Drug delivery system comprising transmembrane domain and use thereof

Abstract

The present invention relates to a drug delivery system platform comprising a transmembrane domain and, more specifically, to extracellular vesicles loaded with a drug delivery system comprising a transmembrane domain and use thereof for treating brain diseases. It was confirmed that the drug delivery system comprising a transmembrane domain, according to the present invention, is maintained in a lipid bilayer when loaded into extracellular vesicles and can bind various active substances to the N-terminus thereof. It was confirmed that the extracellular vesicles loaded with the drug delivery system comprising a transmembrane domain have excellent cellular uptake efficiency, and have excellent effects of cognitive function improvement, neuroprotection, and nerve regeneration when brain disease therapeutic activity-related factors are attached. Therefore, the drug delivery system comprising a transmembrane domain, of the present invention, can have various applications in the drug delivery-related field and the brain disease treatment field.

Description

Drug delivery system comprising a transmembrane domain and use thereof

[0001] The present invention relates to a drug delivery platform comprising a transmembrane domain, and more particularly, to an extracellular vesicle loaded with a drug delivery platform comprising a transmembrane domain and its use in treating brain diseases.

[0002] Extracellular vesicles (EVs) are tiny spheres, each smaller than a cell, enclosed by a phospholipid bilayer and originating from cells. They are present in all body fluids. EVs are a key vehicle for intercellular communication, containing key cellular components like nucleic acids and proteins. Recently, they have attracted attention for their potential as a novel drug delivery method. Significant advantages over other drug delivery methods include their immunity to the individual immune system, their tolerance to natural barriers like the cell membrane, and their ability to target specific cells.

[0003] Extracellular vesicles have a wide variety of names, depending on their cellular origin and method of production. Depending on how they are produced within the cell, they are classified as exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles. Among these extracellular vesicles, exosomes and microvesicles are attracting attention as novel drug delivery vehicles.

[0004] Meanwhile, the blood-brain barrier (BBB) ​​is a cellular barrier composed of tight junctions with a very high electrical resistance of more than 0.1 Ω·m between endothelial cells in contact with related pericytes and astrocytes, and is a highly selective permeability barrier that separates circulating blood from brain extracellular fluid in the central nervous system (CNS), acting as a gateway to protect the CNS by regulating the entry and exit of nutrients and other substances into the brain.

[0005] Thus, the blood-brain barrier functions to prevent blood-borne bacteria, pathogens, and potentially hazardous substances from reaching the brain. However, due to this vascular barrier, most central nervous system drugs exhibit low transcranial delivery efficiency. To compensate, these drugs are administered at high doses, which can cause serious side effects in surrounding organs. Therefore, the development of an efficient drug delivery system capable of penetrating the blood-brain barrier is essential to prevent negative systemic effects while ensuring the therapeutic efficacy of chemodrugs.

[0006] Accordingly, the inventors of the present invention developed a drug delivery platform including a transmembrane domain and confirmed that extracellular vesicles loaded with the drug delivery vehicle have excellent cognitive function improvement, neuroprotection, and neuroregeneration effects, thereby completing the present invention.

[0007] Accordingly, an object of the present invention is to provide a drug delivery system comprising a DE peptide represented by at least one amino acid sequence of SEQ ID NO: 1; a transmembrane domain; and a cell-penetrating peptide motif.

[0008] Another object of the present invention is to provide an extracellular vesicle loaded with the drug delivery system.

[0009] Another object of the present invention is to provide a drug delivery composition comprising the drug delivery vehicle or extracellular vesicle.

[0010] Another object of the present invention is to provide a drug delivery kit comprising the drug delivery vehicle or extracellular vesicle.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating brain diseases, comprising the drug delivery vehicle or extracellular vesicle.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of metabolic diseases, cardiovascular diseases, nervous system diseases, digestive diseases, musculoskeletal diseases, renal and urinary diseases, and skin diseases, comprising the drug delivery system or extracellular vesicles.

[0013] Another object of the present invention is to provide an in vitro composition for promoting nerve regeneration comprising the drug delivery vehicle or extracellular vesicle.

[0014] Another object of the present invention is to provide a method for treating nervous system damage or nervous system disease, comprising the step of administering the drug delivery vehicle or extracellular vesicle to a subject in need thereof.

[0015] Another object of the present invention is to provide a drug delivery method comprising the step of administering the drug delivery vehicle or extracellular vesicle to a subject in need thereof.

[0016] To achieve the above object, the present invention provides a drug delivery system comprising a DE peptide represented by at least one amino acid sequence of SEQ ID NO: 1; a transmembrane domain; and a cell-penetrating peptide motif.

[0017] Additionally, the present invention provides an extracellular vesicle loaded with the drug delivery vehicle.

[0018] The present invention also provides a drug delivery composition comprising the drug delivery vehicle or extracellular vesicle.

[0019] The present invention also provides a drug delivery kit comprising the drug delivery vehicle or extracellular vesicle.

[0020] In addition, the present invention provides a pharmaceutical composition for preventing or treating brain diseases, including the drug delivery vehicle or extracellular vesicle.

[0021] In addition, the present invention provides a pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of metabolic diseases, cardiovascular diseases, nervous system diseases, digestive diseases, musculoskeletal diseases, renal and urinary diseases, and skin diseases, comprising the drug delivery system or extracellular vesicles.

[0022] The present invention also provides an in vitro composition for promoting nerve regeneration comprising the drug delivery vehicle or extracellular vesicle.

[0023] The present invention also provides a method for treating nervous system damage or nervous system disease, comprising the step of administering the drug delivery vehicle or extracellular vesicle to a subject in need thereof.

[0024] The present invention also provides a drug delivery method comprising a step of administering the drug delivery vehicle or extracellular vesicle to an individual in need thereof.

[0025] It was confirmed that the drug delivery system comprising a transmembrane domain according to the present invention is maintained in the lipid bilayer when loaded into an extracellular vesicle and can bind various active substances to the N-terminus. The extracellular vesicle loaded with the drug delivery system comprising a transmembrane domain has excellent intracellular uptake efficiency, and when a factor related to the treatment of brain diseases is attached, it has excellent effects on improving cognitive function, neuroprotection, and nerve regeneration. Therefore, the drug delivery system comprising a transmembrane domain of the present invention can be utilized in various fields related to drug delivery and brain disease treatment.

[0026] Figure 1a is a diagram showing the results of analyzing the loading efficiency of the designed platform peptides APP-LRLLR, NUS-LRLLR, and CHL-LRLLR.

[0027] Figure 1bA is a diagram showing the results of analyzing the EV membrane binding morphology using a STROM high-resolution microscope.

[0028] Figure 1B B is a diagram showing the results of analyzing the mounting location using a STROM high-resolution microscope.

[0029] Figure 2 is a diagram showing the results of analyzing the cellular uptake of the designed platform peptides APP-LRLLR, NUS-LRLLR, and CHL-LRLLR.

