Method of fusing lipid nanoparticles

Abstract

According to one embodiment of the present invention, a method of fusing lipid nanoparticles is provided. In several embodiments, the method comprises the steps of: causing a fibrous structure to capture first lipid nanoparticles; causing the fibrous structure to capture second lipid nanoparticles; and shrinking the fibrous structure.

Description

Technical Field

[0001] The present invention relates to a method for fusing lipid nanoparticles. Background Art

[0002] Techniques for artificially fusing lipid nanoparticles have been under development. In particular, the fusion of lipid nanoparticles has been developed for many years as an effective drug delivery system (DDS) technology for drugs.

[0003] For example, DDSs using liposomes or micelles have been developed. However, liposomes themselves are easily cleared in tissues such as the liver and spleen and lack long-term stability in the blood. It is said that this can be overcome to some extent by modifying the liposome surface (PEGylation) with polyethylene glycol, but there are still the following problems. In addition, the manufacture of liposome preparations requires special machinery.

[0004] It is said that micellized nanoparticles are generally 30 to 100 nm in size and tend to concentrate in diseased sites, so that the drug efficacy and safety can be improved. However, micellized nanoparticles tend to accumulate in specific organs such as the liver and lungs and it is difficult to be specific for specific organs.

[0005] For these DDSs, improvement in drug delivery efficiency is also expected. For example, a method of fusing the lipid bilayer of exosomes derived from a living body, which are attracting attention as a DDS, with liposomes by a freeze-thaw method has been reported (Reference 3). It has been reported that thereby the surface properties of exosomes are changed, the immunogenicity is reduced, the colloidal stability is improved, and the half-life of exosomes in the blood is prolonged. In addition, the efficiency of uptake of fused exosomes into cells is increased, suggesting application in DDS (Non-Patent Document 1).

[0006] In order to promote membrane fusion, for example, calcium ions are sometimes added. The surface of the lipid bilayer is covered with water molecules, which repel each other. It is considered that calcium ions replace the water molecules on the surface of the lipid bilayer and thereby destabilize the lipid bilayer, making it possible for the lipid bilayers to come into contact. However, the addition of calcium ions also causes problems. For example, calcium ions can cause other biological phenomena and can also induce aggregation.

[0007] In addition, sometimes polyethylene glycol (PEG) is added. PEG does not cause problems such as affecting biochemical phenomena and aggregation, and helps with membrane fusion. One theory is that PEG can bind water molecules, and PEG-modified liposomes can improve the blood retention of liposomes and remove water molecules from the lipid bilayer. Utilizing the effect of extravasating through vascular endothelial cells with enhanced permeability in tumor tissues and accumulating in the recipient tissues (enhanced permeability and retention (EPR) effect), it is being developed as a promising drug delivery system (DDS) such as nucleic acid drugs. However, a large number of PEG-based modifications can inhibit contact with target cancer cells, and the problem of reduced intracellular uptake has emerged. In response, various developments are underway. In addition, some methods are also known, but special or expensive devices are required.

[0008] Electrostatic repulsion acts between lipid nanoparticles with the same sign of charge, making it difficult for them to fuse.

[0009] Prior art documents

[0010] Non-patent literature

[0011] Non-patent literature 1: Y.T. Sato, K. Umezaki, S. Sawada, S. Mukai, Y. Sasaki, N. Harada, H. Shiku and K. Akiyoshi, Engineering hybrid exosomes by membrane fusion with liposomes. Scientific Reports. 6, 21933; doi: 10.1038 / srep21933 (2016). Summary of the invention

[0012] According to one embodiment of the present invention, a method for fusing lipid nanoparticles is provided. The method includes the following steps: causing a fibrous structure to capture a first lipid nanoparticle; causing the fibrous structure to capture a second lipid nanoparticle; and causing the fibrous structure to contract. By causing the fibrous structure to contract, the first lipid nanoparticle and the second lipid nanoparticle can be fused. Thereby, fused lipid nanoparticles can be formed.

[0013] Thus, for example, it is not affected by ions (such as calcium ions) around the lipid bilayer, is not restricted by the necessity of modifications using PEG, etc., and the process temperature (the necessity of low-temperature treatment), etc., and high-degree-of-freedom fusion of lipid nanoparticles can be achieved.

[0014] According to an embodiment of the present invention, in a method for fusing lipid nanoparticles, a first lipid nanoparticle encapsulates a first substance, a second lipid nanoparticle encapsulates a second substance, and the step of shrinking the fibrous structure to fuse the first lipid nanoparticle and the second lipid nanoparticle (thereby forming a fused lipid nanoparticle) includes the step of mixing the first substance and the second substance and / or reacting the first substance and the second substance.

[0015] Thus, for example, a desired DDS agent can be manufactured more efficiently with a high degree of freedom. Generally, artificial lipid nanoparticles (LNPs) tend to accumulate in the liver and are easily cleared by the immune response. In contrast, fused lipid nanoparticles have a lipid bilayer membrane derived from the subject and are less likely to cause an immune response. Therefore, fused lipid nanoparticles are easily absorbed by tissues in the body and are suitable for DDS.

[0016] From the following detailed description, which illustrates only exemplary embodiments of the present invention, those skilled in the art can easily identify further aspects and advantages of the present invention. As understood, the present invention can be other different embodiments, and some details thereof can be modified in various obvious aspects without departing from the present invention. Therefore, the drawings and the description should be regarded as illustrative in nature and not as restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A flowchart of a method for fusing lipid nanoparticles using a fibrous structure, showing one embodiment.

[0018] Figure 2 A schematic diagram for explaining the steps of fusing lipid nanoparticles using a fibrous structure, showing one embodiment.

[0019] Figure 3 The size distribution of LNPs in each state obtained by nanoparticle tracking analysis in Example 1.

[0020] Figure 4 A bar graph showing the side scatter light intensity and fluorescence intensity of LNPs in each state obtained by flow cytometry in Example 1.

[0021] Figure 5 The fluorescence intensity of LNPs in each state obtained by microplate reader in Example 1.

[0022] Figure 6 The surface potential of LNPs in each state in Example 1.

[0023] Figure 7Shows the size distribution of LNPs in various states obtained by nanoparticle tracking analysis in Example 2.

[0024] Figure 8 Shows a bar graph of the side scatter light intensity and fluorescence intensity of the fused LNPs obtained by flow cytometry in Example 2.

[0025] Figure 9 Shows the fluorescence intensity of LNPs in various states obtained by microplate reader in Example 2.

[0026] Figure 10 Shows the surface potential of LNPs in various states in Example 2.

[0027] Figure 11 Shows the size distribution of LNPs in various states obtained by nanoparticle tracking analysis in Example 3.

[0028] Figure 12 Shows a bar graph of the side scatter light intensity and fluorescence intensity of the fused LNPs obtained by flow cytometry in Example 3.

[0029] Figure 13 Shows the fluorescence intensity of LNPs in various states obtained by microplate reader in Example 3.

[0030] Figure 14 Shows the surface potential of LNPs in various states in Example 3.

[0031] Figure 15 Shows the size distribution of EVs before fusion obtained by nanoparticle tracking analysis in Example 4.

[0032] Figure 16 Shows the size distribution of the fused LNPs obtained by nanoparticle tracking analysis in Example 4.

[0033] Figure 17 Shows a bar graph of the side scatter light intensity and fluorescence intensity of the fused LNPs obtained by flow cytometry in Example 4.

[0034] Figure 18 Shows the surface potential of LNPs in various states in Example 4.

[0035] Figure 19 Is a schematic diagram for explaining the steps of fusing lipid nanoparticles using a fibrous structure in one embodiment.

[0036] Figure 20 Shows the electron microscope images of each LNP in Example 5.

[0037] Figure 21Shows the size distribution of each LNP obtained by nanoparticle tracking analysis in Example 5.

[0038] Figure 22 Shows the bar chart of the side scatter light intensity and fluorescence intensity of each LNP obtained by flow cytometry in Example 5.

[0039] Figure 23 Shows the fluorescence intensity of each LNP obtained by microplate reader in Example 5.

[0040] Figure 24 Shows the surface potential of each LNP in Example 5.

[0041] Figure 25 Is a figure showing the effect of the addition of the mixture of miR1246 and IL on the expression of IL6 as an example.

[0042] Figure 26 Shows the bar chart of the side scatter light intensity and fluorescence intensity of each LNP obtained by flow cytometry in Example 6.

[0043] Figure 27 Is a schematic diagram for explaining a step of fusing lipid nanoparticles using a fibrous structure in one embodiment.

[0044] Figure 28 Shows the bar chart of the fluorescence intensity of the membrane staining of the fused LNP and the fluorescence intensity of the fluorophore of the molecular beacon obtained by flow cytometry in Example 7.

[0045] Figure 29 Shows the bar chart of the fluorescence intensity of the membrane staining of the fused LNP and the fluorescence intensity of the fluorophore of the molecular beacon obtained by flow cytometry in Example 8. Detailed implementation mode

[0046] Figure 1 Shows the process flow of the method for fusing lipid nanoparticles in one embodiment of the present invention. The fibrous structure captures the first lipid nanoparticle (S101). The fibrous structure captures the second lipid nanoparticle (S102). The fibrous structure contracts in a state where the fibrous structure has captured the first lipid nanoparticle and the second lipid nanoparticle, and fuses the first lipid nanoparticle and the second lipid nanoparticle (S103).

