Composition for transdermal administration comprising exosomes and intracerebral drug delivery method using same

Abstract

The present invention relates to a composition for transdermal administration comprising exosomes and, more specifically, to a method for improving intracerebral drug delivery efficiency using the composition. When the composition for transdermal administration comprising exosomes according to the present invention is administered transdermally, it was experimentally confirmed that substances such as drugs are effectively delivered into the brain through the glymphatic system, compared to conventional administration methods (intravenous administration, nasal administration, etc.). This signifies that the composition for transdermal administration of the present invention significantly increases drug delivery efficiency in the brain, and thus can be effective in treating brain diseases. Therefore, the composition of the present invention can be variously utilized in the field of brain disease research and treatment.

Description

Transdermal composition containing exosomes and method for delivering drugs into the brain using the same

[0001] The present invention relates to a composition for transdermal administration containing exosomes, and more particularly, to a method for improving the efficiency of drug delivery into the brain using the composition.

[0002] Among brain disease treatments under development worldwide, 98% of small molecule therapies and 100% of large molecule therapies are reported to be unable to penetrate the blood-brain barrier (BBB). Consequently, even if drugs with potential therapeutic potential are developed, their practical application in clinical trials is challenging. Currently, treatments for brain diseases involve direct injection of drugs into the brain or spinal cord, the use of vectors such as viruses, and intravenous overdose administration. However, these methods pose limitations, including the risk of local side effects due to invasive administration, concerns about the use of viruses, and the risk of off-target effects.

[0003] Maintaining drug stability is crucial as the drug is exposed to various enzymatic and chemical differentiation processes during its transport to the brain. New strategies are particularly needed for delivering genetic material into the brain. Currently, liposomes and lipid nanoparticles (LNPs) are being used. However, systemic side effects have recently surfaced following the use of LNPs as delivery vehicles for COVID-19 mRNA vaccines. LNPs also have the disadvantages of being non-biologically compatible and lacking cell targeting.

[0004] The glymphatic system is the primary fluid circulatory system that removes waste products from the brain. It is particularly active during sleep and plays a crucial role in removing neurotoxic proteins such as beta-amyloid. This system functions to drain waste products into the venous system through the exchange of cerebrospinal fluid (CSF) and interstitial fluid (ISF). Among them, the meningeal lymphatic vessels, which exist within the dura mater, were first discovered around 2015. Previously, it was thought that the brain did not have a traditional lymphatic system, but modern research has revealed that the meninges (especially the dura mater) have a network of lymphatic vessels and that these serve as important conduits connecting the brain to the systemic lymphatic system.

[0005] The meningeal lymphatic vessels collect waste products removed through the cerebrospinal fluid (CSF) and interstitial fluid (ISF), transport them to the dural lymph nodes, and then drain them through the systemic lymphatic system for disposal. The meningeal lymphatic vessels play a role in regulating immune cell and neuroinflammatory responses. Impaired drainage pathways due to aging or lymphatic dysfunction can increase the risk of neurodegenerative diseases such as Alzheimer's and Parkinson's.

[0006] Meanwhile, exosomes, small particles secreted in vivo, are attracting attention as safer delivery vehicles than existing vectors or lipid nanoparticles. While various methods, including transfection and introduction of targeting particles, have been attempted, their delivery efficiency into the brain remains low. Furthermore, methods for loading genetic material or drugs while maintaining the properties of exosomes for human application have not yet been established.

[0007] Accordingly, the inventors of the present invention completed the present invention by confirming that the glymphatic system can be activated when exosomes are administered through a new route of administration, the deep cervical lymph node, while studying a method for improving the efficiency of drug delivery into the brain.

[0008] Accordingly, the purpose of the present invention is to provide a transdermal composition for intracerebral drug delivery containing exosomes.

[0009] Another object of the present invention is to provide a drug delivery composition for transdermal administration for intracerebral drug delivery comprising exosomes.

[0010] Another object of the present invention is to provide a composition for intracerebral drug delivery for deep cervical lymph node administration, which includes exosomes.

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

[0012] Another object of the present invention is to provide a kit for intracerebral drug delivery comprising a composition for transdermal administration.

[0013] Another object of the present invention is to provide a method for delivering drugs into the brain, comprising the step of transdermally administering exosomes to a subject.

[0014] To achieve the above purpose, the present invention provides a transdermal composition for intracerebral drug delivery containing exosomes.

[0015] The present invention also provides a drug delivery composition for transdermal administration for intracerebral drug delivery comprising exosomes.

[0016] The present invention also provides a composition for intracerebral drug delivery for deep cervical lymph node administration, which comprises exosomes.

[0017] In addition, the present invention provides a pharmaceutical composition for preventing or treating brain diseases, comprising the composition for transdermal administration, the drug delivery composition, or the composition for intracerebral drug delivery.

[0018] The present invention also provides a kit for intracerebral drug delivery comprising a composition for transdermal administration.

[0019] The present invention also provides a method for delivering drugs into the brain, comprising the step of transdermally administering exosomes to a target.

