Method for mixing substances using microfluidic chip, method for synthesizing natural exosome-mimicking nanovesicles using same, and natural exosome-mimicking nanovesicles synthesized thereby

Abstract

Disclosed are a method for mixing substances using a microfluidic chip, a method for synthesizing natural exosome-mimicking nanovesicles using same, and natural exosome-mimicking nanovesicles synthesized thereby. The method for mixing substances using a microfluidic chip comprises a step of mixing two or more groups of substances by using a microfluidic chip including: two or more inlets; one or more outlets; and a flow channel connecting the inlets and the outlets and allowing a fluid to flow therein, wherein the two or more groups of substances are injected into the two or more inlets, respectively, the flow channel includes one or more vortex-generating structures, and the vortex-generating structures may include a main flow channel and one or more sub-flow channels branching from a first point of the main flow channel and merging at a second point spaced apart from the first point in a fluid flow direction at an angle greater than 90° and less than 180°.

Description

A method for mixing materials using a microfluidic chip, a method for synthesizing natural exosome-mimicking nanovesicles using the same, and natural exosome-mimicking nanovesicles synthesized thereby

[0001] The present invention relates to a method for mixing materials using a microfluidic chip, a method for synthesizing natural exosome-mimicking nanovesicles using the same, and natural exosome-mimicking nanovesicles synthesized thereby. In particular, the present invention relates to the mass production of artificial exosomes at ultra-high speed based on a microfluidic mixing channel.

[0002] Exosomes (hereinafter referred to as natural exosomes), derived from natural organisms such as animals, plants, and microorganisms, are vesicles with an average size of 100 nm and are recognized as excellent nanobiomaterials for diagnosis and treatment. Excellent research results in the fields of diagnosis and treatment are continuously being published, and companies are pursuing related businesses accordingly. However, existing production volumes fall short of industrial demand, necessitating mass production technology. To address this issue, research has been conducted to increase bioavailability through methods such as isolating functionally specific exosomes and creating delivery vehicles using hydrogel microparticles. However, low production volumes and batch-to-batch heterogeneity still remain, making these limitations difficult to overcome.

[0003] The present invention proposes a technology for mass-producing, at ultra-high speed, natural exosome-mimicking nanovesicles (hereinafter referred to as artificial exosomes) with secured homogeneity by mixing phospholipids and active substances (proteins, nucleic acids, metabolites, etc.) contained in natural exosomes to overcome the limitations of natural exosomes. When producing artificial exosomes, a microfluidic-based mixing technique is required to control the size while maintaining a size similar to that of natural exosomes, i.e., 50-150 nm. The microfluidic channel used is referred to as a micromixer, and the structure of the micromixer used to produce artificial exosomes is very important.

[0004] The challenge of the present invention lies in developing a technology capable of mass-producing artificial exosomes with an efficient and uniform size distribution, transcending the limitations of natural exosomes. This technology can be utilized to address a variety of medical and biological challenges.

[0005] In one aspect, the present invention provides a method for mixing materials using a microfluidic chip, including a step of mixing two or more groups of materials using a microfluidic chip including two or more inlets (100); one or more outlets (300); and a flow passage (200) connecting the inlets (100) and outlets (300) and allowing fluid to flow therein.

[0006] Specifically, the microfluidic chip may include a micro mixer (10), and the micro mixer (10) may include two or more inlets (100); one or more outlets (300); and a flow passage (200) connecting the inlets (100) and the outlets (300) and allowing fluid to flow therein.

[0007] In one embodiment, hydrophilic treatment may be performed on at least a portion of the interior of the flow passage (200).

[0008] In one embodiment, the two or more groups of materials can be injected into each of the two or more inlets (100).

[0009] In one embodiment, the flow passage (200) may include one or more vortex forming structures (210).

[0010] In one embodiment, the vortex forming structure (210) may include a main flow passage (211) and one or more sub flow passages (212) that branch off from a first point (2111) of the main flow passage (211) and merge at an angle greater than about 90° and less than about 180° at a second point (2112) spaced apart from the first point (2111) in the direction of fluid flow.

[0011] In one embodiment, the flow passage (200) may include a plurality of vortex-forming structures (210) arranged in series.

[0012] In one embodiment, nanovesicles mimicking natural exosomes can be synthesized by mixing two or more of the above-described materials.

[0013] In one embodiment, at least one of the two or more groups of substances may include one or more lipids selected from the group comprising fatty acids, cholesterol or derivatives thereof, and phospholipids.

[0014] More specifically, in some embodiments, the fatty acids include straight chain fatty acids, including butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, hexacosanoic acid, octacosanoic acid, triacosanoic acid and n-dotriacosanoic acid, and those having an odd number of carbon atoms, such as propionic acid, n-valeric acid, enanthic acid, pelargonic acid, hendecanoic acid, tridecanoic acid, pentadecanoic acid, heptadecanoic acid, nonadecanoic acid, heneicosanoic acid, tricosanoic acid, pentacosanoic acid, heptacosanoic acid, or any combination thereof. In some embodiments, branched fatty acids are included, which include isobutyric acid, isocaproic acid, isocaprylic acid, isocapric acid, isolauric acid, 11-methyldodecanoic acid, isomyristic acid, 13-methyl-tetradecanoic acid, isopalmitic acid, 15-methyl-hexadecanoic acid, isostearic acid, 17-methyloctadecanoic acid, isoarachidic acid, 19-methyl-eicosanoic acid, α-ethyl-hexanoic acid, α-hexyldecanoic acid, α-heptylundecanoic acid, 2-decyltetradecanoic acid, 2-undecyltetradecanoic acid, 2-decylpentadecanoic acid, 2-undecylpentadecanoic acid, and Fine oxocol 1800 acid (a product of Nissan Chemical Industries, Ltd.), anteiso fatty acids terminated with an isobutyl group, such as 6-methyl-octanoic acid, 8-methyl-decanoic acid, 10-methyl-dodecanoic acid, 12-methyl-tetradecanoic acid, 14-methyl-hexadecanoic acid, 16-methyl-octadecanoic acid, 18-methyl-eicosanoic acid, 20-methyl-docosanoic acid, 22-methyl-tetracosanoic acid, 24-methyl-hexacosanoic acid, and 26-methyloctacosanoic acid, or any combination thereof.