[0030] Figure 3 is a diagram showing the platform peptide APP-LRLLR with a cargo (BDNF loop2 or T7 transferrin) attached.

[0031] Figure 4 is a diagram showing the results of confirming the effect of extracellular vesicles loaded with BDNF-loop2 attachment platform peptide on TrkB activity in neurons.

[0032] Figure 5 is a diagram showing the results of confirming the nerve regeneration effect of extracellular vesicles loaded with BDNF-loop2 attachment platform peptide in nerve cells.

[0033] Figure 6 is a diagram showing the results of immunostaining of neurons treated with extracellular vesicles loaded with a BDNF-loop2 attachment platform peptide.

[0034] Figure 7 is a diagram showing the results of confirming the neuroprotective effect of extracellular vesicles loaded with a BDNF-loop2 attachment platform peptide in neurons.

[0035] Figure 8 is a diagram showing the results of confirming the cognitive function improvement effect of extracellular vesicles loaded with BDNF-loop2 attachment platform peptide in an Alzheimer's animal model (p<0.05; *, p<0.01; **, p<0.001; ***, p<0.05; #, p<0.01; ##).

[0036] Figure 9 is a diagram showing the results of measuring amyloid beta in an Alzheimer's animal model administered with extracellular vesicles loaded with a BDNF-loop2 attachment platform peptide (p<0.05; *, p<0.01; **, p<0.001; ***, p<0.0001; ****).

[0037] Figure 10A is a diagram showing the results of confirming the expression of Transferrin receptor in HBEC-5i cells, which are BBB vascular endothelial cells; and Hek293 cells; using Western blotting.

[0038] Figure 10B is a diagram showing the results of analyzing the cellular uptake of extracellular vesicles loaded with a T7 attachment platform peptide in HBEC cells.

[0039] Figure 10C is a diagram showing the results of confirming the internalization of extracellular vesicles loaded with T7 attachment platform peptide in HBEC cells.

[0040] Figure 11 is a diagram showing the results of confirming the BBB permeability of extracellular vesicles loaded with a platform peptide in an in vitro BBB model.

[0041] Figure 12 is a diagram showing the results of confirming the BBB permeability of extracellular vesicles loaded with a platform peptide in an in vivo model.

[0042] Hereinafter, the present invention will be described in detail.

[0043] According to an aspect of the present invention, the present invention provides a drug delivery system comprising a DE peptide represented by at least one amino acid sequence of SEQ ID NO: 1; a transmembrane domain; and a cell-penetrating peptide motif.

[0044] In a specific embodiment of the present invention, the transmembrane domain may be one or more peptides selected from the group consisting of SEQ ID NOs: 3 to 5.

[0045] In the present invention, the transmembrane domain is a short amino acid sequence that readily and selectively binds to the double lipid membrane of an extracellular vesicle without special reaction conditions and is stably maintained in the double lipid membrane. The transmembrane domain is composed of 21-23 amino acids and allows a relatively large protein to be maintained in the cell membrane.

[0046] In a specific embodiment of the present invention, the cell-penetrating peptide motif is preferably represented by the amino acid sequence of SEQ ID NO: 2.

[0047] In the present invention, the cell-penetrating peptide motif is a peptide composed of LRLLR, which is a peptide that penetrates the cell membrane without energy requirement at the C-terminus of the transmembrane domain.

[0048] In the present invention, the DE peptide is a peptide composed of a combination of negatively charged amino acids D (aspartic acid) and E (glutamic acid). (DE) n Depending on the desired negative charge repulsion, the DE peptide can be used in a repeating form, such as DE or DEDE. The DE peptide is linked to the N-terminus of the transmembrane domain and causes the drug delivery system to repel the negative charge of the cell membrane phospholipids, thereby preventing its penetration into the cell membrane and maintaining its stability. Furthermore, various active substances can bind to the DE peptide, which is located at the N-terminus of the transmembrane domain.

[0049] In a specific embodiment of the present invention, it is preferable that the drug delivery system is operably linked as shown in the following structural formula.

[0050]

[0051] [constitutional formula]

[0052] (DE peptide)n - transmembrane domain - cell-penetrating peptide motif, where n is an integer greater than or equal to 1.

[0053]

[0054] In a specific embodiment of the present invention, the drug delivery system may be represented by one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 6 to 8.

[0055] The peptides disclosed in the present invention include functional equivalents thereof.

[0056] The above "functional equivalent" refers to a peptide having at least 80%, preferably 90%, and more preferably 95% sequence homology (i.e., identity) with the peptide disclosed in the present invention as a result of addition, substitution, or deletion of amino acids, including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology, and exhibiting substantially the same physiological activity as the peptide disclosed in the present invention. Sequence homology and identity herein are defined as the percentage of amino acid residues in a candidate sequence relative to the amino acid sequence of the present invention after aligning the amino acid sequence of the present invention with the candidate sequence and introducing gaps. Where necessary, conservative substitutions are not considered part of the sequence homology to obtain the maximum percent sequence homology. N-terminal, C-terminal, or internal extensions, deletions, or insertions in the amino acid sequence of the present invention are not construed as sequences that affect sequence homology or homology.

[0057] Additionally, the sequence identity can be determined by standard methods commonly used to compare similar portions of the amino acid sequences of two polypeptides. Computer programs such as BLAST or FASTA align two polypeptides so that each amino acid matches optimally (along the full length of one or both sequences or along predicted portions of one or both sequences). The programs provide default opening penalties and default gap penalties, and provide scoring matrices such as PAM250 (a standard scoring matrix) that can be used in conjunction with the computer program. For example, the percent identity can be calculated as follows: the total number of identical matches is multiplied by 100, and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequence to align the two sequences.

[0058] In specific embodiments of the present invention, the drug delivery system may further comprise a cargo. Additional components, such as an active ingredient or a labeling agent, may be linked to the N- or C-terminus of the drug delivery system of the present invention to achieve the purpose of drug delivery. Furthermore, the active ingredient or labeling agent may be incorporated into the interior of the extracellular vesicle carrying the drug delivery system of the present invention.

[0059] In a preferred embodiment of the present invention, the cargo can be bound to the N-terminus of the transmembrane domain.

[0060] In a specific embodiment of the present invention, the cargo may be at least one selected from the group consisting of proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, nanoparticles, biological agents, contrast agents, drugs, and compounds.

[0061] The above cargo may be a drug for the prevention, improvement, or treatment of a specific disease, without limitation on its type. For example, the drug may be a substance for the treatment of a brain disease.

[0062] Examples of the nucleic acid-based drugs include mRNA (VEGF, TP53 mRNA), siRNA and miRNA (KRAS siRNA, miR-21 inhibitor), and the CRISPR / Cas system (gene editing RNA-guided Cas9 protein delivery system) as gene therapy delivery platforms.

[0063] Examples of the above protein and peptide-based drugs include BDNF loop2, T7 transferrin, anticancer protein peptides (PD-L1 antibodies, TP53 protein), anti-inflammatory and immune-regulating proteins (IL-10, TGF-beta), etc.

