Composition for delivering functional substance and uses thereof

Abstract

The present invention relates to a composition for delivering functional substances and uses thereof. The composition for delivering functional substances using non-vesicular extracellular particles according to the present invention can effectively load genetic materials such as proteins, mRNA, and / or pDNA and efficiently deliver same to cells, and thus can be used as a pharmaceutical composition or a functional cosmetic composition for treating diseases.

Description

Composition for functional material delivery and use thereof

[0001] The present invention relates to a composition for functional material delivery and its use.

[0002]

[0003] Various attempts have been made to develop drug delivery systems to maximize therapeutic efficacy. Representative examples include lipid nanoparticles, viral particles, and various synthetic carriers. However, these carriers have relatively low biocompatibility and high immunogenicity, which significantly limit their safety during repeated administration. Furthermore, when administered systemically, most carriers accumulate in the liver and spleen, reducing drug delivery efficiency to the desired target tissues and cells. Furthermore, mammalian cell membranes act as a biological barrier, making them largely impermeable to externally introduced pharmacologically active substances, a major obstacle to efficient drug delivery.

[0004] Extracellular vesicles (EVs) have recently been attracting attention as an alternative to overcome these limitations. EVs are spherical particles with a lipid bilayer structure naturally secreted by various cells in the body, and possess excellent characteristics such as high biocompatibility and low immunogenicity. EVs are present in most body fluids, including blood and urine, and contain biologically active substances such as proteins, mRNA, and miRNA, playing a crucial role as intercellular signaling mediators. EVs possess the ability to deliver payloads through fusion with the target cell membrane and endocytosis, protecting these endogenous substances from various immune components and enzymes in plasma, enabling stable delivery. Furthermore, EVs can efficiently load a wide range of biologically active substances, including proteins, membrane receptors, and nucleic acids, demonstrating their potential as next-generation therapeutic delivery vehicles.

[0005] However, even when the extracellular vesicle-based drug delivery system is administered systemically, a significant amount accumulates in the liver and spleen, and since the intracellular material transport pathway mainly involves clathrin-mediated endocytosis and macropinocytosis, additional technological approaches are required to avoid capture and degradation by endosomes and lysosomes.

[0006]

[0007] The present inventors completed the present invention by confirming that non-vesicular extracellular particles, particularly supermere, can effectively load functional substances, such as external genes, and efficiently deliver them to cells.

[0008]

[0009] One object of the present invention is to provide a composition for delivering a functional substance comprising non-vesicular extracellular particles (NVEPs) as an active ingredient.

[0010] Another object of the present invention is to provide a composition for delivering a functional substance comprising a non-vesicular extracellular particle marker protein and a non-vesicular extracellular particle comprising a target functional substance.

[0011] Another object of the present invention is to provide a pharmaceutical composition comprising the composition.

[0012] Another object of the present invention is to provide a treatment method comprising a step of administering the pharmaceutical composition.

[0013] Another object of the present invention is to provide a cosmetic composition comprising the composition.

[0014] Another object of the present invention is to provide a functional substance delivery kit comprising non-vesicular extracellular particles as an active ingredient.

[0015] Another object of the present invention is to provide a method for delivering a functional substance using the composition for delivering a functional substance.

[0016] Another object of the present invention is to provide a functional substance delivery use of a composition for functional substance delivery comprising non-vesicular extracellular particles.

[0017] Another object of the present invention is to provide a use of non-vesicular extracellular particles for functional substance delivery.

[0018]

[0019] The technical problem to be achieved according to the technical idea of ​​the invention disclosed in this specification is not limited to the problem to solve the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0020]

[0021] According to one embodiment of the present invention, a composition for functional material delivery using non-vesicular extracellular particles of the present invention can effectively load genetic material such as protein, mRNA and / or pDNA and deliver it well to cells, and thus can be utilized as a pharmaceutical composition for treating diseases or a functional cosmetic composition.

[0022]

[0023] Figure 1 illustrates the classification system of extracellular particles and their subclassification.

[0024] Figure 2 shows the manufacturing process of sub-classified substances of extracellular particles.

[0025] Figure 3 shows the expression of protein markers of supermere, which are distinguished from exomere and small extracellular vesicles.

[0026] Figure 4 shows the results of treating recipient cells with extracellular particles isolated from cells transduced with eGFP, confirming that expression of eGFP protein was induced in recipient cells regardless of whether RNase pretreatment was performed.

[0027] Figure 5 shows the results of confirming that among the extracellular particles isolated from cells transduced with eGFP, the particles capable of inducing eGFP expression in recipient cells are small extracellular particles, not large extracellular vesicles.

[0028] Figure 6 shows the results confirming that among the small extracellular particles isolated from cells transduced with eGFP, the main carriers that can effectively induce eGFP expression in recipient cells are non-vesicular extracellular particles, not small extracellular vesicles.

[0029] Figure 7 shows the results confirming that, regardless of the type of transduced plasmid, non-vesicular extracellular particles are the main carriers that can more effectively induce eGFP expression in recipient cells compared to small extracellular vesicles and the entire extracellular particle group.

[0030] Figure 8 shows the results confirming that supermere among non-vesicular extracellular particles is the main carrier that can induce eGFP expression in recipient cells more effectively than small extracellular vesicles and exomeres, regardless of the type of transduced plasmid.

[0031] Figure 9 shows the results of confirming the content of endogenous eGFP-mRNA in small extracellular vesicles, exomeres, and supermeres isolated from cells transduced with eGFP.

[0032] Figure 10 shows the results of confirming the level of endogenous eGFP protein expression in small extracellular vesicles, exomeres, and supermeres isolated from cells transduced with eGFP.

[0033] Figure 11 shows the results demonstrating that supermeres isolated from eGFP-transduced cells are the main delivery vehicles that can more effectively deliver eGFP mRNA into recipient cells than small extracellular vesicles and exomeres.

[0034] Figure 12 shows the results demonstrating that supermeres isolated from eGFP-transduced cells are the main delivery vehicles that induce eGFP protein expression in recipient cells more effectively than small extracellular vesicles and exomeres.

[0035] Figure 13 shows the results of confirming the internalization of supermere into cells through lipid rafts (Figures 13a and 13b: Cy5.5 expression results in recipient cells 30 minutes, 3 hours, and 24 hours after treating recipient cells with each drug and supermere, Figure 13c: Changes in the proportion of cells containing supermere stained with Cy5.5 for 24 hours after treating recipient cells with each drug and supermere).

[0036] Figure 14 shows the results confirming that supermere can deliver exogenous eGFP mRNA to recipient cells more effectively than small extracellular vesicles and exomeres.

[0037] Figure 15 shows the results of confirming intracellular delivery of GFP pDNA using Supermere.

[0038] Figure 16 shows the results of ex vivo imaging of GFP fluorescence expressed in intestinal tissue to compare the intestinal exogenous eGFP mRNA delivery and protein translation efficiency of supermere and lipid nanoparticles after intravascular injection.

[0039] Figure 17 shows the results of confirming eGFP protein expression in intestinal tissue to compare the intestinal exogenous eGFP mRNA delivery and protein translation efficiency of supermere and lipid nanoparticles after intravascular injection.

[0040] Figure 18 shows the results of confirming the distribution in each organ after a certain period of time after intravascular injection of supermembrane and small extracellular vesicles derived from HEK293FT cells labeled with fluorescent dye.

[0041] Figure 19 is an image comparing the accumulation of supermere and small extracellular vesicles derived from HEK293FT cells in each organ by isolating only liver, spleen, and kidney tissues in the experiment of Figure 18.

[0042] Figure 20 shows the results of confirming the distribution by organ after a certain period of time after intravascular injection of supermembrane and small extracellular vesicles derived from human bone marrow-derived mesenchymal stem cells (MSCs) labeled with fluorescent dye.

[0043] Figure 21 shows the results of confirming the expression level of a functional substance in a supermere isolated from cells transduced in a form in which the supermere marker protein TGFBI and the target functional substance are fused.

[0044]

[0045] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0046] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the present invention described herein. Furthermore, it should be understood that such equivalents are encompassed by the present invention.

[0047] Additionally, numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety, thereby providing a clearer understanding of the technical field to which this application pertains and the content of this application.

[0048]

[0049] The present invention is based on the identification of a novel use for non-vesicular extracellular particles (NVEPs).

[0050] Conventionally, cells release vesicles of various membrane types depending on the extracellular environment. These released vesicles are usually referred to as extracellular vesicles (EV). These extracellular vesicles enable the exchange of materials such as proteins, lipids, and genetic materials between cells, and act as mediators that transmit physiological / pathological signals, and various studies are being conducted using them.

[0051] However, there are still few studies utilizing non-vesicular extracellular particles, especially exomeres and supermeres (Supernatant of Exomere), and in particular, research on compositions that deliver functional substances using non-vesicular extracellular particles is insufficient.

[0052] The present invention is based on the novel demonstration that non-vesicular extracellular particles, particularly supermelas, can effectively load functional substances, such as external genes, and efficiently deliver them to cells.

[0053] Specifically, one aspect according to one embodiment of the present invention provides a composition for functional material delivery comprising a non-vesicular extracellular particle (NVEP) as an active ingredient.