[0020] It was experimentally confirmed that when a transdermal composition containing exosomes according to the present invention is administered transdermally, substances such as drugs are delivered into the brain more effectively via the glymphatic system than conventional administration methods (intravenous administration, nasal administration, etc.). This suggests that the transdermal composition of the present invention significantly increases the efficiency of drug delivery into the brain, thereby being effective in the treatment of brain diseases. Therefore, the composition of the present invention can be utilized in various fields of brain disease research and treatment.

[0021] This diagram shows the results of confirming the expression of LRRK2 protein in the substantia nigra and hippocampus regions following administration via the glymphatic route through subcutaneous injection of loaded exosomes.

[0022] Figure 2 is a diagram showing the results of confirming the nerve regeneration ability of LRLLR-BDNF peptide-loaded exosomes in a hypoxic nerve cell model (p<0.01; **, p<0.001; ***).

[0023] Figure 3 is a diagram showing the results of confirming the cognitive function improvement effect of exosomes loaded with BDNF-LRLLR (loop2 TD) peptide in an Alzheimer's zebrafish model (p<0.05; *, p<0.01; **, p<0.001; ***, p<0.05; #, p<0.01; ##).

[0024] Figure 4a is a diagram showing the results of confirming the effect of stem cell-derived exosomes on the expression of aging-related proteins in a neural stem cell-aging model using western blot.

[0025] Figure 4b is a diagram showing the results of confirming the effect of stem cell-derived exosomes on the expression of anti-aging-related mRNA in a neural stem cell-aging model using real-time qPCR.

[0026] Figure 4c shows the results of flow cytometry to confirm the effect of stem cell-derived exosomes on the cell cycle in a neural stem cell-aging model (p<0.05; *, p<0.01; **).

[0027] Figure 5a is a diagram showing the results of confirming the drug delivery ability into the brain when exosomes are administered transdermally using fluorescent molecular imaging analysis (p<0.001; ***).

[0028] Figure 5b is a diagram showing the results of real-time imaging of percutaneously administered exosomes using a confocal intravital microscopy (IVIM) system.

[0029] Figure 6 is a diagram showing the results of a comparative analysis of the drug delivery capacity in the brain according to the exosome administration route through fluorescence molecular imaging analysis (ns: not-significant, p<0.01; **)

[0030] Figure 7 is a diagram showing the results of confirming drug delivery into the brain according to the coordinates of transdermal administration of exosomes through fluorescence molecular image analysis (p<0.05; *, p<0.01; **, p<0.001; ***, p<0.0001; ****).

[0031] Figure 8 is a diagram showing the administration locations of the AX (Axillary Lymph node) and IN (Inguinal Lymph node) of a mouse.

[0032] Figure 9 is a diagram showing the results of confirming the intracerebral delivery of exosomes according to the lymph node site in mice (ns: not-significant, p<0.01; **, p<0.0001; ****).

[0033] Figure 10 is a diagram showing the transdermal injection site of a mouse and the transdermal administration site of exosomes when applied to a human body.

[0034] Figure 11a is a schematic diagram of an experiment to confirm the intracerebral delivery of exosomes loaded with BDNF-LRLLR (CPP-TD) peptide according to the administration route.

[0035] Figure 11b is a diagram showing the results of confirming the intracerebral delivery of exosomes loaded with BDNF-LRLLR (CPP-TD) peptide according to the administration route via IVIS.

[0036] Figure 11c is a graph showing a result created based on the results of Figure 11b (p<0.01; **, p<0.01; ##).

[0037] Figure 12 is a diagram showing the results of confirming the intracerebral delivery of exosomes loaded with BDNF-LRLLR (CPP-TD) peptide according to the administration route in various parts of the brain (p<0.01; **, p<0.001; ***).

[0038] Figure 13 is a diagram showing the results of confirming TrkB activation and BDNF expression according to transdermal administration of exosomes loaded with TrkB activating peptide (comparison with control, p<0.05; *, p<0.01; **, p<0.001; ***, p<0.0001 ****, comparison with IV and SC, p<0.05; #).

[0039] Hereinafter, the present invention will be described in detail.

[0040] According to an aspect of the present invention, the present invention provides a composition comprising exosomes. The composition comprising exosomes of the present invention is a composition for transdermal administration for intracerebral drug delivery; a drug delivery vehicle composition for transdermal administration for intracerebral drug delivery; or a composition for intracerebral drug delivery for deep cervical lymph node administration.

[0041] In the present invention, transdermal administration refers to a method of administering drugs, etc. through the skin, and preferably refers to transdermal administration so that drug-loaded exosomes are delivered through the glymphatic system. In specific examples of the present invention, unless otherwise stated, transdermal administration refers to transdermal administration to the deep cervical lymph node region, and the terms 'lymphatic system administration', 'transdermal administration', or 'glimphatic transdermal administration' refer to 'delivery to the brain through the intracranial glymphatic system via percutaneous administration to the deep cervical lymph nodes.'

[0042] The composition for transdermal administration of the present invention may further include one or more auxiliary agents selected from the group consisting of carriers, excipients, disintegrants, sweeteners, coating agents, swelling agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants. Specifically, carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0043] The composition for transdermal administration of the present invention may be formulated for topical skin application, and more specifically, may be formulated as a polymer matrix, cream, gel, emulsion, or ointment, but is not limited thereto.