[0047] The step of the fibrous structure capturing the first lipid nanoparticle (S101) and the step of the fibrous structure capturing the second lipid nanoparticle (S102) can be Figure 1In the reverse order of that shown, or it can also be simultaneous. That is, in some embodiments, the fibrous structure can capture the first lipid nanoparticle (S101), and then the fibrous structure can capture the second lipid nanoparticle (S102). In some embodiments, the fibrous structure can capture the second lipid nanoparticle (S102), and then the fibrous structure can capture the first lipid nanoparticle (S101). In some embodiments, the fibrous structure can capture the first lipid nanoparticle and the second lipid nanoparticle substantially simultaneously.

[0048] The step of having the fibrous structure capture the lipid nanoparticle can be the step of allowing a solution containing the lipid nanoparticle to be absorbed or infiltrated into the fibrous structure, or it can include this step.

[0049] <Lipid Nanoparticle>

[0050] The "lipid nanoparticle" (LNP) used in this specification can be used interchangeably with solid lipid nanoparticle (SLNP / SLN), and generally refers to nanoparticles of nanoscale size composed of lipids.

[0051] The lipid nanoparticle can be a biomolecule from nature, and can be a biomolecule collected from animals. The lipid nanoparticle can be an organelle and can be a vesicle. The vesicle can be, without limitation, a vacuole, lysosome, transport vesicle, secretion, gas vesicle, extracellular matrix vesicle, extracellular vesicle, etc., and can also contain two or more of them. The extracellular vesicle (EV) can be, without limitation, an exosome, exosome complex, ectosome, shedding microvesicle, microvesicle, membrane particle, plasma membrane, apoptotic body, etc.

[0052] The lipid nanoparticle can be, without limitation, a cell, and can also contain cells. The cell can be a red blood cell, white blood cell, immune cell, etc. The lipid nanoparticle can be a virus, bacterium, etc.

[0053] The lipid nanoparticle can be a liposome of natural origin or artificially manufactured.

[0054] Combinations of lipid nanoparticles for fusion include, for example, without limitation, the following combinations: liposome and liposome; liposome and extracellular vesicle; liposome and cell; liposome and virus; extracellular vesicle and extracellular vesicle; extracellular vesicle and cell; extracellular vesicle and virus; cell and cell; cell and virus; virus and virus. Combinations of liposome and liposome, extracellular vesicle and extracellular vesicle, cell and cell, virus and virus can be composed of two same types or two different types.

[0055] In some embodiments, one (the first or second) lipid nanoparticle may be from the subject or patient (autologous lipid nanoparticle), and the other (the second or first) lipid nanoparticle may be non-autologous (allogeneic or artificial) lipid nanoparticle. As a non-limiting example, the first lipid nanoparticle may be exosomes from the subject or patient, and the second lipid nanoparticle may be artificial liposomes.

[0056] <Substances encapsulated in lipid nanoparticles, substances present on their membranes>

[0057] Lipid nanoparticles may contain a target substance or other substances. For example, extracellular vesicles such as exosomes contain lipids, proteins, etc. from the cell membrane on their surface and contain nucleic acids (microRNA, messenger RNA, DNA, etc.), proteins, body fluids, etc. inside.

[0058] The nucleic acid may be ribonucleic acid (RNA) or may contain RNA. RNA may be, without limitation, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), non-coding RNA (ncRNA), microRNA (miRNA), ribozyme, double-stranded RNA (dsRNA), siRNA (small interfering RNA), etc., or may contain two or more of them. RNA may be modified. RNA and miRNA may be related to the onset or development of cancer, cardiovascular diseases, neurodegenerative diseases, mental diseases, chronic inflammatory diseases, etc. miRNA may be of the type that promotes carcinogenesis or is positively regulated (onco miRNA (oncogenic miRNA, cancer-promoting type miRNA)), or may be of the type that inhibits carcinogenesis or is negatively regulated (Tumor Suppressor miRNA (cancer-suppressing type miRNA)).

[0059] The nucleic acid may be deoxyribonucleic acid (DNA) or may contain DNA.

[0060] In some embodiments, the lipid nanoparticles to be fused may contain a medicament (anticancer agent, nucleic acid drug, anti-aging substance, anesthetic, etc.) inside, and the lipid nanoparticles formed by fusion (fused lipid nanoparticles) may contain the above-mentioned medicament inside. In some embodiments, such fused lipid nanoparticles may be used as a DDS medicament. In some embodiments, the fused lipid nanoparticles may be used as an anticancer agent.

[0061] In some embodiments, the lipid nanoparticles can encapsulate a substance that recognizes, binds to, or reacts with the substance encapsulated in another lipid nanoparticle (the target lipid nanoparticle) for fusion. For example, as another lipid nanoparticle (the target lipid nanoparticle), it can contain a nucleic acid that hybridizes with the nucleic acid encapsulated in extracellular vesicles such as exosomes or a molecular beacon having the nucleic acid sequence. Thus, the internal nucleic acid of the lipid nanoparticles of a subject, for example, can be detected or analyzed. Furthermore, the biological information of the subject (such as the type, presence or absence, condition, future risk, and location of a disease) can be inferred or obtained thereby.

[0062] In some embodiments, one lipid nanoparticle (e.g., the second lipid nanoparticle) for fusion has a protein on its membrane and fuses with another lipid nanoparticle (e.g., the first lipid nanoparticle). The fused lipid nanoparticle has the proteins of the first lipid nanoparticle and the proteins of the second lipid nanoparticle on its membrane. For example, the protein on the membrane of the second lipid nanoparticle can be PEG. In some embodiments, one lipid nanoparticle for fusion can have an inorganic substance. The inorganic substance can be, for example, a substance used in quantum sensors such as nanodiamonds and / or quantum dots.

[0063] In some embodiments, one lipid nanoparticle for fusion can have a ligand targeting cancer on its membrane. The fused lipid nanoparticle using it can be used, for example, as an actively targeted lipid nanoparticle without limitation.

[0064] <Lipid nanoparticle solution>

[0065] The lipid nanoparticles can be provided by being contained in a solution or a liquid. The solution containing the lipid nanoparticles can be introduced into a fibrous structure and absorbed and infiltrated therein. The "solution" used in this specification includes a liquid containing the target substance, a liquid considered to contain the target substance, or a liquid that does not contain the target substance but is used for the purpose of enabling the fibrous structure to capture the target substance or its related purposes (hereinafter sometimes referred to as "the solution containing lipid nanoparticles").

[0066] In some embodiments, the first solution containing the first lipid nanoparticles and the second solution containing the second lipid nanoparticles can be provided separately or introduced into the fibrous structure. In some embodiments, the first solution and the second solution can be introduced into the fibrous structure simultaneously or at different times. The first solution and the second solution can be provided in the form of the same solution. That is, a certain solution can contain the first lipid nanoparticles and the second lipid nanoparticles. In some embodiments, a solution containing both the first lipid nanoparticles and the second lipid nanoparticles can be provided.

[0067] The solution containing lipid nanoparticles can be a body fluid, a liquid from a body fluid (diluent, processing solution, etc.). The solution can be a solution that is not a body fluid (non-body fluid source), can also be an artificially prepared liquid, or can be a mixture of a body fluid or a solution from a body fluid and a solution from a non-body fluid source. The solution can be a solution used in sample determination and can be a solution used in calibration determination. The solution can be used in the state of a stock solution, or can be a liquid obtained by diluting or concentrating the stock solution. The solution can be a standard solution or a calibration solution. The specimen to be measured can be a test subject. The solution can include physiological buffer solutions such as phosphate buffered saline (PBS) and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES) containing the substance to be recovered. The body fluid can contain additives. In the additives, stabilizers, pH regulators, etc. can be added, for example.

[0068] The body fluid can be, without limitation, blood, serum, plasma, lymph fluid, can be tissue fluid such as interstitial fluid, intercellular fluid, stromal fluid, can be body cavity fluid, serous cavity fluid, pleural effusion, ascites, pericardial fluid, cerebrospinal fluid (spinal fluid), synovial fluid (joint fluid), aqueous humor (eye chamber fluid). The body fluid can be digestive fluids such as saliva, gastric juice, bile, pancreatic juice, intestinal juice, can be sweat, tears, nasal discharge, urine, semen, vaginal fluid, amniotic fluid, milk.

[0069] The body fluid can be a human body fluid. The body fluid can be an animal body fluid. The animal can be a reptile, a mammal, an amphibian. The mammal can be a dog, a cat, a cow, a horse, a sheep, a pig, a hamster, a mouse, a squirrel, and primates such as monkeys, gorillas, chimpanzees, bonobos, and humans.

[0070] In some embodiments, the solution of lipid nanoparticles can be an aqueous solution. In some embodiments, the solution of lipid nanoparticles can be a non-aqueous solution.

[0071] <Fibrous structure>

[0072] The term "fibrous structure" used in this specification generally refers to a member with pores formed by mechanically and / or chemically processing fibers. The fibrous structure includes woven fabrics, non-woven fabrics, papers, etc. The fibrous structure of the present invention preferably has a hydrophilicity such that an aqueous solution can penetrate into the interior by capillary blood phenomenon or higher. The fibrous structure can be formed into a sheet shape, that is, a flat shape (also called a fiber sheet). In this specification, the fiber sheet is mainly used as an example for description. However, the present invention is not limited to a sheet structure and also includes fibrous structures that are substantially three-dimensional shapes.

[0073] In some embodiments, the fiber can be a chemical fiber, a natural fiber, or a fiber formed by mixing two or more fibers.

[0074] In some embodiments, the natural fibers may be plant fibers such as cellulose, cotton, hemp, and linen. In some embodiments, they may be animal fibers such as wool, silk, and cashmere.

