TWI880092B - Uses of urinary formulations - Google Patents

Abstract

Disclosed herein are uses of a formulation in the preparation of a medicament for treating diseases, wherein the formulation comprises an urinary extracellular vesicle and a plurality of active factors. According to one embodiments of the present disclosure, the urinary extracellular vesicle is derived from a human at age 35 or less or a pregnant woman, and may mix with one or more active agents to produce the present formulation.

Description

Urine preparation uses

This disclosure relates to the field of biotransmission systems. Specifically, this disclosure relates to a preparation comprising extracellular vesicles and a plurality of active molecules and its use.

Urine therapy is a treatment method with a long history, which can be traced back to ancient Egypt, China, Japan and India. It can be seen that urine preparations have great potential in treating diseases, and the midstream urine of children under 10 years old has the best therapeutic effect. However, its treatment principle has not yet been clarified.

Extracellular vesicles (EVs) are cell-derived biological membrane vesicles that carry contents for intercellular signaling. Generally speaking, a cell (i.e., a donor cell) can release different forms of EVs, including exosomes (diameters between 30 and 150 nanometers) and microvesicles (diameters between 100 and 800 nanometers). A recipient cell will absorb the released EVs through endocytosis, membrane fusion, or specific ligand-receptor internalization reactions, thereby transferring the contents of the EVs (e.g., nucleic acids, lipopolysaccharides, proteins, and/or lipids from donor cells) to the recipient cells to cause effects.

Given that EVs are commonly found in different body fluids (e.g., blood, urine, cerebrospinal fluid, malignant ascites, breast milk, bronchoalveolar lavage fluid, and saliva), and their contents reflect the status of donor cells, they can be used as a biomarker for the treatment or diagnosis of diseases and epidemiology. For example, EVs derived from cancer cells or released by their surrounding stromal cells or leukocytes will enter the circulatory system and guide cancer cell invasion and metastasis; vice versa, mesenchymal stem cells and/or immune cells will release EVs in response to cancer cells to antagonize inflammation and cancer invasion and metastasis. Furthermore, pregnant women, placenta, and fetuses communicate through different EVs, which are used as messages about fetal growth and development and anti-rejection, and enter the urine of pregnant women through the circulatory system. Therefore, EVs in urine can be used in cancer diagnosis and treatment, anti-inflammatory diseases, maternal and fetal diseases, degenerative diseases, regenerative or cosmetic medicine, etc.

In addition, based on the characteristics of EVs such as double lipid layer, high biocompatibility, low immunogenicity, nanoscale size and ability to penetrate major biological barriers (including the blood-brain barrier), they can also be used as a candidate for organelle therapy or drug delivery. EVs in cell culture medium, plasma or urine can promote cell fusion, carry proteins or microRNAs (miRNAs) for intercellular communication, reach remote organs such as the brain, liver and central nervous system through major biological membranes, or circulate to the kidneys and be directly or indirectly secreted into urine as biomarkers or for tissue regeneration; however, their efficacy often varies with factors such as the type and severity of the disease and the characteristics of the EVs (such as cell origin, size and contents).

In view of this, there is an urgent need in this field for a drug whose active ingredients can be adjusted according to individual needs so as to be suitable for different applications such as disease treatment, regeneration or cosmetic medicine.

The content of the invention is intended to provide a simplified summary of the disclosure so that readers can have a basic understanding of the disclosure. This content of the invention is not a complete overview of the disclosure, and it is not intended to point out the important/key elements of the embodiments of the invention or to define the scope of the invention.

The present disclosure relates to a method for treating a disease using a preparation comprising an extracellular vesicle and a plurality of active molecules, wherein the extracellular vesicle is isolated from urine of an individual, and the plurality of active molecules are encapsulated in the extracellular vesicle. According to a specific embodiment of the present disclosure, the individual is a human being less than or equal to 35 years old, or a pregnant woman.

According to certain embodiments of the present disclosure, the drug is used to treat a disease, wherein the disease is an inflammatory disease, maternal and infant disease, autoimmune disease, cardiovascular disease, dry eye, diabetes, acute and chronic brain damage, acute and chronic kidney disease, degenerative disease, cosmetology, regenerative medicine, radiation injury, stubborn wounds, cancer or infection.

According to the implementation method of the present disclosure, the drug is administered topically through the skin, conjunctiva, nasal cavity, trachea, oral administration, spinal cord, vein, artery, muscle, subcutaneous, intra-articular, intraventricular, intracerebral ventricle, abdominal cavity or middle ear.

According to certain embodiments of the present disclosure, the plurality of active molecules are a group consisting of growth factors, immunomodulatory factors, anti-cancer factors, anti-inflammatory factors, anti-infective factors, anti-aging factors, antioxidant factors, anti-radiation factors, nucleic acids and combinations thereof.

According to certain embodiments of the present disclosure, the nucleic acid is messenger ribonucleic acid (mRNA), long noncoding RNA (lncRNA), small interfering RNA (siRNA), small hairpin RNA (shRNA) or microRNA (miRNA).

According to certain embodiments of the present disclosure, the extracellular vesicles encapsulate growth factors, anti-inflammatory factors, or both. According to a preferred embodiment of the present disclosure, the extracellular vesicles encapsulate granulocyte colony-stimulating factor (G-CSF), epidermal growth factor (EGF), interleukin-1 receptor antagonist (IL1-RA) and miRNA let-7i

According to certain embodiments of the present disclosure, the preparation further comprises an active agent, and the active agent is bound to the extracellular vesicles, wherein the active agent is aspirin, shrimp oil, melatonin, metformin, lipopolysaccharide, phytochemical, curcumin, dehydroe-piandrosterone (DHEA), tea phenol, polyphenol, tripterone or a combination thereof.