[0054] The term extracellular particle (or extracellular particle) of the present invention refers to all particles naturally derived from cells.

[0055] The above extracellular particles are classified into vesicular and non-vesicular, and vesicles include extracellular vesicles (EV). The extracellular vesicles are natural nanoparticles composed of all lipid bilayers derived from cells, and are distinguished from the non-vesicular extracellular particles (NVEPs) used as the active ingredient in the present invention.

[0056] The above extracellular vesicles are divided into large extracellular vesicles (large EVs) with an average diameter of 200 nm or more and small extracellular vesicles (small EVs) with an average diameter of 200 nm or less.

[0057] The term "Non-Vesicular Extracellular Particle (NVEP)" of the present invention refers to a natural nanoparticle derived from a cell and without a lipid bilayer.

[0058] The above non-vesicular extracellular particles are classified into exomeres, which are natural nanoparticles without a lipid bilayer of 50 nm or less, and supermeres (Supernatant of Exomere), which are contained in the supernatant (sup) obtained by ultracentrifuging exomeres.

[0059] The above supermere has characteristics that distinguish it from extracellular vesicles or exomeres in terms of size, RNA and protein profiles, and total RNA quantity.

[0060] In addition, the supermere can be distinguished from extracellular vesicles or exomeres using a marker. For example, the marker is a protein that exists specifically in large numbers in the supermere and can be named a “supermere marker protein.” In a specific example, the supermere marker protein may be at least one selected from Transforming growth factor, beta-induced (TGFBI), Enolase-1 (ENO1), Enolase-2 (ENO2), Heat shock 70 kDa protein 13 (HSPA13), GPI (Glucose-6-phosphate isomerase), LDHA (Lactate Dehydrogenase A), TPI1 (Triosephosphate Isomerase 1), HK1 (Hexokinase 1), MDH1 (Malate Dehydrogenase 1), NUCB1 (Nucleobindin-1), and PDIA4 (Protein Disulfide Isomerase Family A Member 4), but is not limited thereto.

[0061] In one specific example, the supermembrane can be distinguished by the marker regardless of the type of derived cell.

[0062] The above cells may be, for example, human embryonic kidney cells (HEK cells) or stem cells, but are not limited thereto.

[0063] In one embodiment of the present invention, the stem cells may be embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or adult stem cells.

[0064] In one embodiment of the present invention, the adult stem cells may be selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, multipotent stem cells, and amniotic epithelial cells.

[0065] In one embodiment of the present invention, the mesenchymal stem cells may be derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.

[0066] In one embodiment of the present invention, the non-vesicular extracellular particle may be, but is not limited to, a supermere.

[0067] The above extracellular particles and their subclassification system are illustrated in Figure 1.

[0068] In one embodiment of the present invention, the “functional substance delivery” may be delivery of a functional substance possessed by a donor cell to a recipient cell, organ, or individual, or may be delivery of a functional substance existing outside a cell or to be delivered to a target, such as a recipient cell, organ, or individual, using the functional substance delivery composition of the present invention, but is not limited thereto.

[0069] In one embodiment of the present invention, the functional material may be in the form of a protein.

[0070] In one embodiment of the present invention, the functional material may be in the form of RNA.

[0071] In one embodiment of the present invention, the functional material may be in the form of DNA.

[0072] The functional material may be, for example, a gene, and may be selected from the group consisting of, but not limited to, mRNA, shRNA, miRNA, gRNA, pri-miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA, IncRNA, ribozyme, mini-circle DNA, or plasmid DNA (pDNA) as a specific example.

[0073] In the present invention, the genes may be naturally occurring or synthetic, and may vary in size from oligonucleotides to chromosomes. These genes may originate from, but are not limited to, humans, animals, plants, bacteria, viruses, etc.

[0074] For example, the gene may be a newly synthesized synthetic gene, and may be a gene synthesized for various purposes, such as a gene as a drug, a gene as an inhibitor, or a gene having a cosmetic effect, and may be delivered by being loaded onto the non-vesicular extracellular particle of the present invention without limitation.

[0075] These can be obtained using methods known in the art.

[0076] In another embodiment of the present invention, the functional material may include a protein.

[0077] The protein may be, but is not limited to, an antibody or an immunologically active fragment thereof, an intrabody, a single chain variable fragment, an affibody, an enzyme, a transporter, a tumor suppressor, a virus or bacterial inhibitor, a cellular component protein, a DNA or RNA binding protein, a DNA repair inhibitor, a nuclease, a proteinase, an integrase, a transcription factor, a growth factor, an apoptosis inhibitor or inducer, a toxin, a structural protein, a neurotrophic factor, a membrane transporter, a nucleotide binding protein, a heat shock protein, or a CRISPR-associated protein.

[0078] In another embodiment of the present invention, the functional material may include a drug.

[0079] The above drug has no special restrictions as long as it is a drug that can move into cells and exert an effect. Such a drug may be any small molecule compound drug such as a cytotoxic anticancer agent, any biopharmaceutical such as a recombinant protein, siRNA, ASO, mRNA, gRNA, tRNA, etc., and in terms of efficacy, it may be an anti-inflammatory agent, analgesic, anti-arthritic agent, antispasmodic agent, antidepressant agent, antipsychotic agent, tranquilizer, anti-anxiety agent, narcotic antagonist, anti-Parkinson's disease drug, cholinergic agonist, anticancer agent, angiogenesis inhibitor, immunosuppressant, immunostimulant, antiviral agent, antibiotic, appetite suppressant, anticholinergic agent, antihistamine, antimigraine agent, hormonal agent, coronary vascular agent, vasodilator, contraceptive, antithrombotic agent, diuretic, antihypertensive agent, cardiovascular disease treatment agent, diagnostic agent such as contrast medium, etc., but is not limited thereto.

[0080] The functional substance may be a therapeutic agent, a diagnostic agent, or a combination thereof.

[0081] The above functional material may be selected from the group consisting of nucleic acids, proteins, polypeptides, low molecular weight compounds and carbohydrates.

[0082] The functional material may comprise one or more nucleic acid sequences, one or more polypeptides, a combination of nucleic acid sequences and / or polypeptides, one or more cellular organelles, or any combination thereof. In some embodiments, the functional material may comprise one or more cellular components. In some embodiments, the functional material may comprise one or more cytoplasmic and / or nuclear components.

[0083] The functional material may be a nucleic acid, for example, a transcription factor, DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein-coding DNA, a gene, an operon, a chromosome, a genome, a transposon, a retrotransposon, a viral genome, an intron, an exon, a modified DNA, ssDNA (single-stranded DNA), dsDNA (double-stranded DNA), mRNA (messenger RNA), sgRNA (single guide RNA), gRNA (guide RNA), pegRNA (prime editing guide RNA), tRNA (transfer RNA), a modified RNA, microRNA (miRNA), siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (small nuclear RNA), small nucleolar RNA (snoRNA), SmY RNA (mRNA trans-splicing RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (cis-native antisense transcript), CRISPR RNA (crRNA), tracrRNA (trans-activating CRISPR RNA), lncRNA (long noncoding RNA), piRNA (piwi interacting RNA), tasiRNA (trans-acting siRNA), eRNA (enhancer RNA), satellite RNA, pcRNA (protein coding RNA), RNAi (interfering RNA), circRNA (circular RNA), reprogramming RNA, aptamers, antisense oligonucleotides, shRNA (short hairpin RNA), dsRNA (double-stranded RNA), antisense RNA, ribozymes, and any combination thereof. In some embodiments, the nucleic acid is a wild-type nucleic acid. In some embodiments, the nucleic acid is a mutant nucleic acid. In some embodiments, the nucleic acid is a fusion or chimera of a plurality of nucleic acid sequences.

[0084] In some embodiments, the nucleic acid may contain a nuclear localization signal (NLS) that can enhance editing efficiency within the nucleus. An NLS is an amino acid sequence that tags a protein for nuclear transport into the cell nucleus.

[0085] In some embodiments, the functional material may comprise a nucleic acid. For example, the target material may comprise RNA that enhances the expression of an endogenous protein (e.g., endogenous to the cell producing the lipid bilayer particle, and endogenous to the target cell, in some embodiments), or siRNA or miRNA that inhibits protein expression of an endogenous protein.

[0086] In some embodiments, the functional material is a polypeptide, for example, an enzyme, a structural polypeptide, a signaling polypeptide, a regulatory polypeptide, a transport polypeptide, a sensory polypeptide, a motor polypeptide, a defense polypeptide, a storage polypeptide, a transcription factor, an antibody, a cytokine, a hormone, a catabolic polypeptide, an anabolic polypeptide, a proteolytic polypeptide, a metabolic polypeptide, a kinase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an enzyme modulator polypeptide, a protein binding polypeptide, a lipid binding polypeptide, a membrane fusion polypeptide, a cell differentiation polypeptide, an epigenetic polypeptide, an apoptotic polypeptide, a nuclear transport polypeptide, a nucleic acid binding polypeptide, a reprogramming polypeptide, a DNA editing polypeptide, a DNA repair polypeptide, a DNA recombination polypeptide, a transposase polypeptide, a DNA integrating polypeptide Polypeptides, targeting endonucleases (e.g., zinc finger nucleases, transcription activator-like nucleases (TALENs), cas9 and their homologs), recombinases, and any combination thereof. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein. In some embodiments, the protein is a fusion or chimeric protein.