[0075] The term "cellulose fiber structure" used in this specification generally refers to a fibrous structure mainly composed of cellulose fibers or containing cellulose fibers. The fibrous structure cellulose fiber structure may be a cellulose fiber sheet. The "cellulose fiber" used in this specification refers to a fiber mainly composed of cellulose. The cellulose fiber may be cellulose nanofiber (CNF). Cellulose nanofibers generally have a diameter of several nanometers (nm) to dozens of nm. In most cases, as a non-limiting example, the width of cellulose nanofibers is about 3 nm to 100 nm.

[0076] Cellulose fibers can be obtained, for example, from wood pulp or other raw materials by chemical and / or mechanical means. The general method for manufacturing cellulose nanofibers is as follows. First, wood fibers (cellulose fibers) are taken out from wood chips and pulped. The cellulose fiber is composed of countless cellulose nanofibers bundled together. Then, the cellulose fiber is made to collide with each other under high pressure in a solvent in the presence of a TEMPO catalyst to untangle the bundled cellulose fibers. Thus, cellulose nanofibers can be obtained. The above manufacturing methods of the cellulose fiber and the cellulose fiber sheet are non-limiting examples, and other manufacturing methods can also be adopted.

[0077] The solvent containing cellulose nanofibers obtained is subjected to suction filtration, whereby the cellulose nanofibers aggregate or form a film due to surface tension. The cellulose nanofiber solvent can be water or the like. In one mode, the produced film can be a non-woven fabric.

[0078] Regarding the nano-pores, a liquid with a low surface tension (hereinafter sometimes referred to as "low surface tension solvent") is added to the wet cellulose nanofibers that have been suction filtered and aggregated. Suction is continued, and the solvent contained in the aggregated cellulose nanofiber mass is replaced with the low surface tension solvent or dried. Thus, nano-pores are formed inside the aggregate of cellulose nanofibers.

[0079] The size of the nano-pores can be adjusted, for example, by the added low surface tension solvent. The surface tension of the low surface tension solvent can be in a range less than the surface tension of water (20 °C, 72.75 mN / m) and capable of creating nano-pores. For example, the surface tension of the solvent at 20 °C can be a value below 35 mN / m, 30 mN / m, 25 mN / m, 20 mN / m, etc. The low surface tension solvent non-limitingly includes tert-butanol (20.7 mN / m), ethanol (22.55 mN / m), isopropanol (20.8 mN / m), etc.

[0080] Taking the formation of the above-mentioned nanopores and the method for adjusting their sizes as an example, other methods can also be used. For example, high-pressure treatment conditions for unraveling cellulose fibers can be adopted. For example, the type of pulp can be changed. For example, cellulose from other sources (such as microorganisms such as acetic acid bacteria (Acetobacter spp., etc.); animals such as ascidians) can be used. Thereby, the width of the cellulose nanofibers can be changed and the sizes of the nanopores can be adjusted.

[0081] In some embodiments, the fibrous structure can be formed from cellulose fibers (pulp). By dispersing the cellulose fibers (pulp) in a solvent, the fibrous structure can be manufactured in substantially the same manner as in the case of cellulose nanofibers.

[0082] The gaps (pore sizes) between the cellulose fibers can be adjusted in substantially the same manner as in the case of cellulose nanofibers. The width (diameter) of the cellulose fibers is mostly about 20 μm to 40 μm. Therefore, the sizes of the gaps are several nm to several μm, and can be about 10 nm to about 1000 nm and about 1 μm to 100 μm.

[0083] In some embodiments, the fibers can be chemical fibers. In some embodiments, the chemical fibers can be polymer fibers and can be synthetic fibers such as polyvinyl alcohol (PVA, etc.). The chemical fibers can be polyester fibers such as polyethylene terephthalate, polyethylene naphthalate, polyethylene naphthalate glycol, polypropylene terephthalate; polyamide fibers such as nylon; acrylic fibers such as acrylonitrile; polyolefin fibers such as polyethylene, polypropylene; regenerated cellulose fibers such as rayon, cuprammonium fiber, polynosic; cellulose-based semi-synthetic fibers such as acetate fiber; protein-based semi-synthetic fibers such as promix; polyurethane fibers; vinylon fibers; glass fibers; carbon fibers (including nanotubes, etc.). In some embodiments, the fibers can be hydrolyzable polymers, biodegradable polymers, absorbent polymers, absorbent filaments such as poly(p-dioxanone) (PDO, PDS).

[0084] As used in this specification, the "pore size" or "feature size" refers to the size of the pores of the fibrous structure. The pore size can be values such as 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, 20 nm, 30 nm, etc. or larger. The pore size can be values such as 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, etc. or smaller. The pore size can be in ranges such as about 4 nm to about 200 nm, about 40 nm to about 200 nm, about 200 nm to about 500 nm, about 4 nm to about 100 nm, about 5 nm to about 90 nm, about 10 nm to about 80 nm, etc.

[0085] For example, a fibrous structure having a pore size of about 40 nm to about 200 nm is suitable for capturing small EVs of the same size. For example, a fibrous structure having a pore size of about 200 nm to about 500 nm is suitable for capturing large EVs of the same size. These are examples, and fibrous structures having other pore sizes can also be used to capture EVs of the same size.

[0086] <Shrinkage of fibrous structure>

[0087] In some embodiments, the step of shrinking the fibrous structure may include the step of removing at least a part of the water held in the fibrous structure. In some embodiments, the step of shrinking the fibrous structure may include the step of removing the solution containing lipid nanoparticles. In some embodiments, the step of shrinking the fibrous structure may include the step of drying it. For example, the fibrous structure that has absorbed the solution containing lipid nanoparticles can be dried and / or heated. Thereby, the solution or solvent can be evaporated.

[0088] In some embodiments, the fibrous structure may be configured such that during manufacturing or when absorbing the target solution, the target substance (lipid nanoparticles) substantially enters or passes through the gaps of the fibrous structure, and during cleaning (after drying or before cleaning), the target substance (lipid nanoparticles) substantially remains in the fibrous structure and / or does not flow out to the outside.

[0089] When the fibrous structure shrinks, the pore size of the fibrous structure becomes smaller. The fibrous structure is preferably manufactured in such a way that in the wet state, the pore size is equal to or larger than the size of the target substance (lipid nanoparticles) to be captured, and after drying, the pore size becomes smaller than the size of the capture target.

[0090] Multiple lipid nanoparticles captured by the same or adjacent pores of the fibrous structure come into contact with and press against each other as the pore size becomes smaller and the fiber spacing becomes smaller, thereby causing fusion. Two lipid nanoparticles fuse, and the substances contained within each of them are mixed with each other.

[0091] <Embodiment 1>

[0092] Refer to Figure 2 , and schematically illustrate the process of fusion of lipid nanoparticles based on the shrinkage of the fibrous structure, as well as the mixing and reaction of the substances inside them, in one embodiment.

[0093] As Figure 2As shown in A, the fibrous structure 10 captures the first lipid nanoparticle 20 and the second lipid nanoparticle 30 within its pores 11. The size of the pores 11 is schematically represented by d1 and is large enough to contain two lipid nanoparticles 20, 30. Furthermore, the size of the pores 11 or the surface properties of the fibers of the fibrous structure 10 enable it to continuously capture two lipid nanoparticles 20, 30 without escape.

[0094] Figure 2 In the example of, the first lipid nanoparticle 20 is an exosome. Exosomes encapsulate various substances inside. Figure 2 One RNA 21 is illustratively shown. Figure 2 In, the exosome 20 illustratively has a protein 22 expressed on its surface. The second lipid nanoparticle 30 is an artificially prepared liposome and has a molecular beacon 31 designed to bind to the RNA 21 encapsulated in the exosome 10 inside.

[0095] Figure 2 As shown by d1 - d3, it schematically shows the distance between two fibers that clamp the captured lipid nanoparticles and are almost parallel. This is Figure 2 an illustrative parameter used in the schematic diagram to explain the size change of the pores 11 and should not be construed restrictively. It can also be a parameter other than the schematic.

[0096] As Figure 2 shown in B, when the fibrous structure 10 is gradually dried, the solvent evaporates and the size of the pores 11 becomes smaller (d2 < d1). The two lipid nanoparticles, the exosome 20 and the liposome 30, are compressed from the outside by the fibers of the fibrous structure 10, come into contact with each other and start to fuse, and start to form a fused lipid nanoparticle 40a. Then the substances located inside the two lipid nanoparticles start to mix due to membrane fusion.

[0097] As Figure 2 shown in C, when the fibrous structure 10 is dried, the size of the pores 11 becomes smaller (d3 < d2). Along with this, the exosome 20 and the liposome 30 are completely fused to form a fused lipid nanoparticle 40.

[0098] Inside the fused lipid nanoparticle 40, the molecular beacon 31 encapsulated in the liposome 30 binds to the target RNA 21 encapsulated in the exosome 20. As a result, the fluorophore of the molecular beacon 31 moves away from the quencher and emits fluorescence ( Figure 2 C).

[0099] The membrane of the fused lipid nanoparticle 40 has a protein from the exosome 20 ( Figure 2 C). Furthermore, the membrane of the fused lipid nanoparticle 40 also has a protein from the liposome 30 (not shown).

[0100] <Example>

[0101] The following describes several examples. The steps for the production and fusion of LNPs are the same in any example and are as described below.