[0015] In some embodiments, the composition comprises unsaturated fatty acids, which are selected from the group consisting of 4-decenoic acid, caprolactam, 4-dodecenoic acid, 5-dodecenoic acid, laurolic acid, 4-tetradecenoic acid, 5-tetradecenoic acid, 9-tetradecenoic acid, palmitoleic acid, 6-octadecenoic acid, oleic acid, 9-octadecenoic acid, 11-octadecenoic acid, 9-eicosenoic acid, cis-11-eicosenoic acid, cetoleic acid, 13-docosenoic acid, 15-tetracosenoic acid, 17-hexacosenoic acid, 6,9,12,15-hexadecatetraenoic acid, linoleic acid, linolenic acid, α-eleostearic acid, β-eleostearic acid, punicic acid, 6,9,12,15-octadecatetraenoic acid, Parinaric acid, 5,8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid, 7,10,13,16,19-docosapentaenoic acid, 4,7,10,13,16,19-docosahexaenoic acid, or any combination thereof.

[0016] In some embodiments, the composition comprises a hydroxy fatty acid, which comprises α-hydroxylauric acid, α-hydroxymyristic acid, α-hydroxypalmitic acid, α-hydroxystearic acid, ω-hydroxylauric acid, α-hydroxyarachidic acid, 9-hydroxy-12-octadecenoic acid, ricinoleic acid, α-hydroxybehenic acid, 9-hydroxy-trans-10,12-octadecadienoic acid, camolenic acid, ifurolic acid, 9,10-dihydroxystearic acid, 12-hydroxystearic acid, or any combination thereof.

[0017] In some embodiments, the polycarboxylic acid is comprised of oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, D,L-malic acid, or any combination thereof.

[0018] For example, phospholipids can be used without limitation as they are commonly used in the relevant technical field, but for example, phosphatidylcholine (PC) series such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), soybean phosphocholine (SOY PC), and hydrogenated soybean phosphocholine (hydrogenated SOY PC, HSPC); Phosphatidylethanolamine (PE) series such as 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), Phosphatidylserine (PS) series such as 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), and 1-palmitoyl-oleoyl-sn-glycero-3-phosphoserine (POPS); Phosphatidic acid series such as 1,2-dimyristoyl sn-glycero-3-phosphate (DMPA), 1,2-dipalmitoyl- sn-glycero-3-phosphate (DPPA), 1,2-disteroyl- sn-glycero-3-phosphate (DSPA), 1,2-dioleoyl- sn-glycero-3-phosphate (DOPA), and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphate (POPA);Sphingomyelin series such as N-stearoyl-D-erythritol-sphingosylphosphorylcholine (DSSM) may be selected from, but is not limited to, one or more thereof.

[0019] The derivative of cholesterol can be, for example, ergosterol, 7-dehydrocholesterol, 24S-hydroxycholesterol, lanosterol, cycloartenol, fucosterol, saringosterol, campesterol, β-sitosterol, sitostanol, coprostanol, avenasterol, stigmasterol, or a functional cholesterol derivative such as 3β-(N(N',N'-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol), or any combination thereof.

[0020] In one embodiment, at least one of the two or more groups of materials may comprise one or more active agents selected from the group comprising proteins, small molecules, and nucleic acids. The active agents may also be considered payloads.

[0021] Here, the nucleic acid may be at least one selected from the group consisting of antisense oligonucleotides (ASOs), DNA, mRNA, miRNA, lcRNA, antagomir, tRNA, and siRNA.

[0022] In one embodiment, at least one of the two or more groups of substances may comprise one or more tetraspanins selected from the group comprising CD9, CD63, CD81, integrin beta1, and alexandrin.

[0023] In one embodiment, the material mixing method may include a step of mixing two groups of materials using a microfluidic chip including two inlets. Here, one group of the two groups of materials may include lipids, and the other group may include an effective agent. Through the material mixing method, natural exosome-mimetic nanovesicles can be produced, which include an outer lipid membrane and an effective agent encapsulated therein.

[0024] In one embodiment, the material mixing method may include a step of mixing two groups of materials using a microfluidic chip including two inlets. Here, one group of the two groups of materials may include lipids having tetraspanins attached thereto, and the other group may include an effective agent. Through the material mixing method, natural exosome-mimetic nanovesicles may be prepared, which include a lipid outer membrane; an effective agent embedded therein; and a tetraspanin formed on the lipid outer membrane.

[0025] In one embodiment, the material mixing method may include a step of mixing three groups of materials using a microfluidic chip including three inlets. Here, one group of the three groups of materials may include lipids, another group may include an effective substance, and yet another group may include tetraspanins. Through the material mixing method, natural exosome-mimetic nanovesicles may be prepared, which include a lipid outer membrane; an effective substance encapsulated therein; and a tetraspanin formed on the lipid outer membrane.

[0026] In one embodiment, the sub-flow passage (212) of the vortex forming structure (210) can merge with the main flow passage (211) at an angle of about 130 to 140°.

[0027] In one embodiment, the main flow passage (211) may have a width of about 80 to 120 μm.

[0028] In one embodiment, the sub-flow passage (212) may include a first portion (2121) branching and extending from a first point (2111) of the main flow passage (211); a second portion (2122) cylindrically curved from an end of the first portion (2121) toward the main flow passage (211); and a third portion (2123) extending from an end of the second portion (2122) and joining the second point (2112) of the main flow passage.

[0029] In one embodiment, the sub-flow passage (212) may be formed alternately left and right along the main flow passage (211).

[0030] In one embodiment, the main flow passage (211) may be formed as a serpentine structure including a plurality of rows.

