Tissue-targeting nanoparticles and method for preparing same

Abstract

The present invention provides: a method for preparing tissue-targeting nanoparticles, the method comprising a step for generating phospholipid nanoparticles, a step for generating mesenchymal stem cell-derived cell membrane nanoparticles, and a step for fusing the generated phospholipid nanoparticles and cell membrane nanoparticles to generate tissue-targeting nanoparticles; and tissue-targeting nanoparticles which are prepared by the method and comprise bisphosphonate-containing phospholipid nanoparticles and mesenchymal stem cell-derived cell membrane nanoparticles.

Description

Tissue-targeting nanoparticles and methods for manufacturing the same

[0001] The present invention relates to tissue-targeting nanoparticles, which are nanoparticles capable of selectively targeting tissue, namely bone tissue, and a method for manufacturing the same.

[0002] Nanotechnology refers to the technology of manipulating materials of nanometer scale, encompassing the synthesis, assembly, and control of atomic, molecular, and supramolecular materials, as well as the measurement and characterization of their properties. Nanotechnology has a very wide range of applications, encompassing various scientific fields such as surface science, organic chemistry, molecular biology, semiconductor physics, and microfabrication. Nanotechnology can create new materials with extensive applications in fields such as medicine, electronics, biomaterials, energy production, and consumer products.

[0003] Since the COVID-19 pandemic, the use of such nanotechnology in the pharmaceutical and bio industries has significantly increased due to the risks of infectious diseases and phenomena such as population aging. More specifically, a Drug Delivery System (DDS) refers to a series of materials and technologies capable of safely and effectively delivering substances with pharmacological activity to target sites within the body, such as cells, tissues, organs, and tissues, and controlling their release. DDS is attracting attention as a very important technology in the medical field because it can drastically enhance drug efficacy while simultaneously reducing the side effects of existing drugs. To date, various nanomaterials have been studied in areas such as disease diagnosis, drug delivery, and molecular medical imaging. Furthermore, nanoparticles incorporating various forms of nanotechnology—such as liposomes, proteins, polymers, micelles, emulsions, nanocapsules, dendrimers, and nanoparticles—are being researched and developed. Currently, some nanoparticles are approved by the U.S. FDA and are being marketed and applied in clinical settings; these include encapsulated mRNA (siRNA) or DNA (in gene therapy), inorganic metals and metal complexes, or chemotherapy agents with pharmacological capabilities.

[0004] However, in the medical field, nanotechnology still faces limitations in that safety aspects, such as toxicity, need to be verified. More specifically, nanoparticles are captured by the mononuclear phagocyte system within the body. Furthermore, the increased surface area of ​​nanoparticles enhances their chemical reactivity; this increased reactivity generates reactive oxygen species that can cause oxidative stress, inflammation, and damage to DNA, proteins, and cell membranes, potentially leading to adverse effects in the body. Additionally, when nanoparticles are administered, they can pass through cell membranes via capillaries and invade areas other than the target site, potentially exerting abnormal pharmacological effects. Moreover, since nanoparticles can cause effects not observed in conventional medicine, such as damage to various cellular organelles including the nucleus and mitochondria, the carrier system itself may induce toxicity. Therefore, further research is required to verify the safety and efficacy of nanotechnology for drug carriers, specifically Drug Delivery Systems (DDS).

[0005] The background description of the invention is provided to facilitate a better understanding of the present invention. The matters described in the background description should not be construed as an acknowledgment that they exist as prior art.

[0006] Lipid-based nanoparticles in DDS are widely utilized as drug delivery systems due to their ease of surface modification and drug loading. However, as artificial phospholipid nanoparticles, such as synthetic liposomes currently used as vaccine carriers, are known to cause various side effects including immune rejection, there is a demand for the development of biocompatible drug delivery systems.

[0007] Accordingly, the inventors of the present invention focused on cell membranes to overcome the limitations of the aforementioned lipid-based DDS. More specifically, cell membranes composed of a phospholipid bilayer can be used as lipid-based DDS, and such cell membrane-based nanoparticles can target specific cells or tissues or evade attacks by immune cells depending on the origin of the cell membrane. However, cell membrane-based nanoparticles have the disadvantage of having lower utility value as a DDS due to lower surface modification and drug loading efficiency compared to artificial phospholipid nanoparticles.

[0008] Meanwhile, the inventors of the present invention noted the characteristic that phospholipids can self-assemble into liposomal forms, and accordingly recognized that self-assembly can be induced when phospholipid nanoparticles and cell membrane nanoparticles are decomposed and then mixed.