According to certain embodiments of the present disclosure, the preparation containing the active agent is prepared by the following method, wherein the method comprises: (a) isolating extracellular vesicles from urine; (b) combining the extracellular vesicles separated in step (a) with the active agent in an environment of pH 3.0-10.0; and (c) isolating and purifying the product of step (b) to obtain the preparation. According to certain embodiments, step (a) is to separate the extracellular vesicles by centrifugation, filter membrane, hollow fiber filter membrane, dialysis, chromatography or affinity binding. According to certain embodiments, step (c) is to separate and purify the preparation by centrifugation, filtration membrane, hollow fiber filtration membrane, dialysis, chromatography or affinity binding.

After reading the implementation method below, a person with ordinary knowledge in the technical field to which the present invention belongs can easily understand the basic spirit and other invention purposes of the present invention, as well as the technical means and implementation methods adopted by the present invention.

In order to make the above and other purposes, features, advantages and embodiments of the present invention more clearly understandable, the attached figures are described as follows: Figure 1 is a scatter plot drawn according to Embodiment 1.1 and Embodiment 8.1 of the present disclosure, which is used to illustrate the size difference between the UEV of urine and the preparation of the present invention. Panel A is the size distribution of UEV; Panel B is the size distribution of curcumin-UEV; Panel C is the size distribution of shrimp-UEV; Panel D is the size distribution of metformin-UEV. UEV: urine extracellular vesicle.

Figure 2 is a scatter plot drawn according to Example 2.1 of the present disclosure, which illustrates the differences in the content of different miRNAs in UEV, wherein the Ct value is used to represent the content of miRNA. **: p<0.01; ***: p<0.005.

Figure 3 is a bar graph drawn according to Example 2.2 of the present disclosure, which is used to illustrate the expression of CD163, an indicator of different immune macrophage positive reactions (M1), and CD206, an indicator of macrophage negative reactions (M2), in macrophages treated with carriers, aluminum hydroxide or UEV alone and aluminum hydroxide and UEV together. Panel A shows the CD206 content representing M2 in macrophages treated with different treatments; Panel B shows the CD163 content representing M1 in macrophages treated with different treatments. UEV: urine extracellular vesicles. The experimental results show that UEV has the effect of regulating the expression of macrophages representing immune macrophage positive reactions (M1).

Figure 4 is a bar graph drawn according to Example 3 of the present disclosure, which is used to illustrate the analysis results of the effects of extracellular vesicles from different sources on the growth of C2C12 myoblasts. Ctr: control group; MEV: extracellular vesicles of umbilical cord mesenchymal stem cells; UEV-Y: extracellular vesicles of urine of young people; UEV-O: extracellular vesicles of urine of the elderly. The results show that UEV-Y can more effectively promote the growth of C2C12 myoblasts than UEV-O. *: p<0.05.

Figure 5 is a scatter plot and a bar graph drawn according to Example 4 of the present disclosure, which is used to illustrate the analysis results of UEV regulation of helper T cell differentiation into positive (Th1; CD183), negative (Th2; CD194), regulatory T cells (regulatory T cells, Treg) (CD25/CD127) and autoimmune response (Th17; CD196). Panels A-C are representative experiments; Panel D is the result of 4 different experiments. *: p<0.05.

FIG. 6 is a bar graph drawn according to Example 5 of the present disclosure, which illustrates the analysis results of the reactive oxygen species (ROS) content in neutrophils that were untreated, treated with the immunostimulant PMA (phorbol myristate acetate), or treated with UEV, and treated with PMA and UEV. The experimental results show that UEV has a significant inhibitory effect on the oxygen release production of neutrophils induced by PMA. PMA: phorbol-12-tetradecanoyl-13-acetate; UEV: urine extracellular vesicles. *: p<0.05.

FIG. 7 is a scatter plot according to Example 6 of the present disclosure, which is used to illustrate the analysis results of UEV inhibiting the formation of neutrophil extracellular trap (NETosis) after neutrophil inflammation or death; Panel A shows the significant NETosis effect of neutrophils treated with PMA; Panel B shows the reduction of NETosis of neutrophils treated with UEV; Panel C shows the increase of NETosis of neutrophils treated with miRNA let-7i antagonist nucleic acid. The experimental results show that UEV uses miRNA let-7i to inhibit the NETosis effect of neutrophils; NETosis is known to be a defense mechanism of the human body against foreign infections, and it can also cause vascular embolism or adult respiratory distress syndrome. UEV has the effect of inhibiting neutrophil NETosis and has the potential to be used for anti-inflammatory prevention and treatment of vascular embolism or adult respiratory distress syndrome. PMA: phorbol-12-tetradecanoyl-13-acetate; UEV: urinary extracellular vesicles.

FIG. 8 is a bar graph drawn according to Example 7 of the present disclosure, which is used to illustrate the difference in the content of active factors between UEV from a pregnant subject (UEV-P) and UEV from the elderly (UEV-O). Panel A shows the difference in the content of G-CSF; Panel B shows the difference in the content of interferon-α2 (IFN-α2); Panel C shows the difference in the content of IL-6; Panel D shows the difference in the content of monocyte chemoattractant protein-1 (MCP-1; also known as CCL2). The experimental results show that compared with UEV-O, UEV-P has higher content of G-CSF and IFN-α2, but lower content of IL-6 and MCP-1. UEV-P: Extracellular vesicles in urine of pregnant women; UEV-O: Extracellular vesicles in urine of the elderly.

Figure 9 is a scatter plot and a bar graph drawn according to Example 8.2 of the present disclosure, which illustrates the binding efficiency of UEV with different active agents of different concentrations; and the different effects of UEV-Y, curcumin-UEV and shrimp-UEV on ROS generation in C2C12 myoblasts. Panel A is the binding efficiency of curcumin and UEV; Panel B: shrimp-UEV binding efficiency; Panel C is the effect of UEV-Y and the preparation of the present invention on ROS generation in C2C12 myoblasts. *: p<0.05.

