show that at an ultrasonic exposure time of 15s, the vesicle yield decreased significantly when the ultrasonic amplitude exceeded 25%. Figure 2E shows transmission electron micrographs of sIVs obtained according to this embodiment at an ultrasonic exposure time of 15s for exposure times of 20%, 25%, 30%, 35%, and 40%. FIG2F shows transmission electron micrographs of sIVs obtained according to this implementation procedure at an ultrasonic amplitude of 20% and ultrasonic times of 5 s, 10 s, 15 s, 20 s, 25 s, 30 s, and 60 s.

[0222] This optimization process showed that at a 20% sonication amplitude, vesicle yield dropped sharply when the sonication duration exceeded 10 or 20 seconds. At a sonication duration of 15 seconds, vesicle yield dropped sharply when the sonication amplitude exceeded 25%. A 20% amplitude and a sonication duration of 15 seconds were the optimal parameters for collecting intracellular nanovesicles.

[0223] For the preparation of cell vesicles, reference may be made to the Chinese patent application with publication number CN118207158A (publication date: 2024.06.18).

[0224] Example 4: sIVs have unique physical characteristics and high thermal stability

[0225] 1. Experimental instruments and materials

[0226] 1.1 Experimental Reagents

[0227] Table 1 Experimental reagents

[0228] 1.2 Experimental instruments

[0229] Table 2 Experimental instruments

[0230] 2. Experimental methods

[0231] 2.1 Transmission electron microscopy observation of the morphology of sIVs and sIVs

[0232] The centrifuged sEVs and sIVs (prepared in Example 2) were resuspended in 200 μL PBS solution and mixed evenly. 10 μL of the sEVs and sIVs solution was mixed with 4% PFA in a volume ratio of 1:1 and dropped onto a clean plastic film to form droplets. The front side of the carbon mesh was then placed on the droplets and allowed to stand for 20 minutes. 10 μL of phosphotungstic acid was used for negative staining for 90 seconds. The carbon mesh was dried and observed using a HitacW-7500 transmission electron microscope.

[0233] 2.2 Nanoparticle size analysis to detect the particle diameter of sEVs and sIVs

[0234] PBS-resuspended sEVs and sIVs were diluted to 1 ml with PBS and analyzed using Nanoparticle Tracking Analysis (NTA) 3.3Dev Build 3.3.104 software. The temperature was set to 25°C, the laser to Blue 488, the flow rate to 50, and the mode to automatic detection. Three injections and three analyses were performed, and the peak value average was used as the particle size result. The camera mode was set to sCMOS, the laser type to Blue 488, and the viscosity to 0.9 cP.

[0235] 2.3 Analysis of protein composition of sEVs and sIVs by Coomassie Brilliant Blue staining

[0236] After BCA quantification, take an equal amount of protein sample and dilute to 20 μl with PBS. Add 5 μl of protein loading buffer (5×) and heat at 95°C for 5 minutes. Electrophoresis should be performed at 100V for 90 minutes. After electrophoresis, add Coomassie Brilliant Blue Ultrafast staining solution and incubate at room temperature for 2 hours. Rinse with pure water until clear. Photograph the gel.

[0237] 2.4 Western Blot Analysis of Exosomal Marker Protein Composition of sIVs and sEVs

[0238] After protein detection using the BCA kit, take an equal amount of each group of protein samples, add PBS to make up to equal volume, add protein loading buffer (5×), and heat at 95°C for five minutes. Prepare the gel according to the instructions of the SDS-PAGE kit and insert the electrophoresis comb. Let it stand and wait for 25 minutes at room temperature for solidification. Place the prepared SDS gel in the pre-prepared electrophoresis tank. Remove the electrophoresis comb and inject the denatured protein into the SDS-PAGE loading tank. Add 1-4μl of marker on each side. Adjust the voltage to 60V. After the upper gel has run, change the voltage to 100V and stop electrophoresis when the bottom bromophenol blue indicator line is 1-2cm away from the bottom of the glass plate. Place the activated PVDF membrane on the SDS-PAGE surface, and then place filter paper and sponge pads on both sides of the gel and membrane. Use the column to gently roll to remove bubbles in the system and clamp the electrophoresis clamp. Set the black electrode as SDS-PAGE and the red electrode as PVDF membrane, and transfer the membrane under constant voltage while placing ice packs in the electrotransfer tank to cool it down. After electrotransfer, place the PVDF membrane in a TBST solution containing 5% skim milk or 1% BSA and block it at room temperature for 2 hours. After blocking, add the primary antibody and incubate overnight on a shaker in a 4°C refrigerator. The next day, remove the primary antibody and add TBST solution to wash the PVDF membrane. Then, add the secondary antibody and incubate on a shaker at room temperature for 2 hours. After incubation, remove the secondary antibody again and add TBST solution to wash the PVDF membrane 3 times, 10 minutes each time. Finally, use ECL supersensitive luminescent liquid to develop the PVDF membrane.

[0239] 3. Statistical processing

[0240] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0. One-way ANOVA was used for analysis of variance, with least significant difference (LSD) analysis for post hoc testing. Nonparametric tests were used for nonnormally distributed data and data with unequal variances, and P values ​​< 0.05 were considered statistically significant.

[0241] 4. Experimental results

[0242] 4.1 Transmission electron microscopy reveals the morphology of sEVs and sIVs

[0243] MSC-derived sIVs were enriched and examined using transmission electron microscopy. As shown in Figure 3, MSC-derived sEVs were round or horseshoe-shaped, with diameters ranging from 100 to 200 nm. sIVs were more numerous, round in shape, and less than 100 nm in diameter. Electron microscopy revealed that sIVs were significantly smaller in diameter than sEVs.

[0244] 4.2 Nanoparticle tracking analysis reveals the size distribution of sEVs and sIVs

[0245] The results of nanoparticle size detection are shown in Figure 4 . The particle size distribution of sEVs from MSCs cells is wide and the particle size is large, while the particle size distribution of sIVs is narrow and the particle size is small.

[0246] After statistical analysis, the results are shown in Figure 5. The average particle size of MSC sEVs was 123.1±4.453 nm, and the average particle size of sIVs was 75.28±9.067 nm; the particle size of sIVs was smaller than that of sEVs.

[0247] 4.3 Comparison of sEV and sIV yields at equal cell counts

[0248] To compare the yields of the two types of vesicles, we collected sEVs and sIVs from cell culture supernatants and adherent cells. 7 The number of sIVs vesicles produced by cells was 10- to 20-fold higher than that of sEVs (Figure 6A). 7 The protein production in cellular sIVs was 20- to 40-fold higher than that in sEVs (Figure 6B), indicating that the production of sIVs was much higher than that of sEVs.

[0249] 4.4 Differences in protein distribution between sEVs and sIVs

[0250] Total proteins from cells, sEVs, and sIVs were separated by SDS-PAGE and stained with Coomassie Brilliant Blue to visualize total protein distribution. The staining results showed that cellular proteins exhibited the most abundant bands, with multiple high-abundance protein bands. sEVs contained fewer protein species, with high-abundance proteins located around 200 kD and 70 kD. sIVs contained more protein species than sEVs, with high-abundance proteins located around 250 kD and 55 kD (Figure 7). The differences in protein distribution between different cells preliminarily suggest that sEVs and sIVs have distinct protein compositions and differ from the total protein distribution of both cells and sEVs.

[0251] To further analyze the protein expression characteristics of cells, their sEVs, and sIVs, Western blot was used to examine the expression of exosomal marker proteins, including Alix, HSP70, TSG101, CD63, and CD81, in cells, their sEVs, and sIVs under isoprotein conditions. The results are shown in Figure 8. Cellular sEVs expressed the most exosomal marker proteins, and the cells themselves also expressed a certain amount of Alix, HSP70, TSG101, and CD63. However, sIVs expressed little Alix and CD81, and the expression levels of HSP70, TSG101, and CD63 were much lower than those of cells and sEVs. This further demonstrates that sIVs lack exosome characteristics and are not the intracellular precursors of exosomes.

[0252] 4.5 Comparison of the stability of sEVs and sIVs at different temperatures

[0253] To evaluate the stability of the two vesicles at different temperatures, the sIVs and sEVs suspensions were divided into three equal parts and stored at different temperatures (-80°C, 4°C, and 37°C). After 24 hours, the morphology, size, and protein amount of sEVs and sIVs were evaluated. Both vesicles were stable at -80°C and 4°C. However, transmission electron microscopy images showed that the morphology of sEVs was impaired at 37°C, with irregular shapes, broken vesicles, and rough boundaries (Figure 9), and the number of sEVs was also reduced (Figure 10B), while the morphology and particle number of sIVs remained stable at 37°C. The above results indicate that sIVs have higher thermal stability than sEVs.

[0254] 4.6 Super-resolution imaging reveals that the distribution of sIVs in cells is different from that of sEVs

[0255] To gain a deeper understanding of the intracellular distribution of sIVs, we used proteomic analysis to identify proteins uniquely expressed in sIVs compared to sEVs, namely IV characteristic proteins. We made the characteristic proteins carry green fluorescent protein and performed intracellular imaging to visualize the morphology of sIVs in cells. By performing proteomic analysis on sIVs and sEVs, we identified proteins uniquely expressed in sIVs. The protein expression abundance was ranked from high to low, and the top 50 proteins were displayed (Figure 11). We observed that the expression abundance of TMEM214 protein was the highest in MSCs. TMEM214 is a transmembrane protein involved in cellular processes such as vesicle transport and protein transport (Zhao J., Xu J., Wang Y., et al. Membrane Localized GbTMEM214s Participate in Modulating Cotton Resistance to Verticillium Wilt. Plants (Basel). 2022 Sep 8; 11(18): 2342.). Therefore, we used green fluorescent protein (GFP) to label TMEM214 to visualize the intracellular status of sIVs, and used GFP-labeled CD63 as a marker of sEVs in cells.

[0256] Super-resolution microscopy combined with total internal reflection fluorescence structured illumination microscopy (TIRF-SIM) showed that CD63 was visible on the cell membrane (Figure 12A, left), while TMEM214 was not expressed on the cell membrane (Figure 12A, right). This finding excludes the possibility that sIVs originate from cell membrane remodeling and confirms that the origin and activity site of sIVs are located within the cell. Super-resolution microscopy using Wildfield-2DSM scanning mode provides an overview of protein expression throughout the cell, and images show that both CD63 and TMEM214 are significantly expressed within the cell (Figure 12B). Subsequently, under wide-field conditions, we observed the dynamic changes in the structures of these protein markers in living cells. Pictures were taken every 10 seconds for 15 minutes to form a dynamic video. In the video screenshot, the dynamic release of sEVs from the cell membrane to the extracellular space can be observed (Figure 12C, left, white arrow), while TMEM214-labeled sIVs are diffusely distributed within the cell and are not released extracellularly (Figure 12C, right). Intuitive microscopic imaging shows that sIVs are diffusely distributed in the cell in a cloud-like manner and are not released extracellularly.

[0257] 5. Summary

[0258] In this example, we took MSCs cells as an example to collect and characterize sIVs and sEVs. Transmission electron microscopy and nanoparticle size analysis revealed that sIVs were significantly smaller than sEVs; the total protein expression patterns of cells, sEVs, and sIVs were different, with sIVs expressing low exosome marker proteins; and at the same cell number, the yield of sIVs was significantly greater than that of sEVs. At -80°C, the stability of sIVs and sEVs was comparable, but at 37°C, the stability of sIVs was significantly better than that of sEVs. Super-resolution imaging observed that sEVs were released from the cell membrane to the outside of the cell, while sIVs were active inside the cell. In general, the sIVs vesicles collected by the method of the present invention contain a unique protein composition, are highly stable at physiological temperature, and have a yield much higher than that of extracellular vesicles.

[0259] Example 5: Quantitative proteomic analysis reveals that sIVs have unique protein expression profiles

[0260] 1. Experimental instruments and materials

[0261] 1.1 Experimental Reagents

[0262] Table 3 Experimental reagents

[0263] 1.2 Experimental instruments

[0264] Table 4 Experimental instruments

[0265] 2. Experimental methods

[0266] 2.1 Sample preparation for protein profiling of cells and their sEVs and sLVs

[0267] 2.1.1 Protein extraction

[0268] 1) Add 220 μl of urea lysis buffer (8 M urea, 50 mM NH 4 HCO 3 , protease inhibitors) to each of the cells, sEVs, and sIVs (prepared in Example 2) and lyse at room temperature for 5 minutes.