[0102] <LNP Production>

[0103] LNPs are produced using an iLiNP (invasive lipid nanoparticle production) device. The lipid solutions for anionic and cationic LNPs are 5 mM DSPC / DOPS / Chol (45 / 10 / 45 mol%) in EtOH:MeOH = 8:2 and 10 mM DOTAP / DSPC / Chol / PEG-amine (50 / 10 / 38.5 / 1.5 mol%) in EtOH:MeOH = 8:2, respectively. The aqueous solution contains a buffer of 13.4 mM CaCl 2 / 10 mM Tris-HCl (pH 8.0) and 10 μM of the molecule to be encapsulated. The lipid solution and the aqueous solution are simultaneously flowed into the device at a flow rate ratio of 2:1. The produced LNPs are dialyzed in PBS at 4°C overnight to remove the molecules outside the LNP membrane.

[0104] 10 μL of a suspension of LNPs encapsulating molecular beacons (labeled LNPs) is coated on a cellulose nanofiber (1 cm × 1 cm) and dried in a dryer for 3 days. Then, 10 μL of a suspension of LNPs or EVs (target LNPs) encapsulating miRNA is coated and dried in the dryer again for 3 days. Fusion is thus performed. Then, the cellulose nanofiber is added to a tube containing PBS and vigorously stirred to extract the fused LNPs.

[0105] <Example 1: Fusion of Positively Charged LNPs with Positively Charged LNPs>

[0106] Prepare two types of LNPs (LNP(+)) both having a positively charged surface. A detection miRNA (miR3960) is encapsulated in one type of LNP (target LNP) (LNP(+, miR3960)). The size of the prepared LNPs is measured using nanoparticle tracking analysis (NTA, NanoSight, Malvern Instruments Ltd.)( Figure 3 A). It is confirmed that the particle size has a maximum value at approximately 120 nm and is distributed from 80 nm to 240 nm.

[0107] In another LNP (labeled LNP), a molecular beacon (LNP(+, amiR3960)) designed to hybridize with the detection miR3960 and having Cy5 as a fluorophore is encapsulated. Similarly, the size of the prepared LNP was measured ( Figure 3 B). It was confirmed that the particle size had a maximum value at approximately 120 nm and was distributed from 80 nm to 240 nm. It can be said that Figure 3 the distribution shown in B, although there are some differences compared to Figure 3 A, is generally the same.

[0108] A cellulose fiber sheet was made to absorb an aqueous solution containing two LNPs, and the two LNPs were fused by the method of the present invention to produce a fused LNP. Thereafter, the fused LNP was recovered from the cellulose fiber. The size of the recovered fused LNP was measured ( Figure 3 C). The size of the prepared LNP was hardly detected. Instead, multiple peaks were observed between 200 nm and 700 nm. This indicates that, through the fusion process, not only two but also more than two LNPs were fused.

[0109] Figure 4 The results of flow cytometry of the target LNP ( Figure 4 A) and the fused LNP ( Figure 4 B) are shown. Flow cytometry was performed using a NanoAnalyzer (nanoFCM). The horizontal axis (SS-H) represents the intensity of the side-scattered light. Information related to the particle size was obtained therefrom. The vertical axis (Cy5-A) represents the fluorescence intensity.

[0110] Figure 4 In A, almost all the measurement points had a fluorescence intensity of approximately 2×10 1 , and were located further below. These points are data that were counted as particles but for which the optical label was not counted. That is, these are noises unrelated to the Cy5 of the molecular beacon that is the measurement object. On the other hand, in Figure 4 B, there were two clearly separated distributions with a fluorescence intensity of approximately 2×10 1 . The lower distribution is the noise corresponding to the distribution in Figure 4 A. The upper distribution is not noise, that is, it corresponds to the Cy5 of the molecular beacon that is the measurement object.

[0111] Figure 5The fluorescence intensity showing the state of each LNP obtained using a microplate reader is presented. A microplate reader (Spark (registered trademark), a multimode microplate reader, TECAN) and a plate (Thermo Scientific (TM) black 96-well immunoplate) were used. 50 μL of the sample was added dropwise to each well, and the fluorescence intensity was measured at Ex / Em = 630 / 675 (nm). The horizontal axis represents respectively: the case of measuring only the labeled LNP before fusion (only (+, a3960)); the case of first mixing the labeled LNP with the target LNP, then introducing them into cellulose nanofibers and performing the fusion process ((+, a3960)+(+, 3960)); the case of first introducing the labeled LNP into cellulose nanofibers, then introducing the target LNP into cellulose nanofibers, and then performing the fusion process ((+, a3960)→(+, 3960)).

[0112] The fluorescence intensity of only the labeled LNP before fusion (only (+, a3960)) being 5000 can be considered noise or background.

[0113] The fluorescence intensity of the case of mixing the labeled LNP with the target LNP and introducing them into cellulose nanofibers to perform the fusion process ((+, a3960)+(+, 3960)) is 9000. This indicates that through the fusion process, the labeled LNP and the target LNP were fused.

[0114] The fluorescence intensity of the case of first introducing the labeled LNP into cellulose nanofibers, then introducing the target LNP into cellulose nanofibers, and then performing the fusion process ((+, a3960)→(+, 3960)) is 14000. This indicates that the labeled LNP and the target LNP were fused. Compared with the case of introducing them after mixing into cellulose fibers, the fusion efficiency is high. The scientific reason for this was not clear at the time of this application. Various mechanisms were considered, but this tendency should not be explained by being limited to a certain mechanism.

[0115] These results indicate that the molecular beacon located inside the labeled LNP recognized miR3960 located inside the target LNP. That is, it was confirmed that through the fusion process, any positively charged target LNP and the labeled LNP were fused.

[0116] Figure 6Results of the surface potential (ζ potential (mV)) showing the states of each LNP. The surface potential was measured using a ζ potential measurement system (ELSZ-2000Z, Otsuka Electronics). The measurement was performed at room temperature using a standard cell. The horizontal axis represents the target LNP before fusion ((+, miR3960)), the labeled LNP before fusion ((+, amiR3960MB)), and the fused LNP ((+, miR3960)+(+, amiR3960MB)) generated by fusing after mixing and introducing into cellulose fibers, respectively.

[0117] The surface potentials of the target LNP ((+, miR3960)) before fusion and the labeled LNP ((+, amiR3960MB)) after fusion were approximately 1 mV and approximately 9 mV, respectively. On the other hand, the surface potential of the fused LNP ((+, miR3960)+(+, amiR3960MB)) after fusion was approximately 4 mV. That is, the charge of the fused LNP has the average value of the two types of LNP before fusion. This result indicates that the target LNP and the labeled LNP, both having positive charges, underwent membrane fusion through the fusion process.

[0118] <Example 2: Fusion of negatively charged LNP with negatively charged LNP>

[0119] In this example, two types of LNP (LNP(-)) both having a negatively charged surface were prepared and fused. A miRNA for detection (miR21) was encapsulated in one LNP (target LNP) (LNP(-, miR21)). A molecular beacon designed to hybridize with the detection miR21 and having Cy5 as a fluorophore was encapsulated in the other LNP (labeled LNP) (LNP(-, amiR21)). The conditions for the fusion process, NTA measurement, flow cytometry measurement, and surface potential measurement were the same as those in Example 1.

[0120] Figure 7 The size distributions of each LNP measured using nanoparticle tracking analysis are shown. It was confirmed that although there were some differences in the sizes of the target LNP and the labeled LNP before fusion, the maximum value was at approximately 150 nm and the distribution was from 80 nm to 400 nm ( Figure 7 A and Figure 7 B).

[0121] Although the size of the fused LNP had a distribution at 150 nm, multiple new peaks were observed from 150 nm to 600 nm.

[0122] Figure 8 The results of flow cytometry of the fused LNP are shown. There was a distribution at approximately 10 4 or more in terms of Cy5-A.

[0123] Figure 9 The fluorescence intensities showing the states of each LNP obtained using a microplate reader are presented. The horizontal axis represents, respectively: the case of measuring only the labeled LNP before fusion (only(-, a21)); the case of first mixing the labeled LNP with the target LNP, then introducing them into cellulose nanofibers and performing the fusion process ((-, a21)+(-, 21)); the case of first introducing the labeled LNP into cellulose nanofibers, then introducing the target LNP into cellulose nanofibers, and then performing the fusion process ((-, a21)→(-, 21)).

[0124] The fluorescence intensity of the labeled LNP before fusion (only(-, a21)) at 5000 can be considered as noise or background. The fluorescence intensity after mixing the labeled LNP with the target LNP and introducing them into cellulose nanofibers ((-, a21)+(-, 21)) is 2000. This indicates that the LNP did not fuse. Different from Example 1 where both were negatively charged LNPs, no substantial fusion was observed. The fluorescence intensity after the fusion process ((-, a21)→(-, 21)) is 32000. This value is much larger than the other values, proving that the molecular beacon binds to the target miRNA through the fusion process.

[0125] The following difference was observed between Example 1 and Example 2: when introduced into cellulose fibers after mixing, the positively charged LNPs fused, while the negatively charged LNPs did not fuse. The scientific reason for this was not clear at the time of this application. Various mechanisms were considered, but this tendency should not be explained by a single mechanism.

[0126] Figure 10 The results of the surface potential (ζ potential (mV)) showing the states of each LNP are presented. The horizontal axis represents, respectively, the target LNP before fusion ((-, miR21)), the labeled LNP before fusion ((-, amiR21MB)), and the fused LNP after fusion ((-, miR21)+(-, amiR21MB)).

[0127] The surface potentials of the target LNP before fusion ((-, miR21)) and the labeled LNP before fusion ((-, amiR21MB)) are approximately -10 mV and approximately -18 mV, respectively. On the other hand, the surface potential of the fused LNP after fusion ((-, miR21)+(-, amiR21MB)) is approximately -9 mV. This result indicates that through the fusion process, the target LNP and the labeled LNP, both having negative charges, underwent membrane fusion.