In order to make the description of the disclosure more detailed and complete, the following provides an illustrative description of the implementation and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The implementation covers the features of multiple specific embodiments and the method steps and their sequence for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences.

I Definition

Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as those understood and used by persons with ordinary knowledge in the technical field to which the present invention belongs.

Unless otherwise inconsistent with the context, a singular term used in this specification includes the plural form of the term; and a plural term used in this specification also includes the singular form of the term.

Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations caused by individual testing methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the mean, depending on the consideration of a person of ordinary skill in the art to which the present invention belongs. Except for experimental examples, or unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used herein (for example, to describe material usage, time duration, temperature, operating conditions, quantity ratios and the like) are modified by "about". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the attached patent application are approximate values and can be changed as needed. At least these numerical parameters should be understood as the indicated number of significant digits and the values obtained by applying the general rounding method. Here, the numerical range is expressed from one end point to another or between two end points; unless otherwise stated, the numerical range described here includes the end points.

In the present disclosure, the term "formulation" refers to a combination of different contents, which can be produced by conditioning cells with active molecules during cell culture to produce different EV contents, or by encapsulating or embedding different active agents in the EV after obtaining the EV. The formulation of the present disclosure includes an EV having a liposome structure (i.e., a hydrophilic core encapsulated in a double layer of lipids), and at least one different content or different active agents encapsulated in the EV.

In the present disclosure, the term "encapsulate" refers to the process of containing, encapsulating or otherwise connecting two or more endogenous or exogenous coatings, thereby including endogenous active molecules or exogenous active agents in or on a structure. The term "encapsulate" in the present disclosure refers to the inclusion of one or more molecules (such as active molecules and active agents of the present invention) in an extracellular vesicle particle (for example, the lipophilic/hydrophilic membrane of EV). Generally speaking, the active agent on the surface of the EV can be surrounded or covered by the double lipid layer of the EV.

In the present disclosure, the term "treat" includes partial or complete prevention, improvement, alleviation and/or management of a symptom, secondary disease or symptom of a disease. The term "treat" in this specification also refers to the application or administration of a preparation of the present disclosure to an individual to achieve partial or complete alleviation, alleviation, cure of a disease, delay onset, inhibit progression of the disease, reduce the severity of the disease, and/or reduce the occurrence of one or more symptoms, secondary diseases or symptoms associated with sarcopenia. "Treatment" can also be administration to an individual suffering from such early signs or symptoms to reduce the occurrence of symptoms, secondary diseases or symptoms associated with sarcopenia. When one or more symptoms or clinical markers are reduced, the treatment is effective. Alternatively, treatment is effective when the progression of a symptom, secondary disease or condition is slowed or stopped.

In this disclosure, the term "subject" refers to animals, including humans, that can be treated with the methods of the present invention. Unless otherwise specified, the term "subject" refers to both males and females.

II Specific implementation methods

In modern medicine, the application of extracellular vesicles has become increasingly widespread; for example, as a biomarker to treat or diagnose diseases, as a carrier for drug delivery, and in organelle treatment methods. Part of the present disclosure is based on the discovery that the types and contents of extracellular vesicles in plasma and urine (e.g., active molecules such as growth factors and anti-inflammatory factors) are different, and that the urinary extracellular vesicles (UEV) of young people contain higher concentrations of active molecules than those of the elderly. Accordingly, urine from young people aged 35 or younger, or pregnant women, is prepared into a preparation for the treatment of different diseases.

Therefore, one aspect of the present disclosure is related to the use of a preparation comprising an extracellular vesicle and a plurality of active molecules for preparing a drug. According to the implementation method of the present disclosure, the extracellular vesicle is separated from the urine of an individual, and the plurality of active molecules are encapsulated in the extracellular vesicle; preferably, the individual is a human being less than or equal to 35 years old, or a female individual in pregnancy.

According to certain embodiments of the present disclosure, the drug is used to treat a disease, wherein the disease includes, but is not limited to, inflammatory diseases, maternal and infant diseases, autoimmune diseases, cardiovascular diseases, dry eyes, diabetes, acute and chronic brain damage, acute and chronic kidney diseases, degenerative diseases, cosmetology, regenerative medicine, radiation damage, stubborn wounds, cancer or infections.

Exemplary inflammatory diseases include, but are not limited to, hay fever, periodontitis, encephalitis, keratitis, conjunctivitis, rhinitis, otitis media, gingivitis, pharyngitis, tonsillitis, pneumonia, hepatitis, enteritis, dysentery, prostatitis, endometritis, cervicitis, pelvic inflammatory disease, or groinitis.

Exemplary autoimmune diseases include, but are not limited to, antiphospholipid syndrome, connective tissue disease, lupus erythematosus, celiac disease, type 1 diabetes, diffuse toxic thyroiditis (or Graves' disease), psoriasis (also known as tinea pedis or psoriasis), rheumatoid arthritis, achalasia, Addison's disease, agammaglobulinemia, benign mucosal pemphigoid, bullous pemphigoid, Chagas' disease, cold agglutinin disease, dermatitis herpetiformis, chronic discoid lupus, lupus), eosinophilic fasciitis.

Exemplary cardiovascular diseases include, but are not limited to, coronary syndrome, stroke, hypertensive heart disease, rheumatic heart disease, aneurysm, cardiomyopathy, atrial fibrillation, congenital heart disease, endocarditis, or peripheral arterial obstructive disease.

Exemplary degenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (also known as frozen man), dementia, cerebellar ataxia, progressive supranuclear palsy, osteoporosis, cystic fibrosis, Ehlers-Danlos syndrome (also known as congenital skin and subcutaneous vascular fragility), Huntington's disease, infantile neuroaxonal dystrophy, keratoglobus, leukodystrophy, macular degeneration, degeneration), osteoarthritis or Tay-Sachs disease.