[0009] Ultimately, the inventors of the present invention generated hybrid nanoparticles incorporating the respective advantages by degrading phospholipid-based nanoparticles and cell membrane-based nanoparticles, then inducing self-assembly and fusing them. Furthermore, by loading drugs onto the generated hybrid nanoparticles, the inventors developed a novel drug delivery system capable of delivering drugs to target tissues with high efficiency.

[0010] Accordingly, the problem to be solved by the present invention is to provide a method for manufacturing tissue-targeting nanoparticles, comprising the steps of generating phospholipid nanoparticles, generating cell membrane nanoparticles derived from mesenchymal stem cells, and fusing the generated phospholipid nanoparticles and the cell membrane nanoparticles so as to generate tissue-targeting nanoparticles, and to provide tissue-targeting nanoparticles generated based thereon.

[0011] The problems of the present invention are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0012] To solve the problem described above, the present invention provides a method for producing tissue-targeting nanoparticles comprising the steps of generating phospholipid nanoparticles, generating cell membrane nanoparticles derived from mesenchymal stem cells, and fusing the generated phospholipid nanoparticles and cell membrane nanoparticles to produce tissue-targeting nanoparticles.

[0013] According to the features of the present invention, the tissue may include bone tissue, but is not limited thereto.

[0014] According to another feature of the present invention, phospholipid nanoparticles comprise bisphosphonate (BP), tetracyclines, CXC chemokine receptor type 4 (CXCR4), Ephrin type B receptor 4 (EphB4), Dentin Matrix Protein 1 (DMP1), anti-sclerostin antibody, anti-type I collagen antibody, acidic oligopeptide, TRAP binding peptide, Ser-Asp-Ser-Ser-Asp (SDSSD) peptide, (DSS)6 peptide, Asp-rich peptide ((Asp) 14 It may be selected from the group consisting of (AspSerSer)6) and CH6 aptamers, but is not limited thereto.

[0015] According to another feature of the present invention, the step of generating phospholipid nanoparticles comprises: mixing a first solution containing DSPE-PEG-MAL (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)2000]) and a second solution containing Thiol-BP to generate DSPE-PEG-BP; dissolving a mixture containing DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, DSPE-PEG, and the generated DSPE-PEG-BP in a nonpolar solvent to generate a lipid membrane; and inducing self-assembly of the generated lipid membrane to generate phospholipid nanoparticles.

[0016] According to another feature of the present invention, the step of producing DSPE-PEG-BP comprises the step of mixing a first solution and a second solution to obtain a first reaction solution, the step of dialyzing the obtained first reaction solution to obtain a purified second reaction solution so as to remove residual thiol-BP in the first reaction solution, and the step of freeze-drying the second reaction solution to produce DSPE-PEG-BP.

[0017] According to another feature of the present invention, the step of obtaining the first reaction solution may be performed for 50 to 70 minutes, but is not limited thereto.

[0018] According to another feature of the present invention, the first solution comprises 30 to 35 mg / ml of DSPE-PEG-MAL.

[0019] According to another feature of the present invention, the second solution contains 35 to 45 mg / ml of Thiol-BP.

[0020] According to another feature of the present invention, the step of generating a lipid membrane may be performed for 40 to 56 hours, but is not limited thereto.

[0021] According to another feature of the present invention, the mixture comprises DSPC, cholesterol, DSPE-PEG, and the generated DSPE-PEG-BP in a ratio of 65 to 75 : 15 to 25 : 3 to 7 : 3 to 7 mol %, respectively.

[0022] According to another feature of the present invention, the step of generating phospholipid nanoparticles includes the step of ultrasonically treating the generated lipid membrane and the step of extruding the ultrasonically treated lipid membrane.

[0023] According to another feature of the present invention, the step of generating phospholipid nanoparticles further includes, after the extrusion step, a step of removing byproducts using a filter.

[0024] According to another feature of the present invention, the step of generating cell membrane nanoparticles includes the step of crushing mesenchymal stem cells to a size of 0.5 to 1.5 μm, and the step of extruding the crushed mesenchymal stem cells so as to induce self-assembly of the crushed mesenchymal stem cells.

[0025] According to another feature of the present invention, the step of generating cell membrane nanoparticles further includes, after the extrusion step, a step of removing byproducts using a filter.

[0026] According to another feature of the present invention, the grinding step comprises the step of preparing a first cell suspension by suspending mesenchymal stem cells in a culture medium, and the step of freezing and thawing the first cell suspension so that the mesenchymal stem cells are physically crushed.

[0027] According to another feature of the present invention, the step of freezing and thawing the first cell suspension may be performed five or more times, but is not limited thereto.