[0064] Examples of the aforementioned drugs and chemical agents include anticancer agents (docetaxel, paclitaxel, doxorubicin, cisplatin), and anti-inflammatory and analgesic agents such as curcumin, dexamethasone, and ibuprofen. Additionally, natural products and bioactive compounds include resveratrol, caffeine, and epigallocatechin gallate.

[0065] Examples of the above contrast agent may be a fluorescent substance, and the fluorescent substance may be a fluorescent protein, a molecule that emits light in an excited state, a metal ion, a complex compound, an organic dye, a conductor, a semiconductor, an insulator, a quantum dot, a quantum wire, etc., but the type thereof is not limited.

[0066] Examples of the fluorescent proteins include enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), enhanced yellow fluorescent protein (EYFP), and red fluorescent protein (RFP).

[0067] In addition, examples of the fluorescent material include pyrene or derivatives thereof, cyanine (Cy) series, Alexa Fluor series, BODIPY series, DY series, rhodamine or derivatives thereof, fluorescein or derivatives thereof, coumarin or derivatives thereof, acridine homodimer or derivatives thereof, acridine orange or derivatives thereof, 7-aminoactinomycin D (7-AAD) or derivatives thereof, actinomycin D or derivatives thereof, ACMA (9-amino-6-chloro-2-methoxyacridine) or derivatives thereof, DAPI or derivatives thereof, Dihydroethidium or a derivative thereof, Ethidium bromide or a derivative thereof, Ethidium homodimer-1 (EthD-1) or a derivative thereof, Ethidium homodimer-2 (EthD-2) or a derivative thereof, Ethidium monoazide or a derivative thereof, Hexidium iodide or a derivative thereof, Bisbenzimide (Hoechst 33258) or a derivative thereof, Hoechst 33342 or a derivative thereof, Hoechst 34580 or a derivative thereof, Hydroxystilbamidine or a derivative thereof, LDS 751 751) or a derivative thereof, Propidium Iodide (PI) or a derivative thereof, Calcein or a derivative thereof, Oregon Green or a derivative thereof,Magnesium Green or a derivative thereof, Calcium Green or a derivative thereof, JOE or a derivative thereof, Tetramethylrhodamine or a derivative thereof, TRITC or a derivative thereof, N,N,N',N'-tetrametyl-6-carboxyrhodamine (TAMRA) or a derivative thereof, Pyronin Y or a derivative thereof, Lissamine or a derivative thereof, ROX or a derivative thereof, Calcium Crimson or a derivative thereof, Texas Red or a derivative thereof, Nile Red or a derivative thereof, Thiadicarbocyanine or a derivative thereof, dansylamide or a derivative thereof, cascade blue, DAPI (4',6-diamidino-2-phenylindole), etc. there is.,

[0068]

[0069] According to another aspect of the present invention, the present invention provides an extracellular vesicle loaded with the drug delivery vehicle.

[0070] In a specific example of the present invention, the extracellular vesicle may be an extracellular vesicle derived from at least one selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells, preferably stem cells, more preferably mesenchymal stem cells, but the present invention is not limited thereto.

[0071] In a specific embodiment of the present invention, the mesenchymal stem cells may be derived from umbilical cord, umbilical cord blood, Wharton's jelly, bone marrow, fat, muscle, nerve, skin, amniotic membrane, teeth, hair root cells, or placenta, and the present invention is not limited thereto.

[0072] The extracellular vesicle of the present invention may be incorporated into a cell and internalized when treated with a cell, and when incorporated into a cell and internalized, it may effectively deliver a clinically significant substance highly expressed in the extracellular vesicle to the cell, so that it is highly expressed in the cell, and it may effectively deliver a cargo attached to the drug delivery system of the present invention, thereby exhibiting effects of nerve regeneration, nerve protection, and cognitive function improvement.

[0073] In a specific embodiment of the present invention, the extracellular vesicles contain a drug carrier in an amount of 1 to 50 nmol / 6x10 8 It is preferable to load particles at a concentration of 5 to 30 nmol / 6x10 8 It may be loaded at a particle concentration of 27 nmol / 6x10 8 particles or 11 nmol / 6x10 8 It can be loaded with particles concentration.

[0074] In a specific embodiment of the present invention, it is preferable that the drug delivery system is loaded onto the lipid bilayer of an extracellular vesicle.

[0075] The extracellular vesicles loaded with the cargo-attached drug delivery system of the present invention have the drug delivery system retained within the lipid bilayer of the extracellular vesicle, and the cargo is displayed either inside or outside the cell. The extracellular vesicles are effectively delivered into cells and can exhibit the activity of the cargo within the cell.

[0076]

[0077] According to another aspect of the present invention, the present invention provides a drug delivery composition comprising the drug delivery vehicle or the extracellular vesicle; and a drug delivery kit.

[0078] The above drug delivery may be intracerebral drug delivery. In an embodiment of the present invention, it was confirmed that extracellular vesicles loaded with the drug delivery system of the present invention can not only effectively deliver cargo to neurons, but also efficiently deliver drugs into the brain by crossing the blood-brain barrier.

[0079] The drug delivery composition and kit of the present invention may further include additional components for preserving and maintaining the drug delivery vehicle or extracellular vesicle.

[0080] In order to monitor drug delivery, the drug delivery kit may include tools, reagents, etc. commonly used in the art for immunological analysis. Examples of the tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, etc. When the labeling substance is an enzyme, the kit may include a substrate and a reaction terminator capable of measuring enzyme activity. The carrier may be a soluble carrier or an insoluble carrier. An example of a soluble carrier is a physiologically acceptable buffer known in the art, such as PBS, and an example of an insoluble carrier may be a polymer such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, a fluororesin, cross-linked dextran, a polysaccharide, a latex-plated magnetic microparticle, other paper, glass, metal, agarose, and combinations thereof.

[0081] Since the kit of the present invention comprises the above-described composition as a component, redundant descriptions are omitted to avoid excessive complexity of the present specification.

[0082]

[0083] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating brain diseases, comprising the drug delivery vehicle or the extracellular vesicle.

[0084] In a specific example of the present invention, the brain disease may be at least one selected from the group consisting of brain tumor, traumatic brain injury, hypoxic brain injury, ischemic brain disease, degenerative brain disease, bacterial or viral brain infection, Parkinson's disease, encephalitis, stroke, apoplexy, Alzheimer's disease, Lou Gehrig's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, epilepsy, thrombosis, embolism, cerebral infarction, stroke, small infarction, and cerebral circulation metabolic disorder.

[0085] When the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be formulated and used in various forms according to conventional methods. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

[0086] The composition of the present invention may contain one or more known effective ingredients having a preventive or therapeutic effect on brain diseases together with the drug delivery system and extracellular vesicles of the present invention.

[0087] The composition of the present invention may further include a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may be starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, etc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0088] The composition of the present invention can be administered in various oral or parenteral dosage forms during actual clinical administration. When formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. It is preferable to use suitable formulations known in the art as disclosed in the literature (Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA).