Exemplary cancers include, but are not limited to, brain cancer, lung cancer, breast cancer, oral cancer, esophageal cancer, stomach cancer, liver cancer, bile duct cancer, pancreatic cancer, colon cancer, kidney cancer, gastric cancer, cervical cancer, ovarian cancer, or prostate cancer.

According to certain embodiments of the present disclosure, the drug is administered to a subject in need thereof topically, intraconjunctival, intranasal, intratracheal, oral, intraspinal, intravenous, intraarterial, intramuscular, subcutaneous, intraarticular, intraventricular, intracerebral, abdominal or middle ear.

According to the embodiments of the present disclosure, the drug is prepared from a preparation comprising extracellular vesicles and a plurality of active molecules, wherein the preparation is prepared by the following method, comprising (a) isolating extracellular vesicles from urine; (b) combining the extracellular vesicles separated in step (a) with the active agent in different buffer solutions (pH 3.0-10.0); and (c) isolating and purifying the product of step (b) to obtain the preparation of the present invention.

In step (a), a person skilled in the art can utilize the difference in size, density and/or affinity between extracellular vesicles and other pollutants in urine (such as uric acid, creatinine, sodium, ammonia, etc.) to separate extracellular vesicles from urine by any known method for separating the desired substance, such as differential centrifugation, size exclusion chromatography, sucrose density gradient centrifugation, immunoaffinity capture, etc. According to the operational implementation of the present disclosure, centrifugation, filter membrane, hollow fiber filter membrane, dialysis, chromatography or affinity binding are used to separate extracellular vesicles.

According to certain embodiments of the present disclosure, the isolated urinary extracellular vesicles (UEV) encapsulate a plurality of endogenous active molecules (i.e., active molecules derived from an individual), such as growth factors, immunomodulatory factors, anticancer factors, anti-inflammatory factors, anti-infective factors, anti-aging factors, antioxidant factors, anti-radioactive factors, nucleic acids, or a combination thereof.

Exemplary growth factors include, but are not limited to, angiopoietin, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), placental growth factor (PLGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), bone morphogenetic protein (BMP-1), and fibroblast growth factor (GF-2). protein, BMP), endoglin, endothelin, leptin, follistatin, hepatocyte growth factor (HGF), insulin-like growth factor (IGF), keratinocyte growth factor (KGF), nerve growth factor (NGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), cartilage growth factor (CGF), stem cell factor (SCF), brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF), interleukin (IL) and ephrin.

Exemplary immunomodulatory factors include, but are not limited to, peptides, nucleic acids (e.g., small interfering ribonucleic acid (siRNA), small hairpin ribonucleic acid (shRNA), and micro-ribonucleic acid (miRNA)), interleukins (e.g., IL-2, IL-7, IL-12), cytokines (e.g., G-CSF and interferon (IFN)), chemokines (e.g., CXC motif chemokine ligand 13, CXCL13), CC motif chemokine ligand 26 (CCL26), and CXCL7), immune checkpoint antagonists (e.g., anti-cytotoxic T lymphocyte associated antigen 4 (cytotoxic T-lymphocyte-associated antigen 4, CTLA-4), anti-programmed death 1 (PD1) or anti-PD-L1 (PD-1 ligand), anti-lymphocyte-activation gene 3 (LAG3) and anti-B7 homolog 3 (B7-H3).

Exemplary anti-inflammatory factors include, but are not limited to, IL1-RA, IL-1β, IL-4, IL-6, IL-10, IL-11, IL-13, IL-18, tumor necrosis factor-α (TNF-α), VEGF, and TGF-β.

According to certain embodiments of the present disclosure, the active molecule is a nucleic acid, wherein the nucleic acid can be DNA or RNA. In an exemplary embodiment of the present disclosure, the nucleic acid is RNA, for example, mRNA, lncRNA, siRNA, shRNA or miRNA. Alternatively, the nucleic acid can be a DNA aptamer or an RNA aptamer. Exemplary miRNAs include miRNA precursors (agomir) and miRNA antagonists (antagomir).

According to certain embodiments of the present disclosure, the extracellular vesicles encapsulate growth factors, anti-inflammatory factors, nucleic acids or a combination thereof. According to certain embodiments of the present disclosure, the extracellular vesicles encapsulate G-CSF, EGF, IL1-RA and miRNA let-7i.

In order to optimize the type and content of active factors encapsulated in the extracellular vesicles, the preparation may further include an exogenous active agent (i.e., an active substance not derived from an individual), wherein the exogenous active agent is in an environment of pH 3.0-10.0 (e.g., pH 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.9, 9.0, 9.1, 9.2, 9.3, 9.5 7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10.0) binds to extracellular vesicles (step (b)).

According to certain embodiments of the present disclosure, the exogenous active agent may be an anti-inflammatory agent, an anti-infective agent, an anti-aging agent, an antioxidant, an anti-cancer agent or a combination thereof. According to certain embodiments of the present disclosure, the exogenous active agent is a plant component extracted from a plant component, wherein the plant component is selected from the group consisting of leaves, stems, roots, flowers, tubers, pollen, fruits and seeds. Alternatively, the exogenous active agent may be an extract extracted from fungi or bacteria.