[0028] According to another feature of the present invention, thawing may be performed for 30 to 50 minutes, but is not limited thereto.

[0029] According to another feature of the present invention, the fusing step comprises the steps of: preparing a first nanoparticle suspension by suspending the generated phospholipid nanoparticles and cell membrane nanoparticles in a culture medium; freezing and thawing the first nanoparticle suspension to generate fused particles; and extruding the generated fused particles so that final tissue-targeting nanoparticles are generated from the fused particles.

[0030] According to another feature of the present invention, the step of preparing a first nanoparticle suspension includes suspending the generated phospholipid nanoparticles and cell membrane nanoparticles in a medium at a 1:1 ratio.

[0031] According to another feature of the present invention, the step of generating fusion particles may be performed 10 or more times, but is not limited thereto.

[0032] According to another feature of the present invention, thawing may be performed for 30 to 50 minutes, but is not limited thereto.

[0033] According to another feature of the present invention, the fusing step further includes a step of removing by-products using a filter after the extrusion step.

[0034] According to another feature of the present invention, the step of generating phospholipid nanoparticles further includes the step of loading a drug containing genetic material onto the generated phospholipid nanoparticles.

[0035] According to another feature of the present invention, the loading step comprises the steps of preparing a second nanoparticle suspension by suspending phospholipid nanoparticles in an organic solvent, preparing a dielectric material suspension by suspending a dielectric material in a buffer solution, and dual-injecting the second nanoparticle suspension and the dielectric material suspension into a single container.

[0036] According to another feature of the present invention, the organic solvent and buffer solution have a pH of 3.5 to 4.5.

[0037] According to another feature of the present invention, the genetic material may include mRNA and plasmid DNA, but is not limited thereto.

[0038] To solve another problem as described above, the present invention provides a tissue-targeting nanoparticle manufactured by a method for manufacturing tissue-targeting nanoparticles, wherein the tissue-targeting nanoparticle is fused from a phospholipid nanoparticle containing bisphosphonate (BP) and a cell membrane nanoparticle derived from a mesenchymal stem cell.

[0039] According to another feature of the present invention, phospholipid nanoparticles may include phospholipids and sterol-based lipids, but are not limited thereto.

[0040] According to another feature of the present invention, the phospholipids include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol) 2000]), DSPE-PEG-MAL(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)2000]), DOPE(1,2-diacyl-sn-glycero-3-phosphoethanolamine), DOPE-mal(dioleoyl-sn-glycero-3-phosphoethanolamine-N-(maleimidomethyl)), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine), It may include at least one of DMPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPG (1,2-dipalmitoyl-sn-glycero-3-Phospho-rac-(1-glycerol)), DLPC (1,2-dilauroylsn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), but is not limited thereto.

[0041] According to another feature of the present invention, BP may be in a form combined with DSPE-PEG.

[0042] According to another feature of the present invention, the sterol lipid may include at least one of cholesterol, sitosterol, stigmasterol, campesterol, and ergosterol, but is not limited thereto.

[0043] According to another feature of the present invention, tissue-targeting nanoparticles may have a diameter of 1 to 200 nm, but are not limited thereto.

[0044] According to another feature of the present invention, the phospholipid nanoparticles and cell membrane nanoparticles may be in a 1:1 ratio, but are not limited thereto.

[0045] According to another feature of the present invention, tissue-targeting nanoparticles can target tissues containing Ca, and preferably bone tissues containing Ca.

[0046] According to another feature of the present invention, fusion can be performed by a physical method.

[0047] According to another feature of the present invention, the tissue-targeting nanoparticles may further include at least one drug among genetic material drugs, ionic drugs, and protein drugs, but are not limited thereto.

[0048] According to another feature of the present invention, the genetic material may include at least one of mRNA and plasmid DNA, but is not limited thereto.

[0049] According to another feature of the present invention, mRNA is a tissue-targeting nanoparticle 1×10 10 More than 16 ng can be loaded per piece.

[0050] According to another feature of the present invention, plasmid DNA is a tissue-targeting nanoparticle 1×10 10 More than 29 ng can be loaded per piece.

[0051] The present invention will be explained in more detail below through examples. However, since these examples are merely illustrative of the present invention, the scope of the present invention should not be interpreted as being limited by these examples.

[0052] The effects according to the present invention are not limited to those exemplified above, and various other effects are included in this specification.

[0053] FIG. 1a is a flowchart of a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention.

[0054] FIG. 1b is a flowchart of a method for producing phospholipid nanoparticles in a method for producing tissue-targeting nanoparticles according to one embodiment of the present invention.