[0087] In some embodiments, the functional material is a small molecule, for example, an ion (e.g., Ca 2+ , Cl - , Fe 2+ ), carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, hemes, polypeptide cofactors, electron accepting compounds, electron donating compounds, metabolites, ligands, and any combination thereof.

[0088] In some embodiments, the functional agent comprises a mixture of proteins, nucleic acids, or metabolites, e.g., multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g., Cas9-gRNA complexes); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any combination thereof.

[0089] In some embodiments, the functional material comprises one or more organelles, such as chondrosomes, mitochondria, lysosomes, nuclei, cell membranes, cytoplasms, endoplasmic reticulum, ribosomes, vacuoles, endosomes, spliceosomes, polymerases, capsids, acrosomes, autophagosomes, centrosomes, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, myofibrils, nematodes, peroxisomes, proteasomes, vesicles, stress granules, networks of organelles, and any combination thereof.

[0090] In some embodiments, the functional agent may be a DNA digesting agent capable of digesting DNA. This refers to an agent capable of cleaving bonds between nucleotide subunits of a nucleic acid (e.g., phosphodiester bonds).

[0091] In one embodiment, the DNA digesting agent is a nuclease. A nuclease is an enzyme that hydrolyzes nucleic acids. Nucleases can be classified as endonucleases or exonucleases. Endonucleases are a group of enzymes that catalyze the hydrolysis of bonds between nucleic acids within a DNA or RNA molecule. Exonucleases are a group of enzymes that catalyze the hydrolysis of single nucleotides at the ends of DNA or RNA chains. Nucleases can also be classified based on whether they specifically digest DNA or RNA. Nucleases that specifically catalyze the hydrolysis of DNA can be referred to as deoxyribonucleases, or DNases, while nucleases that specifically catalyze the hydrolysis of RNA can be referred to as ribonucleases, or RNases. Some nucleases are specific for single-stranded or double-stranded nucleic acid sequences. Some enzymes have both exonuclease and endonuclease properties. Additionally, some enzymes can digest both DNA and RNA sequences.

[0092] In some embodiments, the functional agent may be an endonuclease. Non-limiting examples of endonucleases include, but are not limited to, zinc finger nucleases (ZFNs), ZFN dimers, ZFNickases, transcription activator-like effector nucleases (TALENs), meganucleases, or RNA-guided DNA endonucleases (e.g., the CRISPR / Cas system).

[0093] In one embodiment, the endonuclease may be engineered, chimerized, or isolated from an organism. The endonuclease may be engineered to recognize a specific DNA sequence, for example, by mutagenesis.

[0094] In some embodiments, the functional agent may be an RNA-guided DNA endonuclease (e.g., a CRISPR-Cas system). In this case, the lipid bilayer particle may additionally include gRNA, crRNA, tracrRNA, etc. The terms "gRNA," "guide RNA," and "CRISPR guide sequence" may be used interchangeably and refer to a nucleic acid comprising a sequence that determines the specificity of the Cas DNA binding protein of the CRISPR / Cas system. The gRNA hybridizes (partially or fully complementarily) to a target nucleic acid sequence in the host cell genome. The gRNA or portion thereof that hybridizes to the target nucleic acid may be 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length.

[0095] In some embodiments, the length of the gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. A "scaffold sequence," also called tracrRNA (trans-activating CRISPR RNA), refers to a nucleic acid sequence that recruits a Cas endonuclease to the target nucleic acid bound to (hybridized with) the complementary gRNA sequence.

[0096] In some embodiments, the gRNA sequence does not include a scaffold sequence, and the scaffold sequence is expressed as a separate transcript. In such embodiments, the gRNA sequence further includes an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence and recruit an endonuclease to the target nucleic acid.

[0097] In some embodiments, the RNA-guided DNA endonuclease is a Cas enzyme, i.e., a CRISPR-associated endonuclease. The Cas enzyme can be a naturally occurring Cas enzyme or a functional derivative thereof. In certain embodiments, the Cas enzyme can comprise one or more mutations. The Cas enzyme can be a type II, type I, type III, type IV, or type V CRISPR system enzyme. In some embodiments, the Cas enzyme is a Cas9 enzyme (also known as Cas5, Csn1, or Csx12). Cas9 can be wild-type or mutant.

[0098] In some embodiments, the endonuclease is a Cas9 homolog or ortholog. Cas9 may be any variant disclosed in U.S. Patent Publication No. US 2014 / 0068797 A1, which is incorporated herein by reference.

[0099] In one embodiment, the Cas9 enzyme can be type II-A, type II-B or type II-C.

[0100] In one embodiment, the Cas9 enzyme may be derived from various species. Non-limiting examples of Cas9 enzymes may include Cas9 derived from Streptococcus pyogenes (S. pyogenes), Streptococcus pneumoniae (S. pneumoniae), Staphylococcus aureus, Neisseria meningitidis, Streptococcus thermophilus (S. thermophilus), or Treponema denticola. The Cas9 enzyme may also be derived from a microorganism of the genus Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma or campylobacter.

[0101] In some embodiments, the Cas enzyme is Cas9, Cpf1, C2c1, C2c2, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Cs f1, Csf2, Csf3, Csf4, It may be a homologue, an ortholog thereof, or a variant thereof.

[0102] In some embodiments, the functional material may comprise a diagnostic agent, including but not limited to a radiotracer or radionuclide used in positron emission tomography (PET) (e.g., carbon-11, nitrogen-13, oxygen-15, and fluorine-18). These agents can be more precisely delivered to the tissue of interest by loading them into engineered extracellular particles that target the tissue of interest, i.e., the tissue being scanned, either artificially or naturally, thereby reducing off-target delivery of the diagnostic agent.

[0103] In some embodiments, the functional material may be a targeting moiety.

[0104] In some embodiments, the non-vesicular extracellular particle may additionally comprise at least one targeting moiety.

[0105] In some embodiments, the targeting moiety can be used to target the non-vesicular extracellular particle to a specific organ, tissue, or cell for delivery of the functional agent using the non-vesicular extracellular particle.

[0106] In certain embodiments, the targeting moiety can bind to a marker (or target molecule) expressed in a cell or cell population.

[0107] In certain embodiments, the marker may be expressed on various cell types, for example, all antigen-presenting cells (e.g., dendritic cells, macrophages, and B lymphocytes).

[0108] In some embodiments, the marker may be expressed only in a particular cell population (e.g., dendritic cells). Non-limiting examples of markers expressed in a particular cell population (e.g., dendritic cells) include C-type lectin domain family 9 member A (CLEC9A) protein or dendritic cell-specific intercellular adhesion molecule-3-grafted non-binding protein: integrin (DC-SIGN), CD207, CD40, Clec6, dendritic cell immunoreceptor (DCIR), DEC-205, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, DC-asialoglycoprotein receptor (DC-ASGPR), DC immunoreceptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-1 (CD303, Clec4c), Dectin-2, Bst-2 (CD317), and any combination thereof.

[0109] In some embodiments, the targeting moiety can be an antibody or an antigen-binding fragment thereof. Antibodies and antigen-binding fragments thereof include whole antibodies, polyclonal, monoclonal, and recombinant antibodies, and fragments thereof, and may further include single chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, mouse-human, mouse-primate, and primate-human antibodies. Monoclonal antibodies, anti-idiotypic antibodies, antibody fragments (e.g., scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments), diabodies, and antibody-related polypeptides. Antibodies and antigen-binding fragments thereof can include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0110] In one embodiment of the present invention, the supermere may have improved functional substance delivery capacity compared to extracellular vesicles or exomeres.

[0111] In one embodiment of the present invention, the supermere may accumulate in a smaller amount in the liver compared to extracellular vesicles or exomeres.

[0112] In one embodiment of the present invention, the supermere may accumulate in a smaller amount in the spleen compared to extracellular vesicles or exomeres.

[0113] In one embodiment of the present invention, the supermere may accumulate in a larger amount in the kidney compared to extracellular vesicles or exomeres.

[0114] In one embodiment of the present invention, the composition may comprise a vector comprising the gene.

[0115] As used herein, the term "vector" refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence may be exogenous or heterologous. Examples of vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages).

[0116] In one embodiment of the present invention, the vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like. Suitable vectors include, in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and can be manufactured in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible.

[0117] In one specific example, the vector may be, but is not limited to, pcDNA3.1, pcDNA3.4, pMXs-IRES, pDisplay, pcDNa3.4, pRG2-GG, prp[exp] or pCMV.

[0118] Another aspect according to one embodiment of the present invention provides a composition for functional substance delivery comprising a non-vesicular extracellular particle comprising a non-vesicular extracellular particle marker protein and a target functional substance.

[0119] The terms 'non-vesicular extracellular particle', 'functional substance' and 'functional substance delivery' are as described above.

[0120] In one embodiment of the present invention, the target functional material may be in a form fused with a non-vesicular extracellular particle marker protein.