[0089] The above solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, the above liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.

[0090] The above-mentioned parenteral administration formulations include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0091] The dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and / or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field, and a person having ordinary knowledge in the relevant technical field can easily determine and prescribe an effective dosage for the desired treatment.

[0092] The dosage of the pharmaceutical composition of the present invention is preferably administered at a concentration of, for example, 0.05 to 5 mg / kg, more preferably 0.1 to 0.4 mg / kg, even more preferably 0.2 to 0.35 mg / kg, and even more preferably 0.25 mg / kg, but the dosage does not limit the scope of the present invention in any way.

[0093] The route and method of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in their method, and any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area.

[0094] The pharmaceutical composition may be administered orally or parenterally. Parenteral administration methods include, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration, or subcutaneous administration.

[0095] The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of brain diseases.

[0096]

[0097] According to another aspect of the present invention, a pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of metabolic diseases, cardiovascular diseases, nervous system diseases, digestive diseases, musculoskeletal diseases, renal and urinary diseases, and skin diseases, comprising the drug delivery vehicle or extracellular vesicles is provided.

[0098] The drug delivery system of the present invention can be loaded with various drugs. Therefore, it can exhibit a therapeutic effect on one or more diseases selected from the group consisting of metabolic diseases, cardiovascular diseases, nervous system diseases, digestive diseases, musculoskeletal diseases, renal and urinary diseases, and skin diseases. For example, metabolic diseases may include diabetes and hyperlipidemia, and cardiovascular diseases may include hypertension, myocardial infarction, stroke, and arteriosclerosis. Furthermore, nervous system diseases may include Alzheimer's disease, Parkinson's disease, epilepsy, and multiple sclerosis, and digestive diseases may include fatty liver disease, cirrhosis, and Crohn's disease. Musculoskeletal diseases may include rheumatoid arthritis, osteoarthritis, and osteoporosis, and skin diseases may include atopy or psoriasis.

[0099]

[0100] According to another aspect of the present invention, the present invention provides an in vitro composition for promoting nerve regeneration comprising the drug delivery vehicle or extracellular vesicle.

[0101] The in vitro composition for promoting nerve regeneration of the present invention can be used for experimental purposes, and can be a composition for the purpose of treating isolated cells or tissues requiring nerve regeneration due to nerve damage.

[0102] The in vitro composition for promoting nerve regeneration of the present invention may be a medium composition, and the medium may include, without limitation, a medium known to those skilled in the art, for example, a medium containing serum (e.g., fetal bovine serum, horse serum, and human serum). The medium that can be used in the present invention may include, for example, RPMI series, EMEM, MEM, Iscove's MEM, 199 medium, CMRL 1066, RPMI 1640, F12, F10, DMEM, a mixture of DMEM and F12, Way-mo, McCoy's 5A, or a medium known in the art suitable for culturing cells requiring nerve regeneration. The nerve regeneration may be used to mean all of inducing nerve regeneration, promoting BDNF expression, inducing neural differentiation, proliferation of neural cells, or inducing neural circuit recovery.

[0103]

[0104] According to another aspect of the present invention, the present invention provides a method for treating a brain disease, comprising the step of administering the extracellular vesicle to a subject in need thereof.

[0105] In a specific example of the present invention, the subject may be an subject expected to develop a brain disease; an individual who has developed a brain disease; or an individual who has been diagnosed as cured; but the scope of the present invention is not limited thereto.

[0106] In addition, the treatment method of the present invention can simultaneously or simultaneously administer a brain disease treatment agent known in the art in addition to the extracellular vesicles.

[0107]

[0108] According to another aspect of the present invention, the present invention provides a drug delivery method comprising the step of administering the drug delivery vehicle or extracellular vesicle to a subject in need thereof.

[0109] The above drug delivery may be intracerebral drug delivery. In an embodiment of the present invention, it was confirmed that extracellular vesicles loaded with the drug delivery system of the present invention can not only effectively deliver cargo to neurons, but also efficiently deliver drugs into the brain by crossing the blood-brain barrier.

[0110]

[0111] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0112] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0113]

[0114] Example 1. Design of an extracellular vesicle membrane binding platform peptide.

[0115] In this example, a platform peptide that binds to the extracellular vesicle membrane was designed. The designed platform peptide consists of a transmembrane domain (TD), an LRLLR peptide, and (DE). n It contains a peptide and is operably linked with the following structural formula.

[0116]

[0117] [constitutional formula]

[0118] (DE) n - Transmembrane domain - LRLLR

[0119]

[0120] The above transmembrane domain (SEQ ID NO: 3, 4, or 5) is a short amino acid sequence that selectively binds to the double lipid membrane of an extracellular vesicle without special reaction conditions and is stably maintained in the double lipid membrane. The LRLLR peptide (SEQ ID NO: 2) is a motif of a cell-penetrating peptide (CPP) and is a peptide that binds to the C-terminus of the transmembrane domain without energy requirement and penetrates the cell membrane. The (DE) n The peptide (SEQ ID NO: 1) is linked to the N-terminus of the transmembrane domain and is a combination of negatively charged amino acids D (aspartic acid) and E (glutamic acid). The above (DE) n The peptide is stably maintained in the double lipid membrane without penetrating the membrane of the extracellular vesicle by having the platform peptide have a negative charge and repulsion with the phospholipid, and various active substances can be bound to its N-terminus.

[0121]

[0122] 1-1. Exploring the transmembrane domain

[0123] We investigated proteins with a single transmembrane domain, resulting in the identification of 15 proteins. The 15 identified proteins are listed in Table 1.

[0124] 서열번호Single tramsmembraneproteinTransmembrane domainTotal lengthsequencelengthCharge at pH710receptor-type tyrosine-protein phosphatase S isoform 7 precursor (PTPRS)1283-LIWVIGPVLAVVFIICIVIAI-130321-0.11,94811tumor necrosis factor receptor superfamily member 27 isoform 1139-LVALVGSLLVVFALAFLGLFF-15921029712Junctional adhesion molecule C242-IGGIIGGVLVVLAVLALITLG-26221031013low-density lipoprotein receptor-related protein 2 precursor (LRP2)4424-AVAVLLTILLIVVIGALAIAGFF-44462304,65514Neural cell adhesion molecule L1-like protein (CHL1)1083-FIGLMCAIALLTLLLLTVCFV-110321-0.11,20815Sarcoplasmic / endoplasmic reticulum calcium ATPase 2787-IPVQLLWVNLVTDGLPATALG-80721-11,04216tyrosine-protein kinase RYK isoform 1 precursor228-VFYISVGVCCAVIFLVAIILA-24821-0.160717fibrocystinisoform 2 precursor3859-IILAASLSSVASWLALSCLVC-387921-0.14,07418leucine-rich repeat transmembrane protein FLRT2 precursor1283-LIWVIGPVLAVVFIICIVIAI-130321-0.1194819transmembrane protein 132D precursor916-LEIGMYALLGVFCLAILVFI-93621-0.11,09920Zeta-sarcoglycan38-FFVLLLLVTMIVNLAMTIWIL-5821029921Triadin48-AAWLLVIALIITWSAVAIVMF-6821072922Nectin-1356-IIGGVAGSILLVLIVVGGIVV-37621057123Dehydrodolichyldiphosphate synthase complex subunit NUS135-WIWRRCCRAASAAVLAPLGFTL-56222.929324Amyloid-beta precursor protein (APP)702-IIGLMVGGVVIATVIVITLVM-722210770.