[0044] In addition, the composition for transdermal administration of the present invention may further include one or more known components for stabilizing, maintaining, and preserving exosomes.

[0045] In a specific embodiment of the present invention, the transdermal administration is preferably (i) deep cervical lymph node administration; or (ii) subcutaneous administration above the sternocleidomastoid muscle area behind the common carotid artery and internal jugular vein; so that the drug-loaded exosomes are delivered via a glymphatic system. In an embodiment of the present invention, it was confirmed that percutaneous administration to the above areas had a significantly higher drug delivery rate than administration to the axillary lymph nodes or intraperitoneal lymph nodes.

[0046] In a preferred embodiment of the present invention, the exosomes are preferably delivered into the brain via the glymphatic system.

[0047] In the present invention, the glymphatic system refers to the main body fluid circulation system that removes neural waste from the brain. The glymphatic system is activated during sleep and plays a crucial role in excreting neurotoxic proteins (e.g., amyloid beta, tau protein, etc.) that are deeply related to brain diseases into the venous system through the exchange of cerebrospinal fluid and interstitial fluid. In a specific embodiment of the present invention, the exosomes may be for activating the glymphatic system. Activating the glymphatic system refers to the transdermally administered exosomes being delivered to the brain through the glymphatic system to degrade neurotoxic proteins related to brain diseases and promote brain nerve regeneration and excretion of toxic substances.

[0048] Therefore, when a composition including the exosome of the present invention is transdermally administered to the above-mentioned area, the active ingredient can be effectively delivered to the glymphatic system in the brain, thereby excreting neurotoxic proteins into the venous system.

[0049] In a specific embodiment of the present invention, the exosome is preferably loaded with a drug, and examples of the drug may be at least one selected from the group consisting of nucleic acids, peptides, compounds, and chemical drug-encapsulated nanoparticles, but the scope of the present invention is not limited thereto. For example, the peptide may be a PROTAC, a TrkB activating peptide that induces enhanced BDNF expression, an LRLLR-BDNF peptide, or a BDNF-LRLLR (loop2 TD) peptide.

[0050] The exosome of the present invention may be an exosome 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. In addition, the mesenchymal stem cells may be derived from umbilical cord, cord blood, Wharton's jelly, bone marrow, fat, muscle, nerve, skin, amniotic membrane, teeth, hair root cells, or placenta, but the present invention is not limited thereto.

[0051] The exosome of the present invention is a concept that includes an exosome mimic (i.e., a similar substance) produced by extruding while applying pressure to a cell.

[0052] The inventors of the present invention experimentally confirmed that transdermal administration of the drug-loaded exosomes, as described above, effectively delivers the drug to the brain's glymphatic system compared to other administration sites and routes. This suggests that the composition of the present invention significantly increases the efficiency of intracerebral drug delivery, thereby potentially being effective in the treatment of brain diseases. Therefore, the composition of the present invention can be utilized in various fields of brain disease research and treatment.

[0053]

[0054] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating brain diseases, comprising the composition for transdermal administration, the drug delivery composition, or the composition for intracerebral drug delivery.

[0055] In an embodiment of the present invention, exosomes loaded with a BDNF-LRLLR (CPP-TD) peptide were prepared and transdermally administered as described above. As a result, it was confirmed that in subjects transdermally administered the drug-loaded exosomes, (i) the brain drug delivery rate and (ii) target protein TrkB activation and BDNF expression were significantly increased compared to the intravenous administration group. The BDNF-LRLLR (CPP-TD) peptide directly binds to the TrkB receptor to increase the expression of BDNF, which is widely known in the art as a therapeutic target for improving the pathophysiology of neurological and brain diseases and promoting nerve cell regeneration. The above results indicate that the pharmaceutical composition of the present invention can have an effective therapeutic effect on brain diseases, particularly brain diseases for which BNDF is a therapeutic target.

[0056] In a specific embodiment of the present invention, the exosome of the present invention can not only activate the glymphatic system by itself, but can also be a carrier loaded with a PROTAC, a Targeted Protein Degrader of LRRK2 (related to Parkinson's disease); an LRLLR-BDNF peptide (related to nerve regeneration and protection); or a BDNF-LRLLR (loop2 TD) peptide (related to Alzheimer's disease); and in this case, the pathology of the related disease can be significantly treated or prevented.

[0057] 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.

[0058]

[0059] The composition of the present invention may contain one or more known effective ingredients having a preventive or therapeutic effect on brain diseases.

[0060] 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.

[0061] The composition of the present invention can be administered in various 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).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066]

[0067] 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.

[0068]

[0069] According to another aspect of the present invention, the present invention provides a kit for intracerebral drug delivery comprising a composition for transdermal administration.

[0070] In order to monitor drug delivery into the brain, the kit for drug delivery into the brain 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, a suitable carrier, a label capable of generating a detectable signal, chromophores, a solubilizer, a detergent, a buffer, a stabilizer, etc. When the label is an enzyme, the kit may include a substrate capable of measuring enzyme activity and a reaction terminator. 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, a cross-linked dextran, a polysaccharide, a magnetic microparticle plated with a metal on latex, other paper, glass, metal, agarose, and a combination thereof.

[0071] 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.