[0031] In one embodiment, the microfluidic chip may be manufactured from at least one selected from the group consisting of polydimethylsiloxane (PDMS), polycarbonate (PC), polymethyl methacrylate (PMMA), cyclic olefin polymer (COP), and cyclic olefin copolymer (COC). Specifically, in this embodiment, the microfluidic chip was manufactured using polydimethylsiloxane (PDMS) and polycarbonate (PC), and it was confirmed that the final product manufactured thereby was non-toxic.

[0032] In another aspect, the present invention provides a material mixing device using a microfluidic chip that mixes materials using the material mixing method using the microfluidic chip.

[0033] In one embodiment, the material mixing device using the microfluidic chip may include: the microfluidic chip; a pump unit that injects the two or more groups of materials into the two or more inlets; and a recovery unit that recovers the mixed material from the outlet.

[0034] According to the present invention, the material mixing device using the microfluidic chip mentioned above has the same features as the device configuration mentioned in the material mixing method using the microfluidic chip mentioned above, and in order to avoid repetitive description, the related description is omitted in the description of the present device.

[0035] In another aspect, the present invention provides a device for synthesizing natural exosome-mimicking nanovesicles by mixing substances through the material mixing method using the microfluidic chip.

[0036] In one embodiment, the natural exosome-mimetic nanovesicle synthesis device may include the microfluidic chip; a pump unit that injects the two or more groups of substances into the two or more inlets; and a recovery unit that recovers the nanovesicles from which synthesis is completed from the outlet.

[0037] In another aspect, the present invention provides a natural exosome-mimetic nanovesicle synthesized through the material mixing method using the microfluidic chip.

[0038] In one embodiment, the natural exosome-mimetic nanovesicles may comprise a lipid outer membrane; and an encapsulated active ingredient.

[0039] In one embodiment, the natural exosome-mimicking nanovesicles may include a lipid outer membrane; an effective active ingredient encapsulated therein; and a tetraspanin formed on the lipid outer membrane.

[0040] In another aspect, the present invention provides natural exosome-mimicking nanovesicles synthesized through the above-described natural exosome-mimicking nanovesicle synthesis device.

[0041] In one embodiment, the natural exosome-mimicking nanovesicles may include a lipid outer membrane; an effective active ingredient encapsulated therein; and a tetraspanin formed on the lipid outer membrane.

[0042] Specifically, in the present invention, artificial exosomes are synthesized using a microfluidic mixer having a link structure.

[0043] As an example, a micromixer consists of two inlets and one outlet, each of which receives a solution containing an active ingredient derived from natural exosomes. The two solutions meet at a cross-neck and are then effectively mixed through multiple vortex-forming structures connected in series.

[0044] As an example, a micromixer consists of three inlets and one outlet, each of which receives a solution containing an active ingredient derived from natural exosomes. These three solutions meet at a cross-neck and are then effectively mixed through multiple vortex-forming structures connected in series.

[0045] The micromixer structure proposed in the present invention is a structure in which fluids move through a main channel and branched streams, then converge again to form vortices, effectively mixing the fluids. This process achieves a high mixing effect within the channel. For example, the main channel of this micromixer can be designed as a serpentine structure comprising multiple rows, allowing for more precise control of fluid flow, thereby enabling the efficient production of artificial exosomes.

[0046] In the present invention, the channel surface is hydrophilically treated to prevent clumping and blocking of particles generated during solution mixing within the designed channel. Comparing clogging before and after applying PEG, the hydrophilic treatment, revealed a significant reduction in channel clogging after PEG treatment.

[0047] In the present invention, the flow rate of the injected solution ranges from approximately 0.1 mL / min to 2 mL / min, and the volume of the solution that can be injected may vary depending on the syringe containing the solution. The artificial exosomes formed after injection have an average size of approximately 110 nm, and the polydispersity index, which can be used to determine uniformity, was confirmed to be approximately 0.11. This is a value close to approximately 0.1, which can generally be considered uniform, and this confirms their potential for use as a therapeutic agent. In addition, the production capacity of the artificial exosomes produced by this method is approximately 1010 or more per minute, which can overcome the limitations of mass production of existing natural exosomes.

[0048] The artificial exosome production technology developed by the present inventors overcomes the production limitations of natural exosomes. Typically, 10 per cell 2 In the treatment requiring exosomes from dogs, the amount of exosomes that one adipose-derived stem cell can produce per day is on average 1-4 Х 10 2 This is only a small number, which significantly limits its use as a drug. The existing extrusion method for producing artificial exosomes processes lipids and active substances using a membrane filter under high pressure. During this process, the materials are not perfectly dispersed, resulting in inconsistent particle distribution and size. On the other hand, the method using the microfluidic mixer of the present invention has a flow rate of 10 times per minute. 10 It can produce more than 100 artificial exosomes, solving the problem of shortage of natural exosomes, reducing the economic burden on companies through mass production, and meeting the demand in medical and other application fields.

[0049] The micromixer structure of the present invention can efficiently replace the process of mixing and reacting various reagents used in diagnostic fields through a link structure. This structure can be used without a separate syringe pump, and the channel width is designed to be small while maintaining low fluid flow rates and volumes within the channel. Furthermore, the inventors calculated and designed the channel resistance using the Hagen-Poiseuille equation to ensure stable operation even at high flow rates by utilizing the link structure. This technology enables highly efficient fluid mixing, contributing to the mass production of artificial exosomes.

[0050] The technology presented in the present invention, based on the high therapeutic efficacy of natural exosomes, developed artificial exosomes mimicking them using a microfluidic mixing channel. During the COVID-19 pandemic, mRNA-loaded lipid nanoparticles were utilized as vaccines, leading to rapid commercialization of related technologies. Building on this experience, the development of lipid nanoparticles and nanoparticle-based drug delivery systems has been emphasized. Existing lipid nanoparticles have limitations, such as allergy and toxicity issues due to non-biological substances, rapid efficacy loss, formulation instability, and production difficulties. The micromixer-based artificial exosomes of the present invention can address these issues and satisfy the unmet needs of the natural exosome and lipid nanoparticle industries. The fabricated artificial exosomes overcome the low yield and batch-to-batch inconsistency issues of natural exosomes, offering a novel approach that overcomes the limitations of existing natural exosome-based therapeutic research and addresses the challenges of mass production.