[0128] <Example 3: Fusion of Negatively Charged LNP and Positively Charged LNP>

[0129] In this example, an LNP with a negatively charged surface (LNP(-)) and an LNP with a positively charged surface (LNP(+)) were prepared and fused together.

[0130] A detection miRNA (miR3960) (LNP(-, miR3960)) was encapsulated in the negatively charged LNP (target LNP) of one of them. A molecular beacon designed to hybridize with the detection miR3960 and having Cy5 as a fluorophore (LNP(+, amiR3960)) was encapsulated in the LNP (labeled LNP) of the other. The conditions for the fusion process, NTA measurement, flow cytometry measurement, and surface potential measurement were the same as in Example 1.

[0131] Figure 11 The size distributions of the respective LNPs measured using nanoparticle tracking analysis are shown. Although there were some differences in the sizes of the target LNP and the labeled LNP before fusion, they had a maximum value at approximately 150 nm and were distributed from 80 nm to 400 nm ( Figure 11 A) and from 80 nm to 300 nm ( Figure 11 B).

[0132] Regarding the size of the fused LNPs, multiple new peaks were observed from 150 nm to 800 nm ( Figure 1 C).

[0133] Figure 12 The results of flow cytometry of the fused LNPs are shown. The distribution above 2x10 1 is not noise but corresponds to Cy5 of the molecular beacon.

[0134] Figure 13 The fluorescence intensities of the states of the respective LNPs obtained using a microplate reader are shown. The horizontal axis represents, respectively: the case where the target LNP and the labeled LNP were sequentially introduced into cellulose nanofibers at a concentration ratio of 1:1 and the fusion process was carried out ((+, a3960)→(-, 3960) 1×); the case of a concentration ratio of 10:1 ((+, a3960)→(-, 3960) 10×); the case of a concentration ratio of 100:1 ((+, a3960)→(-, 3960) 100×); the case of this concentration ratio of 1000:1 ((+, a3960)→(-, 3960) 1000×); the case of only the labeled LNP (only(+, a3960)); the case where the target LNP and the labeled LNP were first mixed at a concentration ratio of 1:1, and then the mixture was introduced into cellulose nanofibers and the fusion process was carried out ((+, a3960)+(-, 3960) 1×); and the case of only PBS being introduced into cellulose nanofibers (PBS).

[0135] The fusion efficiency is higher compared with the case of sequential addition ((+ , a3960) → (- , 3960) 1×) and the case of pre - mixing LNP ((+ , a3960)+(- , 3960) 1×). In the group with sequential addition, when the concentration of labeled LNP is 1:1, the fluorescence intensity is the highest, and the fluorescence intensity decreases as the labeled LNP becomes thinner. It is considered that as the dilution progresses, the amount of the target miRNA (miR3960) decreases, and the molecular beacon encapsulated in the labeled LNP is provided in relative excess.

[0136] Figure 14 Results of the surface potential (ζ potential (mV)) showing the state of each LNP are presented. The horizontal axis represents, respectively: the negatively charged target LNP before fusion (( - , miR3960)); the positively charged labeled LNP before fusion (( + , amiR3960MB)); the fused LNP generated by mixing and then introducing into cellulose fibers for fusion (( - , miR3960)+( + , amiR3960MB)).

[0137] The surface potentials of the target LNP (( - , miR3960)) before fusion and the labeled LNP (( + , amiR3960MB)) after fusion are approximately - 12 mV and approximately + 9 mV, respectively. On the other hand, the surface potential of the fused LNP (( - , miR3960)+( + , amiR3960MB)) after fusion is approximately 0 mV. This result indicates that through the fusion process, the negatively charged target EV and the positively charged labeled LNP have undergone membrane fusion.

[0138] <Example 4: Fusion of EV and LNP>

[0139] In this example, EV and LNP with a positively charged surface (LNP(+)) were prepared and fused.

[0140] Two types of EV were prepared. One is extracellular vesicles from a normal cell line not activated by interleukin (EV(IL - )), and the other is extracellular vesicles from a normal cell line activated by interleukin (EV(IL+)).

[0141] Figure 15 The size distribution of each EV measured by nanoparticle tracking analysis is shown. Regarding the sizes of the target LNP and the labeled LNP before fusion, it was confirmed that EV(IL - ) has a maximum value at approximately 150 nm and is distributed from 80 nm to 450 nm ( Figure 15 A), and EV(IL+) is distributed from 80 nm to 600 nm ( Figure 15 B).

[0142] Regarding the size of the fused LNP, multiple new peaks were observed in the range of 50 nm to 800 nm ( Figure 16A and Figure 16 B).

[0143] Figure 17 Results of flow cytometry showing the fusion of LNPs. Compared with 2x10 1 The upper distribution is not noise but corresponds to Cy5 of the molecular beacon.

[0144] Figure 18 Results of the surface potential (ζ potential (mV)) showing the respective states of EVs before fusion and fused LNPs. The horizontal axis represents: the target EV before fusion (IL-); the target EV before fusion (IL+); the fused LNP after fusion of the target EV (IL-) (EV(IL-)+LNP(+, amiR3960)); the fused LNP after fusion of the target EV (IL+) (EV(IL+)+LNP(+, amiR3960)).

[0145] The surface potentials of the target EV (IL-) before fusion and the target EV (IL-) were approximately -7.5 mV and approximately -2.5 mV, respectively. On the other hand, the surface potentials of the fused LNP (EV(IL-)+LNP(+, amiR3960)) and the fused LNP (EV(IL+)+LNP(+, amiR3960)) after fusion were approximately +7 mV and approximately +5 mV, respectively. This result indicates that through the fusion process, the negatively charged target EV underwent membrane fusion with the positively charged labeled LNP.

[0146] Examples 1 to 4 show that the target LNP and the labeled LNP can fuse regardless of their charge signs, and in particular, the target LNP and the labeled LNP with the same charge sign can fuse. These also show that the contents of both can be mixed within the fused LNP and the lipid membranes of both can fuse in the form of the membrane of the fused LNP.

[0147] <Other examples>

[0148] In some embodiments, the first LNP can be from the subject, and the second LNP can encapsulate a drug. The fusion process can be carried out in vitro of the subject. The fused LNP can be returned to the body of the subject. The subject can be a cancer patient and the drug can be an anticancer agent. In some embodiments, the first LNP can be from the subject, and the second LNP can encapsulate an anesthetic.

[0149] In some embodiments, the first LNP (or the second LNP) can be from an organism, and the second LNP (or the first LNP) can be from an organism or artificially synthesized and adjusted for fusion with the first LNP (or the second LNP).

[0150] In some embodiments, a fiber sheet can be used to fuse the first LNP and the second LNP from a subject and paste the fiber sheet onto the subject, thereby introducing the fused LNP into the subject's body.

[0151] For example, the first LNP from a subject can be extracellular vesicles from the body fluid of the subject. Extracellular vesicles from body fluid can be obtained using various methods. Examples of such methods can non - limitatively include: ultra - centrifugation, use of commercially available kits or devices, utilization of microfluidic devices, utilization of nanowires, utilization of cellulose nanowires, etc. The second LNP can encapsulate an anti - cancer agent, an effective drug prescribed for the subject. The body fluid containing extracellular vesicles or a solution from the body fluid can be absorbed by the fiber sheet or infiltrated, or can also be dropped onto the fiber sheet.

[0152] In some embodiments, the fiber sheet can absorb a solution containing the first LNP and a solution containing the second LNP. In some embodiments, the second LNP encapsulating an effective drug prescribed for the subject can be pre - loaded onto the fiber sheet. The fiber sheet loaded with the second LNP can absorb a solution of body fluid containing extracellular vesicles from the subject. For example, the fiber sheet loaded with the second LNP can be pasted onto the surface of the subject's skin, mucosa, organ, etc. (such as the skin) to allow the fiber sheet to absorb the body fluid. For example, body fluid, a liquid from body fluid, or a culture medium can be dropped onto the fiber sheet loaded with the second LNP. The same fiber sheet can be used to obtain the first LNP from the subject and fuse it with the second LNP containing the drug. The acquisition and fusion of the first LNP can be carried out efficiently or substantially simultaneously in a short time.

[0153] The fused LNP can be dried and / or stored in the same fiber sheet or transferred to other fiber sheets. The fiber sheet holding the fused LNP can be pasted onto the surface of the subject's skin, etc. Thereby, it is introduced into the subject's body or absorbed at the position where the fused LNP is pasted. During the subject's surgery, the fiber sheet holding the fused LNP can be pasted onto the surface of the subject's skin, etc., so as to absorb the subject's body fluid, fuse the LNP, and / or introduce the fused LNP into the subject.

[0154] The fiber sheet holding the first LNP, the second LNP, and / or the fused LNP can be pasted onto the surface of the subject's skin, etc. during or after the surgery. For example, during or after the surgery, the fiber sheet holding the second LNP or the fused LNP containing the anti - cancer agent can be pasted onto the skin, etc. of the body part suspected of tumor metastasis or residue. Thereby, for example, the recurrence, metastasis, etc. of cancer can be effectively inhibited.

[0155] <Embodiment 2>

[0156] Refer to Figure 19Schematically illustrate the fusion of lipid nanoparticles based on the contraction of a fibrous structure, as well as the mixing and reaction processes of the substances inside it. Figure 19 The upper diagrams (a1) to (e) in Figure 19 show schematic diagrams of the structures observed macroscopically, etc.