[0072]

[0073] According to another aspect of the present invention, the present invention provides a method for intracerebral drug delivery, comprising the step of transdermally administering exosomes to a subject.

[0074] In a specific embodiment of the present invention, the subject may be, but is not limited to, a subject expected to develop a disease; a subject that has developed a disease; or a subject that has been judged to be cured.

[0075]

[0076] 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.

[0077] 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.

[0078]

[0079] Example 1. Preparation of drug-loaded stem cell-derived exosomes

[0080] 1-1. Preparation of mesenchymal stem cell-derived exosomes

[0081] Human bone marrow-derived stem cells were cultured to collect exosomes, which are extracellular vesicles (EVs) used in the experiment. Cells were seeded at 2.5 x 10 in a 100 mm culture dish (SPL, 20100). 5Cells were cultured at a concentration of 100 cells / mL in 0.2 μm-filtered low-glucose Dulbecco's modified Eagle's medium (DMEM, Life Technologies Corporation, CA, USA), 10% fetal bovine serum (FBS, Life Technologies Corporation), and 1% antibiotics-antimycotics (Life Technologies Corporation). Subcultures were performed every 80–90% from P2 to P6, and cultured in an incubator at 37°C and 5% CO2.

[0082] After P6, stem cells cultured under the same conditions were cultured for 5 days in a medium consisting of low-glucose DMEM, 10% exosome-depleted fetal bovine serum (System Biosciences, Palo Alto, CA, USA), and 1% antibiotics-antimycotics. The culture medium was then harvested and centrifuged at 4°C, 2500 g, and 10 min, and only the supernatant was filtered with a 0.2 μm sieve. The collected medium was immediately subjected to tangential flow filtration (TFF), and concentration and buffer exchange (diafiltration) were performed, respectively, to achieve a concentration of approximately 10 times the starting volume. The membrane used was a Minimate TFF 300K membrane (Pall Corporation, NY, USA). Exosomes were collected through the above process and used in subsequent experiments.

[0083]

[0084] 1-2. Loading and purifying target substances into exosomes

[0085] The exosomes obtained in Example 1-1 above were loaded with a target substance and purified. The target substance may be RNA or a peptide. Specifically, in order to load a peptide into stem cell-derived exosomes, 100 ng to 5 ug of the peptide and 5 x 10 exosomes were loaded. 9 After mixing, the exosomes were incubated at room temperature for more than 12 hours. Only the peptide-loaded exosomes were isolated through size exclusion chromatography (SEC) or dialysis (100 KDa cut off)).

[0086]

[0087] 1-3. Efficacy Evaluation of Peptide-Loaded Exosomes

[0088] The efficacy of the peptide-loaded exosomes prepared in Examples 1-2 was evaluated in various disease models.

[0089]

[0090] (1) Efficacy of drug-loaded exosomes in a Parkinson's disease neuronal cell model

[0091] LRRK2 (Leucine-rich repeat kinase 2), a gene associated with Parkinson's disease, is a factor that increases the incidence of Parkinson's disease when mutations occur, leading to excessive activation of the kinase. Therefore, substances that reduce LRRK2 enzyme activity could be used as treatments for Parkinson's disease.

[0092] By loading PROTAC, a Targeted Protein Degrader of LRRK2, into exosomes, we confirmed the decrease of LRRK2 in dopaminergic neurons induced by Parkinson's disease, and the results are shown in Fig. 1. As a comparative example, a group administered with naive exosomes and PROTAC alone was used. Specifically, 300 ng of PROTAC and 5 x 10 exosomes were administered. 8After mixing the dogs, they were loaded onto stem cell-derived exosomes. The loading methods included room temperature incubation, heat shock, and liposome fusion, and the specific experimental groups were as follows.

[0093] - PBS: Control group, PBS only

[0094] - naive EV: Administration of stem cell-derived exosomes (5 x 10^8 particles) without drug loading

[0095] -EV+PROTAC(P): passive incubation group. PROTAC-loaded exosomes were prepared by incubating exosomes and PROTAC at room temperature for 12 hours. The PROTAC-loaded exosomes were administered (PROTAC: 300 nm, exosomes: 5 x 10^8 particles).

[0096] -EV+PROTAC(H): HS (Heat shock) group. Exosomes and PROTAC are subjected to heat and chemical conditions at 42 degrees Celsius in a calcium buffer to form exosomes loaded with PROTAC. The above PROTAC-loaded exosomes are administered (PROTAC: 300 nm, exosomes: 5 x 10^8 particles).

[0097] -EV+PROTAC(E): liposome fusion group. PROTAC is loaded onto exosomes using liposome fusion to produce PROTAC-loaded exosomes. The PROTAC-loaded exosomes are administered (PROTAC: 300 nm, exosomes: 5 x 10^8 particles).

[0098]

[0099] Peptide-loaded exosomes prepared using various loading methods were treated with dopaminergic neurons for 4 or 24 hours, respectively. During incubation, degradation of the brain lesion molecule LRRK2 by PROTAC was confirmed, and the results are shown in Figure 1a.

[0100] As shown in Fig. 1a, PROTAC-loaded exosomes were confirmed to reduce LRRK1 in dopaminergic neurons induced by Parkinson's disease in all experimental groups.