[0051] Figure 1 is a diagram illustrating an overview of the artificial exosome production process based on microfluidic mixing technology.

[0052] Fig. 2 is a drawing illustrating an example design of a micromixer. Specifically, (a) to (d) in the upper part of Fig. 2 illustrate various examples of micromixer designs according to the difference in the number of vortex forming structures (16, 24, 20, and 40, respectively), and the lower drawing of Fig. 2 is a drawing illustrating one cycle of a re-Tesla channel.

[0053] Figure 3 is a diagram showing the results of a simulation of a mixing process in a micro mixer.

[0054] Figure 4 is a drawing showing the mixing process and production result of a solution containing lipids and an effective substance in a micro mixer.

[0055] Figure 5 is a diagram showing the size distribution, polydispersity index, and zeta potential of the formed artificial exosomes.

[0056] Figure 6 is a diagram showing the encapsulation efficiency of effective substances in artificial exosomes using a gel retardation assay.

[0057] Figure 7 is a diagram illustrating the results of a comparative analysis of the physicochemical properties of natural exosomes and artificial exosomes.

[0058] Figure 8 is a diagram showing the results of comparison of the cellular uptake capacity of natural exosomes and artificial exosomes.

[0059] Fig. 9 is a drawing illustrating a design example of a micro mixer. Specifically, the upper drawing of Fig. 9 illustrates a design example of a micro mixer with three inlets, and (a) and (b) at the bottom of Fig. 9 illustrate various micro mixer design examples according to the difference in the number of vortex forming structures (16 and 24, respectively) of the micro mixer with three inlets.

[0060] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention may be modified in various ways and may take various forms. Therefore, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing each drawing, similar reference numerals are used to indicate similar components. In the attached drawings, the dimensions of structures are shown larger than actual size to ensure clarity of the present invention.

[0061] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes plural expressions unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof. In the context of this specification, the term "about" or the like can mean about ± 1%, about ± 2%, about ± 3%, about ± 4%, about ± 5%, about ± 6%, about ± 7%, about ± 8%, about ± 9%, or about ± 10% of a numerical value described in the specification.

[0062] Additionally, the description of one aspect of the present invention may be applied identically or similarly to the same or similar configurations or terms in the description of other aspects.

[0063] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0064] A method for mixing substances using a microfluidic chip according to an embodiment of the present invention may include a step of mixing two or more groups of substances using a microfluidic chip including two or more inlets (100); one or more outlets (300); and a flow passage (200) connecting the inlets and outlets and allowing a fluid to flow therein. The role of the mixing step is to effectively mix different substances and evenly distribute them, thereby obtaining a uniform reaction result. In the context of the present specification, the meanings of the inlet and the outlet are defined as 'an inlet through which a fluid flows in' and 'an outlet through which a fluid flows out', respectively. In one embodiment, the two or more groups of substances may be injected into each of the two or more inlets.

[0065] Specifically, the microfluidic chip may include a micro mixer (10), and the micro mixer (10) may include two or more inlets (100); one or more outlets (300); and a flow passage (200) connecting the inlets (100) and the outlets (300) and allowing fluid to flow therein.

[0066] In one embodiment, at least a portion of the interior of the flow passage (200) may be subjected to a hydrophilic treatment. This hydrophilic treatment has the advantage of effectively dispersing particles within the fluid without clumping together, thereby preventing channel clogging and maintaining smooth fluid flow. This treatment is particularly useful when using solutions containing high concentrations of bioactive substances or other fine particles, and can enhance the efficiency of the mixing process, thereby improving the consistency and quality of the final product.

[0067] In one embodiment, the flow path (200) may include one or more vortex-forming structures (210). In the context of this specification, the meaning of a vortex-forming structure is defined as a structure that causes a rotation or swirl of a fluid within a fluid flow. This vortex-forming structure serves two main functions necessary for material mixing. First, the vortex helps the various components within the fluid to mix with each other quickly and effectively. Second, this structure plays a crucial role in ensuring the consistency of the mixing process and improving the quality of the final product by promoting a more uniform distribution and reaction of the fluid.

[0068] In one embodiment, the vortex-forming structure (210) may be a reverse-Tesla structure. In the context of the present specification, the meaning of the reverse-Tesla structure is defined as a structure that forms a vortex in a fluid by utilizing the design of a Tesla valve, but generally utilizing a direction that impedes the flow. While a Tesla valve acts as a check valve that promotes one-way flow of a fluid and impedes the opposite flow, the reverse-Tesla structure utilizes this opposite flow to generate a strong vortex. This vortex promotes rapid and uniform mixing of various components within a microfluidic channel, and in particular, ensures accurate mixing of active ingredients and lipids in the formation of nanovesicles, thereby greatly contributing to achieving uniform size and quality of nanovesicles. Such a structure can significantly improve the efficiency of a microfluidic-based nanovesicle production process.

[0069] In one embodiment, the vortex forming structure (210) may include a main flow passage (211) and one or more sub flow passages (212) that branch off from a first point (2111) of the main flow passage (211) and merge at an angle greater than about 90° and less than 180° at a second point (2112) spaced apart from the first point (2111) in the direction of fluid flow. The main flow passage (211) and the sub flow passages (212) are designed to effectively mix and swirl various substances within the fluid, thereby promoting forced mixing of the fluid and achieving a more uniform material distribution.