[0125] As shown in Table 1, the obtained proteins ranged in size from a minimum of 293 amino acids to a maximum of 4,655 amino acids. The transmembrane domains consisted of 21-23 amino acids, suggesting that large proteins can be retained in the cell membrane by short transmembrane domains. Among these, the transmembrane domains of three proteins were selected based on size: low-density lipoprotein receptor-related protein 2 precursor (LRP2, 4,655 amino acids), amyloid precursor protein (APP, 770 amino acids), and dehydrodolichyldiphosphate synthase complex subunit NUS1 (NUS1, 293 amino acids).

[0126]

[0127] 1-2. Production of an extracellular vesicle membrane-binding platform peptide

[0128] Using the two membrane-transmitting domains selected in Example 1-1, an extracellular vesicle membrane-binding platform peptide was produced. In addition, for further analysis (DE) nBiotin or FITC was loaded at the N-terminus of the peptide. The fabricated platform peptide and its characteristics (hydrophilicity, molecular weight, IE, net charge) are shown in Table 2.

[0129] Peptides NameTMD originAdditionalAmino acidNo. ofamino acidWaterSolubilityMWIENet chargeat pH7.0BiotinBiotin-DEDEGAIIGLMVGGLRLLR-NH2DEDE + APP TM 700-709 + LRLLRDEDELRLLR19Good2252.66pH 3.93-2Biotin-DESAAVLAPLGFTLRLLR-NH2DE + NUS1 45-55 + LRLLRDELRLLR18Good2167.58pH8.040Biotin-DEDEFIGLMCAIALLRLLR-NH2DE + CHL1 1083-1092+ LRLLRDEDELRLLR19Good2459.96pH4.44-1.1FITCFITC-DEDEGAIIGLMVGGLRLLR-NH2DEDE + APP TM 700-709 + LRLLRDEDELRLLR19Good2384.66pH 3.93-2FITC-DESAAVLAPLGFTLRLLR-NH2DE + NUS1 45-55 + LRLLRDELRLLR18Good2299.58pH8.040FITC-DEDEFIGLMCAIALLRLLR-NH2DE + CHL1 1083-1092+ LRLLRDEDELRLLR19Good2591.96pH4.44-1.1

[0130]

[0131] -APP-LRLLR peptide (SEQ ID NO: 6): The APP-LRLLR peptide is a LRLLR peptide conjugated to the APP dimer-forming domain. In the case of peptide derivatives of neurotrophic factors such as BDNF, a platform peptide was produced utilizing APP TD, which has been reported to bind to cell membranes and form dimerization on its own.

[0132] -NUS1-LRLLR peptide (SEQ ID NO: 7): A platform peptide was produced by utilizing the highly hydrophobic domain of the NUS peptide.

[0133] -CHL-LRLLR peptide (SEQ ID NO: 8): A platform peptide was prepared using the TD peptide of CHL consisting of 1208 amino acids.

[0134] -Reverse reverse sequence peptide (SEQ ID NO: 9): Control

[0135]

[0136] Example 2. Analysis of extracellular vesicle loading of an extracellular vesicle membrane-binding platform peptide.

[0137] 2-1. Extracellular vesicle loading with extracellular vesicle membrane binding platform peptide

[0138] Transmembrane peptides (hereinafter, TD peptides) have the property of binding to the double lipid membrane of extracellular vesicles due to their membrane-binding ability when mixed with extracellular vesicles. Utilizing this property, the extracellular vesicle membrane-binding platform peptide prepared in Example 1 was loaded onto extracellular vesicles (EVs). To this end, the platform peptide and extracellular vesicles were incubated at room temperature for 3 to 18 hours to induce membrane binding. Afterwards, dialysis was performed for 4 hours to remove unloaded peptide, and finally, extracellular vesicles loaded with the platform peptide were purified.

[0139]

[0140] 2-2. Analysis of the loading efficiency and loading location of the extracellular vesicle membrane-binding platform peptide.

[0141] In this example, the loading efficiency of three designed platform peptides, APP-LRLLR, NUS-LRLLR, and CHL-LRLLR, was analyzed. Specifically, a platform peptide conjugated to biotin was prepared, and the reverse-LRLLR peptide was used as a control. Biotin is linked to the N-terminal end of the TD peptide, so it is exposed when the platform peptide is loaded onto the EV membrane surface. This was measured using ELISA to determine the directionality and loading amount of the loaded platform peptide.

[0142]

[0143] i) Peptide release test: Measure the time it takes for 100 ng of peptide to completely release using a 20 kDa dialysis bag. Dissolve 100 ng of peptide in 100 ul of PBS, place in a 20 kDa dialysis bag, seal, and place in 10 ml of PBS. Collect samples at 1-hour intervals and measure biotin ELISA.

[0144] ii) EV loading analysis: The residual peptide removal time was set to 4 hours through the experiment in i) above. For this experiment, TD peptide and extracellular vesicles were passively incubated for 3 hours and dialyzed for 4 hours to remove unloaded peptide. Afterwards, biotin ELISA was performed to measure the peptide content loaded in extracellular vesicles.

[0145]

[0146] The results of the above experiments i) and ii) are shown in Fig. 1a.

[0147] As shown in Fig. 1a, the results of quantifying the platform peptide loaded on the extracellular vesicles confirmed that 27 pmol of APP-LRLLR peptide was loaded on 6 X 10^8 extracellular vesicles. It was confirmed that 11.19 and 28.9 pmol of NUS-LRLLR and CHL-LRLLR were loaded, respectively. The results indicate that 11,000 NUS-LRLLR peptides and 29,000 CHL-LRLLR peptides were loaded per extracellular vesicle particle. In addition, 27,000 peptides of APP-LRLLR peptide were loaded per extracellular vesicle particle. In addition, the loading efficiencies of APP-LRLLR, NUS-LRLLR, and CHL-LRLLR were confirmed to be 46.8%, 31.1, and 45.34%, respectively.

[0148]

[0149] To confirm the membrane location of EVs loaded with the T7 APP LRLLR peptide, the EV membrane binding morphology and loading location were analyzed using a STROM high-resolution microscope. The results of analyzing the EV membrane binding morphology are shown in Figure 1B, A, and the results of analyzing the loading location are shown in Figure 1B, B.