[0101]

[0102] In a mouse model of Parkinson's disease, the area where brain lesion substances accumulate is the substantia nigra, a structure located in the midbrain, an area with a large distribution of dopamine-producing neurons. The accumulation and overactivation of brain lesion substances such as LRRK2 (Leucine-Rich Repeat Kinase 2) and serine / threonine kinase contribute to the onset and progression of Parkinson's disease through the loss of dopamine neurons and the accumulation of pathological proteins (e.g., α-synuclein).

[0103] To analyze the LRRK2 degradation capacity of exosomes and PROTAC-loaded exosomes with enhanced brain delivery via transdermal administration, they were administered to MPTP-induced Parkinson's disease model mice. Three days later, brain tissue was extracted, and LRRK2 protein expression in the substantia nigra and hippocampus was analyzed by Western blotting. The Western blotting results are shown in Figure 1b.

[0104] As shown in Figure 1b, we indirectly confirmed that LRRK2 expression was reduced and degraded in the groups administered exosomes and PROTAC-exosomes. This indirectly demonstrates that exosome therapeutics delivered to the brain via the lymphatic system enhanced their delivery to the brain, removing brain lesion material and excreting it via the glymphatic system.

[0105]

[0106] (2) Neuroregenerative and protective effects of peptide-loaded exosomes in hypoxic neuronal cell models

[0107] The neural regenerative potential of exosomes loaded with the LRLLR-BDNF peptide, a cell-penetrating peptide (CPP), was confirmed in a hypoxic neuronal model. After preparing exosomes loaded with the LRLLR-BDNF peptide, their neural regenerative efficacy was verified. The experimental groups were as follows.

[0108] PBS: Administered only saline solution

[0109] MSC-3D EV LRLLR: Administration of MSC-3D EV (5x10^8 / ml) loaded with 75 ng of peptide

[0110] MSC 3D EV: MSC-3D EV (5x10^8 / ml) administered alone

[0111] LRLLR only: 75 ng of peptide administered alone

[0112] reverse peptide: Administration of 75 ng of reverse peptide of LRLLR peptide

[0113] BNDF: Positive control, BNDF (10 ng / ml dose) administered alone

[0114] After treating hypoxic neuronal models with LRLLR-BDNF peptide-loaded exosomes, the cells were cultured for a total of 72 hours. At the 72-hour endpoint, the neuronal length, tip number, and total cell number were measured, and the results are shown in Figure 2.

[0115] As shown in Figure 2, it was confirmed that the peptide-loaded exosome-treated group regulated axon branching, dendrite growth, and promoted synapse regeneration and growth.

[0116]

[0117] (3) Confirmation of improved cognitive function of peptide-loaded exosomes in an Alzheimer's zebrafish model.

[0118] The cognitive function improvement effect was confirmed in a zebrafish model of Alzheimer's disease. The experimental groups were as follows.

[0119] EPPS = Donepezil administration group as positive control group

[0120] SNE#1 = Pure extracellular vesicles (MSC-derived naive 3D-EVs)

[0121] SNE#2 = BDNF-LRLLR(loop2 TD) peptide-loaded MSC 3D-extracellular vesicles (BDNF-LRLLR(loop2 TD)- MSC 3D-EV)

[0122] SNE#3 = Hek 293-2D- extracellular vesicles loaded with BDNF-LRLLR(loop2 TD) peptide (BDNF-LRLLR(loop2 TD) Hek 293 -2D-EV))

[0123] SNE#4 = BDNF NUS TD peptide-loaded MSC 3D-extracellular vesicles (BDNF NUS TD-MSC 3D-EV)

[0124] Each exosome group was prepared with a dose of 1.7 x 10^7 / 10μl and the experiment was performed by administering 2nl each into the vein of zebrafish.

[0125] Cognitive function improvement was assessed using the Red Ball Test. Specifically, the Red Ball Test is a cognitive function test utilizing visual stimuli. Zebrafish were pre-trained to observe the movements of a red ball using a red ball screen in their rearing environment. After learning the movements, the zebrafish's ability to avoid the red ball was tested. The cognitive function of the zebrafish was measured by the frequency of staying in the area exhibiting red ball avoidance behavior, with the average value calculated at 12-second intervals for 6 minutes. The results are shown in Figure 3.

[0126] As shown in Fig. 3, the cognitive function ability was confirmed in the Pre-treatment and Post-treatment groups, where the test drug was injected first before administering Beta-Amyloid(1-42). The Pre-treatment group was able to confirm the efficacy in terms of preventing and defending the deposition of Beta-Amyloid(1-42) and cognitive function decline, and in the case of the positive control group, EPPS, it was confirmed that the cognitive function ability showed a pattern of increase as much as the Control group. In particular, it was confirmed that the cognitive function ability was normalized as much as the Control group in the MSC 3D-extracellular vesicles loaded with the BDNF-LRLLR(loop2 TD) peptide and the Hek 293 2D-extracellular vesicles loaded with the BDNF-LRLLR(loop2 TD) peptide. This means that the peptide-loaded exosomes of the present invention can improve cognitive dysfunction caused by Alzheimer's disease.

[0127]

[0128] (4) Proof of the nerve regeneration efficacy of stem cell-derived exosomes through anti-aging and reverse aging.