[0070] In one embodiment, the sub-flow passage (212) may include a first portion (2121) branching from a first point (2111) of the main flow passage (211) and extending; a second portion (2122) cylindrically curving from an end of the first portion (2121) toward the main flow passage (211); and a third portion (2123) extending from an end of the second portion (2122) and joining the second point (2112) of the main flow passage (211). This configuration utilizes a reverse-Tesla structure to cause the sub-flow passage (212) to encounter the flow of the main flow passage (211) in the opposite direction, thereby forcibly mixing materials in the fluid even in a direction opposite to the main flow and joining them again into the main flow passage (211). The fluid collisions and vortices that occur during this process promote more effective mixing of materials, and the dynamic turbulence that occurs as the mixed materials re-enter the main flow path (211) ensures uniform and efficient mixing results. This is particularly important for achieving the high mixing precision required in microfluidic reactions.

[0071] In one embodiment, the flow path (200) may include a plurality of vortex-forming structures (210) arranged in series. When there are multiple vortex-forming structures, each structure helps to mix various components within the fluid more effectively by repeatedly swirling them. When these structures are arranged in series, the mixing process is gradually strengthened as the fluid passes through each vortex-forming structure, ultimately achieving a very uniform mixing result. This is particularly advantageous in maximizing the dynamic properties of the fluid in microfluidic systems, thereby providing a uniform reaction environment required for complex and diverse chemical or biological reactions. The serial arrangement increases the mixing efficiency at each stage, which results in improved overall reaction efficiency and reaction completion.

[0072] In one embodiment, the sub-flow passages (212) may be formed alternately left and right along the main flow passage (211). The advantage of this alternately left and right structure is that it disperses the fluid flow in multiple directions while simultaneously rejoining the main flow passage at multiple points, thereby enabling more uniform mixing of substances within the fluid. This structure increases the effect of dynamically intersecting and mixing substances in various directions during fluid flow instead of simply moving in a straight line, which is particularly useful for providing a uniform reaction environment required for complex chemical or biological reactions. Furthermore, this method contributes to significantly improving the overall mixing efficiency through appropriate vortices and fluid dynamics formed within the fluid.

[0073] In one embodiment, nanovesicles mimicking natural exosomes can be synthesized by mixing two or more of the above-described materials. Utilizing the reverse-Tesla structure during this process causes collisions of the mixture simultaneously with mixing, which not only ensures highly effective mixing of each component but also promotes rapid and uniform formation of nanovesicles. The reverse-Tesla structure generates strong vortices within the fluid, which significantly increases the opportunities for the materials to sufficiently contact and react with each other. In particular, this structure facilitates more efficient nanovesicle synthesis by allowing the swirling motion of the fluid to finely control the mixture. This plays a key role in the production of nanovesicles mimicking key characteristics of natural exosomes and maximizes their utility in biological and medical applications.

[0074] In one embodiment, at least one of the two or more groups of materials may comprise one or more lipids selected from the group comprising fatty acids, cholesterol, and phospholipids. Lipids are a category of organic molecules including fatty acids, cholesterol, phospholipids, etc., and are major components of cell membranes and essential for the formation of biological membranes. In nanovesicle synthesis, lipids play a crucial role in forming the lipid bilayer of the vesicle, which provides the nanovesicle with the ability to transport active ingredients outside the cell and deliver them into the cell.

[0075] In one embodiment, at least one of the two or more groups of materials may comprise one or more active ingredients selected from the group consisting of proteins, small molecules, and nucleic acids. An active ingredient is an active ingredient that performs a specific biological or chemical function in vivo and can be used as a therapeutic agent, diagnostic agent, or research reagent. The presence of active ingredients in nanovesicle synthesis is essential for targeting specific diseases or enhancing therapeutic effects, and nanovesicles containing these ingredients can exhibit improved bioavailability and efficacy.

[0076] In one embodiment, at least one of the two or more groups of substances may include one or more tetraspanins selected from the group consisting of CD9, CD63, CD81, integrin beta 1, and alexandrin. Tetraspanins are a type of protein present in cell membranes and play an important role in regulating cell-to-cell interactions, signal transduction, and cell movement. Inclusion of tetraspanins in the synthesis of nanovesicles contributes to enhancing the biorecognition and intracellular integration of the vesicles, which helps the nanovesicles to effectively bind to specific cell types and deliver active substances.

[0077] By including lipids, effective compounds, and tetraspanins in the above-described mixed two or more groups of materials, artificial nanovesicles can mimic the biological properties and functions of natural exosomes. Lipids form the structural foundation of the nanovesicles and, similar to the double lipid membrane of natural exosomes, play an essential role in the transport of substances into and out of cells. This membrane protects the vesicles, ensuring their stable existence in vivo and their efficient interaction with cells, while enhancing their targeting and transit capabilities.

[0078] In one embodiment, the material mixing method may include a step of mixing two groups of materials using a microfluidic chip including two inlets. Here, one group of the two groups of materials may include lipids, and the other group may include an effective agent. Through the material mixing method, natural exosome-mimetic nanovesicles can be produced, which include an outer lipid membrane and an effective agent encapsulated therein.

[0079] In one embodiment, the material mixing method may include a step of mixing two groups of materials using a microfluidic chip including two inlets. Here, one group of the two groups of materials may include lipids having tetraspanins attached thereto, and the other group may include an effective agent. Through the material mixing method, natural exosome-mimetic nanovesicles may be prepared, which include a lipid outer membrane; an effective agent embedded therein; and a tetraspanin formed on the lipid outer membrane.

[0080] In one embodiment, the material mixing method may include a step of mixing three groups of materials using a microfluidic chip including three inlets. Here, one group of the three groups of materials may include lipids, another group may include an effective substance, and yet another group may include tetraspanins. Through the material mixing method, natural exosome-mimetic nanovesicles may be prepared, which include a lipid outer membrane; an effective substance encapsulated therein; and a tetraspanin formed on the lipid outer membrane.

[0081] The dimensions of the microfluidic chip, the micro mixer (10) included therein, and the vortex forming structure (210) and the flow passage (200) included therein are not particularly limited, but preferably, the height and width may each be 50 to 500 μm. In one embodiment, the sub-flow passage (212) of the vortex forming structure (210) may be merged with the main flow passage (211) at an angle of about 130 to 140°. In one embodiment, the main flow passage (211) may have a width of about 80 to 120 μm.