[0121] In one embodiment of the present invention, the non-vesicular extracellular particles may be supermeers.

[0122] In one embodiment of the present invention, the target functional material may be in a form fused with a supermere marker protein.

[0123] In one embodiment of the present invention, the supermembrane marker protein may be at least one selected from the group consisting of Transforming growth factor, beta-induced (TGFBI), Enolase-1 (ENO1), Enolase-2 (ENO2), Heat shock 70 kDa protein 13 (HSPA13), Glucose-6-phosphate isomerase (GPI), Lactate Dehydrogenase A (LDHA), Triosephosphate Isomerase 1 (TPI1), Hexokinase 1 (HK1), Malate Dehydrogenase 1 (MDH1), Nucleobindin-1 (NUCB1), Protein Disulfide Isomerase Family A Member 4 (PDIA4), fragments thereof, mutants thereof, mutants of fragments thereof, and fragments of mutants thereof, but is not limited thereto.

[0124] In addition, another aspect according to one embodiment of the present invention provides a pharmaceutical composition comprising the composition for delivering the functional substance.

[0125] The pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Pharmaceutically acceptable carriers may include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion, or into a pill, capsule, granule, or tablet. Accordingly, the pharmaceutical composition of the present invention may be a patch, a liquid, a pill, a capsule, a granule, a tablet, a suppository, or the like. These preparations can be prepared by conventional methods used in formulation in the art or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, and can be formulated into various preparations depending on each disease or ingredient.

[0126] The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route and excretion rate, the duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that achieves the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art. The daily dosage of the compound of chemical formula 1 of the present invention is about 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg, and can be administered once or several times a day.

[0127] The term "administration" in the present invention means introducing a predetermined substance into a patient by an appropriate method, and the administration route of the composition may be administered through any common route as long as it can reach the target tissue. In addition, the pharmaceutical composition of the present invention may be administered by any device that allows the active substance to move to the target tissue. For example, it may be administered by transdermal administration, oral administration, intrathecal administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, inner ear administration, intrauterine epidural administration, sublingual administration, and intracerebrovascular injection, but is not limited thereto.

[0128] In the present invention, the pharmaceutical composition may appropriately contain, if necessary, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc. depending on the administration method or formulation. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, 19th ed., 1995]. The pharmaceutical composition may be manufactured in a unit dose form or may be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets, or capsules.

[0129] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may be used.

[0130] Oral liquid preparations include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0131] 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 withepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin. Meanwhile, injections can include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0132] The route of administration of the pharmaceutical composition of the present invention may be through any general route as long as it can reach the target tissue, but may be through subcutaneous injection using an osmotic pump, intradermal injection, intravenous injection, intraperitoneal injection, intravitreal injection, oral administration, etc.

[0133] The term “prevention” used in the present invention means any act of suppressing a disease or delaying its onset by administering the composition.

[0134] In the present invention, “treatment” means any act in which the symptoms of a disease are improved or beneficially changed by administering the composition.

[0135] The term "subject" in the present invention refers to any animal that has developed or is likely to develop a disease, and typically refers to an animal that can exhibit a beneficial effect from treatment using the composition of the present invention, but includes, without limitation, any subject that has symptoms of the disease or is likely to develop such symptoms. As described above, by administering the pharmaceutical composition of the present invention to a subject, the disease can be effectively prevented or treated. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent, or in combination with existing disease therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents.

[0136] In addition, another aspect according to one embodiment of the present invention provides a treatment method comprising a step of administering the pharmaceutical composition.

[0137] In addition, another aspect according to one embodiment of the present invention provides a cosmetic composition comprising the composition for delivering the functional material.

[0138] In the present invention, the composition can be formulated in various forms by adding various components as auxiliary components for delivery and stabilization, etc.

[0139] In the present invention, the cosmetic composition may have a formulation such as a mist, serum, nourishing toner, emulsifying toner, emulsion, suspension, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, powder, makeup base, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, facial cleanser, treatment, beauty liquid, beauty pack, ointment, gel, liniment, liquid, patch, spray, bath agent, sunscreen, sun oil, and hair product. The scope of the formulation is not limited thereto, and the formulation of the cosmetic composition may be manufactured into any formulation commonly manufactured in the art.

[0140] In the present invention, the cosmetic composition may further comprise a cosmetically acceptable carrier. The type of the cosmetically acceptable carrier of the present invention is not particularly limited, as long as it does not inhibit the activity and properties of the cosmetic composition of the present invention, and any cosmetically acceptable carrier commonly used in the art may be used. Non-limiting examples of the cosmetically acceptable carrier include saline solution, sterile water, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, and ethanol. These may be used alone or in combination of two or more.

[0141] In the present invention, the cosmetically acceptable carrier varies depending on the formulation of the cosmetic composition.

[0142] When the above formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0143] When the above formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, when it is a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0144] When the above formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0145] When the above formulation is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as a carrier component.

[0146] When the formulation of the above cosmetic composition is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto.

[0147] When the formulation of the above cosmetic composition is a pack, it includes all forms of a peel-off pack containing polyvinyl alcohol or the like, a wash-off pack containing pigments such as kaolin, talc, zinc oxide, or titanium dioxide in a general emulsified cosmetic, or a mask sheet pack, but is not particularly limited thereto.

[0148] The ingredients included in the above cosmetic composition may include, in addition to the composition for delivering functional substances as an active ingredient, ingredients commonly used in cosmetic compositions, and may include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, preservatives, moisturizers, colorants, bactericides, antioxidants, surfactants, vitamins, pigments, and fragrances. In addition, the above cosmetic composition may additionally include a skin absorption promoter to enhance its effectiveness.

[0149] In the present invention, the composition may further include one or more active ingredients exhibiting the same or similar efficacy.

[0150] In the present invention, the composition for delivering the functional material is present in an amount of 0.000000001 wt% to 90 wt%, for example, 0.000000001 wt% to 80 wt%, 0.000000001 wt% to 60 wt%, 0.000000001 wt% to 30 wt%, 0.000000001 wt% to 20 wt%, 0.000000001 wt% to 10 wt%, 0.000000001 wt% to 5 wt%, 0.000000001 wt% to 0.001 wt%, 0.000000001 wt% to 0.0001 wt%, 0.000000001 wt% to 0.00001 wt%, 0.0001 wt% to 90 wt%, 0.0001 wt% to 80 wt%, 0.0001 wt% to 60 wt%, 0.0001 wt% to 30 wt%, 0.0001 wt% to 20 wt%, 0.0001 wt% to 10 wt%, 0.0001 wt% to 5 wt%, 0.001 wt% to 90 wt%, 0.001 wt% to 80 wt%, 0.001 wt% to 60 wt%, 0.001 wt% to 30 wt%, 0.001 wt% to 20 wt%, 0.001 wt% to 10 wt%, 0.001 wt% to 5 wt%, 0.01 wt% to 90 wt%, 0.01 wt% to 80 wt%, It may be included in an amount of 0.01 wt% to 60 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.1 wt% to 90 wt%, 0.1 wt% to 80 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt%. When it is included in an amount of less than 0.000000001 wt%, the skin improvement effect, etc. is minimal, and 90.If it exceeds 0 wt%, it is not economical as the efficiency is low compared to the amount of material input.

[0151] In one specific example, the cosmetic composition comprises 10 functional material delivery compositions. 5 10 to 100 pcs / ml 10 dog / ml, for example, 10 5 10 to 100 pcs / ml 9 dog / ml, 10 5 10 to 100 pcs / ml 8 dog / ml, 10 5 10 to 100 pcs / ml 7 dog / ml, 10 5 10 to 100 pcs / ml 6 dog / ml, 10 6 10 to 100 pcs / ml 10 dog / ml, 10 6 10 to 100 pcs / ml 9 dog / ml, 10 6 10 to 100 pcs / ml 8 dog / ml, 10 6 10 to 100 pcs / ml 7 dog / ml, 10 7 10 to 100 pcs / ml 10 dog / ml, 10 7 10 to 100 pcs / ml 9 dog / ml, 10 7 10 to 100 pcs / ml 8 dog / ml, 10 8 10 to 100 pcs / ml 10 dog / ml, 10 8 10 to 100 pcs / ml 9 dog / ml or 10 9 10 to 100 pcs / ml 10 It may be included in a concentration of 100 mg / ml.

[0152] In addition, another aspect according to one embodiment of the present invention provides a method for preparing a composition for functional material delivery, comprising the following steps.

[0153] (a) A step of centrifuging the supernatant obtained by culturing cells;

[0154] (b) a step of filtering and purifying the supernatant obtained by centrifugation;

[0155] (c) A step of centrifuging the purified supernatant several times.

[0156] For example, the centrifugation in step (c) above can be performed initially at 5,000 g to 15,000 g, thereby obtaining large extracellular vesicles or small extracellular particles.

[0157] For example, after the first operation, a second centrifugation may be performed at 100,000 g to 200,000 g, through which small extracellular vesicles or non-vesicular extracellular particles may be obtained.

[0158] For example, after the secondary centrifugation, a tertiary centrifugation can be performed at 150,000 g to 200,000 g, through which exomeres can be obtained.