[0150] As shown in Fig. 1b, EVs were confirmed to have vesicle particles that formed a 3D structure when photographed in cross-section along the X and Y axes (Fig. 1b, A). In addition, the size and loading location were confirmed through Cellbrite staining, and it was confirmed that Cellbrite and the peptide were 107 nm and 103 nm, respectively, and bound to the same location on the cell membrane (Fig. 1b, B).

[0151]

[0152] 2-3. Cellular uptake analysis of extracellular vesicles loaded with extracellular vesicle membrane-binding platform peptides

[0153] In this example, we analyzed the cellular uptake of the designed platform peptides APP-LRLLR and NUS-LRLLR. Specifically, extracellular vesicles loaded with the platform peptides (1.86 x 10^8 particles) and 50 ng of Streptavidin-FITC were mixed and incubated at room temperature for 1 hour to label the extracellular vesicles loaded with the platform peptides. The labeled extracellular vesicles were treated with SY5Y cells, which are neural cells. After 1 hour of treatment, the remaining medium was removed and mounting medium containing DAPI was added. The extracellular vesicles taken up by the cells were observed using a fluorescence microscope. The results of the cellular uptake analysis are shown in Figure 2.

[0154] As shown in Fig. 2, it was confirmed that extracellular vesicles loaded with platform peptides had stronger fluorescence intensity (i.e., cellular uptake) than those treated with only the platform peptide. In addition, it was confirmed that extracellular vesicles loaded with platform peptides APP-LRLLR, NUS-LRLLR, or CHL-LRLLR had significantly higher fluorescence intensity than extracellular vesicles loaded with reverse-LRLLR.

[0155]

[0156] Example 3. Production of a cargo-attached extracellular vesicle membrane-binding platform peptide.

[0157] Among the platform peptides designed in Example 2, APP-LRLLR was confirmed to have the best extracellular vesicle membrane binding efficiency. Accordingly, in this Example, a platform peptide APP-LRLLR was produced in which the cargo BDNF loop2 or T7 transferrin was attached to the designed platform peptide APP-LRLLR, and is shown in Figure 3.

[0158]

[0159] Example 4. Evaluation of the efficacy of extracellular vesicles loaded with BDNF-loop2 attachment platform peptide.

[0160] 4-1. Experiment to confirm TrkB activation in extracellular vesicles loaded with BDNF-loop2 attachment platform peptide

[0161] Extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide were treated with C6 and PC12 cells, which are neuronal cells. After 30 minutes of treatment, cells were lysed in RIPA buffer. Western blotting was performed with the obtained lysates to confirm TrkB activation. Before extracellular vesicle treatment, the culture medium was replaced with serum-free medium 30 minutes in advance to stabilize. As controls, a peptide-only group and a reverse-peptide-loaded extracellular vesicle group with the peptide sequence arranged in the reverse order were added and compared. The total protein amount was measured with an actin antibody and normalized. The results confirming TrkB activation are shown in Figure 4.

[0162] As shown in Fig. 4, it was confirmed that the extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide displayed the loaded peptide on the lipid bilayer and were delivered into the cell to promote the expression of cellular growth factors, i.e., the activity of the TrkB signaling system.

[0163]

[0164] 4-2. Confirmation of the Neurogenesis Efficacy of Extracellular Vesicles Loaded with BDNF-loop2 Attachment Platform Peptide

[0165] SH-SY5Y cells were differentiated into neurons by treating 10uM retinoic acid for 5 days. 1 x 10^4 differentiated neurons were seeded in a 24-well plate, and the number and length of neurite tips and the total number of neurons were measured from the next day. Two days after cell seeding, SY5Y cells were treated with extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide. Afterwards, the number of tips extending from neurons, the length of neurites, and the total number of neurons were determined. The results of determining the number and length of neurite tips extending from neurons, the total number of neurons, and the total number of neurons according to the treatment with extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide are shown in Figure 5.

[0166] As shown in Fig. 5, cells treated with extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide showed significantly higher numbers of neurite tips, length, and total number of neurons compared to the EV-treated group or the peptide-only treated group.

[0167]

[0168] Three days after cell seeding, the outer edges of neurons were stained by immunocytochemistry using Tuj1 primary antibody and Alexa-488 secondary antibody to measure neurofilament. After staining, the cells were stained for nuclei using DAPI mounting dye and mounted on a glass slide. The cells were observed under a fluorescence microscope, and the results are shown in Figure 6.

[0169] As shown in Figure 6, cells treated with extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide showed increased numbers of neurons and axonal length compared to the EV-treated group or the peptide-only treated group. This confirmed the efficacy of nerve regeneration in two or more types of nerve cells.

[0170]

[0171] The above results imply that the efficacy of existing BDNF can be maximized through a drug delivery system using extracellular vesicles, as extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide regulate axonal branching, dendrite growth, and promote synapse regeneration and growth.

[0172]

[0173] 4-3. Neuroprotective efficacy of extracellular vesicles loaded with BDNF-loop2 attachment platform peptides

[0174] In this example, we confirmed the neuroprotective effect of extracellular vesicles loaded with BDNF-loop2 attachment platform peptide in a hypoxic environment. Specifically, SH-SY5Y cells were differentiated into neurons by treating them with 10 μM of retinoic acid for 5 days. Differentiated cells (1 x 10^5 cells) were seeded in a 96-well plate. From the next day, each well was treated with extracellular vesicles (5 x 10^8 / ml) loaded with BDNF-loop2 attachment platform peptide, and then pre-incubated in a 37°C incubator for 24 hours. The following day, the medium was replaced with DMEM without glucose and FBS, and placed in a modular incubator chamber (Billups-Rothenberg, Del Mar, USA) and incubated in a hypoxic environment (95% N2, 5% CO2 mixture bubbling to eliminate oxygen) for 6 hours. After incubation, 5 mg / ml of MTT was treated at 20 ul / final volume of 200 ul. This was incubated for 2 hours in a 37°C incubator. The ratio of areas with live cells and dead cells was quantified using live / dead staining. The above experiment was performed, but after 6 hours of incubation in a hypoxic environment, a live / dead staining kit product (LIVE / DEAD™ Viability / Cytotoxicity Kit. Invitrogen # L3224) was added and incubated for an additional hour. After additional incubation, fluorescence was measured using a fluorescence microscope. Through fluorescence measurement, live active cells can be identified by the emission of GFP, and dead cells can be identified by the emission of TRITC. The results of the live / dead staining experiment are shown in Figure 7.

[0175] As shown in Figure 7, the group treated with extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide showed a higher proportion of living neurons under hypoxic conditions compared to the EV treatment group or the peptide alone treatment group.

[0176]

[0177] 4-4. Confirmation of the cognitive function improvement effect of extracellular vesicles loaded with BDNF-loop2 attachment platform peptide in an Alzheimer's disease animal model.