[0082] In one embodiment, the main flow path (211) may be formed as a serpentine structure comprising multiple rows. In the context of this specification, the serpentine structure refers to a meandering structure. This structure extends the fluid path in a microfluidic system, allowing the fluid to travel a longer distance along the path. The primary advantage of a main flow path with a serpentine structure is that it increases the residence time of the fluid and provides an opportunity for sufficient mixing of substances within the fluid. This structure helps to equalize temperature and / or concentration gradients that may occur within the fluid by forcing the fluid flow through multiple bends. Furthermore, the serpentine structure promotes efficient heat and mass transfer within the fluid, which is particularly important in applications requiring chemical and biological reactions. This structure is designed to ensure complete mixing of the fluid and to consistently maintain the required conditions during the reaction process, thereby contributing to increased reaction efficiency and improved final product quality.

[0083] In one embodiment, the microfluidic chip may be manufactured from at least one selected from the group consisting of polydimethylsiloxane (PDMS), polycarbonate (PC), polymethyl methacrylate (PMMA), cyclic olefin polymer (COP), and cyclic olefin copolymer (COC). Specifically, in this embodiment, the microfluidic chip was manufactured using polydimethylsiloxane (PDMS) and polycarbonate (PC), and it was confirmed that the final product manufactured thereby was non-toxic.

[0084] Meanwhile, a material mixing device using a microfluidic chip according to an embodiment of the present invention can mix materials through the material mixing method using the microfluidic chip. The material mixing method using the microfluidic chip may be the material mixing method using the microfluidic chip according to the embodiment of the present invention described above. In one embodiment, the material mixing device using a microfluidic chip may include: the microfluidic chip; a pump unit that injects the two or more groups of materials into the two or more inlets; and a recovery unit that recovers the mixed materials from the outlets.

[0085] The role of the pump unit is to inject substances into the microfluidic chip through each inlet at a precise ratio and constant flow rate. This ensures accurate dosing of reactants and is essential for enhancing the reproducibility and accuracy of reactions. The pump unit precisely controls various types of fluids and can play a crucial role in promoting desired chemical or biological reactions during the mixing process.

[0086] The role of the recovery unit is to ensure the effective recovery of the mixed fluid through the outlet of the microfluidic chip. This unit ensures the stable collection of the product after the mixing process is complete and appropriately transports the mixture for subsequent processing or analysis. The recovery unit also minimizes mixture loss, maximizes process efficiency, and is crucial for maintaining the quality and purity of the mixed material. This configuration can optimize the performance of the entire microfluidic system.

[0087] Meanwhile, the natural exosome-mimicking nano-vesicle synthesis device according to an embodiment of the present invention can mix materials through the material mixing method using the microfluidic chip. The material mixing method using the microfluidic chip may be the material mixing method using the microfluidic chip according to the embodiment of the present invention described above. In one embodiment, the natural exosome-mimicking nano-vesicle synthesis device may include the microfluidic chip; a pump unit that injects the two or more groups of materials into the two or more inlets, respectively; and a recovery unit that recovers the nano-vesicles that have been synthesized from the outlets.

[0088] Meanwhile, the natural exosome-mimicking nanovesicles according to an embodiment of the present invention can be synthesized through the material mixing method using the microfluidic chip. The material mixing method using the microfluidic chip may be the material mixing method using the microfluidic chip according to the embodiment of the present invention described above. In one embodiment, the natural exosome-mimicking nanovesicles may include a lipid outer membrane; an effective substance encapsulated therein; and a tetraspanin formed on the lipid outer membrane.

[0089] In nanovesicles synthesized according to embodiments of the present invention, the lipid outer membrane serves to provide a structural foundation for the nanovesicles. This outer membrane forms a double lipid layer structure similar to natural exosomes, enabling the nanovesicles to maintain stability in vivo and effectively transport substances between cells. Furthermore, the lipid outer membrane helps minimize immune responses in the biological environment and facilitates recognition and uptake by cells.

[0090] In nanovesicles synthesized according to embodiments of the present invention, the role of the active ingredient is to enable the nanovesicles to perform specific biological or therapeutic functions. These ingredients may include disease-targeting therapeutics, gene regulators, or other biologically active ingredients, and can be precisely delivered to specific cells or tissues via the nanovesicles. Active ingredients are key functional components of the nanovesicles and play a crucial role in the prevention, diagnosis, and treatment of diseases.

[0091] In nanovesicles synthesized according to an embodiment of the present invention, the role of tetraspanins is to promote cell targeting and cell-to-cell interaction. Tetraspanins are cell membrane proteins that play a crucial role in cell-to-cell binding and signaling, facilitating the effective recognition and uptake of nanovesicles by cells. These proteins are crucial factors that enable nanovesicles to specifically bind to specific cell types and effectively induce biological responses.

[0092] Meanwhile, the natural exosome-mimicking nanovesicles according to an embodiment of the present invention can be synthesized through the natural exosome-mimicking nanovesicle synthesis device. The natural exosome-mimicking nanovesicle synthesis device may be the natural exosome-mimicking nanovesicle synthesis device according to an embodiment of the present invention described above. In one embodiment, the natural exosome-mimicking nanovesicles may include a lipid outer membrane; an effective substance encapsulated therein; and a tetraspanin formed on the lipid outer membrane.

[0093] Hereinafter, embodiments of the present invention will be described. However, the embodiments described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[0094] Overview of the Synthesis of Artificial Exosomes Using Microfluidics-Based Technology

[0095] Figure 1 is a schematic diagram illustrating the process for producing artificial exosomes using microfluidic mixing technology. Referring to Figure 1, components such as lipid layers, proteins, and RNA are introduced into a micromixer within a microfluidic chip through an inlet, and these components are mixed within the micromixer to form nanovesicles. Through this process, artificial exosomes are synthesized, and the resulting product possesses structural and functional properties similar to natural exosomes.