According to certain embodiments of the present disclosure, the exogenous active agent is an anti-inflammatory agent. Exemplary anti-inflammatory agents include, but are not limited to, non-steroidal anti-inflammatory drugs (NASIDs), such as aspirin, ibuprofen, and naproxen. Other exemplary anti-inflammatory agents include alclofenac, alclometasone dipropionate, algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride, hydrochloride), bromelains, broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetasone butyrate, clopirac, cloticasone propionate, cormethasone acetate, cortodoxone, decanoate, deflazacort, delatestryl, depo-testosterone, desonide, desoximetasone, dexamethasone dipropionate dipropionate, diclofenac potassium, diclofenac sodium, diflorasonediacetate, diflumidone sodium, diflunisal, difluprednate, diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium sodium), epirizole, etodolac, etofenamate, felbinac, fenamol, fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac, frazalone, fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin, flunixin meglumine, fluocortin butyl, fluorometholone acetate acetate), fluquazone, flurbiprofen, fluretofen, fluticasone propionate, furaprofen, furobufen, halcinonide, halobetasol propionate, halopredone acetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol, ilonidap, indomethacin, indomethacin sodium, indoprofen, intrazole, isoflurane acetate, acetate), isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride, lomoxicam, loteprednol etabonate, meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone, mesterolone, methandrostenolone, methenolone, methenolone acetate, methylprednisolone suleptanate, momiflumate, nabumetone, nandrolone, naproxen, naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin, oxandrolane, oxaprozin, oxyphenbutazone, oxymetholone, paranyline hydrochloride, pentosan polysulfate sodium, phenbutazone sodium glycerate), pirfenidone, piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone, proxazole, proxazole citrate, rimexolone, romazarit, salcolex, salnacedin, salsalate, sanguinarium chloride), seclazone, sermetacin, stanozolol, sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, testosterone, testosterone blends, tetrydamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium sodium), triclonide, triflumidate, zidometacin and zomepirac sodium, and combinations thereof.

According to certain embodiments of the present disclosure, the exogenous active agent is an anti-infective agent. Exemplary anti-infective agents include, but are not limited to, LL37, IFN-α, hydrophilic and hydrophobic antibiotics and aptamers.

According to certain embodiments of the present disclosure, the exogenous active agent is an anti-aging agent. Exemplary anti-aging agents include, but are not limited to, curcumin, coenzyme Q10, xanthophyll (e.g., atractylodesin, fucoxanthin, and zeaxanthin), metformin, L-glutamine, retinoids, α-hydroxy acid, β-hydroxy acid, and lutein.

According to certain embodiments of the present disclosure, the exogenous active agent is an antioxidant. Exemplary antioxidant factors include, but are not limited to, amines (e.g., N,N-diethylhydroxylamine and amino-guanidine), arginine pilolate, ascorbic acid and its salts, ascorbyl esters of fatty acids, bioflavonoids, butylated hydroxy benzoic acid and its salts, dihydroxy fumaric acid and its salts, gallic acid and its alkyl esters (e.g., propyl gallate and uric acid), glycine pilolate, and dapoxetine. pidolate), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, lipoic acid, lysine, melanin, methionine, nordihydroguaiaretic acid, proline, silymarin, sorbic acid and its salts, hydroxyl compounds (e.g., glutathione), superoxide dismutase, catalase, tea extract, grape skin/seed extract, rosemary extract, tocopherol acetate, tocopherol, tocopherol sorbate sorbate) and combinations thereof.

According to certain embodiments of the present disclosure, the exogenous active agent is an anticancer agent. Exemplary anticancer agents include, but are not limited to, curcumin, IFN, cytokines, antibodies (e.g., trastuzumab (HERCEPTIN®), trastuzumab emtansine (Kadcyla®), bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), rituximab (RITUXAN®) and tositumomab (BEXXAR®)), anti-estrogen (e.g., tamoxifen, raloxifene and megestrol), luteinizing hormone agonists (LHRH agonists (e.g., goserelin and leuprolide), anti-androgen (e.g., flutamide and bicalutamide), photodynamic therapies (e.g., vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4 and demethoxy-hypocrellin A (2BA-2-DMHA), nitrogen mustard (e.g., mustards (e.g., cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g., carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g., busulfan and treosulfan), triazenes (e.g., dacarbazine and temozolomide), platinum containing compounds (e.g., compounds, such as cisplatin, carboplatin, and oxaliplatin), vinca alkaloids (such as vincristine, vinblastine, vindesine, and vinorelbine), taxoids (such as paclitaxel or paclitaxel bound to nanoparticle albumin (Abraxane), paclitaxel bound to docosahexaenoic acid (DHA-paclitaxel, Taxoprexin), paclitaxel bound to polyglutamic acid (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and paclitaxel-conjugated to glucose (e.g., etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, and mytomycin C), paclitaxel equivalents), anti-metabolites, DHFR inhibitors, inhibitors (e.g., methotrexate, dichloromethotrexate, trimetrexate, and edatrexate), inosine-5'-monophosphate dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonuclotide reductase inhibitors (e.g., hydroxyurea and deferoxamine), uracil analogs (e.g., analogs (e.g., 5-fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, and capecitabine), cytosine analogs (e.g., cytarabine, ara C or cytosine arabinoside, and fludarabine), purine analogs (e.g., mercaptopurine and thioguanine), vitamin D3 analogs (e.g., EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g., inhibitors (e.g., lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g., staurosporine), actinomycins (e.g., actinomycin D), bleomycins (e.g., bleomycin A2, bleomycin B2, and peplomycin), anthracyclines (e.g., daunorubicin, doxorubicin, pegylated liposomal doxorubicin, doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin and mitoxantrone), MDR inhibitors (e.g. verapamil), Ca2+ ATPase inhibitors (e.g. thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors inhibitors, such as axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTINTM, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib , Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, S U11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin, MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOKTM), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228), protease inhibitors (e.g., bortezomib, Velcade), mammalian target protein inhibitors (mTOR inhibitors, such as rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), rapamycin derivatives (ridaforolimus, AP23573, Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe), and OSI-027 (OSI), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine or procarbazine, prednisolone, dexamethasone, ethasone, campathecin, plicamycin, asparaginase, aminopterin, methotrexate, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, discodermolide, carminomycin and hexamethyl melamine.