[0157] First, an aqueous solution 135 ( Figure 19 A) of the labeled LNP130 containing the encapsulated molecular beacon 131 is coated on the fibrous structure 110 ( Figure 19 a1). After that, the fibrous structure 110 is dried ( Figure 19 a2). The labeled LNP130 is captured inside the pores of the fibrous structure 110 ( Figure 19 A). The molecular beacon 131 is designed to hybridize with the target nucleic acid 121 encapsulated in the target LNP120 shown in the following diagrams.

[0158] Then, for the fibrous structure 110, an aqueous solution 125 of the target LNP120 containing the encapsulated target nucleic acid 121 is coated on the fibrous structure 110 ( Figure 19 b). As a result, the fibrous structure 110 captures both the labeled LNP130 and the target LNP120 ( Figure 19 B).

[0159] It is dried ( Figure 19 c), and the fibrous structure 110 contracts, the pores become smaller, and the labeled LNP130 and the target LNP120 captured therein are compressed from the outside by the fibers of the fibrous structure 110, come into contact with each other and start to fuse, and start to form the fused lipid nanoparticle 140a ( Figure 19 C).

[0160] Further drying it ( Figure 19 d) makes the pores even smaller, and then the labeled LNP130 and the target LNP120 are completely fused to form the fused LNP140 ( Figure 19 D).

[0161] Inside the fused lipid nanoparticle 140, the molecular beacon 131 encapsulated in the target LNP130 binds to the target nucleic acid 121 encapsulated in the target LNP120 ( Figure 19 D).

[0162] The fibrous structure 110 is placed in a container (tube) 151 containing the cleaning solution 150 and stirred ( Figure 19 e), the pores of the fibrous structure 110 open, and the fused LNP140 can be recovered.Figure 19 E).

[0163] Fluorescence observation of the thus obtained fused LNP140 can detect the fluorescence of the fluorophore 131f of the molecular beacon 131 hybridized with the target nucleic acid 121 within the fused LNP140 ( Figure 19 F).

[0164] It should be noted that in this embodiment and subsequent embodiments, the fibrous structure was dried after introducing the labeled LNP, but the present invention is not limited thereto. The fibrous structure may not be dried after introducing the labeled LNP or the target LNP may be introduced in a wet state of the fibrous structure. The fibrous structure may also be dried after introducing both the labeled LNP and the target LNP to shrink the pores. The order of introducing the labeled LNP and the target LNP should not be construed as being limited to these embodiments. The labeled LNP may be introduced after introducing the target LNP. The labeled LNP and the target LNP may be introduced simultaneously. The LNP may be introduced multiple times or repeatedly.

[0165] <Example 5>

[0166] In this example, a target LNP encapsulating the target miRNA and a labeled LNP encapsulating a molecular beacon designed to hybridize with the target miRNA were respectively prepared by the same method as above. The target miRNA was nucleic acid miR1246. The molecular beacon of the labeled LNP was designed to hybridize with this miR1246.

[0167] A PBS dispersion of a target LNP (hereinafter also referred to as "LNP(miR)") encapsulating miR1246 as the target nucleic acid and a PBS dispersion of a labeled LNP (hereinafter also referred to as "LNP(MB)") encapsulating a molecular beacon having Cy5 as the fluorophore and hybridizing with miR1246 were respectively prepared. The concentration of the LNP was appropriately adjusted. Dispersions of 10 μL each were mixed, and the mixture was applied to a 1 cm × 1 cm carbon nanofiber (CNF) sheet. Thereafter, the CNF sheet was dried in a dryer for 2 days or more. Thereby, a fused LNP (hereinafter also referred to as "(miR)+(MB)") was formed.

[0168] The dried CNF sheet was placed in a 1.5 mL tube, washed with 1 mL of PBS, the PBS washing solution was discarded, 50 μL of PBS was added for impregnation, and stirring was performed using a vortex mixer to extract the fused LNP and the remaining LNP.

[0169] Figure 20 Shows LNP(miR)( Figure 20 A), LNP(MB)( Figure 20 B) and (miR)+(MB)(Figure 20 Transmission electron microscopy images of (C). In (miR)+(MB) Figure 20 C), larger LNPs than LNP(miR) Figure 20 A) and LNP(MB) Figure 20 B) were confirmed.

[0170] Figure 21 Shows the particle size distribution of LNP(miR) Figure 21 A), LNP(MB) Figure 21 B) and (miR)+(MB) Figure 21 C) measured by nanoparticle tracking analysis. LNP(miR) has a peak at around 90 nm Figure 21 A), and LNP(MB) has a peak at around 100 nm Figure 21 B). In contrast, (miR)+(MB) was confirmed to have several peaks, with a maximum peak at around 130 nm and a size larger than LNP(miR) Figure 21 A) and LNP(MB) Figure 21 B) Figure 21 C). That is, it was shown that the size increased due to fusion.

[0171] Figure 22 Shows the results of flow cytometry of LNP(miR) Figure 22 A), LNP(MB) Figure 22 B) and (miR)+(MB) Figure 22 C). In the case of LNP(miR) Figure 22 A), almost all measurement points had a fluorescence intensity of approximately 2×10 1 , and were located further below. These points were counted as particles but not as optically labeled data. In the case of LNP(MB) Figure 22 B), only 1.3% was counted as a fluorescent label, and almost all measurement points had a fluorescence intensity of approximately 2×10 1 , and were located further below. In the case of (miR)+(MB) Figure 22 C), 28% was detected not as noise but as a signal corresponding to PC5 of the molecular beacon, which is the measurement object. From this, it was confirmed that as a result of the fusion, the molecular beacon hybridized with the target miR1246 within the fused LNP.

[0172] Figure 23 Shows LNP(miR) Figure 23 A), LNP(MB) Figure 23 B) and (miR)+(MB) Figure 24C) The fluorescence intensity measured by a microplate reader. From this result, it can also be confirmed that as a result of the fusion, the molecular beacon hybridized with the target miR1246 within the fused LNP.

[0173] Figure 24 Results showing the surface potential (ζ potential (mV)) of LNP(miR), LNP(MB), and (miR)+(MB). The surface potentials of LNP(miR) and LNP(MB) were approximately -10 mV and approximately +15 mV, respectively. On the other hand, the surface potential of the fused (miR)+(MB) was approximately +5 mV. This result indicates that through the fusion process, LNP(miR) and LNP(MB), both having negative charges, underwent membrane fusion.

[0174] <Example 6: Fusion of EVs from RPTEC with LNP(MB)>

[0175] Fusion of EVs from human renal proximal tubule epithelial cells (RPTEC) with LNP encapsulating a molecular beacon was performed. The molecular beacon was designed to hybridize with miR1246.

[0176] Cell culture and inflammatory activation were carried out as follows. EVs from activated RPTEC and EVs from non-activated RPTEC were prepared. First, RPTEC immortalized by TERT1 was cultured in Dulbecco's Modified Eagle Medium (DMEM) (high glucose, 10% FBS, 1% penicillin / streptomycin). After 48 hours from seeding, the medium was washed with PBS. It was then cultured with modified DMEM and 1% penicillin. The culture supernatant containing EVs was recovered after 24 hours. Thus, EVs from non-activated RPTEC were prepared. In the case of inflammatory non-activation, interleukin was added when switching to the above-mentioned modified DMEM. Specifically, recombinant human IL-6 100 ng / mL, recombinant human IL-6R 100 ng / mL, and recombinant human IL-17 50 ng / mL were added. Thus, EVs from activated RPTEC were prepared. For the following "miR1246", miR1246 was added to Lipofectamine and added when switching to the above-mentioned modified DMEM. For "miR1246+ILmix", miR1246 was added to Lipofectamine and it and IL-6, IL-6R, and IL-17 were added when switching to the above-mentioned modified DMEM.

[0177] Next, EV purification was carried out. First, the supernatant of RPTEC recovered from 3 15 cm × 15 cm culture dishes (about 50 mL) was centrifuged at 300 × g and 4 °C for 10 minutes to remove debris such as cell sheets. Then it was centrifuged at 2000 × g and 4 °C for 10 minutes, and passed through a 0.22 μm filter (Millipore, registered trademark) to remove coarse particles. Furthermore, it was ultracentrifuged at 110000 × g and 4 °C for 80 minutes, and finally concentrated to about 100 μL.

[0178] <Role of miR1246 in IL6 amplification>

[0179] Figure 25 The IL6 expression levels in the case of non-activation ("no"), the case of adding IL-6, IL-6R and IL-17 ("ILmix"), the case of adding miR1246 ("miR1246"), and the case of adding miR1246 and adding IL-6, IL-6R and IL-17 ("miR1246 + ILmix") are shown.

[0180] It can be seen that in the case of "ILmix", even in EVs from non-immune system cells such as RPTEC, IL6 is overexpressed, that is, IL6 amplification occurs. Overexpression was also confirmed when only miR1246 was added ("miR1246"). That is, it can be known that miR1246 becomes a trigger for IL6 expression. In the case of "miR1246 + ILmix", it can be seen that IL6 amplification is further enhanced.

[0181] From this, it can be known that miR1246 enables IL6 expression. In some embodiments, miR1246 can be used as a marker for IL6 amplification.

[0182] <Fusion process>

[0183] The fusion process is the same as in Example 5. Figure 26 The flow cytometry results of the fusion LNP ((1246)+(IL+)) of EV (EV(IL+)) from RPTEC activated by IL-6, IL-6R and IL-17 and LNP (1246MB) are shown Figure 26 A), and the fusion LNP ((1246)+(IL-)) of EV (IL-)) from non-activated RPTEC and LNP (1246MB) Figure 26 B) are shown.