[0129] The anti-aging efficacy of exosome therapy was confirmed in a mouse-derived neural stem cell-aging model. Specifically, primary NSC cells isolated from mouse fetuses were passaged to induce senescence, creating a senescent cell model. For each passage, p53 and p21, known senescence markers, were quantitatively analyzed by Western blotting. Furthermore, real-time PCR was used to quantitatively analyze mRNA levels of CCK2, CCK4, CCK6, BAX, H2AX, and PCNA. FACs analysis was performed to assess senescence as a cell cycle marker. The results of Western blotting, real-time qPCR, and FACs analysis are presented in Figures 4a to 4c, respectively.

[0130] As shown in Figures 4a to c, Senescence-Associated B-galactosidase staining confirmed that cell lysosomal activity increased in 3D senescent neural stem cells, and decreased when treated with exosome therapy. Anti-aging efficacy was also confirmed by confirming a decrease in SA-b-gal staining positive cells when treated with exosome therapy in 2D monolayer senescent neural stem cells. Western blot confirmed the regulation of anti-aging factors by exosome therapy through the Sirt1-p53 pathway at the protein level. Significant differences in protein expression were also confirmed at the gene level using qPCR.

[0131]

[0132] Example 2. Superiority of drug delivery through glymphatic administration

[0133] Experiments using various disease models from Example 1 demonstrated that exosomes containing neuroprotective agents and Parkinson's or Alzheimer's disease treatments exhibited superior neuroprotective and cognitive function-enhancing effects. Additionally, the effects of different routes of administration were compared to determine the most appropriate route for delivering these agents to the brain, the target site.

[0134] The present inventors sought to more effectively deliver exosomes to the brain's glymphatic system. As a result, they confirmed that when administered percutaneously along the route of the meningeal lymphatic vessels in the 'glymphatic system', more specifically, to the deep cervical lymph nodes, the brain delivery rate of exosomes was higher than that of other administration sites and routes. That is, when exosomes are administered percutaneously to the deep cervical lymph nodes, they are effectively delivered to the brain's glymphatic system along the meningeal lymphatic vessels. Accordingly, in the examples and drawings described below, the terms 'lymphatic system administration', 'percutaneous administration', or 'glymphatic transdermal administration' mean 'delivery to the brain through the brain's glymphatic system via percutaneous administration to the deep cervical lymph nodes.'

[0135]

[0136] 2-1. Bio-Distribution

[0137] To quantitatively compare the exosome drug delivery capacity of the glymphatic transdermal administration method, the non-injection group and the subcutaneous injection group were compared and analyzed. Each group consisted of at least four 8-week-old mice, and the experiment was conducted simultaneously. The subcutaneous administration group was administered 6 x 10^8 fluorescent dye-labeled exosomes percutaneously. IVIS fluorescent molecular imaging analysis was performed 30 minutes after administration. The actual fluorescence value (intensity) and fold change (fold change) of the ROI measurement area were plotted on a graph. The IVIS results and quantitative results are shown in Figure 5a.

[0138] As shown in Fig. 5a, as a result of conducting a comparative experiment with the non-injection group, it was confirmed that the brain drug delivery ability was enhanced by 300 to 400% or more in all subjects receiving glymphatic transdermal administration.

[0139]

[0140] Using a confocal intravital microscopy (IVIM) system, we performed real-time imaging of exosome delivery to the brain's glymphatic system following transdermal administration of glymphatic drugs. To analyze the distribution of EVs within the brain, a surgical procedure called cranial imaging windows is necessary. To confirm delivery to the glymphatic system, lymphatic vessels responsible for brain clearance must be visualized. Therefore, lymphatic vessels were analyzed using Prox1-GFP mice with endogenous fluorescence expression (GFP). Specifically, the cranial imaging widows implantation surgery was performed to more accurately observe the biodistribution of EVs in various brain regions within the skull in vivo. A 3-mm cranial imaging widows implantation was performed at the Bregma level in the lower right side of the mouse brain. To relieve inflammation caused by cranial imaging windows implantation, Rimadyl from Zoetis was used to eliminate the inflammatory mechanism, and exosomes were administered when brain inflammation due to the surgery had resolved (more than 2 weeks after surgery). Mice administered with exosomes were mounted on a biomicroscope, and the exosomes were administered transdermally. The responses in brain tissue were observed. Furthermore, CD31 (Blood Vessel Endothelial Antibody) was administered intravenously (tail vein injection) and imaged to measure the same location in brain tissue in vivo. The imaging results are shown in Figure 5b.

[0141] As shown in Figure 5b, when glymphatic transdermal administration was performed, exosomes were confirmed to be delivered into each brain tissue. However, when administered intravenously, exosomes were confirmed to be trapped within blood vessels.

[0142]

[0143] 2-2. Comparison of brain drug delivery by administration route

[0144] To compare brain delivery by various delivery routes, we performed a quantitative comparative analysis of brain drug delivery with other administration methods. Specifically, mesenchymal stem cell-derived exosomes were fluorescently stained using SBI's Exoglow in-vivo, a non-lipophilic fluorescent dye that complies with the Ministry of Food and Drug Safety guidelines. The exosomes were purified using Exo-disc, and fluorescently stained exosomes were finally obtained. The obtained fluorescently stained exosomes were administered transdermally (glymphatic system delivery), via the tail vein, or intranasally to 5-12 week-old C57 / B6 mice. The mice were sacrificed 30 minutes after exosome administration, and the brain tissues were collected. The Exoglow fluorescence intensity of the brain tissues was analyzed using a Xenogen IVIS-200 device, and the results are shown in Figure 6.