According to a preferred embodiment of the present disclosure, the exogenous active agent is selected from the group consisting of aspirin, shrimp oil, melatonin, metformin, lipopolysaccharide, phytochemicals, curcumin, DHEA, tea phenol, polyphenols, tripterones and combinations thereof. Optimally, the exogenous active agent is curcumin, shrimp oil, metformin or a combination thereof.

Finally, as described in step (c), the product of step (b) is separated and purified to obtain the preparation of the present invention. According to the implementation method of the present disclosure, the preparation containing extracellular vesicles and active agents is separated and purified by centrifugation, filter membrane, hollow fiber filter membrane, dialysis, chromatographic analysis or affinity binding. Those with ordinary knowledge in the field of the present invention can use other techniques known in the field to screen the desired substances with specific sizes according to experimental requirements to obtain the preparation of the present invention.

Several experimental examples are presented below to illustrate certain aspects of the present invention, so as to facilitate the implementation of the present invention by those with ordinary knowledge in the technical field to which the present invention belongs, and these experimental examples should not be regarded as limiting the scope of the present invention. It is believed that after reading the description provided here, the skilled person can fully utilize and implement the present invention without over-interpretation. All public documents cited here are regarded as part of this specification in their entirety.

Implementation example

Materials and methods

1 Preparation of the preparation of the present invention

In order to obtain the preparation of the present invention, first, about 30-300 ml of urine sample is collected from the subject, centrifuged to remove impurities, and then the EV is separated from the sample by a filter membrane, hollow fiber filter membrane, dialysis or magnetic beads, etc., to obtain UEV with a diameter between 0.02-0.8 microns. The collected UEV is concentrated so that each milliliter contains about 1×10 ¹² UEVs, and then packaged and frozen for storage.

Then, the number and size of UEVs were measured using a nanoparticle tracking analyzer (NTA), and the UEVs were identified to carry extracellular vesicle-specific markers CD9 and CD63 using Western blot. Furthermore, hydrophobic shrimp, curcumin (with cationic properties), and metformin were encapsulated in the liposomal structure of EVs by incubating at room temperature in an environment of pH 3.0-10.0. In this study, 0, 15, 30 or 50 μM of prawns; 0.0625, 0.125, 0.25, 0.5 or 1 mM of curcumin; or 0.25, 0.5, 1, 2 mM of metformin were added to the preparation (about 1×10 ¹¹ UEVs per ml) in 10 ml of serum-free medium and reacted at room temperature for 15 minutes. Then, the mixture was filtered through a 0.02 μm filter to obtain preparations with hydrophobic prawns, curcumin or metformin encapsulated in the UEV structure, which were named curcumin-UEV, prawns-UEV and metformin-UEV, respectively.

2 Cell culture

C2C12 myoblasts were purchased from the Global Bioresource Center (ATCC) and cultured in Dulbecco modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) in a humidified incubator at 37°C and 5% CO ₂ .

3. Treatment of C2C12 myoblast growth

The cultured C2C12 myoblasts were placed in a serum-free environment and treated with specific treatments for 24 hours (1x10 ³ cells per well). The cell survival rate and proliferation were analyzed using the trypan blue exclusion test and CCK-8 cell proliferation assay.

4 Analysis of miRNA expression in EVs

A commercially available kit was used to construct a database of miRNA in UEV for sequencing analysis, and then compared with the miRBase database to analyze miRNA in UEV.

In addition, in order to verify the type and content of miRNA in UEV, RNA of UEV was extracted using a commercial kit, and then the miRNA was reverse transcribed into cDNA using a complementary deoxyribonucleic acid (cDNA) synthesis kit and the trunk-loop reverse transcription (RT) primers of each gene in Table 1. The cDNA was used as a template and the forward and reverse primers of each gene (Table 1) were used to perform real-time polymerase chain reaction (qPCR) to verify the expression of the target miRNA in UEV.

5 Effect of UEV on immune T cell differentiation

After stimulation, T cells will differentiate from a quiescent state (Tho) into T cells with different functions. When T cells differentiate into T helper 1 (Th1) or T helper 2 (Th2), their cell surface receptors will show different characteristics, among which CD194 represents Th1; CD183 represents Th2; CD25/CD127 represents regulatory T cells (Treg); CD196 represents T helper 17 (Th17). Flow cytometry and anti-CD183, anti-CD194, anti-CD25/CD127 and anti-CD196 antibodies were used to detect the differentiation of T cells after UEV treatment, representing differentiation into Th1, Th2, Treg or Th17, respectively.

6 Effect of UEV on macrophage differentiation

Macrophages can differentiate into M1 or M2 types according to the stimulation of different microenvironments. When macrophages differentiate into M1 or M2 macrophages, their cell surface receptors will show different characteristics, among which CD163 and CD206 represent M1 and M2 types respectively. Flow cytometry and anti-CD163 and anti-CD206 antibodies are used to detect the M1 and M2 differentiation of macrophages after UEV treatment.

7 Effect of UEV on the release of reactive oxygen species (ROS)

First, neutrophils (1×10 ⁶ cells per ml) or C2C12 myoblasts (1×10 ³ cells per well) were stimulated with phorbol myristoyl acetate (PMA) to produce oxygen free radicals. UEV was then added to the cell culture medium at a ratio of 1:50,000 for 30 minutes. After washing with phosphate buffered solution, the ROS content was analyzed using 1 μM hydroethidium (HE) (10 μM) and flow cytometry.

8 Effect of UEV on neutrophil extracellular trap formation (NETosis).

Neutrophils (1×10 ⁶ cells/mL) were induced to produce NETosis with 100 nanograms (ng) of PMA, and then UEV (at a ratio of 1:5000) or miRNA was added for 6 hours. Finally, fluorescent dye and myeloperoxidase (MPO) were added for staining, and the rate of NETosis in cells was analyzed using excitation light at a wavelength of 485 nm and flow cytometry.