[0159] For example, after the third centrifugation, a fourth centrifugation can be performed at 350,000 g to 400,000 g, through which a supermeer can be obtained.

[0160] In one embodiment of the present invention, the cell of step (a) may be one into which a functional material, for example a gene, has been introduced, but is not limited thereto.

[0161] In one embodiment of the present invention, the manufacturing method may further include a step of mixing the obtained supermembrane with a functional material to be delivered, for example, a gene.

[0162] In addition, another aspect according to one embodiment of the present invention provides a method for delivering a functional material to a cell by a non-vesicular extracellular particle (NVEP).

[0163] The above non-vesicular extracellular particles and functional materials are as described above.

[0164] In addition, another aspect according to one embodiment of the present invention provides a kit for delivering a functional substance, comprising a non-vesicular extracellular particle (NVEP) as an active ingredient.

[0165] The above non-vesicular extracellular particles, functional substances and functional substance delivery are as described above.

[0166] The carrier means is suitable for containing one or more containers, such as bottles or tubes, each container containing independent components used in the method of the present invention.

[0167] Additionally, the kit may include a guide. A guide is a printed document explaining how to use the kit, such as the reaction conditions provided. This guide includes instructions in the form of a pamphlet or leaflet, a label attached to the kit, and instructions on the surface of the package containing the kit. Furthermore, the guide includes information disclosed or provided through electronic media, such as the Internet.

[0168] Another aspect according to one embodiment of the present invention provides a kit for delivering a functional substance, comprising a non-vesicular extracellular particle (NVEP) marker protein; and a non-vesicular extracellular particle (NVEP) comprising a target functional substance.

[0169] The above non-vesicular extracellular particle marker protein may be, but is not limited to, a non-specific protein.

[0170] For example, the non-specific protein may be a protein present in a non-vesicular extracellular particle, or may be a protein present on the outside of a non-vesicular extracellular particle that moves into a non-vesicular extracellular particle when overexpressed in a cell, but is not limited thereto.

[0171] The non-vesicular extracellular particles, non-vesicular extracellular particle marker proteins, target functional substances, functional substance delivery and kits are as described above.

[0172] Another aspect according to one embodiment of the present invention provides a composition for functional substance delivery, comprising a non-vesicular extracellular particle marker protein and a target functional substance.

[0173] The above non-vesicular extracellular particles, non-vesicular extracellular particle marker protein, target functional substance and functional substance delivery are as described above.

[0174] In addition, another aspect according to one embodiment of the present invention provides a method for delivering a functional substance using the composition for delivering a functional substance.

[0175] In addition, another aspect according to one embodiment of the present invention provides a use of a functional substance delivery composition comprising non-vesicular extracellular particles for functional substance delivery.

[0176] In addition, another aspect according to one embodiment of the present invention provides a use of non-vesicular extracellular particles for functional material delivery.

[0177]

[0178] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention more specifically, and the scope of the present invention is not limited by these examples.

[0179]

[0180] Example 1: Collection method of extracellular particle sub-classes

[0181] Extracellular particles, including supermere, exomere, and small extracellular vesicles, were isolated and obtained from HEK293FT cell line and human bone marrow-derived stem cells (Fig. 2). Specifically, donor cells were cultured in a 150 π culture dish at a density of 4.5 × 10 6After seeding with cells, 20 ml of cell culture medium (DMEM high glucose + 10% FBS + 1% AA) was added and cultured for 48 hours. The supernatant was then removed, washed once with DPBS, and replaced with 19 ml of serum-free medium (DMEM high glucose + 1% AA + 1% Glutamax) and pre-cultured for 30 minutes.

[0182] If transduction was required, a transfection reagent was prepared by mixing pcDNA3.1 and PEI in serum-free medium, incubating at room temperature for 15 minutes, and then adding the reagent to the donor cells. The medium was replaced with serum-free medium 6 hours after transduction, and the final supernatant was collected 48 hours later. Collection of extracellular particles obtained without transduction was also performed in the same manner.

[0183] The obtained supernatant was centrifuged at 3000 g for 5 minutes and filtered through a 0.45 μm filter (Sartolab RF500 PES). Subsequently, it was concentrated using Amicon (10 kDa filter, UFC901024, Merck) or TFF (100 kDa, S04-E100-05-N, Repligen). The supernatant was then centrifuged at 10,000 g for 30 minutes and then ultracentrifuged at 150,000 g for 90 minutes at 4 °C (Ultracentrifuge, Beckman). Small extracellular vesicles were isolated using 1X PBS-PIC (kept on ice for at least 20 min, then centrifuged at 13,000 g for 20 min). The supernatant of small extracellular vesicles was transferred to a UC tube (355655) and centrifuged at 167,000 g for 16 h. Exomeres were isolated using 1X PBS-PIC (kept on ice for at least 20 min, then centrifuged at 13,000 g for 20 min). The exomere supernatant was transferred to a 70Ti rotor high speed tube (355618) and ultracentrifuged at 367,000 g for 16 h. Supermeres were isolated from the pellet generated after ultracentrifugation using 1X PBS-PIC (kept on ice for at least 20 min, then briefly centrifuged for 2 min).

[0184]

[0185] Example 2: Confirmation of marker expression using SuperMere

[0186] Supermere was isolated from the HEK293FT cell line according to the collection method described in Example 1, and the marker expression patterns were compared with those of exomere and small extracellular vesicles.

[0187] Automated Western blotting (ProteinSimple) was performed on isolated small extracellular vesicles, exomeres, and supermeres. TGFBI (10188-1-AP-20UL), Enolase-1 (3810T), Enolase-2 (8171S), HSPA13 (sc-398297), and CD81 (EXOAB-CD81A1) antibodies were used at a 1:100 dilution, and protein expression was confirmed by loading 3 μg of each substance.

[0188] As a result, HEK293FT-derived supermere exhibited a protein expression pattern distinct from exomere and small extracellular vesicles (Fig. 3).

[0189] From these results, it was confirmed that supermere can be detected using TGFBI, ENO1, ENO2, HSPA13, and CD81 as markers.

[0190]

[0191] Example 3. Confirmation of cell delivery of functional materials loaded inside extracellular particles (EPs).

[0192] An experiment was conducted to confirm whether functional materials transduced into donor cells were loaded into extracellular particles and delivered to recipient cells. This experiment was conducted according to the process reported in https: / / doi.org / 10.1016 / j.cell.2023.06.013. HEK293FT cells were used as donor and recipient cells, and extracellular particles were isolated and obtained according to the collection method described in Example 1.

[0193] For transduction, HEK293FT cells were treated with a transfection reagent (1 ml) containing pcDNA3.1-eGFP-MS2X2 (60 μg) and PEI (240 μg), and the medium was replaced with serum-free medium after 6 h, and the supernatant was collected after 48 h. The plasmid used was constructed by cloning a polynucleotide (SEQ ID NO: 2) encoding the eGFP-MS2X2 protein (SEQ ID NO: 1) into the pcDNA3.1 vector (ThermoFisher).

[0194] Afterwards, the recipient cells, HEK293FT cells, were seeded in 24-well plates at 2.8 Х 10 5 Cells were seeded at 500 μl per well (cell culture medium). After 24 hours, the supernatant was aspirated and washed once with DPBS. The extracellular particles were well mixed in serum-free medium and incubated for 48 hours in a final volume of 500 μl. In addition, a group treated with RNase A was additionally prepared separately in the same process. The RNase A-treated group was used after incubating the extracellular particles at 37°C for 30 minutes and then treating them with 1 μg / ml of RNase A. After 48 hours, the attached recipient cells were collected using a cell scraper (90020) and automated Western blotting (ProteinSimple) was performed.

[0195] As a result, as confirmed in Fig. 4, when eGFP was overexpressed in cells, extracellular particles were separated, and treated at different concentrations, it was confirmed that eGFP expression increased in the recipient cells 48 hours after all proteins were removed.

[0196] In addition, we confirmed that there was no difference in eGFP expression in recipient cells even when extracellular particles and RNase A were treated together.

[0197] These results suggest that eGFP functional materials loaded into extracellular particles by endogenous loading can be delivered to recipient cells through the extracellular particles, and that the loaded functional materials remain stable despite RNase A treatment.

[0198]

[0199] Example 4. Confirmation of cell delivery of functional substances loaded inside small extracellular particles.

[0200] An experiment was conducted to determine which type of extracellular particle the eGFP-MS2X2 functional material transferred from donor cells to recipient cells was loaded onto. This experiment was confirmed according to the process reported in https: / / doi.org / 10.1016 / j.cell.2023.06.013. HEK293FT cells were used as donor and recipient cells.

[0201] Donor cells were treated with 1 ml of a transfection reagent containing 60 μg of pcDNA3.1-eGFP-MS2X2 and 240 μg of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was collected 48 hours later. The obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular particles were separated and obtained from the supernatant after 10,000 g centrifugation, and large extracellular vesicles were separated and obtained from the pellet after 18,000 g centrifugation.