[0184] In Figure 26In the case of non-activation of B ((1246)+(IL-)), 22.2% was detected as a signal. It is considered that miR1246 also has functions other than IL6 amplification and exists even in the case of non-activation. In contrast, Figure 26 In the case of activation of A ((1246)+(IL+)), 38.7% was detected as a signal. It is shown that the expression level of miR1246 increases upon activation, which can be identified by the hybridization of the molecular beacon labeling the LNP due to the fusion of the LNP.

[0185] <Embodiment 3>

[0186] In some embodiments, the target LNP can be stained with a labeled antibody (e.g., a labeled antibody for immunofluorescent staining). As a result, the fused LNP can have a stained site on its protein membrane and a fluorophore of the molecular beacon inside. Therefore, the fused LNP can be detected and observed by two optical measurements.

[0187] Refer to Figure 27 to schematically illustrate the fusion of lipid nanoparticles based on the contraction of a fibrous structure in one embodiment, and the process of mixing and reacting the substances inside thereof. Figure 27 The upper figures (a1) to (e) in [reference] show schematic diagrams of the structures observed macroscopically. Figure 27 The lower figures (A) to (F) in [reference] show schematic diagrams of the structures observed microscopically corresponding to the figures (a1) to (e).

[0188] First, an aqueous solution 235 ( Figure 27 A) containing labeled LNP 230 encapsulating molecular beacon 231 is coated on the fibrous structure 210 ( Figure 27 a1). Then the fibrous structure 210 is dried ( Figure 27 a2). The labeled LNP 230 is captured inside the pores of the fibrous structure 210 ( Figure 27 A). The molecular beacon 231 is designed to hybridize with the target nucleic acid 221 encapsulated in the target LNP 220 shown in the following figure.

[0189] First, a target LNP 220 encapsulating the target nucleic acid 221 is prepared so that its membrane protein surface is recognized by a labeled antibody 223 (not shown). Then, for the fibrous structure 210, an aqueous solution 225 containing the target LNP 220 is coated on the fibrous structure 210 ( Figure 27 b). As a result, the fibrous structure 210 captures both the labeled LNP 230 and the target LNP 220 ( Figure 27 B).

[0190] It is dried ( Figure 27c), the fibrous structure 210 shrinks, the pores become smaller, and the labeled LNP 230 and the target LNP 220 captured therein are compressed from the outside by the fibers of the fibrous structure 210, come into contact with each other and begin to fuse, and begin to form a fused lipid nanoparticle 240a( Figure 27 C).

[0191] Further drying( Figure 27 d) The pores become further smaller, and accordingly the labeled LNP 230 and the target LNP 220 are completely fused to form a fused LNP 240( Figure 27 D).

[0192] Inside the fused lipid nanoparticle 240, the molecular beacon 231 encapsulated in the target LNP 230 binds to the target nucleic acid 221 encapsulated in the target LNP 220( Figure 27 D). The membrane protein of the fused lipid nanoparticle 240 has a labeled antibody 223 derived from the target LNP 220.

[0193] The fibrous structure 210 is placed in a container (tube) 251 containing a cleaning solution 250 and stirred( Figure 27 e), the pores of the fibrous structure 210 open, and the fused LNP 240 can be recovered( Figure 27 E).

[0194] Fluorescence observation of the thus obtained fused LNP 240 can detect both the fluorescence of the fluorophore 231f of the molecular beacon 231 hybridized with the target nucleic acid 221 in the fused LNP 240 and the fluorescence of the fluorescence label 223f bound to the membrane protein( Figure 27 F).

[0195] <Examples 7 and 8>

[0196] In Example 7, EVs derived from RPTEC were prepared in the same manner as in Example 5. In Example 8, CD6 or LDHB (lactate dehydrogenase B) of the membrane protein was stained with FITC (fluorescein isothiocyanate). Specifically, each labeled antibody was added to the prepared EVs to a concentration of 100 nM, and incubated overnight at 4°C. After that, it was washed with PBS and subjected to ultracentrifugation. The fusion process was the same as in Example 5.

[0197] Figure 28 The results of flow cytometry of Example 7 are shown, Figure 29 The results of flow cytometry of Example 8 are shown. The horizontal axis (FITC-A) represents the fluorescence intensity of FITC modifying the membrane protein. The vertical axis (PC5-A) represents the fluorescence intensity of the fluorophore PC5 of the molecular beacon. The % in the figure is the ratio of the part detected as a signal among both the FITC labeled on the membrane protein and the fluorophore PC5 of the molecular beacon.

[0198] In any case, relative to non-activation ( Figure 28 12.7% in B, Figure 29 7.6% in B), the amount of the signal is increased by activation ( Figure 28 41.2% in A, Figure 29 20.0% in A). This implies two points: the expression level of miR1246 is enhanced by activation and the expression level of the membrane protein is increased. It is considered that as the expression level of the membrane protein increases, the expression level of miR also increases.

[0199] In some embodiments, an antibody label can also be bound to multiple membrane proteins. Thereby, multiple membrane proteins can be efficiently detected. In some embodiments, the labeled LNP can contain multiple molecular beacons that hybridize to different nucleic acids respectively. In some embodiments, multiple labeled LNPs can be provided and different molecular beacons can be encapsulated in each labeled LNP. For example, the first labeled LNP can encapsulate the first molecular beacon that hybridizes to the first nucleic acid, the second labeled LNP can encapsulate the second molecular beacon that hybridizes to the second nucleic acid, and these can be simultaneously introduced into the target LNP. According to these embodiments, multiple nucleic acids in the target LNP can be efficiently detected. In some embodiments, these can be combined to simultaneously detect multiple membrane proteins and multiple target nucleic acids.

[0200] By using these methods, the diseases and their disease sites possessed by the subject can be inferred or determined. The source site can be inferred based on the characteristics of the membrane proteins of the target LNP. According to the nucleic acids encapsulated in the target LNP, diseases can be inferred. For example, miR1246 inside can be detected from EVs obtained from body fluids such as blood, and the type or presence of autoimmune diseases can be inferred based on the presence and / or degree of IL6 amplification. At the same time, the source site of the cells that excrete EVs can be inferred based on the membrane proteins of the EVs. For example, by detecting CD81 as a membrane protein, it can be inferred whether rheumatoid arthritis is present, that is, the site can be inferred as the joint.

[0201] The present invention also provides the following embodiments:

[0202] A001

[0203] A method for fusing lipid nanoparticles, which comprises the following steps:

[0204] Providing a fibrous structure;

[0205] Providing a first solution containing a first lipid nanoparticle;

[0206] Providing a second solution containing a second lipid nanoparticle;

[0207] The above fibrous structure absorbs the above first solution to capture the above first lipid nanoparticles;

[0208] The above fibrous structure absorbs the above second solution to capture the above second lipid nanoparticles; and

[0209] The above fibrous structure is contracted to fuse the above first lipid nanoparticles and the above second lipid nanoparticles (thereby forming fused lipid nanoparticles).

[0210] A001b

[0211] A method for fusing lipid nanoparticles, comprising the following steps:

[0212] A fibrous structure absorbs a first solution containing first lipid nanoparticles and a second solution containing second lipid nanoparticles to capture the above first lipid nanoparticles and the above second lipid nanoparticles; and

[0213] (Removing at least a part of the moisture from the above fibrous structure,) the above fibrous structure is contracted to fuse the above first lipid nanoparticles and the above second lipid nanoparticles (thereby forming fused lipid nanoparticles).

[0214] A001c

[0215] A method for fusing lipid nanoparticles, comprising the following steps:

[0216] A fibrous structure captures first lipid nanoparticles;

[0217] The above fibrous structure captures second lipid nanoparticles; and

[0218] The above fibrous structure is contracted to fuse the above first lipid nanoparticles and the above second lipid nanoparticles (thereby forming fused lipid nanoparticles).

[0219] A011

[0220] According to the method described in any one or any embodiment of A001 to A001c, wherein,

[0221] The first lipid nanoparticles encapsulate a first substance,

[0222] The second lipid nanoparticles encapsulate a second substance,

[0223] The step of fusing the above first lipid nanoparticles and the above second lipid nanoparticles (thereby forming fused lipid nanoparticles) comprises the step of mixing the above first substance and the above second substance and / or reacting the above first substance and the above second substance.

[0224] A012

[0225] The method according to any one of A001 to A011 or any embodiment, wherein the first lipid nanoparticle and the second lipid nanoparticle have the same sign of charge.

[0226] A021

[0227] The method according to any one of A001 to A012 or any embodiment, wherein

[0228] the first lipid nanoparticle is an extracellular vesicle (EV) encapsulating nucleic acid,

[0229] the second lipid nanoparticle encapsulates a lipid nanoparticle encapsulating a molecular beacon that binds (hybridizes) to the target sequence of the nucleic acid,

[0230] The step of fusing the first lipid nanoparticle and the second lipid nanoparticle includes the step of allowing the molecular beacon to recognize the nucleic acid.

[0231] The method further includes the step of measuring the luminescence from the molecular beacon (or the fluorophore of the molecular beacon).

[0232] A021b

[0233] A method for inferring biological information of a subject, comprising the following steps:

[0234] Providing a first solution containing a first lipid nanoparticle from a subject;

[0235] Providing a second solution containing a second lipid nanoparticle encapsulating a molecular beacon that hybridizes with a nucleic acid that may be encapsulated in the first lipid nanoparticle;

[0236] Allowing the fibrous structure to absorb the first solution to capture the first lipid nanoparticle;

[0237] Allowing the fibrous structure to absorb the second solution to capture the second lipid nanoparticle;

[0238] Allowing the fibrous structure to contract to fuse the first lipid nanoparticle and the second lipid nanoparticle (thereby forming a fused lipid nanoparticle), allowing the molecular beacon to recognize the nucleic acid; and

[0239] Measuring the luminescence of the molecular beacon in the fused lipid nanoparticle.