[0269] 2) Ultrasonic disruption on ice (energy 35%, ON 3s, OFF 3s, total ultrasonic time 2 min), insert the sample tube into an ice box, set the centrifuge temperature to 20°C, centrifuge at 14,000g for 10 min, remove the supernatant, and repeat the centrifugation once.

[0270] 3) Protein concentration was determined by BCA assay, and 100 μg protein was collected from each of cells, sEVs, and sIVs.

[0271] 2.1.2 Protein denaturation

[0272] 1) Add DTT to each sample tube of cells, sEVs, and sIVs to a final concentration of 10 mM and incubate at 37°C for 1 hour to reduce the proteins.

[0273] 2) Add IAA to each sample tube containing cells, sEVs, and sIVs to a final concentration of 40 mM and incubate at room temperature for 1 hour in the dark.

[0274] 3) First, label the sample number on the collection tube. Equilibrate a 10 kDa ultrafiltration tube twice with 150 μl of HPLC-grade methanol each time. Centrifuge at 14,000 g for 5 min, then add 300 μl of 50 mM NH4HCO3. Rinse twice, add 100 μg of the reductively alkylated protein sample, centrifuge at 14,000 g for 20 min at 4°C, and rinse three times with 300 μl of 50 mM NH4HCO3. Replace the collection tube with a new one, and add 75 μl of 50 mM NH4HCO3 to the ultrafiltration tube.

[0275] 2.1.3 Protease cleavage

[0276] 1) Add 3 μg of trypsin for mass spectrometry and incubate in a 37°C incubator for 14-16 hours.

[0277] 2) The next day, centrifuge at 14,000 g for 20 minutes at 4°C, add 50 μl of 50 mM NH4HCO3, rinse twice, add 1% (volume ratio) formic acid to the collection tube to terminate the enzyme digestion, and evaporate to dryness in vacuo at 60°C.

[0278] 2.1.4 Library construction and fractionation

[0279] 1) Resuspend the samples in 30 μl of 0.1% formic acid and measure the concentration using a nanodrop. Next, remove approximately 10 μg of peptides from each sample and combine them into one sample, S.

[0280] 2) 6 μg of sample S was taken out for mass spectrometry analysis in DDA mode, and the remaining S sample was fractionated using a homemade high pH reverse phase column.

[0281] 3) For fractionation, prepare the required reagents according to Table 5. Buffer A is 100% acetonitrile (ACN), and Buffer B is 0.1% trifluoroacetic acid (TFA).

[0282] Table 5 Fractionation reagent ratio

[0283] 4) Use a wire to load a C18 membrane into a 200 μl pipette tip. Next, dissolve 30 mg of high-pH-resistant C18 filler in 200 μl of Buffer A and add the filler to the fractionating column. Centrifuge at 3,000 g for 2 minutes at 4°C. Wash once with 200 μl of Buffer A and three times with 200 μl of Buffer B. Finally, set the column aside for future use.

[0284] 5) Load the cleaved peptides onto the fractionation column and repeat the loading process 5 times.

[0285] 6) Wash the column three times with 200 μl of Buffer B. Next, add 150 μl of elution buffer of varying concentrations and perform gradient elution. Combine the 6% and 35% fractions into one fraction, and extract 1.5 μg of peptides from each fraction for DDA mass spectrometry analysis.

[0286] 2.2 Liquid chromatography mass spectrometry parameters of cells and their sEVs and sIVs

[0287] The enzymatically digested peptides were loaded onto a homemade Trap column (100 μm × 2 cm, C18 filler, 3 μm particle size, 120A) using phase A (containing 0.1% formic acid, 2% acetonitrile, and 97.9% water) at a flow rate of 3 μl / min. Subsequently, the Trap column was eluted using different gradients of phase B (containing 97.9% acetonitrile, 2% water, and 0.1% formic acid). These eluted peptides passed through an analytical column (150 μm × 15 cm, C18 filler, 1.9 μm particle size, 120A) to form an electrospray and ultimately entered the mass spectrometer detector.

[0288] The gradient of phase B was set as follows: 0 min to 5%, 2 min to 10%, 65 min to 22%, 91 min to 35%, 92 min to 80%, 105 min to 80%, 106 min to 5%, and 120 min to 5%. The flow rate was maintained at 500 nL / min throughout the process.

[0289] For DDA scans, the mass spectrometer parameters were set as follows: a TOF MS accumulation time of 0.25 seconds, a mass scan range of 300–1500 Daltons (Da), detecting only ions with charges of +2 to +5, and a mass deviation of less than 50 ppm. A maximum of 60 ions were monitored per cycle, and after each detection, the detected ions were isolated for 16 seconds. Dynamic fragmentation mode was used for fragmentation energy. The accumulation time for product ions was 0.04 seconds, and high-sensitivity scanning mode was used.

[0290] During DIA scanning, the mass spectrometer parameters differed: the TOF MS had an accumulation time of 0.05 seconds, and the secondary scan was performed in high-sensitivity mode. The number of variable windows was set to 100, with an accumulation time of 30 milliseconds per window. The mass scan range was also 300–1500 Da. The specific mass range for each variable window was calculated using the SWATH Variable Window Calculator_V1.1 program.

[0291] 2.3 Cell and sEVs / sIVs protein profile data processing and bioinformatics analysis

[0292] Proteinpilot software (version 5.0.1) was used to perform database searches on the raw data collected using the DDA mode, using trypsin as the digestion method. The Uniprot human database, which contains 20,431 annotated proteins and was released in July 2019, was used. The screening criteria were an unused protScore greater than 0.05. The Proteinpilot search results were imported into SWATH software (version 2.0) as a database for quantification of the DIA mode data.

[0293] During quantification, six peptides were selected for each protein, and six transitions (ion pairs) were selected for each peptide. Peptide confidence was set to 99%, and the FDR (false positive rate) was set to 1%. Modified peptides were excluded, the peak extraction window was set to 10 minutes, and the mass deviation was controlled within 50 ppm. Two endogenous peptides were selected every 10 minutes to correct for retention time, and the output peak area was used as the quantitative value.

[0294] Processing of protein expression data involved the following steps: First, raw quantitative values ​​were log2 transformed to conform to a normal distribution and then normalized using the normalize.quantiles function in the preprocessCore package in R. After removing proteins without gene names, differential expression analysis was performed using the stats package in R. Proteins with a P-value less than 0.05 and a fold change greater than 1.5 were selected as differentially expressed proteins. Corrected p-values ​​were set to less than 0.05.

[0295] GO and pathway analyses were performed using the Cytoscape plugin clueGo. For GO enrichment analysis, cellular component (CC), molecular function (MF), and biological process (BP) were selected. For pathway enrichment analysis, the kegg and reactome databases were used.

[0296] Heatmaps, principal component analysis (PCA) scores, Venn diagrams, and volcano plots were performed using R or Hiplot software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using Metascape online analysis software.

[0297] 2.4 Enzyme-linked immunosorbent assay

[0298] MSC-derived sEVs and sIVs were collected, adjusted to equal mass, and performed according to the instructions for the enzyme-linked immunosorbent assay (ELISA) kit. The samples were added to the antibody-coated wells and incubated for 2 hours, and the plate was washed. Biotin was added, incubated for 1 hour, and the plate was washed. HRP was added, incubated for 1 hour, and the plate was washed. TMB substrate was added and incubated for 20 minutes. STOP solution was added until color was apparent. The absorbance of each well in the plate was read using a microplate reader at wavelengths of 450 nm and 540 nm.

[0299] 3. Statistical processing

[0300] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0, and statistical analysis between the two groups was performed using the t test. P values ​​< 0.05 were considered statistically significant.

[0301] 4. Experimental results

[0302] 4.1 Venn diagram showing differences in protein composition between cells and their sEVs and sIVs

[0303] To characterize the molecular composition of sEVs and sIVs, we performed proteomic analysis of sEVs and sIVs derived from MSCs using label-free mass spectrometry and compared the analysis with the cells. In MSCs, 2744 proteins were identified; 1678 proteins were detected in sEVs, and 2066 proteins were found in sIVs (Figure 13). There was some overlap in the protein types between the two, but they were not identical. The protein content in sIVs was more diverse than that in sEVs.

[0304] 4.2 Principal component analysis reveals differences in protein composition of cells and their sEVs and sIVs

[0305] PCA analysis of the protein components of cells, sEVs, and sIVs (Figure 14) revealed distinct protein distribution patterns among cells, sEVs, and sIVs. sEVs and sIVs exhibited significant differences in protein expression, suggesting that sIVs possess unique protein expression profiles distinct from sEVs.

[0306] 4.3 Quantifiable protein differential analysis of cells and their sEVs and sIVs

[0307] Further statistics showed that there were 1425 differentially expressed proteins between sEVs and sIVs in MSC cells, of which 753 were significantly downregulated in sIVs compared with sEVs, 672 were significantly upregulated, 468 of which had a fold difference greater than 10-fold, 227 were significantly downregulated in sIVs compared with sEVs, and 241 were significantly upregulated (Figure 15), further demonstrating the uniqueness of sIVs.

[0308] The most significantly upregulated and downregulated proteins in sEVs and sIVs were not identical (Figure 16). However, membrane-associated proteins were expressed at lower levels in sIVs, while endoplasmic reticulum and ribosome-associated proteins were more abundant in sIVs. This initially suggests that sIVs are distinct entities produced by cells, rather than precursors or fragments of cell lysates or extracellular vesicles.

[0309] 4.4 Differences in the expression of exosomal markers in cells and their sEVs and sIVs

[0310] We extracted the list of exosome markers recommended by MISEV2018 (Théry C., Witwer KW, Aikawa E., et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines [J]. Journal of extracellular vesicles, 2018, 7(1): 1535750.) and compared sEVs and sIVs with these markers. sEVs showed higher expression levels of exosome markers, while sIVs had lower expression levels of most exosome marker proteins (Figure 17). This further indicates that sIVs do not have exosome characteristics and are unique vesicles from the inside of cells.

[0311] 4.5 Differential expression of organelle marker proteins in cells and their sEVs and sIVs

[0312] We also compared the organelle protein expression profiles of sEVs and sIVs. Overall, sIVs contained higher levels of intracellular organelle proteins than sEVs (Figure 18). In particular, sIVs showed elevated expression levels of proteins associated with membrane-enriched organelles such as endosomes, the endoplasmic reticulum, and the Golgi apparatus. In contrast, sEVs contained more abundant cellular membrane proteins (Figure 18). This suggests that sIVs possess intracellular characteristics.

[0313] 4.6 Differential expression of the Clathrin protein family in cells and their sEVs and sIVs

[0314] Among the proteins contained in intracellular vesicles, the Clathrin family of proteins is essential for the organization and activity of vesicles. Clathrin proteins play a key role in intracellular transport by promoting the transport of cargo between organelles such as the endoplasmic reticulum, Golgi apparatus, and endosomes in the secretory and endocytic pathways. Given the important role of the clathrin family, we compared the expression levels of clathrin family proteins in sEVs and sIVs. Notably, we observed that the expression of most clathrin family proteins was upregulated in sIVs, while sEVs showed low expression (Figure 19). This finding suggests that the sIVs we isolated may participate in the communication between different cellular compartments within the cell.

[0315] 4.7 Enrichment analysis of sIVs-specific proteins

[0316] In Example 4, we analyzed 106 proteins that were expressed by sIVs that were different from those expressed by sEVs. These proteins are unique proteins expressed by sIVs and can represent the characteristics of sIVs. We performed gene enrichment analysis on these proteins. In terms of cellular components (CC), these proteins are related to COPII-coated endoplasmic reticulum to Golgi transport vesicles, transport vesicles, coated vesicles, endoplasmic reticulum to Golgi transport vesicle membranes, endoplasmic reticulum to Golgi intermediate compartments, transport vesicle membranes, coated vesicle membranes, etc. (Figure 20). At the same time, in the biological process (BP) category, the terms are glycerophospholipid biosynthesis process, response to endoplasmic reticulum stress, ubiquitin-dependent ERAD pathway, intracellular protein transport, and endoplasmic reticulum to Golgi vesicle-mediated transport, etc. (Figure 21). This result indicates that the sIVs we isolated are intrinsic vesicle components within the cell.