[0202] Afterwards, the recipient cells, HEK293FT cells, were seeded in 24-well plates at 2.8 X 10 5 Cells were seeded at 500 μl per well (cell culture medium), and the supernatant was aspirated after 24 h, and washed once with DPBS. Afterwards, the final 500 μl was mixed with serum-free medium, each containing extracellular particles, small extracellular particles, and large extracellular vesicles, and incubated for 24 h. After incubation, attached recipient cells were collected using a cell scraper (90020) and subjected to automated Western blotting (ProteinSimple).

[0203] As a result, as can be confirmed in Fig. 5, when eGFP-MS2X2 mRNA was overexpressed in cells, extracellular particles (EP), small EP, and large EV were separated and treated at different concentrations, and it was confirmed that eGFP expression in the recipient cells increased more in small EP than in all extracellular particles after 24 hours. On the other hand, it was confirmed that eGFP was not expressed in cells treated with micro-sized large EV.

[0204] These results suggest that the eGFP-MS2X2 functional material overexpressed in cells can deliver other types of extracellular particles to recipient cells more effectively than large extracellular vesicles.

[0205]

[0206] Example 5. Confirmation of cell delivery of functional materials loaded inside non-vesicular extracellular particles (NVEPs).

[0207] An experiment was conducted to determine which particles of small extracellular particles (small EPs) were loaded with the eGFP-MS2X2 functional material transferred from donor cells to recipient cells. HEK293FT cells were used as donor and recipient cells.

[0208] Donor cells were treated with 1 ml of transfection reagent containing 60 μg of pcDNA3.1-eGFP-MS2X2 + 240 μg of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was collected 48 hours later.

[0209] The obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular particles were separated and obtained from the 10,000 g centrifugation supernatant, large extracellular vesicles were separated and obtained from the 18,000 g centrifugation pellet, and non-vesicular extracellular particles and small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation supernatant and pellet, respectively.

[0210] Afterwards, the recipient cells, HEK293FT cells, were seeded in 24-well plates at 2.8 X 10 5 Cells were seeded at 500 μl per well (cell culture medium), and the supernatant was aspirated after 24 h and washed once with DPBS. The final 500 μl was mixed with serum-free medium containing extracellular particles, large extracellular vesicles, small extracellular particles, small extracellular vesicles, and non-vesicular extracellular particles, and incubated for 48 h. After incubation, attached recipient cells were collected using a cell scraper (90020) and subjected to automated Western blotting (JESS Protein Simple).

[0211] As a result, as shown in Fig. 6, after overexpressing eGFP-MS2X2 mRNA in cells, extracellular particles, large extracellular vesicles, small extracellular particles, small extracellular vesicles, and non-vesicular extracellular particles were separated and processed, and it was confirmed that eGFP expression in the recipient cells was in the order of non-vesicular extracellular particles > small extracellular particles > extracellular particles > small extracellular vesicles > large extracellular vesicles.

[0212] In addition, in order to exclude the influence of differences in plasmid sequence, eGFP plasmid without MS2X2 was transfected under the same conditions as pcDNA3.1-eGFP-MS2X2, and then extracellular particles such as non-vesicular extracellular particles and small extracellular vesicles were separated and treated in recipient cells. To prepare pcDNA3.1-eGFP plasmid, a polynucleotide (SEQ ID NO: 4) encoding eGFP protein (SEQ ID NO: 3) was cloned into the pcDNA3.1 vector and used. As a result of the experiment, it was confirmed that in the case of eGFP plasmid, as in the case of eGFP-MS2X2, eGFP expression was the highest in recipient cells treated with non-vesicular extracellular particles compared to other particles (Fig. 7).

[0213] These results suggest that, regardless of the sequence type of the plasmid overexpressed in the cells, the overexpressed functional substances (proteins and mRNA) are mainly loaded into non-vesicular extracellular particles of less than 50 nm in size and are effectively delivered to recipient cells.

[0214]

[0215] Example 6: Confirmation of cell delivery of functional materials loaded inside the supermembrane.

[0216] We investigated whether functional materials transduced into donor cells were delivered via supermembrane loading, even among non-vesicular extracellular particles. HEK293FT cells were used as donor and recipient cells.

[0217] Donor cells were treated with 1 ml of a transfection reagent containing 30 μg of pcDNA3.1-eGFP-MS2X2 or pcDNA3.1-myc MCP + 120 μg of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained after 48 hours. At this time, the pcDNA3.1-eGFP-MS2X2 plasmid was the same as that prepared in Example 2, and a polynucleotide encoding the myc-MCP protein (SEQ ID NO: 5) was cloned into the pcDNA3.1 vector to prepare the pcDNA3.1-myc MCP plasmid.

[0218] The obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular particles were separated and obtained from the 10,000 g centrifugation supernatant, large extracellular vesicles from the 18,000 g centrifugation pellet, non-vesicular extracellular particles and small extracellular vesicles from the 150,000 g ultracentrifugation supernatant and pellet, respectively, and supermeres and exomeres from the 167,000 g ultracentrifugation supernatant and pellet, respectively.

[0219] Afterwards, the recipient cells, HEK293FT cells, were seeded in 24-well plates at 2.8 X 10 5Cells were seeded at 500 μl per well (cell culture medium), and the supernatant was aspirated after 24 hours and washed once with DPBS. Afterwards, each nanoparticle was well mixed with serum-free medium at a concentration based on the protein level of each group, and the final volume of 500 μl was incubated for 48 hours. After incubation, attached recipient cells were collected using a cell scraper (90020) and subjected to automated Western blotting (ProteinSimple).

[0220] As a result, it was confirmed that, among non-vesicular extracellular particles, supermere-loaded functional materials mainly play a role in delivering functional materials to recipient cells by utilizing two types of functional materials, eGFP-MS2 and myc-MCP (Fig. 8).

[0221]

[0222] Example 7: Confirmation of Supermere's Endogenous Drug Delivery Ability in Cells

[0223] 7-1. Analysis of mRNA and protein content, which are functional substances in supermere, exomere, and small extracellular vesicles.

[0224] Experiments were conducted to compare the intracellular drug delivery capabilities of supermere, exomere, and small extracellular vesicles. First, the amount of mRNA contained in each extracellular particle isolated from cells transduced with the eGFP plasmid was compared and analyzed.

[0225] Specifically, HEK293FT cells were treated with 1 ml of a transfection reagent containing 60 μg of pcDNA3.1-eGFP + 240 μg of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained 48 hours later.

[0226] The obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular particles were separated and obtained from the 10,000 g centrifugation supernatant, small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation pellet, exomeres were separated and obtained from the 167,000 g ultracentrifugation pellet, and supermeres were separated and obtained from the 367,000 g pellet.

[0227] Total RNA was extracted from supermeres, exomeres, and small extracellular vesicles using the miRNeasy Mini Kit (271004, Qiagen) according to the manufacturer's protocol. The extracted RNA was synthesized into cDNA using TOPscript™ (EZ005S, Enzynomics). The amount of eGFP mRNA in each material was quantified by absolute RT-qPCR, using eGFP IVT mRNA (Genscript) as the standard.

[0228] Additionally, automated Western blotting (ProteinSimple) was performed on the isolated supermeres, exomeres, and small extracellular vesicles. eGFP antibody (1:100, CAB4211, Invitrogen) was used, and 3 μg of supermeres, exomeres, and small extracellular vesicles were loaded each.

[0229] As a result, we confirmed that small extracellular vesicles had 80-fold and exomeres had 30-fold increased eGFP mRNA levels compared to supermeres (Fig. 9). Protein analysis results confirmed that small extracellular vesicles had 1.25-fold and exomeres had 1.85-fold higher levels of eGFP protein compared to supermeres (Fig. 10).

[0230]

[0231] 7-2. Comparison of the Endogenous Functional Material Delivery Capabilities of Supermere, Exomere, and Small Extracellular Vesicles

[0232] An experiment was conducted to compare the endogenous functional substance delivery capabilities of the supermere, exomere, and small extracellular vesicles obtained in Example 7-1.

[0233] Specifically, HEK293FT cells were treated with 1 ml of a transfection reagent containing 60 μg of pcDNA3.1-eGFP + 240 μg of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained 48 hours later.

[0234] The obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation pellet, exomeres from the 167,000 g ultracentrifugation pellet, and supermeres from the 367,000 g pellet.

[0235] Total RNA was extracted from supermeres, exomeres, and small extracellular vesicles using the miRNeasy Mini Kit (271004, Qiagen) according to the manufacturer's protocol. The extracted RNA was synthesized into cDNA using TOPscript™ (EZ005S, Enzynomics). The amount of eGFP mRNA in each material was quantified by absolute RT-qPCR, using the eGFP plasmid as a standard.

[0236] Afterwards, the recipient cells, HEK293FT cells, were seeded in 24-well plates at 2.8 X 10 5Cells were seeded at 500 μl per well (cell culture medium), and the supernatant was aspirated after 24 hours and washed once with DPBS. Each sample was mixed with serum-free medium to make a final volume of 500 μl and incubated for 48 hours. Afterwards, 100 μl of 1X RIPA + PIC was added and attached recipient cells were collected using a cell scraper (90020). The obtained cell lysate was vortexed four times every 5 minutes and centrifuged at 16,000 g for 20 minutes. The supernatant was collected and automated Western blotting (ProteinSimple) was performed. Protein expression was confirmed using eGFP (1:100, CAB4211) by loading 3 μg of cell lysate.