[0096] Design of a micro mixer

[0097] Fig. 2 is a drawing illustrating an example design of a micromixer. Specifically, (a) to (d) in the upper part of Fig. 2 illustrate various examples of micromixer designs according to the difference in the number of vortex forming structures (16, 24, 20, and 40, respectively), and the lower drawing of Fig. 2 is a drawing illustrating one cycle of a re-Tesla channel.

[0098] Referring to the top drawings of Figure 2, the effects of various micromixer designs on nanovesicle formation can be compared. Each drawing shows the vortex patterns generated as fluid passes through a microfluidic channel. Serpentine-shaped channels and straight channels can be compared to induce fluid flow and mixing. Figure 2(a) shows a micromixer with 16 vortex-forming structures and a serpentine structure. This structure allows the fluid to flow along a long path with multiple meanders, maximizing the mixing effect. Figure 2(b) shows a micromixer with the same serpentine structure as Figure 2(a) but with a larger number of vortex-forming structures (24). The introduction of more vortex-forming structures creates more complex fluid flow patterns, which adds variety to the mixing process. Figure 2(c) shows a micromixer with 20 vortex-forming structures and a serpentine structure combined with a straight channel. This mixing function rapidly rearranges and reorders the fluid flow while enhancing the mixing effect by changing the fluid flow. Figure 2(d) features a 44-vortex formation structure and continuous serpentine channels.

[0099] Referring to the lower drawing of Fig. 2, the micromixer includes a vortex-forming structure (210) of a reverse-Tesla structure. The fluid moves along the main flow (211) and the branch flow (sub-flow passage) (212), and then forms a vortex when rejoining, effectively inducing mixing between the fluids through this structure, and achieving high mixing efficiency within the passage. In addition, referring to the lower drawing of Fig. 2, the height and width of the channel are not particularly limited, but can be used in various combinations, preferably within the range of 50 to 500 μm.

[0100] Fig. 9 is a drawing illustrating a design example of a micro mixer. Specifically, the upper drawing of Fig. 9 illustrates a design example of a micro mixer with three inlets, and (a) and (b) at the bottom of Fig. 9 illustrate various micro mixer design examples according to the difference in the number of vortex forming structures (16 and 24, respectively) of the micro mixer with three inlets.

[0101] Referring to the upper drawing of FIG. 9, an example design of a micromixer having three inlets for mixing three groups of materials can be seen, and referring to the lower drawing of FIG. 9, a micromixer having three inlets and having a vortex forming structure (16 and 24, respectively) and a serpentine structure can be seen.

[0102] Simulation of mixing efficiency using a microfluidic micromixer

[0103] Figure 3 is a diagram illustrating the results of a simulation of the mixing process within a micromixer. Referring to Figure 3, the changes in the mixing pattern that occur when different materials flow along the serpentine structure of the micromixer can be observed. The components of the two fluids gradually mix at specific intervals, visually demonstrating that the degree of mixing of the fluids increases with each bend of the micromixer. This simulation result demonstrates how the structural design of the micromixer contributes to the mixing effect, which achieves a uniform distribution of components within the fluids.

[0104] Production process of artificial exosomes through mixing phospholipids and effective RNA materials using a micromixer

[0105] Figure 4 is a diagram illustrating the mixing process and production results of a solution containing lipids and an effective substance within a micromixer. Referring to Figure 4, two different solutions (tetraspanin-attached lipids and ethanol, and miRNA and buffer) are injected at specific points in the micromixer and mixed along serpentine-shaped channels.

[0106] Lipids used were cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS), N-stearoyl-D-erythrocyte-sphingosylphosphorylcholine (DSSM), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 3β-(N(N',N'-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol). The active ingredient was a nucleic acid (human miRNA mimics (hsa-mir-1290, hsa-mir-3162)). The solution used as a buffer was prepared by mixing 1 mM CaCl2 and 10 mM Tris-HCl buffer in DI water. The amount of miRNA input was determined by the ratio of nitrogen (N) in lipids to phosphate (P) in nucleic acids (N:P ratio), and it was experimentally confirmed that an encapsulation efficiency (or delivery efficiency, etc.) of over 96% was achieved when the N:P ratio was 6:1 or higher. The detailed steps of this mixing process are shown at the bottom of the figure, and the changes in the degree of mixing and fluid flow at each step can be observed. Finally, the artificial exosomes formed through this mixing process can be collected as a centrifuged solution shown in the upper right, suggesting that they are nanovesicles with functions similar to natural exosomes that can be used in research and clinical applications.

[0107] Evaluation of the physicochemical properties of artificial exosomes

[0108] Figure 5 is a diagram illustrating the size distribution, polydispersity index, and zeta potential of the formed artificial exosomes. Referring to the upper diagram of Figure 5, the results showing the size distribution of the artificial exosomes measured through nanoparticle tracking analysis (NTA) can be confirmed. The size distribution graph shows that the artificial exosomes are uniformly distributed within a specific size range. Referring to the lower diagram of Figure 5, the results evaluating the polydispersity index and zeta potential of the artificial exosomes measured through dynamic light scattering (DLS) can be confirmed. This indicates the stability and surface charge characteristics of the nanovesicles, and this information is important for understanding how the nanovesicles will behave in biological systems.

[0109] Encapsulation and analysis of effective substances in artificial exosomes

[0110] Figure 6 is a diagram illustrating the encapsulation efficiency of an effective substance into artificial exosomes using a gel retardation assay. Specifically, a gel retardation assay was performed using the artificial exosome sample (10 pmol of miRNA) prepared above. The sample was mixed with 6X loading dye and loaded onto a 1.5% agarose gel prepared with Tris / borate / EDTA (TBE) buffer. After electrophoresis at 100 V for 25 minutes, the gel was visualized using an iBright imaging system. Referring to Figure 6, the detection and confirmation process of artificial exosomes encapsulating effective substances can be visually confirmed. The electrophoresis image on the left shows a DNA band confirming the successful encapsulation of an effective substance into artificial exosomes, thereby confirming the possibility of CMC. The fluorescence image in the middle is an experiment to confirm whether artificial exosomes bind to a specific effective substance and emit a fluorescence signal, which can be used to evaluate the encapsulation efficiency of the substance. The image on the right is a diagram showing a natural exosome, and it can be seen that the irregular composition causes difficulties in CMC.