[0317] 4.8 Comparison of cytokines contained in sEVs and sIVs

[0318] Intracellular vesicles are involved in the intracellular transport of various secreted factors, while exosomes carry these factors to the extracellular space. Therefore, we compared the levels of cytokines carried by sEVs and sIVs. Proteomic analysis revealed that sIVs contained lower levels of interleukin-1β (IL-1β) and insulin-like growth factor 2 (IGF-2) compared to sEVs (Figure 22). Subsequently, we further quantified low-abundance cytokines using ELISA. The results showed that, based on the same mass, sIVs contained high levels of insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), and interleukin-10 (IL-10) (Figure 23). Furthermore, there was no significant difference in IL-6 levels between sEVs and sIVs (Figure 23D), while sIVs contained lower levels of tumor necrosis factor α (TNFα) (Figure 23E).

[0319] 5. Summary

[0320] In this example, we used proteomics to characterize the protein composition of MSCs, sEVs, and sIVs. The results showed that sIVs had a unique protein expression profile, distinguishing them from cells and sEVs. sIVs expressed low levels of exosome markers and high levels of markers for membrane-rich organelles and the Clathrin family of proteins within the cell, suggesting that sIVs play a role in intracellular cargo transport and mediate intracellular organelle communication. Gene enrichment analysis of sIVs directly implicated sIVs in cargo transport between the endoplasmic reticulum and the Golgi apparatus, including forward vesicle-mediated transport from the endoplasmic reticulum to the Golgi apparatus and retrograde vesicle-mediated transport from the Golgi apparatus back to the endoplasm. These findings strongly suggest that sIVs play a key role in intracellular cargo transport, particularly in mediating inter-organelle cargo communication. Among them, COP-coated vesicles have been widely reported to participate in intracellular cargo transport initiated by the endoplasmic reticulum. During this process, correctly folded and assembled proteins in the endoplasmic reticulum (ER) are encapsulated into COP-coated transport vesicles, which then detach from the ER membrane. Subsequently, the vesicles shed their coating and fuse with each other to form tubular clusters of vesicles. The Golgi apparatus is responsible for modifying the proteins and lipids received from the ER and distributing them to the cell membrane, endosomes, and secretory vesicles. Within the Golgi apparatus, proteins and lipids move in a cis-to-trans direction, completing this process through vesicular transport. Proteomic analysis further confirmed that sIVs are involved in intracellular vesicular transport and are closely associated with the ER, Golgi apparatus, and COP-coated vesicles.

[0321] The above results indicate that sIVs are completely different from sEVs, sIVs play an important role in intracellular substance transport, and sIVs are a unique group of vesicles.

[0322] Example 6: sIVs have unique miRNA expression profiles

[0323] 1. Experimental instruments and materials

[0324] 1.1 Experimental Reagents

[0325] Table 6 Experimental reagents

[0326] 1.2 Experimental instruments

[0327] Table 7 Experimental instruments

[0328] 2. Experimental methods

[0329] 2.1 Data Collection and Analysis

[0330] 2.1.1 RNA isolation, library preparation, and sequencing

[0331] After separation and enrichment of sEVs and sIVs (prepared in Example 2), they were resuspended in PBS and tested for RNA degradation and contamination on a 1% agarose gel. Check RNA purity using a spectrophotometer. RNA concentration was determined using 2.0 Qubit in Flurometer TM RNA was measured using the RNA Nano 6000 assay kit on the Agilent Bioanalyzer 2100 system.

[0332] 2.1.2 Library Preparation for Small RNA Sequencing

[0333] 3 μg of total RNA from each sample was used as the input sample for the small RNA library. Multiplex Software Small RNA Library Prep Set for (Generated, and index codes were added for the purpose of assigning sequences to each sample. Amplification was performed on a PCR instrument using LongAmp Taq 2X Master Mix, SR Primer for illumina, and index (X) primers. PCR products were then purified on an 8% polyacrylamide gel (100 V, 80 min). DNA fragments corresponding to 140 to 160 bp were recovered and dissolved in 8 μL of elution buffer. Finally, library quality was assessed on an Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.

[0334] 2.1.3 Cluster Generation and Sequencing

[0335] The indexed samples were clustered using the TruSeq SR Cluster Kit v3-cBot-HS (Illumia) on the cBot Cluster Generation System according to the manufacturer's instructions. After cluster generation, sequencing was performed on the Illumina HiSeq 2500 / 2000 platform to generate 50 bp single-end reads for library preparation.

[0336] 2.1.4 Data Analysis

[0337] 1) Quality Control

[0338] First, the raw data (raw reads) in fastq format were processed by custom Perl and Python scripts. In this step, clean reads were obtained from the raw data by removing reads containing ploy-N, 5' end joint contamination, no 3' end joint or insertion tag, containing ploy A or T or G or C, and low quality. At the same time, the Q20, Q30 and GC content of the raw data were calculated. Then, a certain length range was selected from the clean reads for all downstream analyses. Bowtie (Langmead B., Trapnell C., Pop M., Salzberg SLU ultrafast and memory-efficient alignment of short DNA sequences to the human genome [J]. Genome biology, 2009, 10 (3): R25.) was used to map the small RNA tags to the reference sequence, without allowing mismatches, to analyze their expression and distribution on the reference sequence.

[0339] 2) Alignment of known miRNAs

[0340] The mapped small RNA signatures were used to search for known miRNAs. Using miRBase20.0 as a reference, the modified software mirdeep2 ( MR, Mackowiak SD, Li N., et al. miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades [J]. Nucleic acids research, 2012, 40(1): 37-52) and srna-tools-cli were used to identify potential miRNAs and map their secondary structures. Custom scripts were used to obtain the miRNA count and base bias at the first position of identified miRNAs of a specific length, and the miRNA count and base bias at each position of all identified miRNAs.

[0341] 3) Summary of small RNA annotation

[0342] Summarize all previously obtained alignments and annotations. In the previous alignments and annotations, some small RNA tags may be mapped to multiple categories. To ensure that each unique small RNA is mapped to only one annotation, we follow the following priority rule: known miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene>new miRNA.

[0343] 4) Data Analysis

[0344] Target gene prediction was performed using miRanda, and miRNA target gene prediction was performed using the intersection of miRanda and RNAhybrid software. Differentially expressed miRNA input data were read count data obtained from miRNA expression level analysis. For samples with biological reproducibility: differential expression analysis between two conditions / groups was performed using the DESeq R package (3.0.3). P values ​​were adjusted using the Benjamini & Hochberg method. By default, the adjusted P value was set to 0.05 as the threshold for significant differential expression. Heat maps, principal component analysis, Venn diagrams, volcano plots, etc. were drawn online using Hiplot and adjusted using Adobe Illustrator.

[0345] 5) GO and KEGG enrichment analysis

[0346] GO enrichment analysis was performed on candidate target genes of differentially expressed miRNAs (hereafter referred to as "target gene candidates"). GO enrichment analysis was performed using GOseq, which is based on the Wallenius noncentered hypergeometric distribution and adjusts for gene length bias. KOBAS software was used to test the statistical enrichment of target gene candidates in Kyoto Encyclopedia of Genes and Genomes pathways.

[0347] 3. Experimental results

[0348] 3.1 Relative RNA abundance in sEVs and sIVs

[0349] First, we analyzed the RNA abundance of sEVs and sIVs using a bioanalyzer ( Figure 24 ).

[0350] 3.2 Distribution of small RNAs in sEVs and sIVs

[0351] Small RNAs associated with cells and extracellular vesicles have been a hot topic of research in recent years, especially microRNAs (miRNAs), which have multiple biological functions and can serve as biomarkers for various diseases. We analyzed the types of small RNAs in sEVs and sIVs. The alignment and annotation of each type of small RNA with total RNA were summarized. Since there are cases where one sRNA is aligned with several different annotation information at the same time, in order to make each unique sRNA have a unique annotation, small RNAs were classified according to the priority order of known miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene>novel miRNA detection, and the proportion of each small RNA in total RNA was calculated. The results showed that in sEVs, YRNA is the most predominant RNA (YRNA is a highly conserved small non-coding RNA (see Xie Yuxin, Chen Tianxing, Wang Li, et al. YRNA: Research progress in cancer and non-cancer [J]. Chinese Journal of Experimental Surgery, 2021, 38(9):1844-1848.)); in sIVs, miRNA is the most predominant RNA. MiRNAs accounted for 29.15% of MSC sEVs and 92.52% of sIVs. sIVs had a richer variety of small RNAs, with miRNAs accounting for a significantly higher proportion than sEVs (Figure 25).

[0352] 3.3 Global miRNA expression characteristics of sEVs and sIVs

[0353] MiRNAs have a rich array of biological regulatory roles and comprise a significant proportion of small RNAs. Therefore, we conducted a subsequent analysis of miRNAs, using a Venn diagram to analyze the miRNA species present in sEVs and sIVs. The results revealed 694 miRNAs detected in sEVs and 989 miRNAs in sIVs (Figure 26). While there was some overlap in miRNA species, they were not identical.

[0354] Because sEVs and sIVs contain commonly expressed miRNAs, principal component analysis was subsequently used to compare the miRNA expression patterns of sEVs and sIVs. The results showed that the miRNA components contained in the two vesicles were quite different and the expression patterns were unrelated (Figure 27), further demonstrating that sIVs are different from sEVs and contain a unique miRNA expression profile.

[0355] 3.4 Highly Abundant miRNAs in sEVs and sIVs

[0356] Analysis of the top 10 most abundant miRNAs in sEVs and sIVs revealed that miR-148a-3p and let-7i-5p were highly expressed in sEVs, while let-7f-5p was highly expressed in sIVs. Furthermore, miRNAs such as miR-148-3p, miR-21-5p, and miR-100-5p were highly expressed in both sEVs and sIVs (Figure 28).

[0357] 3.5 Analysis of differentially expressed miRNAs in sEVs and sIVs

[0358] To further compare the differences in miRNA expression between sEVs and sIVs, we performed differential expression analysis. The results showed 70 differential miRNAs in MSCs between sEVs and sIVs, of which 22 were significantly downregulated and 48 were significantly upregulated in sIVs compared to sEVs (Figures 29-30), further demonstrating the differences between sIVs and sEVs.

[0359] 3.6 Enrichment analysis of differentially expressed miRNA target genes in sEVs and sIVs

[0360] MiRNAs exert their biological effects by regulating downstream target genes. Therefore, after comparing the differentially expressed miRNAs in each group, we performed gene enrichment analysis on the target gene sets of these miRNAs, including GO analysis and KEGG analysis. For the sake of convenience, we will refer to "target genes of differentially expressed miRNAs" as "candidate target genes." The results of GO enrichment analysis showed that the candidate target genes of sEVs and sIVs of MSCs were related to intracellular metabolic processes, their cellular localization was intracellular membrane-related organelles, and their molecular functions were related to protein binding and enzyme metabolic reactions (Figure 31A). KEGG pathway analysis showed that the candidate target genes of sEVs and sIVs of MSCs were related to pathways such as axon guidance, cell differentiation, endocytosis, and immune regulation (T cell receptor signaling pathway, B cell receptor signaling pathway) (Figure 31B).

[0361] 4. Summary

[0362] In this example, we used small RNA sequencing technology to analyze the small RNA composition of sEVs and sIVs using MSCs, and further explored the expression patterns of miRNAs. Intracellular miRNAs are produced in the nucleus and transported to the cytoplasm, where they participate in the regulation of target genes. Therefore, miRNAs produced intracellularly must primarily perform their biological functions by regulating gene expression and participating in various cellular biological processes, such as cell proliferation, differentiation, and apoptosis. Many miRNAs have been found to be specifically expressed in different stem cell types, regulating cell differentiation and the maturation of specific cell lineages. Other miRNAs may promote or inhibit cell death signaling pathways, thereby affecting cell survival and apoptosis. They can maintain cellular homeostasis by regulating apoptosis-related genes, such as BCL2 family members, caspases, and p53. Within cells, miRNAs can act as signaling regulators to regulate cellular biological processes, such as growth factor signaling, responses to oxidative stress, and inflammatory responses. They can target key molecules in specific signaling pathways, thereby affecting the entire signaling pathway. Our experimental results indicate that sIVs are rich in miRNAs, suggesting that they may play a key role in regulating gene expression, cell proliferation, differentiation, growth, apoptosis, and signaling pathways, and thus possess significant potential for application. Our results revealed that sIVs exhibited a unique miRNA expression profile, significantly different from that of sEVs, with a higher abundance of miRNAs in sIVs. This finding suggests that sIVs have potential biological regulatory roles and may facilitate communication between different intracellular organelles. Candidate gene enrichment analysis of differentially expressed miRNAs in sEVs and sIVs further revealed that sIVs are closely associated with intracellular membrane-like organelles. In summary, sEVs and sIVs differ in their small RNA composition, particularly miRNAs. This miRNA enrichment analysis further confirms that sIVs play an important role in intracellular cargo transport. Compared to sEVs, sIVs contain a more diverse miRNA population and may possess a wider range of biological functions. These findings provide important insights into the functions of sIVs in cell biology.