Example 1 Characteristic Analysis of UEV

1.1 Concentration and size

In this embodiment, a nanotracker is used to analyze the concentration and size of UEV.

The analysis results showed that the average size of UEVs was between 106-110 nm, with the majority of them having a diameter of 74 nm (Panel A in Figure 1 and Table 2). After concentration, the concentration of UEVs increased from approximately 5-11×10 ⁹ UEVs per ml to approximately 1-1.3×10 ¹² UEVs per ml (Table 2).

1.2 Analysis of active molecules

This example will detect the biomarkers of UEV and the active molecules contained therein. The experimental results are summarized in Table 3.

Flow cytometry and Western blot analysis respectively demonstrated that UEVs expressed three types of CD9, CD63, and CD81 (data not shown). These proteins belong to the transmembrane 4 superfamily and can be used as indicators of EVs. UEVs are rich in active factors such as EGF, FGF-2, G-CSF, GM-CSF, growth-regulated oncogene (GRO), MDC, IL-1α, IL1-RA, and IL-10 (Table 3).

In addition, the separated UEVs were divided into a young group (aged less than or equal to 35 years old, hereinafter referred to as UEV-Y) and an elderly group (aged greater than 60 years old, hereinafter referred to as UEV-O) according to the age of the subjects, and the difference in the content of active factors in the two groups of UEVs was compared. The results are summarized in Table 4.

*: P<0.05; n=10

From the results in Table 4, we can see that compared with the UEV of the elderly, the UEV of young people contains more growth factors (EGF and G-CSF) and anti-inflammatory factors (IL1-RA), and fewer inflammatory factors (GRO, IL-6, IL-8, IP10 and GM-CSF), indicating that the UEV of young people has better anti-inflammatory and growth-promoting effects.

Example 2 Anti-inflammatory effect of UEV

2.1 Analysis of miRNA expression in UEV

In this embodiment, the expression levels of regeneration and anti-inflammation-related miRNAs (miR-148-3p, miR-660-5p, let-7i-5p, miR-30a-5p, miR-21-5p, miR-125a-5p, Let-7a-5p, and miR423-5p) in UEV were analyzed, and U6 non-coding small nuclear RNA was used as a control group.

The results show that UEV is rich in many anti-inflammatory related miRNAs, among which miR-148-3p and let-7i-5p have the highest expression levels; followed by miR-660-5p and miR423-5p (Figure 2). In addition, these miRNAs are also involved in regulating the development of neural cells, indicating the applicability of UEV in the fields of anti-inflammatory and regenerative medicine.

2.2 UEV regulates macrophage differentiation

Macrophages differentiate (or polarize) into M1 or M2 macrophages according to the stimulation of the tissue microenvironment. Among them, M1 macrophages secrete many pro-inflammatory factors (such as interferon γ, IL-12, IL-23, TNF, IL-6, IL-1, etc.) to induce inflammatory response to fight intracellular bacteria or viruses; M2 macrophages secrete anti-inflammatory factors (such as IL1-RA, TGF-β, CCL17, etc.) to inhibit inflammatory response and help tissue repair.

According to the results of panels A and B in Figure 3, administration of UEV promotes the expression of CD163 and CD206, and CD163 is a biomarker of M1 and CD206 is a biomarker of M2 macrophages. Therefore, it can be seen that administration of UEV can regulate the expression of macrophages, which represents the positive response (M1) of immune macrophages.

Example 3 UEV promotes the growth of C2C12 myoblasts

In this embodiment, in order to detect the effect of extracellular vesicles on cell growth, extracellular vesicles (EV of mesenchymal stem cells, MEV) isolated from umbilical cord mesenchymal stem cells and extracellular vesicles (UEV-Y and UEV-O) from urine were co-cultured with myoblasts, and the effects of different extracellular vesicles on cell growth were observed.

The experimental results show that EVs from different biological specimens have the effect of promoting myoblast growth, and UEV-Y has a better effect of promoting myoblast growth than UEV-O. Based on this, UEVs from young people are more suitable for the treatment of muscle nerve regeneration (Figure 4).

Example 4 UEV regulates the differentiation of helper T cells (T helper cells, Th)

Under the action of phytohaemagglutinin (PHA, 1ug/ml), helper T cells were stimulated to differentiate into Th1, Th2, Treg or Th17, and UEV was administered at the same time. Anti-CD183, anti-CD194, anti-CD25/CD127 and anti-CD196 antibodies were used to represent differentiation into Th1, Th2, Treg or Th17, respectively. Flow cytometry was then used to detect the analysis results of UEV regulating the differentiation of helper T cells into positive (Th1; CD183), negative (Th2; CD194), regulatory (Treg; CD25/CD127) and autoimmune reactions (Th17; CD196).

The experimental results show that UEV affects the differentiation of Th1 and Th2, and significantly inhibits the effects of Th17 and Treg (Figure 5, panels A-D).

Example 5 UEV inhibits the generation of oxygen free radicals in neutrophils

Neutrophils were treated with PMA, PMA+UEV or UEV, and after HE staining, the content of reactive oxygen species (ROS) in neutrophils was detected by flow cytometry.

According to the results of Figure 6, administration of UEV can reduce the ROS content of the control group and can also significantly inhibit the generation of ROS induced by PMA (statistical analysis by Mann-Whitney U test, *: p<0.05; n=6,); therefore, UEV can effectively inhibit the generation of oxygen free radicals by neutrophils and achieve an antioxidant effect.

Example 6 UEV inhibits NETosis formation

NETosis is known to be the body's defense mechanism against foreign infections. It is also the culprit of vascular embolism or adult respiratory distress syndrome. Studying the regulatory effect of UEV on NETosis can be used as a model for finding anti-inflammatory or anti-embolic agents. Furthermore, NETosis is known to be involved in the pathogenesis of various diseases such as diabetes, systemic lupus erythematosus, cardiovascular diseases (such as vascular embolism or adult respiratory distress syndrome) and rheumatoid arthritis. Therefore, by observing the effect of UEV on NETosis formation, the possible immunopathogenicity of UEV on these immune diseases can be verified.