[0178] - Creation of an Alzheimer's zebrafish model

[0179] Donepezil and test drug (extracellular vesicles) used as positive controls were diluted to a final concentration (1.7 x10^7 / 10ul) in E3 medium (containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4, adjusted to pH 7.0-7.2) immediately before use. 2nl (1500 extracellular vesicles) were administered intravenously (IV) to zebrafish in the morning on 2 dpf for pre-treatment and in the afternoon on 3 dpf for post-treatment. To induce Alzheimer's disease, 1 mg / ml Beta-Amyloid (1-42), HiLyte™ Fluor 488-labeled, which was cultured at 37°C for 7 days and converted to oligomer form, was administered (2 nl) into the cerebral ventricle of 2 dpf zebrafish fry.

[0180] The control group received 2 nl of 0.5% phenol red intravenously and intraventricularly.

[0181] The experimental group was divided as follows:

[0182] SNE#1 = Pure extracellular vesicles (MSC-derived naive 3D-EVs)

[0183] SNE#2 = MSC-derived extracellular vesicles loaded with BDNF-loop2 attachment platform peptide (BDNF-loo2 TD - MSC 3D-EV)

[0184] SNE#3 = Hek293-derived extracellular vesicles loaded with BDNF-loop2 attachment platform peptide (BDNF-loo2 TD - Hek 293 2D-EV)

[0185] SNE#4 = MSC-derived extracellular vesicles loaded with reverse BDNF-loop2 attachment platform peptide (Reverse TD - MSC 3D-EV)

[0186]

[0187] - Red ball avoidance experiment

[0188] In a zebrafish model of Alzheimer's disease, we confirmed the cognitive function-enhancing effect of extracellular vesicles loaded with a BDNF-loop2 attachment platform peptide. Cognitive function of zebrafish was confirmed through a red ball avoidance experiment. The red ball avoidance experiment is a cognitive function test using visual stimuli. The zebrafish were pre-trained to observe the movement of the red ball using a red ball screen in the zebrafish rearing environment. After learning the movement of the red ball, zebrafish with improved cognitive ability exhibited avoidance behavior patterns. Specifically, after inducing the Alzheimer's disease model, 6 dpf zebrafish fry were transferred to a 6-well plate (5 larvae per well, N=20 in quadruplicate) to measure the avoidance response. The avoidance ability of the zebrafish fry was then confirmed by visual stimulation. The avoidance response to visual stimulation was calculated by counting the percentage of larvae in the non-stimuli area that avoided a red ball (stimuli area) moving left and right in the upper half of the well area and moved in the lower half (non-stimuli area) at 12-second intervals for 6 minutes, and calculating the average value. The results of the red ball avoidance experiment are shown in Figure 8.

[0189] As shown in Figure 8, the pre-treatment group was found to be effective in preventing and defending against deposition of Beta-Amyloid (1-42) and decline in general intelligence. In the case of the positive control group, EPPS, it was confirmed that cognitive function ability showed a pattern of increase as much as that of the control group.

[0190] In particular, it was confirmed that cognitive function was normalized to the level of the control group in the MSC-derived extracellular vesicle group (SNE#2) loaded with the BDNF-loop2 attachment platform peptide; and the Hek293-derived extracellular vesicle group (SNE#3) loaded with the BDNF-loop2 attachment platform peptide. These results indicate that the extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide exhibit significant cognitive function improvement and neuroprotective properties.

[0191]

[0192] 4-5. Intraventricular amyloid beta clearance test of extracellular vesicles loaded with BDNF-loop2 attachment platform peptide in an Alzheimer's disease animal model.

[0193] In this study, we investigated the intraventricular amyloid beta clearance capacity of extracellular vesicles loaded with the BDNF-loop2 attachment platform peptide in an Alzheimer's disease animal model. For the experiment, the experimental groups were divided as follows.

[0194] - Positive control: EPPS

[0195] - SNE#1 = Pure extracellular vesicles (MSC-derived naive 3D-EVs)

[0196] - SNE#2 = MSC-derived extracellular vesicles loaded with BDNF-loop2 attachment platform peptide (BDNF-loo2 TD - MSC 3D-EV)

[0197] - SNE#3 = Hek293-derived extracellular vesicles loaded with BDNF-loop2 attachment platform peptide (BDNF-loo2 TD - Hek 293 2D-EV)

[0198] Zebrafish fry were anesthetized with Tricaine on the day (0 dpi) and 5 days later (5 dpi) of injection with Beta-Amyloid(1-42), HiLyte™ Fluor 488-labeled, mounted in 1% low-meting agarose, and imaged under a fluorescence microscope. The area of ​​the fluorescence-expressing brain region corresponding to the ventricle was measured using Image J, and the clearance index was calculated using the following formula.

[0199]

[0200] Reduction rate(%) =(Area of ​​fluorescence at 0dpi - Area of ​​fluorescence at 5dpi) / (Area of ​​fluorescence at 0dpi)

[0201]

[0202] After dividing by the calculated value (formula = 2 dpf - 7 dpf / 2 dpf), the average value was graphed. The graph is shown in Fig. 9.

[0203] As shown in Fig. 9, the MSC-derived extracellular vesicle group (SNE#2) loaded with the BDNF-loop2 attachment platform peptide; and the Hek293-derived extracellular vesicle group (SNE#3) loaded with the BDNF-loop2 attachment platform peptide; showed a significant decrease in 488-Aβ1-42 in both the pre-treatment and post-treatment groups. This suggests that the pre-treatment group protects and prevents the deposition of Beta-Amyloid(1-42) in the brain, and the results in the post-treatment group indicate that the deposited Beta-Amyloid(1-42) is discharged and eliminated into the cerebrospinal fluid.

[0204]

[0205] Example 5. Investigation of BBB permeability of extracellular vesicles loaded with T7 attachment platform peptide.

[0206] 5-1. Confirmation of BBB permeability of extracellular vesicles loaded with T7 attachment platform peptide (in vitro)

[0207] - Confirmation of transferrin receptor expression

[0208] Transferrin receptor expression was confirmed in HBEC-5i cells, which are BBB vascular endothelial cells; and Hek293 cells. The Western blotting results are shown in Figure 10A.

[0209] As shown in Figure 10A, it was confirmed that HBEC-5i cells expressed Transferrin receptor.

[0210]

[0211] - Cell uptake analysis

[0212] HBEC cells were treated with extracellular vesicles loaded with the T7 attachment platform peptide. Cell uptake was then analyzed by immunostaining the cells. The results are shown in Figure 10B.

[0213] As shown in Figure 10B, it was confirmed that extracellular vesicles loaded with the T7 attachment platform peptide were absorbed into cells.

[0214]

[0215] Additionally, we analyzed the internalization of extracellular vesicles loaded with the T7 attachment platform peptide in HBEC cells treated with temperature-dependent extracellular vesicles. Specifically, HBEC cells were treated with extracellular vesicles loaded with the T7 attachment platform peptide and then incubated at 4°C to block internalization. Cells were then incubated at 37°C to determine whether the extracellular vesicles were internalized. Controls were untreated or treated with the T7 peptide alone. The results of the internalization analysis are shown in Figure 10C.