[0363] Example 7: sIVs have unique lipidomic characteristics

[0364] 1. Experimental instruments and materials

[0365] 1.1 Experimental Reagents

[0366] Table 8 Experimental reagents

[0367] 1.2 Experimental instruments

[0368] Table 9 Experimental instruments

[0369] 2. Experimental methods

[0370] 2.1 Metabolite extraction

[0371] After separation and enrichment of sEVs and sIVs (prepared in Example 2), the samples were resuspended in PBS. 200 μL of water was added to the container, followed by 480 μL of an extract, which was a mixture of MTBE and MeOH in a ratio of 5:1 and contained an internal standard substance. The mixed solution was quickly placed in a liquid nitrogen tank and frozen for 1 minute, then taken out to thaw and mixed by a vortex mixer for 30 seconds to make the solution uniform. The above freezing, thawing and mixing steps were repeated 3 times, followed by ultrasonic treatment in an ice-water bath for 10 minutes. The treated sample was allowed to stand at -40°C for 1 hour. Then, the sample was separated by centrifugation at 3000 rpm (centrifugal force 900 × g, radius 8.6 cm) for 15 minutes at 4°C. 300 μL was taken out from the supernatant, transferred to an EP tube, and vacuum dried. 100 μL of a complex solution (DCM:MeOH=1:1) was added to the dried sample, vortexed for 30 seconds, and ultrasonically treated again in an ice-water bath for 10 minutes. Finally, centrifuge at 13,000 rpm (centrifugal force 16,200 × g, radius 8.6 cm) for 15 min at 4°C, and transfer 75 μL of the supernatant to a sample injection bottle for testing on an instrument.

[0372] 2.2 Metabolite Detection

[0373] The target compounds were separated chromatographically using a Vanquish ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) column. Phase A consisted of 10 mmol / L ammonium formate in 40% water and 60% acetonitrile; phase B consisted of 50 mL / 1000 mL (10 mmol / L) ammonium formate in 10% acetonitrile and 90% isopropanol. The following gradient elution program was used: 40% B from 0 to 1.0 min; linear increase to 100% B from 1.0 to 12.0 min; hold at 100% B from 12.0 to 13.5 min; linear decrease to 40% B from 13.5 to 13.7 min; and hold at 40% B from 13.7 to 18.0 min. The mobile phase flow rate was set at 0.3 mL / min, the column temperature was 55 °C, the sample tray temperature was 4 °C, and the injection volume was 2 μL (positive and negative ion modes).

[0374] Primary and secondary mass spectrometric data were collected simultaneously using a Thermo Q Exactive HFX mass spectrometer under the control of Xcalibur control software (version 4.0.27, Thermo). Specific parameters were as follows: sheath gas flow rate of 10 Arb, capillary temperature of 350°C, full MS resolution of 120,000, MS / MS resolution of 7500, collision energy of 10 / 30 / 60 in NCE mode, and spray voltage of 4 kV (positive ion mode) or -3.8 kV (negative ion mode).

[0375] 2.3 Data Analysis

[0376] ProteoWizard software was used to convert mass spectra into mzXML format. Retention time correction, peak identification, peak extraction, peak integration, and peak alignment were then performed using XCMS, with a minfrac value of 0.5 and a cutoff value of 0.3. Lipid identification was performed using XCMS, a custom R package, and the lipidblast database. Bioinformatics plots were created online using Hiplot and adjusted using Adobe Illustrator.

[0377] 3. Experimental results

[0378] The ionization source of the Orbitrap platform is electrospray ionization, which has two ionization modes: positive ion mode (POS) and negative ion mode (NEG). Combining the two modes when detecting metabolites can achieve higher metabolite coverage and better detection effects. During data analysis, one ion mode is generally selected. This study uses the positive ion mode as an example for analysis.

[0379] 3.1 Proportion of various metabolites in sEVs and sIVs

[0380] Secondary spectra in lipidomics are often haphazard, so only lipid species identified across all groups within a set of comparisons are reliable. Therefore, we categorized and analyzed the metabolites identified in different cell types based on their chemical classification. The percentages of each metabolite are shown in Figure 32. MSCs identified 31 metabolites. Differential expression levels of various lipid species were observed in sEVs and sIVs. PC and PE are common lipid components of cell membranes. Figure 32 shows a high percentage of both PE and PC in vesicles, indicating the presence of abundant biomembrane structures in sEVs and sIVs.

[0381] 3.2 Total lipid expression characteristics in sEVs and sIVs

[0382] Metabolomics data are high-throughput, and principal component analysis can effectively highlight the overall distribution trends of metabolomics data and the degree of differences between sample groups. The results showed that MSC cell sEVs and sIVs have different lipid distribution patterns (Figure 33), indicating that sIVs are a unique vesicle population, distinct from sEVs, with significantly different lipid expression patterns.

[0383] 3.3 Differential lipid expression characteristics in sEVs and sIVs

[0384] Heat maps can visually display the overall distribution of metabolite differences between groups. We visualized the results of screening for differential metabolites in the form of heat maps. The results for the sIVs group versus the sEVs group are shown in Figure 34.

[0385] 3.4 Variation of Differential Lipid Content and Classification Information in sEVs and sIVs

[0386] The lipidome histogram visualizes metabolite content changes and classification information. The results for the sIVs group versus the sEVs group are shown in Figure 35. Each bar in the lipidome histogram represents a metabolite. PC, PI, PE, PG, and OxPI were significantly overexpressed in MSCs compared to sIVs, with PC being over 200-fold higher in sIVs.

[0387] 4. Summary

[0388] Lipidomics identifies and quantifies various lipid molecules. Lipids are divided into eight major categories: fatty acyl groups, glycerolipids, phospholipids, sterol lipids, allyl lipids, sphingolipids, glycolipids, and polyketides. Cell membranes primarily contain various phospholipids, which can be further divided into glycerophospholipids and sphingomyelins, which have distinct differences. Glycerophospholipids are primarily located in the inner leaflet of the phospholipid bilayer of the cell membrane and, along with cholesterol, constitute the main components of the cell membrane. In this example, sIVs contain a high concentration of glycerophospholipids, such as PC and PE. Sphingomyelins are a class of phospholipids containing a sphingosine group. Sphingomyelins are located in the outer leaflet of the cell membrane and are primarily involved in neuronal activity and signal transduction. In this example, sEVs contain a high concentration of sphingomyelins, such as SM. Furthermore, glycerophospholipids are involved in many other physiological processes in the body, such as energy metabolism and hormone synthesis; sphingomyelins play a relatively minor role in these processes. sIVs express high levels of PC and PE, with PC being over 200-fold more highly expressed in sIVs. Among them, PC, also known as lecithin, is known as the "third nutrient" alongside proteins and vitamins, and plays a variety of important biological roles. Lecithin can increase axonal growth of neurons, promote brain development, enhance memory, and prevent Alzheimer's disease. Furthermore, PE is one of the main molecules that make up the skeleton of biological membranes. Its unique structure, consisting of a phosphate group, a glycerol group, an acyl group, and an ethanolamine, enables it to form stable non-lamellar and multilamellar liposome vesicles in biological membranes. This structure provides a stable foundation for biological membranes and helps maintain the normal structure and function of cells.

[0389] Interestingly, PC and PE, which are highly expressed in sIVs, are both glycerophospholipids. Since the endoplasmic reticulum is the site of glycerophospholipid synthesis, it is reasonable that sIVs contain more glycerophospholipids, further confirming that sIVs are intracellular components that mediate intracellular material transport and communication between organelles. However, sEVs contain more sphingomyelin. sEVs originate from invaginations of the cell membrane and are secreted to the extracellular space through the cell membrane, thus containing more components outside the cell membrane. This also indicates that sIVs lack an external membrane structure, and the differences in multiple lipids distinguish sIVs from sEVs. Previous studies have shown that the transport of proteins and lipids within cells is related to membrane curvature and lipid distribution effects. Glycerophospholipids can regulate membrane curvature and fluidity by adjusting chain length and cooperating with cholesterol, endowing sIVs with greater activity, thereby continuously participating in membrane fusion and fission events within the cell.

[0390] In this example, lipidomics data further verified that sIVs are different from sEVs, providing an important basis for in-depth exploration of the unique characteristics of sIVs in cell and tissue compatibility.

[0391] Example 8: Cells cultured in vitro and retinal tissue in vivo have higher absorption efficiency for sIVs

[0392] 1. Experimental instruments and materials

[0393] 1.1 Experimental Reagents

[0394] Table 10 Experimental reagents

[0395] 1.2 Experimental instruments

[0396] Table 11 Experimental instruments

[0397] 2. Experimental methods

[0398] 2.1 Cell culture

[0399] Human HRMECs were purchased from Angioproteomie, Inc., USA. Complete cell culture medium preparation: 5 ml of fetal bovine serum, 1 ml of growth factor, and 1 ml of penicillin-streptomycin were added to 93 ml of ECM basal medium to obtain a complete culture medium containing 5% serum. Complete RPE cell culture medium preparation: 5 ml of fetal bovine serum and 0.5 ml of penicillin-streptomycin were added to 44.5 ml of DMEM basal medium to obtain a complete culture medium containing 10% serum. Cell growth density was checked under a low-magnification microscope. Under a high-magnification microscope, cells were observed to be round, oval, or polygonal flat cells with abundant cytoplasm and small intracellular vacuoles. Cells were seeded in culture flasks and cultured in a 5% CO2 incubator at 37°C. The culture medium was changed every 3 days. When cell confluence reached 80%, cells were passaged at a 1:2 ratio, and P3 to P5 cells were used for experiments.

[0400] 2.2 Experimental animals

[0401] Healthy male C57BL / 6 mice, 8 weeks old, weighing 21-22 g, SPF grade, were purchased from Beijing Weitonglihua Co., Ltd. (Animal Production License: SCXK (Beijing) 2016-0006). All experimental animals were fed a normal diet at room temperature and maintained under a 12-h light and dark cycle. The animal husbandry and experimental procedures complied with the Regulations on the Administration of Laboratory Animals of the State Science and Technology Commission of China and were approved by the Animal Ethics Committee of our institution (Ethics Number: TJYY2019091124). Mice were randomly assigned to groups for subconjunctival and subretinal injections.

[0402] 2.3 Co-culture of DiD-labeled sIVs and sEVs with cells

[0403] The vesicles were incubated with a lipid-soluble tracer solution of DiD at 37°C for 30 minutes. Ultra centrifugal filter to remove excess DiD. Cells were seeded into pre-placed circular pieces in 24-well culture plates and incubated for 24 hours. DiD-labeled vesicles were added to the culture medium of the cells and incubated at 37°C for different times: 3 hours, 12 hours, 24 hours or 48 hours. After removing the culture medium, the cells were washed with PBS at room temperature. The cells were fixed with 4% PFA for 10 minutes and then incubated with 0.1% Triton X-100 for 5 minutes at room temperature to permeabilize the cells. Subsequently, the cells were fixed with 4% PFA for 10 minutes and then incubated with 0.1% Triton X-100 at room temperature for 5 minutes to permeabilize the cells. The cells were then stained with a phalloidin antibody conjugated to Plus 488 for 30 minutes. DAPI was used to label the nuclei. Finally, cells were imaged using a confocal laser scanning microscope, and the amount of endocytosed vesicles was analyzed using Image J.

[0404] 2.4 Subconjunctival and intravitreal injection of DiD-labeled sIVs and sEVs

[0405] Vesicles were stained with DiD as described above. DiD-labeled vesicles were injected subconjunctivally into mice at a volume of 5 μL and a mass of 3 μg. Eyeballs were harvested for observation 24 and 48 hours after subconjunctival injection. DiD-labeled vesicles were injected into the vitreous cavity of mice at a volume of 1 μL and a mass of 1 μg. Eyeballs were harvested for evaluation 8 and 48 hours after intravitreal injection.