[0240] A025

[0241] The method according to any one of A001 to A021 or any embodiment, further comprising the following steps:

[0242] Label the membrane protein of the first lipid nanoparticle with a (e.g., optical) labeled antibody; and

[0243] Determine the labeled antibody possessed by the fusion nanoparticle described above.

[0244] A026

[0245] A method for inferring biological information of a subject, comprising the following steps:

[0246] Provide a first lipid nanoparticle from the subject;

[0247] Label the membrane protein of the first lipid nanoparticle with a (e.g., optical) labeled antibody (e.g., an antibody having an optical label);

[0248] Provide a first solution containing the labeled first lipid nanoparticle;

[0249] Provide a second solution containing a second lipid nanoparticle encapsulating a molecular beacon hybridizable with a nucleic acid that may be encapsulated in the first lipid nanoparticle;

[0250] Cause the fibrous structure to absorb the first solution and capture the first lipid nanoparticle;

[0251] Cause the fibrous structure to absorb the second solution and capture the second lipid nanoparticle;

[0252] Cause the fibrous structure to contract to fuse the first lipid nanoparticle and the second lipid nanoparticle (thereby forming a fusion lipid nanoparticle), and cause the molecular beacon to recognize the nucleic acid; and

[0253] Measure the luminescence of the molecular beacon within the fusion lipid nanoparticle; and

[0254] Measure the above-mentioned (optical) label of the membrane protein of the fusion lipid nanoparticle.

[0255] A027

[0256] The method according to A026 or any embodiment, wherein the first lipid nanoparticle is an extracellular vesicle (EV) from the subject.

[0257] A028

[0258] The method according to A027 or any embodiment, wherein

[0259] the labeled antibody is a fluorescently labeled antibody,

[0260] the step of measuring the labeled antibody includes the step of measuring the luminescence from the fluorescently labeled antibody.

[0261] A029

[0262] The method according to any one of A027 or A028, or any embodiment, further comprises the following steps:

[0263] Inferring the disease of the subject based on the measurement of the luminescence from the above-mentioned molecular beacon; and

[0264] Inferring the location of the disease of the subject based on the measurement of the above-mentioned labeled antibody.

[0265] A029b

[0266] The method according to any one of A027 or A028, or any embodiment, further comprises the following steps:

[0267] Inferring one or both of the type and location of the disease of the subject based on any one, at least one, or both of the measurement of the luminescence from the above-mentioned molecular beacon and the measurement of the above-mentioned labeled antibody.

[0268] A031

[0269] The method according to any one of A001 to A012 or any embodiment, wherein

[0270] The above-mentioned first lipid nanoparticle is an extracellular vesicle (EV) from the subject,

[0271] The above-mentioned second lipid nanoparticle is a medicament.

[0272] A032

[0273] The method according to A31 or any embodiment, wherein

[0274] The above-mentioned first lipid nanoparticle is an extracellular vesicle (EV) from a cancer patient (subject),

[0275] The above-mentioned second lipid nanoparticle is an anticancer agent (medicament) for the above-mentioned cancer.

[0276] A033

[0277] The method according to A032 or any embodiment, further comprises the following step: administering the above-mentioned fusion nanoparticle to the above-mentioned cancer patient.

[0278] A041

[0279] The method according to any one of A001 to A033 or any embodiment, wherein the above-mentioned first lipid nanoparticle and the above-mentioned second lipid nanoparticle have the same sign of charge.

[0280] B001

[0281] An artificial lipid nanoparticle encapsulating a molecular beacon having a complementary sequence (hybridizing with the target nucleic acid) to the target nucleic acid encapsulated in the extracellular vesicles of the subject.

[0282] B002

[0283] The artificial lipid nanoparticle according to B001 or any embodiment, wherein the target nucleic acid is a microRNA.

[0284] B011

[0285] An artificial lipid nanoparticle containing a medicament.

[0286] B012

[0287] The artificial lipid nanoparticle according to B011 or any embodiment, wherein the medicament is an anticancer agent or contains an anticancer agent.

[0288] B013

[0289] The artificial lipid nanoparticle according to B001 or B002, or any embodiment, which is configured to fuse with the extracellular vesicles of the subject using a fibrous structure.

[0290] B101

[0291] The artificial lipid nanoparticle according to any one of B001 to B013 or any embodiment, which is a medicament or a DDS medicament.

[0292] B102

[0293] Use of the artificial lipid nanoparticle according to any one of B001 to B013 or any embodiment as a medicament or a DDS medicament.

[0294] Preferred embodiments of the present invention are shown and described in this specification, but such embodiments are provided only by way of example, which will be apparent to those skilled in the art. The present invention is not intended to be limited by the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments of this specification are not meant to be construed in a limiting sense. Those skilled in the art will envision various modifications, changes, and substitutions without departing from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative ratios described in this specification that depend on various conditions and variables. It should be understood that various alternative ways of the embodiments of the present invention described in this specification can be used when implementing the present invention. Therefore, the present invention is also intended to cover such alternatives, changes, modifications, or equivalents. The scope of the present invention is intended to be defined by the claims of this application, and such methods and structures within the claims and their equivalents are hereby covered.

Claims

1. A method for fusing lipid nanoparticles, comprising the following steps: Capturing a first lipid nanoparticle with a fibrous structure; Capturing a second lipid nanoparticle with the fibrous structure; and Contracting the fibrous structure to fuse the first lipid nanoparticle and the second lipid nanoparticle.

2. The method according to claim 1, wherein the first lipid nanoparticle encapsulates a first substance, the second lipid nanoparticle encapsulates a second substance, and the step of fusing the first lipid nanoparticle and the second lipid nanoparticle comprises the steps of mixing the first substance and the second substance and / or reacting the first substance and the second substance.

3. The method according to claim 1, wherein the first lipid nanoparticle and the second lipid nanoparticle have the same-sign charge.

4. The method according to claim 1, wherein the first lipid nanoparticle is an extracellular vesicle (EV) encapsulating nucleic acid, the second lipid nanoparticle is a lipid nanoparticle encapsulating a molecular beacon that binds (hybridizes) to the target sequence of the nucleic acid, the step of fusing the first lipid nanoparticle and the second lipid nanoparticle comprises the step of allowing the molecular beacon to recognize the nucleic acid, and the method further comprises the step of measuring the luminescence from the molecular beacon.

5. The method according to claim 1, wherein the first lipid nanoparticle is an extracellular vesicle (EV) from a subject, and the second lipid nanoparticle is a medicament.

6. The method according to claim 5, wherein the first lipid nanoparticle is an extracellular vesicle (EV) from a cancer patient (subject), and the second lipid nanoparticle is an anticancer agent (medicament) for the cancer.

7. An artificial lipid nanoparticle encapsulating a molecular beacon having a sequence complementary to a target nucleic acid encapsulated in a target extracellular vesicle.

8. The artificial lipid nanoparticle according to claim 7, wherein the target nucleic acid is microRNA.

9. An artificial lipid nanoparticle containing a medicament.

10. The artificial lipid nanoparticle according to claim 8, wherein the medicament is an anticancer agent or contains an anticancer agent.

11. The artificial lipid nanoparticle according to any one of claims 7 to 10, which is configured to fuse with a target extracellular vesicle from a subject using a fibrous structure.

12. A method for inferring biological information of a subject, comprising the following steps: Providing a first solution containing a first lipid nanoparticle from the subject; Providing a second solution containing a second lipid nanoparticle encapsulating a molecular beacon that hybridizes with a nucleic acid that may be encapsulated in the first lipid nanoparticle; Absorbing the first solution with the fibrous structure to capture the first lipid nanoparticle; Absorbing the second solution with the fibrous structure to capture the second lipid nanoparticle; Contracting the fibrous structure to fuse the first lipid nanoparticle and the second lipid nanoparticle, forming a fused lipid nanoparticle, and allowing the molecular beacon to recognize the nucleic acid; and Measure the luminescence of the molecular beacon within the fusion lipid nanoparticles.

13. A method for inferring biological information of a subject, comprising the following steps: Provide a first lipid nanoparticle from the subject; Label the membrane protein of the first lipid nanoparticle with an optically labeled antibody; Provide a first solution containing the labeled first lipid nanoparticle; Provide a second solution containing second lipid nanoparticles encapsulating a molecular beacon that hybridizes with a nucleic acid that may be encapsulated in the first lipid nanoparticle; Cause the fibrous structure to absorb the first solution and capture the first lipid nanoparticle; Cause the fibrous structure to absorb the second solution and capture the second lipid nanoparticle; Cause the fibrous structure to contract to fuse the first lipid nanoparticle and the second lipid nanoparticle, forming fusion lipid nanoparticles, and cause the molecular beacon to recognize the nucleic acid; and Measure the luminescence of the molecular beacon within the fusion lipid nanoparticles; and Measure the optical label of the membrane protein of the fusion lipid nanoparticles.

14. The method according to claim 12 or 13, wherein the first lipid nanoparticle is an extracellular vesicle (EV) from the subject.

15. The method according to claim 12, further comprising the following step: Infer one or both of the type and location of the disease of the subject based on the measurement of the luminescence from the molecular beacon.

16. The method according to claim 13, further comprising the following step: Infer one or both of the type and location of the disease of the subject based on either or both of the measurement of the luminescence from the molecular beacon and the measurement of the labeled antibody.

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