[0145] As shown in Figure 6, the transdermal administration group via the glymphatic system was confirmed to have a significantly higher drug delivery rate into the brain than the other administration groups (tail vein, nasal).

[0146]

[0147] 2-3. Establishing coordinates for transdermal glymphatic administration and comparing intracerebral delivery

[0148] Through Example 2-2, it was confirmed that transdermal administration via the glymphatic system has a much higher drug delivery rate into the brain compared to other administration routes. Therefore, in order to specifically define the transdermal administration site for drug delivery via the glymphatic system,

[0149] Coordinates were set and drug delivery was analyzed upon administration to these sites. Specifically, in this example, nine other coordinates were set around the administration site of 8-week-old mice, and the difference in delivery power and coordinates within the range of coordinate groups upon transdermal administration were set. A total of nine transdermal administration sites around the mouse neck were specified, and the specified coordinates were 3 to 6 mm caudal from the cistserna mganum and 5 to 8 mm lateral to both ears. 6 x 10^8 exosomes labeled with fluorescent dyes were transdermally administered to the specified coordinates. IVIS fluorescent molecular imaging analysis was performed 30 minutes after exosome administration, and the results are shown in Fig. 7.

[0150] As shown in Figure 7, all nine established coordinates were administered to the deep cervical lymph nodes and delivered via the glymphatic system when administered percutaneously, confirming high exosome delivery efficiency to the brain. Therefore, we confirmed that exosomes can be effectively delivered to the brain via the glymphatic system when administered percutaneously to the deep cervical lymph nodes.

[0151]

[0152] 2-4. Analysis of the effects of percutaneous administration to various lymphatic sites

[0153] It was confirmed that the drug was effectively delivered to the brain when administered percutaneously at the deep cervical lymph node location. An experiment was conducted to compare whether the brain delivery ability was superior when administered percutaneously to other lymph node sites in the mouse. There are six major mouse lymph nodes (LN: lymph node, AX: Axillary Lymph node, IL: iliac Lymph node, IN: Inguinal Lymph node, PO: Popliteal Lymph node, RE: Renal Lymph node), and among them, the sites that are most frequently applied to humans in clinical practice are the AX, IN, and PO sites in mice. An experiment was conducted to confirm whether EVs were effectively delivered to the brain by administering them percutaneously to two of the six lymph node sites (AX and IN lymph nodes). Figure 8 shows the administration sites and anatomical descriptions of the AX and IN lymph nodes.

[0154] As shown in Figure 8, the AX lymph node of the mouse corresponds to the armpit of a human, and the IN lymph node corresponds to the abdominal cavity of a human.

[0155]

[0156] Brain delivery was confirmed upon percutaneous or intravenous administration to the deep cervical lymph node and other lymph nodes (AX, IN). Specifically, 6 x 10^8 fluorescent dye-labeled exosomes were percutaneously administered to the axillary lymph node and inguinal lymph node of 8-week-old mice for comparison with each lymph node present in the body. IVIS fluorescent molecular imaging analysis was performed 30 minutes after percutaneous administration. The results comparing brain delivery according to lymph node site are shown in Figure 9.

[0157] As shown in Figure 9, except for the deep cervical lymph nodes corresponding to the glymphatic system in the mouse anatomy, no drug was delivered into the brain through other axillary LNs (human axillary equivalent) and inguinal LNs (human intraperitoneal equivalent). In addition, the drug delivery rate into the brain in the subcutaneous injection group via the glymphatic system was significantly higher than that in the tail vein injection group.

[0158]

[0159] Therefore, we confirmed that percutaneous administration to the deep cervical lymph node area is the most effective way to deliver exosomes to the brain, rather than intravenous administration, nasal administration, or percutaneous administration to other lymph node locations, and that exosomes can be delivered to the glymphatic system through deep cervical lymph node administration. When applied to humans, as shown in Figure 10, by injecting exosomes into the subcutaneous tissue above the sternocleidomastoid muscle area behind the common carotid artery and internal jugular vein, they can be delivered to the meningeal lymphatic vessels, i.e., the surrounding deep cervical lymph nodes, and thus, exosomes can be delivered into the brain via glymphatic delivery.

[0160]

[0161] Example 3. Confirmation of intracerebral delivery of peptide-loaded exosomes via transdermal administration

[0162] Through the above Example 1, it was confirmed that drug-loaded exosomes have a brain disease treatment effect, and through Example 2, it was confirmed that transdermal administration from the deep cervical lymph nodes to the glymphatic system has the best brain drug delivery efficiency. Therefore, in this Example, the brain delivery and drug effect of the peptide-loaded exosomes prepared in the above Examples 1-2 were confirmed through transdermal administration.