[0406] 2.5 Retinal Slices

[0407] Frozen retinal sections (8 μm thick) were used for immunofluorescence analysis. Retinal sections were fixed with PFA for 15 minutes at room temperature, then the slides were washed with PBS and the cell nuclei were stained with DAPI. Finally, the sections were mounted with anti-fluorescence attenuation mounting medium and observed using a confocal laser scanning microscope.

[0408] 3. Statistical processing

[0409] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0. One-way ANOVA was used for analysis of variance, with least significant difference (LSD) analysis for post hoc testing. Nonparametric tests were used for nonnormally distributed data and data with unequal variances, and P values ​​< 0.05 were considered statistically significant.

[0410] 4. Experimental results

[0411] 4.1 Cells have a better ability to internalize sIVs than sEVs

[0412] In order to evaluate the phagocytic ability of cells to two types of vesicles, RPE cells and HRMECs were selected to co-culture with these MSC-sEVs (sEVs for short in this embodiment) and MSC-sIVs (sIVs for short in this embodiment). In short, an equal amount of DiD-labeled vesicles were incubated with cells, and at different time points, the distribution of DiD was observed using a confocal microscope to observe the cell uptake of vesicles. The results showed that after 3 hours of co-culture, cells began to internalize sEVs and sIVs, and over time, their uptake continued to increase (Figure 36A and B, Figure 37A and B). At 24 hours, the amount of sEVs and sIVs internalized by cells reached a peak, then gradually decreased within 24-48 hours (Figure 36C, Figure 37C). It is worth noting that in RPE cells and HRMECs, during 12 to 48 hours, the internalization rate of cells to sIVs always exceeded sEVs (Figure 36C, Figure 37C).

[0413] 4.2 The retina has a better ability to internalize sIVs than sEVs

[0414] To evaluate the phagocytic ability of the retina for the two types of vesicles, equal amounts of DiD-labeled vesicles were administered to the eyes by two methods: subconjunctival injection and intravitreal injection. Eyeball samples were collected at different time points, and frozen sections were prepared, and confocal microscopy was used to observe vesicle uptake in the retina. The results showed that 24 hours after subconjunctival injection, sIVs penetrated the sclera and entered the subretinal and retinal spaces, while sEVs accumulated between the conjunctiva and sclera, with fewer sEVs reaching the subretinal area (Figure 38A). After 48 hours, the retina showed significant uptake of sEVs and sIVs (Figure 38C). Specifically, sEVs were distributed in the RPE layer, while sIVs were widely distributed in the outer nuclear layer, inner nuclear layer, and ganglion cell layer. After intravitreal injection of DiD-labeled vesicles, the vesicles spread from the vitreous cavity to the retina. The results showed that 8 hours after injection, sEVs were distributed in the vitreous cavity and had not yet reached the retina, while sIVs had reached the entire retinal layer and were internalized by cells in the ganglion cell layer and inner nuclear layer, with higher sIVs accumulation observed on the RPE layer (Figure 38B). 24 hours after intravitreal injection, the retina effectively absorbed sEVs and sIVs, and both types of vesicles were diffusely distributed throughout the retina (Figure 38B). Compared with sEVs, sIVs showed more significant uptake (Figure 38D) and were widely distributed in all retinal layers, including the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, and RPE layer (Figure 38B).

[0415] 5. Summary

[0416] This example demonstrates that cells cultured in vitro have a better ability to internalize sIVs than sEVs. Subconjunctival and intravitreal injections in mice revealed that the retina internalizes sIVs more effectively than sEVs, demonstrating the tissue compatibility of sIVs.

[0417] Example 9: Protective effect of sIVs on retinal RGCs and retinal structure in ONC mice

[0418] 1. Experimental methods

[0419] 1.1 Establishment of the mouse optic nerve crush (ONC) model

[0420] Mice in the ONC group were anesthetized and then topically anesthetized with oxybuprocaine hydrochloride eye drops. Under a surgical dissecting microscope, the conjunctiva of one eye was cut open at approximately the 4 o'clock position on the temporal side of the eyeball using spring scissors. The orbital muscles were gently deflected to expose the white optic nerve. A forceps was applied to the optic nerve 2 mm from the eyeball for approximately 5 seconds. After the compression injury was complete, the incision was sutured. Postoperative model inclusion criteria included: no opacity in the mouse lens; no retinal hemorrhage or detachment; normal fundus blood supply; and normal recovery of the eyeball muscles, with normal retraction, no protrusion, and free rotation.

[0421] The groups included normal control group (normal group, only optic nerve was exposed without clamping), ONC group + PBS injection into the vitreous after injury, ONC group + sEVs injection into the vitreous after injury, and ONC group + sIVs injection into the vitreous after injury.

[0422] 1.2 Immunofluorescence staining of retinal flat mounts

[0423] The mouse eyeballs were removed and fixed in paraformaldehyde at room temperature for 15 minutes, placed in cold PBS for 5-10 minutes, and the eyeballs were transferred to gauze. An incision was made at the limbus of the cornea and sclera under an operating microscope. The sclera was gently torn open with two toothed forceps, and the lens was removed. The retina was cut into four petals with small scissors, and obvious blood vessel clusters were cut off. Cold anhydrous methanol was gently dripped on the retina for fixation. After the retina turned white, it was transferred to a 2 ml flat-bottom EP tube.

[0424] The anhydrous methanol in the EP tube was aspirated, and the slides were washed twice with PBS on a shaker at 4°C. 1 ml of blocking buffer was added and shaken at 4°C for 2 hours. The primary antibody was then added and incubated in a refrigerator at 4°C for 24 hours (primary antibody RBPMS dilution: 1:200). The slides were washed three times for 10 minutes each time and incubated with a fluorescent secondary antibody for 4 hours (secondary antibody Dylight 488 dilution: 1:1000). After washing, the slides were mounted with a drop of anti-fluorescence quencher. The slides were observed and photographed under a fluorescence microscope. Three images of each retina distal to the optic disc were taken. The number of RGCs in each image was counted using the StarDist2D plugin in ImageJ (v153) software, and the mean RGC density was calculated.

[0425] 1.3 Optical Coherence Tomography (OCT)

[0426] After anesthesia and pupil dilation, imaging and analysis were performed after applying clear eye gel to the corneas of both eyes, keeping the corneas moist at all times. Images of the mouse retina surrounding the optic nerve head were captured and measured using a UK-based Optoprobe. The thickness of the retinal ganglion cell complex (GCC), encompassing the RNFL, ganglion cell layer (GCL), and inner plexiform layer (IPL), was measured. Built-in software was used to segment the GCC and quantify its thickness.

[0427] 1.4 Hematoxylin-Eosin Staining (HE)

[0428] Mouse eyeballs were removed, fixed, and paraffin sections were prepared for routine HE staining. The extent of retinal tissue damage was recorded under an optical microscope, and the number of RGCs was further statistically analyzed.

[0429] 1.5 TUNEL staining of retinal frozen sections

[0430] Ten discontinuous frozen sections were taken from the peripheral and central regions of the eyeballs of mice in the experimental and control groups, with the optic nerve as the central reference point. TUNEL staining was performed according to the instructions of the apoptosis kit. Three to four fields of view of each section were photographed using a fluorescence microscope, and the number of TUNEL-positive cells in the sections was counted using computer software to evaluate retinal cell apoptosis in the different groups of mice.

[0431] 1.6 Western blotting

[0432] The eyeballs of the test mice in each group were collected and the retinas were isolated. The total protein in the retina was extracted, and the protein expression levels of inflammation-related factors (GFAP and iba1), anti-apoptotic protein Bcl-2, and pro-apoptotic protein Bax, caspase-3, and c-caspase-3 were detected by Western Blot.

[0433] 2. Statistical processing

[0434] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0. One-way ANOVA was used for analysis of variance, with least significant difference (LSD) analysis for post hoc testing. Nonparametric tests were used for nonnormally distributed data and data with unequal variances, and P values ​​< 0.05 were considered statistically significant.

[0435] 3. Experimental results

[0436] 3.1 Successful construction of ONC mouse model

[0437] The optic nerve of ONC mice was clamped for varying lengths, 2 mm from the posterior pole. Retinal flat mounts were taken 21 days after modeling, and the number of retinal ganglion cells was counted. The results showed that significant loss of retinal ganglion cells occurred throughout the retina after clamping for varying lengths, with similar levels of damage after clamping for 3, 5, and 8 seconds. The ONC mouse model was successfully established, and a 3-second clamping time was selected for subsequent experiments (Figure 39).

[0438] 3.2 sIVs significantly reduced the loss of retinal ganglion cells in ONC mice

[0439] ONC mice were treated with intravitreal injections of various concentrations of sIVs and sEVs. Retinal flat mounts were taken 21 days after administration and the number of retinal ganglion cells was stained and counted. The results showed that at a low concentration (1.25 mg / ml), the therapeutic effects of sIVs and sEVs were not significantly different from those of the intravitreal PBS injection group. However, at a high concentration (2.5 mg / ml), sIVs reduced retinal ganglion cell loss, so high concentrations of sIVs and sEVs were selected for subsequent experiments (Figure 40).

[0440] Retinal flat mounts were taken from ONC mice in each treatment group, and the number of retinal ganglion cells was counted at different time points after treatment. The results showed that sIVs reduced retinal ganglion cell loss 7 days after intravitreal injection, demonstrating the same short-term therapeutic effect as sEVs. However, sIVs demonstrated a superior therapeutic effect 21 days after administration, maintaining retinal ganglion cell count for a longer period (Figure 41).

[0441] 3.3 sIVs maintain the thickness of the retinal ganglion cell complex in ONC mice

[0442] Whole-retinal optical coherence tomography (OCT) scans of ONC mice 21 days after administration analyzed changes in the structure and thickness of each retinal layer. The results showed that intravitreal injection of sIVs and sEVs had no effect on the morphology or light reflectivity of the retinal layers. The ganglion cell complex (GCC), which includes the nerve fiber layer, ganglion cell layer, and inner plexiform layer, reflects neuronal damage. sIVs significantly slowed the reduction in GCC thickness, demonstrating a superior therapeutic effect (Figure 42).

[0443] 4. Summary

[0444] In retinal flat mount staining 7 days after treatment in the ONC mouse model, sEVs and sIVs inhibited ganglion cell apoptosis to the same extent. Notably, in whole-retinal optical coherence tomography scans 21 days after treatment in the ONC mouse model, sIVs better slowed the reduction in GCC thickness, indicating a superior therapeutic effect.

[0445] Example 10: Neuroprotective Effects of MSC-sIVs on Stroke-Induced Mice

[0446] 1. Experimental reagents

[0447] Table 12 Experimental reagents

[0448] 2. Experimental methods

[0449] 2.1 Establishment of a mouse stroke model

[0450] The mouse model of photochemical embolism was induced using Rose Bengal. Rose Bengal is a light-sensitive dye that, when exposed to light, undergoes lipid peroxidation with vascular endothelial cells, releasing free radicals that damage endothelial cells, triggering platelet adhesion, and forming thrombi. To better simulate the acute ischemic stroke model, we subjected 7-8 week old C57 male mice to a specific cold light source. The specific steps are as follows:

[0451] (1) Anesthesia: Induce anesthesia in mice using 3.5% isoflurane. After successful anesthesia, place the mice in a prone position on the experimental operating table. After trimming the hair on the head, perform local skin disinfection. After disinfection, use ophthalmic surgical scissors to gently cut the skin along the midline of the mouse's head to fully expose the stroke window (based on the mouse's anterior fontanelle, 2 mm lateral to the sagittal suture).

[0452] (2) Light Intervention: After completing the above preparations, a concentration of 75 mg / kg of Rose Bengal was injected intraperitoneally into the mice. Because Rose Bengal is somewhat toxic, the injection should be slow and completed within 5 minutes. Then, a 4 mm diameter optical fiber was fixed to the stroke window and a green cold light source was used to illuminate this area for 10 minutes at the maximum intensity. During this period, the changes in the mice's vital signs were observed.

[0453] (3) Suturing: After 15 minutes of irradiation, the scalp wound of the mouse was sutured with medical sutures. After suturing, the mouse was returned to the cage. During the entire surgical process and after the operation, the body temperature of the mouse was maintained at around 37°C using a mouse electric heating blanket. In order to reduce the error caused by the experimental modeling, all the above experimental steps were completed by the same experimenter on the same day.

[0454] (4) Animal grouping: After the model mice woke up, they were carefully observed for half an hour, and the stroke mice with similar vital signs were randomly divided into PBS group, sEVs group, and sIVs group.