According to the results of Figure 7, administration of PMA induces neutrophils to form NETosis (increased MPO content) (panel A); in contrast, the group administered with UEV can effectively reduce the MPO content in neutrophils, that is, UEV can inhibit NETosis formation (panel B). In addition, administration of miRNA let-7i antagonist nucleic acid inhibits the inhibitory effect of UEV (panel C), indicating that UEV contains miRNA let-7i, and can achieve anti-inflammatory purposes by inhibiting leukocyte activity through miRNA let-7i, and further inhibit NETosis formation, which can be used for anti-inflammatory prevention and treatment of chronic diseases or vascular embolism.

Example 7 Differences in active factors between UEV-P and UEV-O

The results in Figure 8 show that the levels of G-CSF and IFN-α2 in UEV-P are significantly higher than those in UEV-O (Panels A and B in Figure 8); while the levels of inflammatory factors IL-6 and monocyte chemoattractant protein-1 (MCP-1; also known as CCL2) in UEV-P are significantly lower than those in UEV-O (Panels C and D in Figure 8). The characteristics of UEV-P containing more growth factors and lower levels of inflammatory factors can just make up for the difference of insufficient growth factors and more inflammatory factors in UEV-O.

Example 8 Analysis of the properties of the preparation of the present invention

The preparation of the present invention was prepared according to Materials and Methods 1, wherein curcumin-UEV had a binding rate of 54-85% at 0.25-0.5 mM; and curcumin-UEV had a binding rate of 54% at 50 μM.

8.1 Concentration and size

The average sizes of curcumin-UEV, shrimp-UEV and metformin-UEV (i.e. the preparation of the present invention) are 109, 104 and 112 nm, respectively, indicating that the size of UEV does not change significantly after being coated with different active agents (Figure 1, panels B-D and Table 5).

After UEV was coated with different active agents, the concentration of recovered curcumin-UEV was about 5.7-9.1×1011 UEV per milliliter; the concentration of schizocyanin-UEV was about 9.1-10.2×1011 UEV per milliliter; and the concentration of metformin-UEV was about 9.4-10×1011 UEV per milliliter (Table 5).

8.2 Different UEV preparations of the present invention inhibit the generation of oxygen free radicals in C2C12 myoblasts

In order to explore the difference in the activity of the preparation of the present invention and UEV-Y without adding active agent in inhibiting the generation of oxygen free radicals, the concentration of the preparation of the present invention (i.e., curcumin-UEV and shrimp-UEV) was calculated with or without binding, and then co-cultured with C2C12 myoblasts for 1 day. After staining with 10μM HE for 30 minutes, the content of oxidative free radicals (ROS) in the cells was measured by flow cytometry. The study found that UEV-Y, curcumin-UEV and shrimp-UEV had different effects on inducing free radicals (Figure 9, panel C).

As shown in the experimental results of panels A and B in Figure 9, the present invention can effectively bind small molecule preparations of different concentrations. UEV has a binding rate of 54-85% with 0.25-0.5mM curcumin, and UEV has a binding rate of 54% with 50μM styrax.

In addition, the results of panel C show that the effects of UEVs combined with different active agents (i.e., the preparations of the present invention, curcumin-UEV and schizonepeta-UEV) on ROS generation in C2C12 myoblasts are different from those of UEV-Y without active agents, indicating that the preparations of the present invention can produce a variety of effects by encapsulating different active agents into UEVs, thereby increasing the applicability of different UEV preparations.

In summary, the present disclosure provides a novel UEV preparation for treating diseases. According to the embodiments of the present disclosure, UEV from young people aged 35 or younger or pregnant women can be separated and mixed with an active agent to obtain a drug with wide applications (e.g., treating inflammatory diseases, maternal and infant diseases, autoimmune diseases, cardiovascular diseases, degenerative diseases, radiation injuries, stubborn wounds, cancer or infectious diseases, regenerative or cosmetic medicine).

Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention belongs can make various changes and modifications without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined in the accompanying patent application.

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Claims (6)

A use of a preparation comprising an extracellular vesicle and a plurality of active molecules in the preparation of a drug, wherein the extracellular vesicle is separated from the urine of an individual, and the plurality of active molecules are encapsulated in the extracellular vesicle, wherein the individual is a human being less than or equal to 35 years old, and the plurality of active molecules at least contain granulocyte colony-stimulating factor (G-CSF), epidermal growth factor (EGF), interleukin-1 receptor antagonist (IL1-RA) and miRNA let-7i. The use as described in claim 1, wherein the preparation further comprises an active agent, and the active agent is bound to the extracellular vesicle, wherein the active agent is selected from the group consisting of aspirin, shrimp oil, melatonin, metformin, curcumin, dehydroisoandrostenone and tea phenol. The use as described in claim 2, wherein the preparation is prepared by the following method, comprising: (a) isolating the extracellular vesicles from the urine; (b) combining the extracellular vesicles separated in step (a) with the active agent in an environment of pH 3.0-10.0; and (c) isolating and purifying the product of step (b) to obtain the preparation. The use as described in claim 3, wherein step (a) and step (c) are to separate and purify the extracellular vesicles and the preparation by centrifugation, filter membrane, hollow fiber filter membrane, dialysis, chromatography or affinity binding. The use as described in claim 1, wherein the drug is administered topically into the skin, into the conjunctiva, into the nasal cavity, into the trachea, orally, into the spinal cord, into the vein, into the artery, into the muscle, subcutaneously, into the joint, into the ventricle, into the cerebral ventricle, into the abdominal cavity or into the middle ear. The use as described in claim 1, wherein the drug is used to treat inflammatory diseases or degenerative diseases.