[0163]

[0164] 3-1. Intracerebral delivery of peptide-loaded exosomes by administration route

[0165] An in vivo distribution assay using IVIS equipment that can quantitatively measure brain delivery after transdermal administration of fluorescently tagged peptide-loaded exosomes was performed as shown in Fig. 11ㅁ. MSC-3D EVs loaded with BDNF-LRLLR (CPP-TD) peptide were labeled with fluorescent dye for biodistribution experiment, and 6 x 10^8 exosomes were administered transdermally or intravenously to 8-week-old mice. Brain drug delivery was analyzed using IVIS equipment 30 minutes after exosome administration. The results of confirming brain delivery of peptide-loaded exosomes through IVIS are shown in Fig. 11b, and the graph created based on this is as shown in Fig. 11c.

[0166] As shown in Figures 11b and c, it was confirmed that the intracerebral delivery of peptide-loaded exosomes was significantly increased in the glymphatic transdermal administration group compared to the intravenous administration group.

[0167]

[0168] Additionally, we analyzed the intracerebral delivery of peptide-loaded exosomes according to brain tissue site. Specifically, MSC-3D EV (6 x 10^8) exosomes loaded with 75 ng of peptide were fluorescently labeled and administered transdermally or intravenously to mice. After administration, mouse brain tissue was obtained, divided into five regions as shown below, and subjected to fluorescence analysis using IVIS equipment.

[0169] - Area 1: Frontal cortex

[0170] - Area 2: cerebral cortex, striatum

[0171] - Area 3: hippocampus, thalamus, hypothalamus

[0172] - Area 4: midbrain, pons

[0173] - Area 5: cerebellum, meddula

[0174] The results of confirming intracerebral delivery of peptide-loaded exosomes according to brain tissue site are shown in Figure 12.

[0175] As shown in Figure 12, when peptide-loaded exosomes were administered transdermally via glymphatic route, the brain delivery rate was significantly higher than that of the intravenous administration group, and it was confirmed that they were evenly distributed and delivered.

[0176] The above results confirmed that when exosomes loaded with cargo are administered via glymphatic transdermal administration, not only is the delivery efficiency into the brain higher than other administration routes, but they can also be delivered and distributed throughout the brain.

[0177]

[0178] 3-2. Confirmation of enhanced therapeutic efficacy following transdermal administration of peptide-loaded exosomes.

[0179] Brain-derived neurotrophic factor (BDNF) holds significant significance as a therapeutic target for improving the pathophysiology of brain and psychiatric disorders and promoting nerve cell regeneration and recovery. Therefore, therapeutic strategies utilizing drugs that regulate BDNF levels are central to the treatment of various conditions, including depression, Alzheimer's disease, Parkinson's disease, and brain injury.

[0180] In this example, MCS-3D exosomes loaded with a TrkB-activating peptide that induces enhanced BDNF expression were administered transdermally via glymphatic glucagon to evaluate whether they were effectively delivered into the brain and induced the expression of target proteins (BNDF, TrkB) in the brain. Specifically, exosomes loaded with 75 ng of a TrkB-activating peptide were prepared. The peptide-loaded exosomes (6 X 10 8) were administered glymphatically or intravenously to 8-week-old mice. Brain tissue was then extracted from each region, and the tissue was dissolved in RIPA buffer to obtain proteins. The obtained proteins were analyzed by Western blotting. The results of analyzing the expression of target proteins TrkB and BNDF are shown in Figure 13.

[0181] As shown in Figure 13, the peptide (75 ng) loaded on exosomes directly binds to the TrkB receptor, enhancing the expression of BDNF. The glymphatic transdermal administration group showed a significant increase in target protein TrkB activation and BDNF expression compared to the intravenous administration group.

[0182] In summary, this means that exosomes loaded with brain disease treatment substances can be delivered to the brain's glymphatic system with high efficiency when administered percutaneously to the deep cervical lymph nodes of mice, and can exhibit further improved therapeutic effects in the brain.

[0183]

[0184] 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

1. A composition for transdermal administration for intracerebral drug delivery containing exosomes.

2. A composition according to claim 1, wherein the percutaneous administration is deep cervical lymph node administration.

3. A composition according to claim 2, wherein the exosome is delivered into the brain via the glymphatic system.

4. In the first paragraph, the composition is administered in the subcutaneous tissue above the sternocleidomastoid muscle area behind the common carotid artery and the internal jugular vein.

5. A composition according to claim 1, wherein the exosome is loaded with a drug.

6. A composition according to claim 5, wherein the drug is at least one selected from the group consisting of nucleic acids, peptides, compounds, and chemical drug-encapsulated nanoparticles.

7. A drug delivery composition for transdermal administration for intracerebral drug delivery containing exosomes.

8. A drug delivery composition according to claim 7, wherein the exosome is for activating the glymphatic system.

9. A composition for intracerebral drug delivery for deep cervical lymph node administration containing exosomes.

10. A pharmaceutical composition for preventing or treating brain diseases, comprising the composition for transdermal administration of paragraph 1, the drug delivery composition of paragraph 7, or the composition for intracerebral drug delivery of paragraph 9.

11. In the 10th paragraph, 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.

12. A kit for intracerebral drug delivery comprising the composition for transdermal administration of paragraph 1.

13. A method for delivering drugs into the brain, comprising the step of transdermally administering exosomes to a target.

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