[0455] 2.2 Behavioral testing

[0456] Two behavioral tests, the fatigue transfer rod test and the forelimb grip strength test, were used to assess the degree of motor recovery. The fatigue transfer rod test is primarily used to assess the animal's motor function and balance and coordination abilities, while the forelimb grip strength test is primarily used to assess the neuromuscular function of the animal's forelimbs.

[0457] 2.2.1 Rotarod test

[0458] During behavioral testing, all mice underwent two days of pretraining on the fatiguing transfer rod before surgery to select mice that could adapt to the fatigue transfer rod under the same conditions. The rotarod test involves placing the animal on a rotating rod and requiring it to adjust its center of gravity to maintain balance and avoid falling. The length of time the animal remains on the rotating rod is measured by adjusting parameters such as the speed, angle, and duration of the rotation to assess its locomotion and balance abilities. Stroke models were administered 1.5 μg / μL of MSC-sEVs (in PBS) or MSC-sIVs (in PBS) in a total volume of 20 μL, along with an equal volume of PBS, via nasogastric administration on the day after surgery and on days 1, 2, and 3 after surgery. Rotarod testing was performed on days 1, 3, 5, and 7 after surgery.

[0459] 2.2.2 Grip strength test

[0460] The forelimb grip strength test is used to assess and evaluate forelimb neuromuscular function. A tension sensor is used to test forelimb grip strength. After the mouse's forelimb grasps a gripping bar on a grid, the tail is gently pulled backward. The grip strength reaches its maximum at the moment the forelimb releases. Data is recorded by the Bio-Signal System software. Each mouse is tested three times, and the mean value is used for statistical analysis.

[0461] After the behavioral test, the mice were subjected to anatomical and pathological examinations. The specific experimental procedures were as follows:

[0462] 2.3 TTC staining of mouse brain tissue

[0463] TTC staining utilizes the principle of redox reaction. TTC can undergo a specific chemical reaction with succinate dehydrogenase in cells. After cerebral infarction occurs, the succinate dehydrogenase in the cells of the infarcted area decreases significantly, and thus cannot fully react chemically with TTC. After the redox reaction occurs, the brain tissue in the infarcted area can be stained white, while the brain tissue in the non-infarcted area can appear red after staining due to the sufficient redox reaction. Therefore, the color change of the brain tissue can clearly distinguish between the infarcted area and the non-infarcted area. We usually calculate the volume of brain tissue in the white area, which is the volume of cerebral infarction. The specific steps are as follows:

[0464] (1) Brain removal: After the neurological function evaluation experiment of stroke mice is completed, the stroke mice are deeply anesthetized with chloral hydrate. The PBS solution is placed in the refrigerator in advance. After the mice are fully anesthetized, ice PBS is used for cardiac perfusion until the lungs and liver of the mice turn white (in order to speed up the perfusion, the lungs and liver can be cut in advance). After cardiac perfusion, the skull of the mouse is quickly removed and the intact brain tissue is removed. The removed brain tissue is quickly placed in a -20℃ refrigerator (about 10 minutes), and then the mouse brain is cut into 2mm thick coronal brain slices on ice.

[0465] (2) TTC staining: Prepare 1.5% TTC solution in advance. Soak the above-cut coronal brain slices in TTC solution for a full chemical reaction. The reaction conditions are to place them in a 37°C constant temperature incubator away from light for 20 minutes. During the placement period, turn the brain slices over several times to allow for sufficient redox reaction, thereby achieving better staining. After staining, place the brain slices in 4% paraformaldehyde for 30 minutes, then place the fixed brain slices on a black background with a ruler next to them, and then take pictures.

[0466] 2.4 TUNEL assay for neuronal apoptosis

[0467] To evaluate the effects of sIVs and sEVs treatment on neuronal apoptosis in stroke mice, we used the TUNEL method to detect neuronal apoptosis in this study. The main principle of the TUNEL method for detecting cell apoptosis is that during cell apoptosis, double-strand breaks in chromosomal DNA will produce a large number of sticky 3'-OH ends. Under the action of terminal deoxyribonucleotide transferase (TdT), deoxyribonucleotides and fluorescent dye can be labeled to the 3'-OH end of DNA, thereby enabling the detection of apoptotic cells. The specific steps are as follows:

[0468] (1) Brain extraction: Stroke mice were deeply anesthetized with chloral hydrate and perfused through the heart using ice-cold PBS. Perfusion was successful when the lungs and liver turned white. After the perfusion, the skull was quickly removed and the brain tissue was completely removed. The brain tissue was then immersed in 4% paraformaldehyde solution and refrigerated at 4°C overnight.

[0469] (2) Dehydration: After the brain tissue is fixed with 4% paraformaldehyde overnight, the residual formaldehyde liquid on the brain tissue is blotted dry. Prepare 15% sucrose aqueous solution and 30% sucrose aqueous solution in advance. Soak the brain tissue in a centrifuge tube containing 15% sucrose aqueous solution for initial dehydration. When the brain tissue completely sinks to the bottom of the centrifuge tube, place the brain tissue in a centrifuge tube containing 30% sucrose aqueous solution for further dehydration. When the brain tissue completely sinks to the bottom of the centrifuge tube again, remove the brain tissue and blot the residual sucrose solution on the brain tissue for the next step.

[0470] (3) Embedding: Prepare the items needed for embedding in advance (tweezers, OCT compound, embedding box, blade, -80℃ ultra-low temperature refrigerator). Use a pre-refrigerated blade to cut off brain tissues such as the brainstem and cerebellum that are not needed later. Slowly squeeze the OCT compound into the embedding box (to avoid bubbles that affect subsequent sectioning) so that it slowly covers the bottom of the embedding box. After slowly placing the brain tissue in the middle of the embedding box, continue to slowly add the OCT compound until the OCT compound covers the entire brain tissue. Finally, place the embedding box containing the OCT compound and brain tissue into a -80℃ ultra-low temperature refrigerator to promote OCT fixation through low temperature. At the same time, the embedded brain tissue can be stored in a -80℃ ultra-low temperature refrigerator.

[0471] (4) Sectioning: When the temperature in the freezing microtome operating room drops to -20°C, fix the embedded brain tissue. First, perform preliminary trimming of the brain tissue. After the brain layer containing the cerebral infarction lesion is seen, adjust the slice thickness to 8 μm and slice. Use a polylysine-coated slide to stick the stretched brain slices, and then place the cut brain slices in a section box. Finally, the section box can be placed in a -20°C refrigerator for storage.

[0472] (5) Antigen repair: Take the brain slices to be stained out of the -20°C refrigerator in advance and place them at room temperature for 30 minutes for antigen repair.

[0473] (6) Fixation: After the frozen sections were circled with an immunohistochemical pen and fixed with 4% paraformaldehyde at room temperature for 30 minutes, they were washed with PBS three times, each time for 5 minutes.

[0474] (7) Penetration promotion: add 0.3% TritonX-100 to the slices for 10-20 minutes at room temperature to promote penetration, and then wash three times, each time for 5 minutes.

[0475] (8) TUNEL reagent incubation: After the permeation is completed, the brain slices are washed again with PBS solution for 5 minutes, and the washing is repeated 5 times. Prepare the TUNEL reaction mixture in advance. Cover the brain slices with the TUNEL reaction mixture and place them in a 37°C constant temperature incubator away from light for 1 hour. After the incubation is completed, the brain slices are immersed in PBS and quickly washed on a shaker for 5 minutes, and this is repeated 5 times. After the washing is completed, DAPI is covered on the brain slices and the coverslip is used to seal the slices. After the sealing is completed, the slices are immediately observed under a fluorescence microscope, and the image data is obtained and then the acquired images are analyzed using Image software.

[0476] 2.5 Immunofluorescence staining to observe microglial activation in each group of stroke mice

[0477] In order to observe the changes in the number and morphology of microglia in different groups after stroke, immunofluorescence staining was used to mark the microglia in the damaged area of ​​each group using IBA1, and it was found that microglia were significantly activated after stroke.

[0478] The preparation, fixation, and permeabilization steps for brain tissue immunofluorescence frozen sections were the same as those described in Example 1.3.

[0479] (1) Blocking: After fixation and permeabilization, blocking solution (PBST + 5% normal goat serum + 2% BSA) was added to the frozen sections of brain tissue and placed in a humidified box in the dark for 1.5 h.

[0480] (2) Antibody incubation: Add 1:100 Iba-1 antibody and incubate overnight in a 4°C refrigerator.

[0481] (3) The next day, the sections were removed and washed with PBST three times for 5 minutes each. 1:300 Alexa Fluor 488-labeled goat anti-rabbit secondary antibody was added and incubated in a humidified chamber at room temperature for 2 hours in the dark. The sections were then washed with PBS three times for 5 minutes each.

[0482] (4) Counterstaining the cell nucleus: Add DAPI staining solution and react at room temperature in the dark for 10 min. Wash off the DAPI staining solution with PBS, seal the slides with anti-fluorescence quencher, and store the slides in the dark.

[0483] (5) Observation: Microglial activation was observed using an Olympus fluorescence microscope. A 10× field of view was set for each slice, and at least three areas were randomly selected for photography.

[0484] 2.6 Immunofluorescence staining to observe astrocyte activation in stroke rats in each group

[0485] Related literature (see, Ni XC, Wang HF, Cai YY, et al. Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke [J]. Redox biology, 2022, 54: 102363.; Hasel P, Rose IVL, Sadick JS, et al. Neuroinflammatory astrocyte subtypes in the mouse brain [J]. Nature neuroscience, 2021, 24 (10): 1475-1487.) reports that astrocytes are reactively activated in an acute ischemic stroke model, and the effect of related treatments on reducing cerebral infarction volume is closely related to inhibiting the activation of astrocytes. The preparation of brain tissue immunofluorescence frozen sections and the immunofluorescence staining steps are the same as those described in Examples 1.3 and 1.4.

[0486] 2.7 Immunofluorescence staining to observe the expression of β-tubulin III in the brain tissue of stroke rats in each group

[0487] Brain tissue was fixed in 4% paraformaldehyde, then frozen and sectioned and incubated with antibodies using the same procedures as described in 1.3 and 1.4 of this Example. Immunofluorescence images were acquired and analyzed using ImageJ software, and the fluorescence intensity of the immunofluorescence images was calculated. β-tubulin III is a recognized neuron-specific marker commonly used to identify neural stem cell proliferation and differentiation.

[0488] 2.8 Immunofluorescence staining to observe the expression of occludin and ZO-1 in the brain tissue of stroke rats in each group

[0489] Brain microvascular endothelial cells actively interact with extracellular matrix proteins that form the basement membrane, pericytes, astrocytes, microglia, and neurons, forming the neurovascular unit. This unit couples neuronal activity with vascular function by controlling regional cerebral blood flow and blood-brain barrier parameters. Ischemic stroke triggers rapid neurovascular unit damage, impairing neurovascular coupling. Oxygen and nutrient deprivation activate proteolytic enzymes, leading to the secretion of matrix metalloproteinases (MMPs) by brain microvascular endothelial cells, which degrade tight junction proteins and increase blood-brain barrier permeability. We examined the expression of CD31, a key marker for microvascular endothelial cells and also known as platelet endothelial cell adhesion molecule-1 (PECAM-1), in the infarcted area of ​​stroke patients using immunohistochemical staining.

[0490] 3. Statistical processing

[0491] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0. One-way ANOVA was used for analysis of variance, with least significant difference (LSD) analysis for post hoc testing. Nonparametric tests were used for nonnormally distributed data and data with unequal variances, and P values ​​< 0.05 were considered statistically significant.

[0492] 4. Experimental results

[0493] MSC-sIVs improve motor coordination and neuromuscular function in stroke-induced mice

[0494] The rotarod fatigue test was used to assess the motor coordination of mice. As shown in Figure 43, the time it took for stroke-affected mice to fall from the rotarod was significantly earlier than in the MSC-sEVs and MSC-sIVs groups. The time it took for the MSC-sEVs and MSC-sIVs groups to fall from the rotarod was significantly longer than that in the PBS group at various time points. Therefore, the results of the rotarod test suggest that MSC-sIVs can improve motor and coordination abilities in animals after ischemic stroke, potentially providing neuroprotection.

[0495] The forelimb grip strength test was used to assess forelimb neuromuscular function. As shown in Figure 44 , ischemia induced a decrease in forelimb grip strength at various time points within 7 days after ischemia, whereas administration of MSC-sIVs and MSC-sEVs significantly increased forelimb grip strength 3 days after ischemia.