[0111] Comparison of nanoparticle tracking analysis (NTA), particle dispersion index (PDI), and transmission electron microscopy (TEM) images of artificial and natural exosomes.

[0112] Figure 7 is a diagram illustrating the results of a comparative analysis of the physicochemical properties of natural exosomes and artificial exosomes. Specifically, the left diagram of Figure 7 shows the particle size and distribution of natural exosomes and artificial exosomes confirmed through nanoparticle tracking analysis (NTA). Referring to the left diagram of Figure 7, the artificial exosomes and natural exosomes show similar size distributions, indicating that the artificial exosomes effectively mimic the size of natural exosomes. Referring to the upper right diagram of Figure 7, the particle dispersity index (PDI) indicating the particle size distribution and consistency of each sample can be confirmed. This graph shows that the distribution of natural exosomes and artificial exosomes is narrow and concentrated, indicating high uniformity. To observe the morphology of natural and artificial exosomes, 10 μL of the sample was placed on a formvar / carbon-coated copper grid for 10 minutes and then photographed using a transmission electron microscope, and the photographed images are shown in the lower right corner of Fig. 7. Referring to the lower right drawing of Fig. 7, the TEM images show that both samples are spherical nanovesicles with similar shapes and sizes, suggesting that the artificial exosomes successfully reproduce the morphological characteristics of natural exosomes.

[0113] Comparison of intracellular natural and artificial exosome uptake by confocal microscopy and flow cytometry.

[0114] Figure 8 is a diagram illustrating the results of a comparison of the cellular uptake of natural and artificial exosomes. Referring to the left diagram of Figure 8, an image comparing natural and artificial exosomes absorbed into cells can be seen using a confocal microscope. Blue represents cell nuclei, yellow represents exosomes, and it can be visually confirmed that artificial exosomes are absorbed into cells similarly to natural exosomes. Referring to the right diagram of Figure 8, the results of a quantitative comparison of the degree of cellular uptake of natural and artificial exosomes using flow cytometry (FACS) can be seen. The graph shows the percentage of exosomes absorbed by cells, and it can be seen that artificial exosomes exhibit a higher uptake rate than natural exosomes. This data supports the idea that artificial exosomes can be effectively delivered and function within cells.

[0115] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

Claims

A step of mixing two or more groups of substances using a microfluidic chip including 1.2 or more inlets; 1 or more outlets; and a flow passage connecting the inlets and outlets and through which a fluid can flow inside; The above two or more groups of substances are injected into each of the above two or more inlets, The above flow path includes one or more vortex forming structures, The above vortex forming structure includes a main flow passage and one or more sub flow passages branching from a first point of the main flow passage and merging at an angle greater than 90° and less than 180° at a second point spaced apart from the first point in the direction of fluid flow. Method for mixing materials using a microfluidic chip.

2. In paragraph 1, The above flow path includes a plurality of vortex-forming structures arranged in series. Method for mixing materials using a microfluidic chip.

3. In paragraph 1, Nano-vesicles mimicking natural exosomes are synthesized by mixing two or more of the above substances. Method for mixing materials using a microfluidic chip.

4. In paragraph 3, At least one of the above two or more groups of substances comprises one or more lipids selected from the group comprising fatty acids, cholesterol, and phospholipids. Method for mixing materials using a microfluidic chip.

5. In paragraph 3, At least one of the above two or more groups of substances comprises one or more effective substances selected from the group comprising proteins, small molecules and nucleic acids. Method for mixing materials using a microfluidic chip.

6. In paragraph 3, At least one of the above two or more groups of substances comprises one or more tetraspanins selected from the group comprising CD9, CD63, CD81, integrin beta1, and alexandrin. Method for mixing materials using a microfluidic chip.

7. In paragraph 1, The sub-flow passage of the above vortex forming structure merges with the main flow passage at an angle of 130 to 140°. Method for mixing materials using a microfluidic chip.

8. In paragraph 1, The above main flow passage has a width of 80 to 120 μm, Method for mixing materials using a microfluidic chip.

9. In paragraph 1, The sub-flow passage includes a first portion extending from a first point of the main flow passage; a second portion curved in a cylindrical shape from an end of the first portion toward the main flow passage; and a third portion extending from an end of the second portion and joining the second point of the main flow passage. Method for mixing materials using a microfluidic chip.

10. In paragraph 1, The above sub-flow passage is formed alternately left and right along the above main flow passage. Method for mixing materials using a microfluidic chip.

11. In paragraph 1, The above main flow passage is formed as a serpentine structure including multiple rows. Method for mixing materials using a microfluidic chip.

12. Mixing a substance using a material mixing method using a microfluidic chip according to any one of clauses 1 to 12, The above microfluidic chip; A pump unit that injects the above two or more groups of substances into the above two or more inlets; and A recovery unit for recovering the mixed material from the outlet; Material mixing device using a microfluidic chip.

13. Mixing a substance using a material mixing method using a microfluidic chip according to any one of clauses 3 to 6, The above microfluidic chip; A pump unit that injects the above two or more groups of substances into the above two or more inlets; and A recovery unit for recovering nano-vesicles whose synthesis is completed from the above outlet; A device for synthesizing natural exosome-mimetic nanovesicles.

14. Synthesized through a material mixing method using a microfluidic chip according to any one of clauses 3 to 6, A lipid outer membrane; an effective active substance embedded within the lipid outer membrane; and a tetraspanin formed on the lipid outer membrane; Natural exosome-mimetic nanovesicles.

15. Synthesized through a natural exosome-mimicking nanovesicle synthesis device according to Article 13, A lipid outer membrane; an effective active substance embedded within the lipid outer membrane; and a tetraspanin formed on the lipid outer membrane; Natural exosome-mimetic nanovesicles.

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