[0237] As a result, it was confirmed that Supermere showed mRNA delivery efficiency up to 20 times higher than that of small extracellular vesicles and exomeres in recipient cells (Fig. 11), and while small extracellular vesicles and exomeres did not induce eGFP protein expression in recipient cells, it was confirmed that Supermere induced eGFP protein expression in recipient cells (Fig. 12).

[0238] These results suggest that supermere has an excellent ability to efficiently deliver endogenous functional substances through excellent delivery efficiency, maximizing functional drug delivery, despite containing less eGFP protein and mRNA than small extracellular vesicles or exomeres.

[0239]

[0240] Example 8: Confirmation of intracellular internalization of Supermere via the lipid raft pathway.

[0241] Since understanding the internalization pathway of Supermere provides important clues for maximizing the characteristics and gene transfer efficiency of Supermere and developing new applications, we conducted experiments to confirm the internalization pathway of Supermere into cells.

[0242] Specifically, HEK293FT cells were treated with 1 ml of a transfection reagent containing 30 μg of pcDNA3.1 + 120 μg of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained 48 hours later.

[0243] The obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation pellet, exomeres from the 167,000 g ultracentrifugation pellet, and supermeres from the 367,000 g pellet.

[0244] To stain the obtained supermere, Cy5.5-NHS ester (Lumiprobe) was mixed at a ratio of 1 mg / ml: 1 μg / ml, pipetted, and incubated overnight at 4°C to induce fluorescence-supermere reaction. Zeba TM Unbound fluorescent dye was removed using a Spin Desalting column (40K MWCO, Thermo Fisher Scientific).

[0245] HEK293FT cells, which are recipient cells, were seeded at 2.5 X 10 in a 24-well plate containing serum-free medium (500 μl). 5Cells were seeded at 10 cells / well and incubated at 37°C for 24 h. Three inhibitors, CPZ (Chloropromazine, clathrin-mediated endocytosis inhibitor) 10 μM, EIPA (5-(N-Ethyl-N-isopropyl)-Amiloride, macropinocytosis inhibitor) 50 μM, and MβCD (Methyl-β-Cyclodextrin, lipid raft-mediated endocytosis inhibitor) 10 mM, were treated respectively, and incubated at 37°C for 30 min. The cells were washed with DPBS and replaced with serum-free DMEM. Cy5.5-labeled SuperMere 50 μg / ml was treated to each well, followed by incubation, and the Cy-5.5 relative MFI was measured after 30 min, 3 h, and 24 h.

[0246] Additionally, to confirm Cy5.5 expression in recipient cells, cells were washed once with cold DPBS (Ca², Mg²-free, Welgene), detached using Trypsin-EDTA, and pelleted by centrifugation at 300 g for 3 minutes. The pelleted cells were resuspended in 200 μl of DPBS, and Cy5.5 expression in single cells was confirmed by flow cytometry.

[0247] As a result, supermere uptake was reduced when treated with CPZ, EIPA, and MβCD, and in particular, supermere uptake was reduced the most when treated with MβCD, confirming that supermere was internalized into cells mainly through lipid rafts (Fig. 13). Fig. 13a and Fig. 13b show the results of confirming Cy5.5 expression in recipient cells 30 minutes, 3 hours, and 24 hours after treating recipient cells with each drug and supermere. Fig. 13c shows the change in the proportion of cells containing supermere stained with Cy5.5 for 24 hours after treating recipient cells with each drug and supermere.

[0248]

[0249] Example 9: Confirmation of Supermere's intracellular exogenous drug delivery ability

[0250] In the field of mRNA-based therapeutics, research on methods for exogenously loading externally synthesized mRNA into a delivery vehicle is attracting attention, and the possibility of delivering mRNA exogenously loaded into a supermembrane was evaluated.

[0251] Specifically, HEK293FT cells were treated with 1 ml of a transfection reagent containing 30 μg of pcDNA3.1 + 120 μg of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained 48 hours later.

[0252] The obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation pellet, exomeres from the 167,000 g ultracentrifugation pellet, and supermeres from the 367,000 g pellet.

[0253] For recipient cell treatment, cells were seeded in 24-well plates at 2.8 х 10 5 After seeding cells / well (cell culture medium), the supernatant was aspirated after 24 hours and washed once with DPBS. Afterwards, small extracellular vesicles, exomeres, and supermeres were divided into groups as shown in Table 1 below and experiments were conducted.

[0254]

[0255] Category Group Name Capacity eGFP mRNA added Free mRNA Free mRNA - 6 μg / ml Small extracellular vesicles Dose 16 μg (12 μg / ml) 0.75 μg (1.5 μg / ml) Dose 212 μg (24 μg / ml) 1.5 μg (3 μg / ml) Dose 324 μg (48 μg / ml) 3 μg (6 μg / ml) Exomere Dose 16 μg (12 μg / ml) 0.75 μg (1.5 μg / ml) Dose 212 μg (24 μg / ml) 1.5 μg (3 μg / ml) Dose 324 μg (48 μg / ml) 3 μg (6 μg / ml) Supermere Dose 130 μg (60 μg / ml) 1 μg (2 μg / ml)

[0256]

[0257] Samples were mixed according to the conditions of each group and incubated at room temperature for 15 minutes. Each group was mixed in serum-free medium to make a final volume of 500 μl and incubated for 48 hours. After that, 100 μl of 1X RIPA + PIC was added and attached recipient cells were collected using a cell scraper (90020). The obtained cell lysate was vortexed four times every 5 minutes and centrifuged at 16,000 g for 20 minutes. The supernatant was collected and subjected to automated Western blotting (ProteinSimple).

[0258] Protein expression in each group was confirmed by loading 3 μg of cell lysate using eGFP (1:100, CAB4211, Invitrogen) and beta-actin (1:500, MAB8929, R&D Systems) antibodies.

[0259] As a result, when small extracellular vesicles and exomeres were used as delivery vehicles to deliver mRNA into cells, protein expression was not observed, confirming that delivery did not occur. In contrast, in the case of supermere, target protein expression was confirmed, confirming that mRNA intracellular delivery via supermere is possible (Fig. 14).

[0260]

[0261] Example 10: Confirmation of Supermere's ability to deliver exogenous pDNA into cells

[0262] Experiments were performed to determine whether SuperMere could deliver not only mRNA but also exogenously loaded pDNA.

[0263] Supermere was isolated and obtained according to the collection method described in Example 1. Then, HEK293FT cells, which are recipient cells, were seeded in 6-well or 24-well plates at a density of 2.8 X 10 5 Cells were seeded at 500 μl per well (medium: DMEM, high glucose + 10% FBS + 1% AA), and the supernatant was aspirated after 24 hours and washed once with DPBS. Then, groups were prepared according to the conditions shown in Table 3 below.

[0264]

[0265] Group (6 well)PlasmidSupermerePEINon-treatedNoneNoneNoneFree DNAeGFP 3 μg (1.5 μg / ml)NoneNonePositiveNone12 μgDose 16 μg (3 μg / ml)NoneDose 212 μg (6 μg / ml)NoneDose 324 μg (12 μg / ml)NoneGroup (24) well)PlasmidSupermereNon-treatedNoneNoneDose 1eGFP pDNA0.375 μg(0.375 μg / ml)3 μg(6 μg / ml)Dose 2eGFP pDNA0.75 μg(1.5 μg / ml)6 μg(12 μg / ml)Dose 3eGFP pDNA1.5 μg(3 μg / ml)12 μg g (24 μg / ml)

[0266]

[0267] Each prepared group was incubated at room temperature for 15 minutes, mixed with the above medium to make a final volume of 500 μl, and incubated for 48 hours. After that, 100 μ μl of 1X RIPA + PIC was added, adherent cells were collected using a cell scraper (90020), and the obtained cell lysate was vortexed every 5 minutes, repeated 4 times, and centrifuged at 16,000 g for 20 minutes. Automated Western blotting (ProteinSimple) was performed with the obtained supernatant.

[0268] As a result, expression of the target protein, pDNA, was confirmed in the recipient cells, confirming that intracellular delivery of pDNA through the supermere was possible (Fig. 15).

[0269]

[0270] Example 11: Comparison of gene delivery capabilities of supermembrane and lipid nanoparticles

[0271] We conducted experiments to verify the exogenous mRNA delivery ability of Supermere confirmed at the cellular level in vivo and to compare its gene delivery ability with that of lipid nanoparticles (LNPs), which are widely used as mRNA delivery vehicles.

[0272] Specifically, small extracellular vesicles and supermems were isolated and obtained from HEK293FT cells according to the collection method described in Example 1. Then, C57BL / 6, male, 7-week-old mice were prepared and fed alfalfa-free feed, and samples as shown in Table 2 below were prepared, and fasting was performed one day before sample injection.

[0273]

[0274] Distinctive capacity eGFP mRNA added amount Free mRNA-20 μg Lipid nanoparticles 20 μg 1 μg 200 μg 10 μg Supermere 8 μg 1 μg 80 μg 10 μg 160 μg 20 μg

[0275]

[0276] Each group in Table 3 was intravascularly injected into mice, and 18 hours later, the mice were sacrificed and their organs were removed. The fluorescence values ​​of the target proteins translated in vivo were measured using an IVIS spectrum (Caliper Life Sciences, IVIS® Lumina Series III).