[0496] The study conducted fatigue transfer rod tests and forelimb grip strength tests within 7 days of modeling. Results showed a significant decrease in motor ability on the first day of modeling, possibly related to impaired cerebral cortical motor function and surgical anesthesia. Subsequently, motor ability gradually recovered, with the MSC-sIVs intervention group showing significantly greater recovery than the PBS group.

[0497] Effects of MSC-sIVs on brain tissue of stroke mice

[0498] TTC staining results can visually demonstrate the area of ​​cerebral ischemia in each group of mice. Normal brain tissue is the dark area, the white area is the infarct, and the dark and white brain tissue area is the penumbra. After TTC staining, it can be seen that the white infarct area in the PBS group is the largest, which is significantly different from that of MSC-sEVs and MSC-sIVs (Figure 45).

[0499] 4.3 Effects of MSC-sIVs on neural cells in stroke mice

[0500] The contrast of the fluorescent staining marked area is brighter, and the part that is not fluorescently marked (ie, the negative area) is a black background. The TUNEL staining data results show that the light white bright spots in the PBS group, that is, apoptotic cells, are the largest in number and occupy the largest area. In contrast, the background of the MSC-sIVs and MSC-sEVs treatment groups is uniformly black, and it is almost difficult to observe the light bright spot area. This clearly shows that the MSC-sIVs and MSC-sEVs groups can significantly reduce the number of neuronal apoptosis in the cerebral cortex of mice with photochemical stroke, and the difference is statistically significant (P < 0.05) (Figure 46). In summary, MSC-sIVs can significantly reduce the degree of neuronal pathological damage in mice after stroke and effectively reduce the relative number of apoptotic cells.

[0501] Effects of MSC-sIVs on microglia in stroke-induced mice

[0502] In the ischemic area of ​​stroke mice, the number of activated microglia increased significantly. The contrast of the fluorescent staining marked area is brighter, and the part not fluorescently marked (ie, the negative area) is a black background. Immunofluorescence staining found that the PBS group showed significantly more fluorescent bright spots, that is, activated microglia, and the coverage area was the largest. In comparison, the intensity of the fluorescent bright spots in the MSC-sIVs group and the MSC-sEVs group was significantly reduced, and the coverage area was also significantly smaller than that of the PBS group. After statistical analysis, the difference was significant (P < 0.05). Further comparison found that compared with the MSC-sEVs group, the MSC-sIVs group showed fewer fluorescent bright spots. This clearly shows that MSC-sIVs is more prominent than MSC-sEVs in reducing the number of activated microglia (see Figure 47). The above results fully demonstrate that MSC-sIVs can effectively reduce the activation of microglia in the ischemic area after stroke in mice, thereby significantly reducing the inflammatory stress response to neural tissue.

[0503] Effects of MSC-sIVs on astrocytes in stroke-induced mice

[0504] GFAP, a specific marker for astrocytes, can directly reflect the degree of damage to the central nervous system. To investigate the effects of MSC-sIVs on astrocyte activation, we examined the expression of GFAP, a marker of astrocyte activation. Fluorescently stained areas have a brighter contrast, while unlabeled areas (i.e., negative areas) have a black background. Immunofluorescence staining revealed that the light-colored, brightly fluorescent areas in the brain tissue of mice in the PBS group, representing GFAP protein expression levels, were significantly higher than those in the MSC-sIVs and MSC-sEVs groups. Further comparison revealed that the light-colored, brightly fluorescent areas were smaller in the MSC-sIVs group than in the MSC-sEVs group. This result clearly demonstrates that MSC-sIVs are more effective than the MSC-sEVs group in reducing astrocyte activation (see Figure 48 for details). In summary, administration of MSC-sIVs after stroke modeling effectively inhibited astrocyte activation in the brain tissue of mice with photochemically induced ischemic stroke.

[0505] Effect of MSC-sIVs on β-tubulin III protein expression in brain tissue of stroke mice

[0506] The contrast of the fluorescent staining marked area is brighter, and the part not fluorescently marked (ie, the negative area) is a black background. Immunofluorescence staining experiments showed that the light-colored bright spot fluorescent area (β-tubulinⅢ protein expression) in the mouse brain tissue after modeling was significantly less than that in the MSC-sIVs and MSC-sEVs groups. Further comparison shows that the area of ​​the light-colored bright spot area in the MSC-sIVs group is larger than that in the MSC-sEVs group. This clearly shows that MSC-sIVs can not only effectively increase the expression of β-tubulinⅢ, but also is more significant in the improvement effect than MSC-sEVs (Figure 49).

[0507] Effects of MSC-sIVs on the Expression of Occludin and ZO-1 in the Brain Tissue of Stroke-Induced Mice

[0508] We used double immunofluorescence staining to stain the tight junction proteins Occludin and ZO-1 to determine the permeability of the blood-brain barrier. The fluorescent staining marked area has a brighter contrast, and the unfluorescently labeled part (i.e., the negative area) has a black background. The results are shown in Figure 50. The fluorescent area and intensity of the light-colored bright spot in the PBS group (Figure 50A: Occludin; Figure 50B: ZO-1) were significantly reduced compared with the MSC-sIVs group and the MSC-sEVs group. This indicates that after stroke modeling, the expression of Occludin and ZO-1 in the mouse brain tissue was significantly reduced. Compared with the PBS group, the levels of Occludin and ZO-1 in the brain tissue were significantly increased after treatment with MSC-sIVs and MSC-sEVs. This result indicates that MSC-sIVs may have a promoting effect on angiogenesis after ischemic injury in stroke.

[0509] 5. Summary

[0510] TUNEL immunofluorescence staining in mice with ischemic stroke showed that both MSC-sEVs and MSC-sIVs inhibited ganglion cell apoptosis. Notably, compared with MSC-sEVs, MSC-sIVs significantly slowed the activation of astrocytes and microglia and increased β-tubulin III expression, demonstrating superior therapeutic efficacy.

[0511] Example 11: Neuroprotective Effects of MSC-sIVs on Alzheimer's Disease Mice

[0512] 1. Experimental reagents

[0513] Table 13 Experimental reagents

[0514] 2. Experimental methods

[0515] 2.1 Experimental animals and groups

[0516] Alzheimer's disease (AD) model mice were selected from Nanjing Junke Biotechnology Co., Ltd., using 4-month-old 5xFAD transgenic mice with a C57 / BL6J background. The experiment began after 2 weeks of adaptive feeding. The mice were treated by nasogastric administration every other day for a total of 2 months. MSC-sEVs (PBS solvent) and MSC-sIVs (PBS solvent) were administered via nasogastric administration at a concentration of 1.5 μg / μL in a total volume of 20 μL, along with an equal volume of PBS. The Normal group received no drug treatment except hyaluronidase. Hyaluronidase (100 U / 5 μL) was administered intranasally to each mouse before administration to promote better drug absorption.

[0517] 2.2 Water maze experiment

[0518] The Morris water maze is a cognitive behavioral experiment designed to assess spatial learning and memory in rodents. The experimental apparatus consists of a circular pool with a radius of 60 cm and a height of 50 cm, evenly divided into four quadrants. Cards of varying shapes and colors are affixed to the walls of each quadrant for easy location and identification. A camera is mounted above the pool, providing a full field of view. Using the water maze image and video capture system, every detail of the experimental process is accurately recorded. To eliminate the influence of external factors such as light on the experimental results, the area surrounding the pool is enclosed with blackout curtains. During the experiment, the pool is filled with approximately 30 cm of water, with a temperature precisely controlled at 21.2°C. In the third quadrant of the pool, a cylindrical platform with a diameter of 10 cm is placed, submerged 1 cm below the water surface.

[0519] The Morris water maze test consists of two parts: a navigation test to evaluate the mice's learning ability, that is, how well they can find the platform hidden underwater through trial and error; and a spatial exploration test to evaluate the mice's spatial memory, that is, whether they can use their memory to find the original platform's location after the platform is removed. The specific test steps are as follows:

[0520] 1) Navigation Test: Each day, a different quadrant was selected and the animal was slowly lowered into the water, facing the pool wall. The time it took for the mouse to find the platform hidden beneath the surface was recorded, and this time was defined as the escape latency. Once the mouse successfully found the platform, it was given a 10-second rest period. However, if the mouse failed to find the platform within 60 seconds, the escape latency was recorded as 60 seconds. At this point, the mouse was gently guided to the platform and allowed to remain on it for 10 seconds, hoping to strengthen its memory of the platform. After completing these steps, the mouse was gently dried with a towel and briefly heated with an incandescent lamp before being returned to its cage. This training process lasted for 5 days, with four training sessions conducted daily at specific times, with an interval of approximately 20 minutes between each session. To more accurately assess the mouse's spatial learning ability, the average of the four escape latencies was calculated and used as the statistical analysis for that day's performance. This method provides a more comprehensive understanding of the mouse's spatial learning ability.

[0521] 2) Spatial Exploration Test: 24 hours after completing the navigation test, the hidden platform in the third quadrant of the pool was removed. The mice were then released from the first quadrant of the pool. The mice's swimming path and direction within the first 60 seconds of entry were closely monitored and recorded, along with the frequency with which they crossed the original platform position. Furthermore, the proportion of time the mice spent swimming in each quadrant was calculated. Through these observations and analyses, we can gain a comprehensive understanding of the mice's spatial memory abilities.

[0522] 2.3 Open field experiment

[0523] The open field test is a method used to detect the autonomous behavior of mice in a new environment and to explore their behavior and mental stress. It can reflect the anxiety and exploratory behavior of mice. The open field test consists of three parts: an open field reaction box and an automatic data acquisition and processing system. The background noise in the laboratory is controlled below 65dB, and the room temperature is around 20°C. The experiment is carried out in a quiet environment. Place the animal in the center of the bottom of the box, and take photos and time it at the same time. Clean the inner wall and bottom of the box to prevent the remaining information of the animal from the last time (such as the animal's defecation, urine, and odor) from affecting the results of the next test. Replace the animal and continue the experiment. Observe the number of times the mouse stands and the movement trajectory during the exploration process.

[0524] 2.4 Y-maze experiment

[0525] The Y-maze alternation test was used to assess the animals' short-term memory. The order in which the mouse entered each arm and the total number of arm entries (N) were recorded. One alternation was considered when the animal entered three different arms consecutively, with the maximum number of arm entries defined as N-2. The spontaneous alternation rate (%) was calculated as [number of alternations / (N-2)] × 100%.

[0526] 2.5 Brain tissue sections and pathological observation

[0527] After the water maze test, three mice in each group were anesthetized with an intraperitoneal injection of 0.3% sodium pentobarbital. The thorax was opened, and normal saline was perfused through the apex of the heart to flush out the blood. Then, 4% paraformaldehyde was irrigated until the liver turned white. The mice were decapitated and the brain tissue was removed. The tissue was fixed in 4% paraformaldehyde for more than 24 hours. Dehydration: The sample tissue was soaked in a gradient of 70%-80%-95%-100% alcohol and xylene solutions. Paraffinization: The tissue blocks transparentized by xylene were treated with pure paraffin twice, each time for 1 hour. Embedding: The paraffin-soaked tissue blocks were embedded and fixed in paraffin. Sectioning: Sagittal sectioning, continuous paraffin sections with a thickness of 4 μm. Baking: The paraffin sections were kept in a constant temperature baking machine at 60°C overnight.

[0528] 2.5.1 Nissl staining

[0529] Dewaxing: Immerse the paraffin sections in xylene for dewaxing twice, 10 minutes each time. Alcohol gradient hydration: 100% (5 min, 2 times) - 95% (5 min, 2 times) - 90% (5 min, 2 times) - 85% (5 min, 2 times) - 75% (5 min, 2 times). Elute in distilled water for 5 minutes. Stain with 1% toluidine blue at a constant temperature of 50°C for 20 minutes, differentiate with 70% ethanol for several minutes, and differentiate with 95% ethanol for several minutes. Five sections from each group were randomly selected for pathological observation to observe the general morphology of neurons. The average number of neurons in the dorsal cell band of the hippocampal DG was calculated by the automatic image analysis system.