[0216] As shown in Figure 10C, the group treated with extracellular vesicles loaded with the T7 attachment platform peptide had internalization blocked at 4°C, with extracellular vesicles observed on the cell membrane surface. However, at 37°C, the extracellular vesicles were confirmed to be internalized into the cells. In contrast, when treated with only the T7 peptide, internalization was not observed under any conditions. These results indicate that extracellular vesicles loaded with the T7 attachment platform peptide are internalized via the transferrin receptor, thereby delivering the peptide into cells.

[0217]

[0218] Example 6. Confirmation of BBB permeability of extracellular vesicles loaded with platform peptides in an in vitro BBB model.

[0219] In this example, the BBB permeability of extracellular vesicles loaded with a BNDF attachment platform peptide or extracellular vesicles loaded with a T7 attachment platform peptide was confirmed in an in vitro BBB model.

[0220] Specifically, an in vitro BBB bilayer model was constructed using HBEC-5i (cerebral microvascular endothelial cells) and C6 (glial cell line). C6 cells were seeded on the outer membrane of the upper chamber on day 1, and HBEC cells were seeded on the inner membrane of the upper chamber on day 2, followed by an additional culturing for 1 day. BBB formation was confirmed by dextran permeability. The in vitro BBB model was then treated with extracellular vesicles loaded with a BNDF-attached platform peptide or with a T7-attached platform peptide. Controls were treated with 70 kDa dextran or T7 peptide. To measure BBB permeability, the fluorescence value of an equivalent volume of media in the lower chamber was measured, taking advantage of the properties of the FITC-labeled peptide. The fluorescence value of an equivalent volume of media in the lower chamber was also measured at 1 h, 3 h, and 5 h. The permeability was calculated by checking the fluorescence values ​​compared to the mock at each time point. The results comparing the BBB permeability of extracellular vesicles loaded with a BNDF attachment platform peptide; extracellular vesicles loaded with a T7 attachment platform peptide; T7 peptide; and 70 kDa dextran in an in vitro BBB model are shown in Figure 11.

[0221] As shown in Figure 11, extracellular vesicles loaded with BNDF attachment platform peptides and extracellular vesicles loaded with T7 attachment platform peptides were confirmed to have superior BBB permeability compared to 70 kDa dextran. In particular, the extracellular vesicles loaded with T7 attachment platform peptides showed a significant increase in BBB permeability after 3 hours. In addition, the extracellular vesicles loaded with T7 attachment platform peptides were confirmed to have significantly superior BBB permeability compared to the group treated with T7 peptide alone.

[0222]

[0223] Example 7. Confirmation of BBB permeability of extracellular vesicles loaded with platform peptides in an in vivo model.

[0224] In this example, we investigated the BBB permeability of extracellular vesicles loaded with a T7-attached platform peptide in an in vivo model. Specifically, CD 31-FSD 647-labeled antibody was injected into the tail vein 3 hours prior to the experiment to stain the vascular wall. Two hours later (1 hour prior to the experiment), extracellular vesicles loaded with a T7-attached platform peptide were administered into the tail vein of the mice. The extracellular vesicles administered into the tail vein contained 1.3 μg of the T7 peptide, comprising 1.4 x 10^9 particles. The mice were euthanized and brain tissue was isolated. The isolated brain tissue was fixed in 4% PFA for 1 day and subjected to a buffer exchange process containing 30% sucrose for 3 days. Afterwards, brain sections were prepared by molding with OCT compound solution and cryosectioning at 40 μm. After washing the OCT compound within the section, the sections were mounted with a fixative solution containing DAPI and observed using a confocal microscope. The control group was treated with extracellular vesicles not loaded with peptide. The observation results are shown in Figure 12.

[0225] As shown in Figure 12, it was confirmed that extracellular vesicles loaded with the T7 attachment platform peptide penetrated the BBB and exited the brain blood vessels to reach neurons.

[0226]

[0227] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

A DE peptide represented by the amino acid sequence of sequence number 1 or more; transmembrane domain; and A drug delivery system comprising a cell-penetrating peptide motif.

2. A drug delivery system according to claim 1, wherein the transmembrane domain is at least one peptide selected from the group consisting of sequence numbers 3 to 5.

3. A drug delivery system according to claim 1, wherein the cell-penetrating peptide motif is represented by the amino acid sequence of SEQ ID NO:

2.

4. A drug delivery system in the first paragraph, wherein the drug delivery system is operably connected as shown in the following structural formula. [constitutional formula] (DE peptide)n - transmembrane domain - cell-penetrating peptide motif, where n is an integer greater than or equal to 1.

5. In the fourth paragraph, the drug delivery system is a drug delivery system represented by one or more amino acid sequences selected from the group consisting of sequence numbers 6 to 8.

6. A drug delivery system according to claim 1, wherein the drug delivery system further comprises a cargo.

7. A drug delivery system in claim 6, wherein the cargo is bound to the N-terminus of the transmembrane domain.

8. A drug delivery system in accordance with claim 6, wherein the cargo is at least one selected from the group consisting of proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, nanoparticles, biological agents, contrast agents, drugs, and compounds.

9. Extracellular vesicles loaded with the drug delivery system of paragraph 1.

10. An extracellular vesicle in claim 9, wherein the drug delivery system is loaded on the lipid bilayer of the extracellular vesicle.

11. A drug delivery composition comprising the drug delivery vehicle of claim 1; or the extracellular vesicle of claim 9.

12. A drug delivery kit comprising the drug delivery vehicle of paragraph 1; or the extracellular vesicle of paragraph 9.

13. A pharmaceutical composition for preventing or treating brain diseases, comprising the drug delivery system of paragraph 1; or the extracellular vesicle of paragraph 9.

14. A pharmaceutical composition according to claim 13, wherein the brain disease is at least one selected from the group consisting of brain tumor, traumatic brain injury, hypoxic brain injury, ischemic brain disease, degenerative brain disease, bacterial or viral brain infection, Parkinson's disease, encephalitis, stroke, apoplexy, Alzheimer's disease, Lou Gehrig's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, epilepsy, thrombosis, embolism, cerebral infarction, stroke, small infarction, and cerebral circulation metabolic disorder.

15. A drug delivery system according to paragraph 1; or an extracellular vesicle according to paragraph 9; comprising; A pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of metabolic diseases, cardiovascular diseases, nervous system diseases, digestive diseases, musculoskeletal diseases, renal and urinary diseases, and skin diseases.

16. An in vitro composition for promoting nerve regeneration, comprising the drug delivery system of paragraph 1; or the extracellular vesicle of paragraph 9.

17. A method for treating nervous system damage or nervous system disease, comprising the step of administering the drug delivery system of paragraph 1; or the extracellular vesicle of paragraph 9; to a subject in need thereof.

18. A drug delivery method comprising a step of administering the drug delivery vehicle of paragraph 1; or the extracellular vesicle of paragraph 9; to an individual in need thereof.

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