[0277] As a result, it was confirmed that SuperMere exhibited a stronger GFP fluorescence signal in the intestine compared to LNP (Fig. 16) and delivered mRNA better, resulting in stronger protein expression (Fig. 17).

[0278]

[0279] Example 12: Long-term accumulation analysis of Supermere

[0280] 12-1. Comparison of in vivo accumulation of supermembranes and small extracellular vesicles derived from HEK293FT cells.

[0281] Lipid nanoparticles, which are mainly used as mRNA delivery vehicles, have the problem of accumulating in large quantities in the liver and spleen and not being systematically distributed in the body. Therefore, SuperMere also conducted an in vivo distribution experiment to confirm whether this side effect exists.

[0282] Specifically, small extracellular vesicles and supermembranes were isolated from HEK293FT cells according to the collection method described in Example 1, and 1 μg of sulfo-Cyanine5.5 NHS ester (Cy5.5; Lumiprobe, 17320) was mixed per 100 μg of small extracellular vesicles and supermembranes, respectively, and incubated overnight at 4°C. Zeba TM Unbound Cy5.5 was removed using Spin Desalting Columns (A57761), and fluorescence values ​​were standardized between samples using a microplate reader.

[0283] C57BL / 6, male, 7-week-old mice were fed an alfalfa-free diet and fasted the day before sample injection. Free dye (200 μl), Cy5.5-labeled small extracellular vesicles (200 μg), or Cy5.5-labeled supermere (200 μg) were injected intravascularly, and the mice were sacrificed 18 h later, and organs were removed. The removed organs were measured using IVIS Spectroscopy (Caliper Life Sciences, IVIS® Lumina Series III) to determine the Cy5.5 fluorescence remaining in the organs.

[0284] As a result, it was confirmed that supermere accumulates relatively less in the liver and spleen and accumulates well in the kidney compared to small extracellular vesicles (Figs. 18 and 19).

[0285]

[0286] 12-2. Comparison of in vivo accumulation of stem cell-derived supermembrane and small extracellular vesicles

[0287] To confirm that supermeres derived from various cells, not just HEK293FT cells, can be utilized as drug delivery vehicles, small extracellular vesicles, exomeres, and supermeres derived from human bone marrow stem cells were obtained and their accumulation in vivo was confirmed.

[0288] Specifically, small extracellular vesicles, exomeres, and supermeres were isolated from human bone marrow stem cells according to the collection method described in Example 1, and 100 μg of small extracellular vesicles, exomeres, or supermeres were mixed with 1 μg of sulfo-Cyanine5.5 NHS ester (Cy5.5; Lumiprobe, 17320) and incubated overnight at 4°C. Zeba TM Unbound Cy5.5 was removed using Spin Desalting Columns (A57761), and fluorescence values ​​were standardized between samples using a microplate reader.

[0289] C57BL / 6, male, 7-week-old mice were fed an alfalfa-free diet and fasted the day before sample injection. Free dye (100 μl), Cy5.5-labeled small extracellular vesicles (100 μl), Cy5.5-labeled exomere (100 μg), or Cy5.5-labeled supermere (100 μg) were injected intravascularly, and 6 hours after injection, the mice were sacrificed and organs (brain, heart, lung, liver, kidney, spleen, intestine, bladder) were removed. The removed organs were measured with IVIS spectrum (Caliper Life Sciences, IVIS® Lumina Series III) to determine the Cy5.5 fluorescence values ​​remaining in the organs.

[0290] As a result, similar to HEK293FT, it was confirmed that supermere derived from human bone marrow stem cells accumulated significantly less in the liver and spleen and accumulated well in the kidney compared to small extracellular vesicles (Fig. 20).

[0291]

[0292] Example 13: Confirmation of selective loading of target substances into supermere through supermere marker-functional substance fusion

[0293] By fusing a marker and a target functional protein to a supermere and expressing them within the supermere, it was experimentally confirmed that the target functional substance could be specifically loaded onto the supermere. The supermere was isolated and obtained from HEK293FT cells according to the collection method described in Example 1.

[0294] For transduction, HEK293FT cells were treated with a transfection reagent (1 ml) containing pcDNA3.1-TGFBI-Myc (60 μg), pcDNA3.1-T7-TGFBI-Myc (60 μg), and PEI (240 μg), respectively, and the medium was replaced with serum-free medium after 6 hours, and the supernatant was collected after 48 hours. The plasmids used were constructed by cloning a polynucleotide encoding the TGFBI-Myc protein (SEQ ID NO: 6) into the pcDNA3.1 vector (ThermoFisher) and a polynucleotide encoding the T7-TGFBI-Myc protein (SEQ ID NO: 7) into the pcDNA3.1 vector (ThermoFisher).

[0295] Six hours after transduction, the medium was replaced with serum-free medium, and the final supernatant was obtained 48 hours later. The cell supernatant was centrifuged at 3000 g for 5 minutes and filtered through a 0.45 μm filter (Sartolab RF500 PES). After concentration with Amicon (10 kDa filter, UFC901024, Merck), the supernatant was centrifuged at 10,000 g for 30 minutes. Subsequently, the supernatant was ultracentrifuged at 150,000 g for 90 minutes at 4 °C (Ultracentrifuge, Beckman), and the supernatant was transferred to a UC tube (355655) and centrifuged at 167,000 g for 16 hours. The supernatant was then transferred to a 70Ti rotor high speed tube (355618) and centrifuged at 367,000 g for 16 hours. Supermere was then isolated from the resulting pellet using 1X PBS-PIC (stored on ice for at least 20 minutes, then briefly centrifuged for 2 minutes).

[0296] Automated Western blotting (ProteinSimple) was performed on the isolated supermembrane. Myc (AB9106), Enolase-1 (3810T), Enolase-2 (8171S), and CD81 (EXOAB-CD81A1) antibodies were used at a 1:100 dilution, and 3 μg of each substance was loaded to confirm protein expression.

[0297] As a result, the T7-TGFBI-Myc supermembrane showed a protein expression pattern similar to that of the TGFBI-Myc supermembrane (Fig. 21).

[0298] These results suggest that supermere markers such as TGFBI function as proteins involved in the supermere formation process and enable selective and effective loading of fused functional proteins of interest into supermeres.

[0299]

[0300] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the claims described below, and their equivalent concepts, rather than the detailed description above.

Claims

1. A composition for delivering a functional substance, comprising non-vesicular extracellular particles (NVEP) as an active ingredient.

2. In paragraph 1, A composition for delivering a functional substance, wherein the above non-vesicular extracellular particles are supermeres.

3. In paragraph 2, The above supermere is a functional material delivery composition having improved material delivery capability compared to an extracellular vesicle or exomere.

4. In paragraph 1, A composition for delivering a functional material, wherein the functional material is in the form of a protein.

5. In paragraph 1, A composition for delivering a functional material, wherein the functional material is in the form of RNA.

6. In paragraph 1, A composition for delivering a functional material, wherein the functional material is in the form of DNA.

7. In paragraph 2, The above supermere is a composition for delivering a functional substance, characterized in that it accumulates in a small amount in the liver compared to extracellular vesicles.

8. In paragraph 2, The above supermere is a composition for delivering a functional substance, characterized in that it accumulates in a small amount in the spleen compared to extracellular vesicles.

9. A composition for delivering a functional substance, comprising a non-vesicular extracellular particle marker protein; and a non-vesicular extracellular particle containing a target functional substance.

10. In paragraph 9, The above non-vesicular extracellular particles are supermeer, a composition for delivering functional substances.

11. In paragraph 10, A composition characterized in that the supermere marker protein is at least one selected from the group consisting of Transforming growth factor, beta-induced (TGFBI), Enolase-1 (ENO1), Enolase-2 (ENO2), Heat shock 70 kDa protein 13 (HSPA13), GPI (Glucose-6-phosphate isomerase), LDHA (Lactate Dehydrogenase A), TPI1 (Triosephosphate Isomerase 1), HK1 (Hexokinase 1), MDH1 (Malate Dehydrogenase 1), NUCB1 (Nucleobindin-1), PDIA4 (Protein Disulfide Isomerase Family A Member 4), fragments thereof, mutants thereof, mutants of fragments thereof, and fragments of mutants thereof.

12. In paragraph 11, A composition for delivering a functional substance, wherein the above-mentioned supermembrane marker protein is in a fused form with the above-mentioned functional substance.

13. A pharmaceutical composition comprising a composition according to any one of claims 1 to 12.

14. A cosmetic composition comprising a composition according to any one of claims 1 to 12.

15. A kit for delivering functional substances, containing non-vesicular extracellular particles (NVEP) as an active ingredient.

16. In paragraph 15, A functional substance delivery kit wherein the above non-vesicular extracellular particles are supermeres.

17. A composition for delivering functional substances, comprising non-vesicular extracellular particles, for use in delivering functional substances.

18. Use of non-vesicular extracellular particles for functional substance delivery.

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