[0530] 2.5.2 Hematoxylin-eosin staining

[0531] (1) Brain removal: Take the whole brain tissue and fix and dehydrate it (same steps as above). (2) Frozen section: When the hippocampal tissue appears, adjust the thickness to 10μm and make continuous sections. (3) Hematoxylin staining: Put the brain slice on the slide, stain with hematoxylin solution at room temperature for 5 minutes, and rinse with tap water. (4) Differentiation: Treat with differentiation solution for 30 seconds, observe the degree of differentiation under a microscope, and rinse with tap water. (5) Eosin staining: Stain with eosin solution at room temperature for 1 minute, and rinse with tap water. (6) Dehydration and transparentization: Put the slide into 70%, 85%, 95% and 100% ethanol solution for dehydration for 5 minutes in sequence, and transparentize with xylene for 5 minutes. (7) Sealing: Put a drop of neutral resin next to the brain slice, seal it with a cover glass, and observe it under a microscope.

[0532] 2.5.3 TUNEL assay

[0533] Apply an anti-fluorescence quencher, cover with a coverslip, and protect from light. Observe the test results under a 400× optical microscope. Green TUNEL represents apoptotic cells, and DAPI represents in situ cell nuclei.

[0534] 2.6 Enzyme-linked immunosorbent assay

[0535] Because amyloid plaque accumulation is a key pathological hallmark of AD, an enzyme-linked immunosorbent assay (ELISA) was used to investigate whether MSC-sIVs treatment affected Aβ deposition in mouse brain tissue. Forty-eight hours after the final behavioral experiment, animals were euthanized, and brain tissue was harvested. After homogenization with PBS, the tissue was centrifuged at 4000 rpm and 4°C. The homogenate was then stored at -80°C for subsequent testing. ELISA assays were performed according to the kit instructions.

[0536] 2.7 Determination of GSH content, SOD activity and MDA content

[0537] Oxidative stress plays a role in the pathogenesis of AD. Decreased GSH levels, elevated MDA content, and fluctuations in SOD activity are associated with the development of Aβ plaques and Alzheimer's disease. To investigate whether MSC-sIVs affect GSH, MDA, and T-SOD, we quantitatively analyzed GSH, MDA levels, and T-SOD activity in the cerebral cortex of 5xFAD mice. Following behavioral experiments, mice were sacrificed, and brain tissue was immediately removed. Residual blood was rinsed with cold saline, dried with filter paper, weighed, minced, and added to 9x ice-cold saline to prepare a 10% aqueous tissue homogenate. The homogenate was centrifuged (3500 rpm) for 10 minutes, and the supernatant was stored at 4°C for further analysis. GSH content, SOD activity, and MDA content were determined strictly according to the kit instructions.

[0538] 3. Statistical processing

[0539] The experimental data are expressed as mean ± standard deviation All experimental data were tested for normality. All quantitative data were analyzed using SPSS 22.0. One-way ANOVA was used for analysis of variance, with least significant difference (LSD) analysis for post hoc testing. Nonparametric tests were used for nonnormally distributed data and data with unequal variances, and P values ​​< 0.05 were considered statistically significant.

[0540] 4. Experimental results

[0541] MSC-sIVs improve spatial learning impairment in 5xFAD mice

[0542] The results of the Morris water maze test showed that the escape latency of all test groups gradually decreased. In the positioning navigation experiment (finding the platform), the escape latency of the PBS group mice from the 4th to the 6th day was significantly higher than that of the normal group, while the escape latency of the 5xFAD mice treated with MSC-sIVs and MSC-sEVs on the 4th to 6th day was significantly lower than that of the 5xFAD mice treated with PBS. The MSC-sIVs group had a significantly shorter latency to reach the platform compared with the PBS group on the 4th and 6th day of positioning navigation, and the difference was statistically significant (Figure 51A). During the positioning navigation stage on the 6th day, we found that the PBS mice had difficulty finding the platform and swam around the edge compared with the normal, MSC-sIVs and MSC-sEVs groups, indicating that the PBS group mice had more severe spatial memory impairment (Figure 51B). In the spatial exploration experiment, compared with the normal group (approximately 5 platform crossings and approximately 10.746 seconds spent in the target quadrant), the PBS group showed significantly fewer platform crossings (approximately 2.47 times) and a shorter target quadrant dwell time (approximately 8.63 seconds). When 5xFAD mice were treated with MSC-sIVs and MSC-sEVs, both the number of platform crossings and the percentage of time spent in the target quadrant were improved, demonstrating excellent learning ability. Specifically, the MSC-sIVs group showed a higher number of platform crossings (approximately 3.9 times) than the PBS group and almost the same as the normal group. Furthermore, the time spent in the target quadrant (approximately 13.19 seconds) was longer than both the PBS and normal groups. These data suggest that 5xFAD mice suffer from neurological damage, and that MSC-sIVs and MSC-sEVs intervention can alleviate this damage to varying degrees. Specifically, MSC-sIVs significantly improved the spatial learning impairment and alleviated memory deficits in 5xFAD mice.

[0543] 4.2 MSC-sIVs improve spontaneous exploratory behavior and tension in 5xFAD mice

[0544] Two months after the experimental treatment, an open field test was performed to detect the autonomous behavior, exploratory behavior, and tension of 5xFAD mice in a new environment. The activity trajectories of mice in the PBS group tended to be more in the peripheral area of ​​the open field, while the activity trajectories of mice in the normal, MSC-sIVs, and MSC-sEVs groups tended to be more in the central area, especially the MSC-sIVs group (Figure 52). Compared with the PBS group, the number of standing times in the normal, MSC-sIVs, and MSC-sEVs groups increased significantly, indicating that they had stronger spontaneous exploratory behavior and better cognitive function. In particular, the number of standing times in the MSC-sIVs group (about 64.3 times) increased significantly, which was significantly higher than that in the PBS group (about 36 times), and almost equivalent to that of the normal group (about 65 times), indicating that MSC-sIVs can significantly stimulate the autonomous exploratory behavior of 5xFAD mice.

[0545] MSC-sIVs improve learning and memory dysfunction in 5xFAD mice

[0546] Representative Y-maze trajectory results are shown in Figure 53. Calculation of the spontaneous alternation rate showed that the spontaneous alternation rate in the PBS group (approximately 22%) was significantly lower than that in the normal group (approximately 30%) (P < 0.05). Compared with the PBS group, the spontaneous alternation rate in the MSC-sIVs and MSC-sEVs mice was significantly increased, indicating that learning and memory impairments in these mice were alleviated after treatment. In particular, the spontaneous alternation percentage in the MSC-sIVs group (approximately 34%) was significantly higher than that in the PBS group (P < 0.01), and it also showed an upward trend compared to the normal group.

[0547] MSC-sIVs alleviate oxidative stress in 5xFAD mice

[0548] Oxidative stress plays a reciprocal role in the pathogenesis of AD. Decreased GSH levels, elevated MDA content, and fluctuations in SOD activity are associated with the accumulation of Aβ plaques and AD. To investigate whether MSC-sIVs affect GSH, MDA, and T-SOD, we quantitatively analyzed GSH, MDA levels, and T-SOD activity in the cerebral cortex of 5xFAD mice.

[0549] The brain GSH level of 5xFAD mice in the PBS group (approximately 52 μmol / gprot) was significantly lower than that in the normal group (approximately 62 μmol / gprot) (P < 0.05), indicating that the PBS group mice had higher levels of free radicals and cell damage. Furthermore, the GSH level of 5xFAD mice showed an upward trend after treatment with MSC-sIVs and MSC-sIVs. The MDA content of mice in the PBS group (approximately 12.69 μmol / mg) was significantly higher than that in the normal group (approximately 7.2 μmol / mg) (P < 0.05), indicating that the PBS-treated 5xFAD mice had higher levels of free radicals and cell damage. Furthermore, the MDA level of 5xFAD mice showed a significant downward trend after treatment with MSC-sIVs and MSC-sIVs, with statistically significant differences (P < 0.05). The SOD activity of mice in the PBS group (approximately 0.23 U / μg) was significantly lower than that in the normal group (0.39 U / μg) (P < 0.05), indicating that 5xFAD mice treated with PBS had higher levels of free radicals and cell damage. Furthermore, the SOD activity of 5xFAD mice treated with MSC-sIVs and MSC-sIVs was significantly increased, with statistically significant differences (P < 0.05). In particular, the GSH level (approximately 59.5 μmol / gprot) and SOD activity (approximately 0.39 U / μg) of 5xFAD mice treated with MSC-sIVs were significantly higher than those in the PBS group and comparable to those in the normal group. Furthermore, the MDA content (6.5 μmol / mg) was significantly lower than that in the PBS group (12.69 μmol / mg).

[0550] 4.5 MSC-sIVs reduce Aβ levels in 5xFAD mice

[0551] The results showed that the amyloid protein content observed in 5xFAD mice in the PBS group (approximately 19.2 pg / mg) was significantly higher than that in the normal group (approximately 12.3 pg / mg) (P < 0.05). Furthermore, the amyloid protein content in 5xFAD mice treated with MSC-sIVs and MSC-sIVs was significantly reduced, with the difference being statistically significant (P < 0.05). In particular, MSC-sIVs significantly reduced the Aβ plaque burden in 5xFAD mice (approximately 11 pg / mg).

[0552] 5. Summary

[0553] After treatment with MSC-sIVs and MSC-sEVs, Aβ amyloid levels in the Alzheimer's disease mouse model were significantly reduced, oxidative stress responses were effectively alleviated, neuronal apoptosis was inhibited, and the activation of microglia and astrocytes was weakened. Even more significant differences were observed during the navigation phase of the water maze behavioral experiment: compared with the PBS group, the escape latency of mice in the MSC-sIVs-treated group was significantly shortened on days 4 and 6 of the experiment, and the reduction in escape latency was even greater in the MSC-sIVs-treated group than in the MSC-sEVs-treated group. This result suggests that MSC-sIVs may have a more significant effect in improving spatial learning and memory abilities in Alzheimer's disease mice.

[0554] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0555] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience, and preference of those skilled in the art.

[0556] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. Use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating nervous system diseases; The vesicles are prepared by a method comprising the following steps: (1) Dispersing mesenchymal stem cells in a suspension solvent and performing ultrasonic treatment; (2) centrifuging the liquid obtained in step (1) once or multiple times, discarding the cell membrane and organelle debris, and taking the supernatant; (3) The supernatant obtained in step (2) is subjected to ultracentrifugation, and the precipitate is obtained as the intracellular nanovesicles.

2. The use according to claim 1, characterized in that The amplitude of the ultrasonic treatment in step (1) is 20%-25%; and / or, the time of the ultrasonic treatment is 10-20s.

3. The use according to claim 2, characterized in that The amplitude of the ultrasonic treatment in step (1) is 20%; and / or, the time of the ultrasonic treatment is 15 seconds, with a running time of 2 seconds and a pause time of 2 seconds.

4. The use according to claim 1, wherein The number of centrifugation treatments in step (2) is two times, and the respective parameters are: 1000-3000g, 5-20 minutes; 10000-30000g, 20-40 minutes; and / or, The parameters of the ultracentrifugation treatment in step (3) are 100,000-180,000 g, 50-100 minutes.

5. The use according to claim 1, characterized in that The mesenchymal stem cells are selected from the group consisting of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, placenta and amniotic fluid, and amniotic membrane mesenchymal stem cells.

6. The use according to claim 1, wherein The average particle size of the vesicles is 50-100 nm.

7. The use according to any one of claims 1 to 6, characterized in that The nervous system disease is selected from the group consisting of optic nerve disease, cerebrovascular disease, traumatic brain injury disease, and nervous system degenerative disease.

8. The use according to any one of claims 1 to 6, characterized in that The nervous system disease is an optic nerve disease.

9. The use according to claim 8, characterized in that The optic nerve disease is optic nerve damage.

10. The use according to any one of claims 1 to 6, characterized in that The nervous system disease is ischemic cerebrovascular disease.

11. The use according to claim 10, characterized in that The ischemic cerebrovascular disease is cerebral infarction.

12. The use according to any one of claims 1 to 6, characterized in that The nervous system disease is a degenerative nervous system disease.

13. The use according to claim 12, characterized in that The neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

14. The use according to any one of claims 1 to 6, characterized in that The dosage form of the drug is selected from: oral dosage form, injection dosage form, subcutaneous dosage form, skin dosage form, eye dosage form, and nasal dosage form.

15. The use according to any one of claims 1 to 6, characterized in that The dosage form of the drug is intravitreal injection, nasal administration, intravenous injection or intraperitoneal injection.

16. The use according to any one of claims 1 to 6, characterized in that The drug is in the form of a cream, foam, cream, ointment, emulsion, liquid solution, eye drops, injection, powder injection, gel, spray, suspension, microemulsion, eye patch or contact lens.

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