[0055] FIG. 1c is a flowchart of a method for generating cell membrane nanoparticles in a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention.

[0056] FIG. 1d is a flowchart illustrating a method for fusing tissue-targeting nanoparticles in a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention.

[0057] FIG. 1e is a flowchart illustrating a method for loading a drug containing genetic material onto phospholipid nanoparticles in a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention.

[0058] FIG. 2 is a schematic diagram of a tissue-targeting nanoparticle according to one embodiment of the present invention.

[0059] Figure 3 shows the results regarding the size and distribution of tissue-targeting nanoparticles according to one embodiment of the present invention.

[0060] Figure 4 is a result of confirming the fusion of tissue-targeting nanoparticles according to one embodiment of the present invention.

[0061] FIGS. 5a to 5d are the results of ex vivo tissue targeting verification of tissue-targeting nanoparticles according to one embodiment of the present invention with and without BP.

[0062] Figures 6a to 6c show the results of in vivo tissue targeting verification of tissue-targeting nanoparticles according to one embodiment of the present invention.

[0063] Figure 7 is the result of an analysis of the intracellular delivery efficiency of tissue-targeting nanoparticles according to one embodiment of the present invention.

[0064] Figure 8 shows the results regarding the loading efficiency of genetic material (mRNA, plasmid DNA) of tissue-targeting nanoparticles according to one embodiment of the present invention.

[0065] Figure 9 shows the results regarding the gene delivery efficiency of tissue-targeting nanoparticles loaded with mRNA according to one embodiment of the present invention.

[0066] Figure 10 shows the results regarding the gene delivery efficiency of tissue-targeting nanoparticles according to one embodiment of the present invention loaded with plasmid DNA.

[0067] Figure 11 shows the results regarding the bone differentiation efficiency of tissue-targeting nanoparticles according to one embodiment of the present invention loaded with mRNA.

[0068] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0069] In this specification, expressions such as “have,” “may have,” “include,” or “may include” indicate the presence of such features (e.g., numerical values, functions, operations, or components such as parts) and do not exclude the presence of additional features.

[0070] In this specification, expressions such as “A or B,” “at least one of A or / and B,” or “one or more of A or / and B” may include all possible combinations of items listed together. For example, “A or B,” “at least one of A and B,” or “at least one of A or B” may refer to cases including (1) at least one A, (2) at least one B, or (3) both at least one A and at least one B.

[0071] The expression “configured to” as used in this specification may be replaced, depending on the context, with, for example, “suitable for,” “having the capacity to,” “designed to,” “adapted to,” “made to,” or “capable of.”

[0072] As used in this specification, the term "or" means "and / or" unless otherwise stated.

[0073] The term 'pharmaceuticalally acceptable salt' as used in the present invention refers to an acidic or basic salt that may be present in the hyaluronic acid and carboxymethylcellulose of the present invention, unless otherwise indicated. For example, the pharmaceutically acceptable salt includes sodium salts, potassium salts, calcium salts, etc., and preferably includes sodium salts.

[0074] As used herein, the term “about” refers to a normal margin of error for each value that is readily known to those skilled in the art. In this specification, the designation of an “about” value or parameter includes an example relating to the value or parameter itself. Furthermore, unless otherwise stated or otherwise evident from the context, the term “about” indicates a range of values ​​corresponding to within 10% in either direction (greater than or less than) a mentioned reference value.

[0075] As used herein, the term “patient or individual” refers interchangeably to any single animal requiring treatment, more preferably a mammal (including such non-human animals, e.g., cats, dogs, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates). In various embodiments of this specification, the patient referred to may be a human.

[0076] The terms used in this document are used merely to describe specific embodiments and are not intended to limit the scope of other embodiments. Singular expressions may include plural expressions unless the context clearly indicates otherwise. Terms used herein, including technical or scientific terms, may have the same meaning as generally understood by those skilled in the art described in this document. Terms used in this document that are defined in general dictionaries may be interpreted as having the same or similar meaning as they have in the context of the relevant technology, and are not to be interpreted in an ideal or overly formal sense unless explicitly defined in this document. In some cases, even terms defined in this document may not be interpreted to exclude the embodiments of this document.

[0077] The features of each of the various embodiments of the present invention may be combined or combined with one another, either partially or wholly, and as will be fully understood by those skilled in the art, various technical interlocking and operation are possible, and each embodiment may be implemented independently of one another or together in an interrelationship.

[0078] Tissue-targeting nanoparticles according to one embodiment of the present invention and a method for manufacturing the same

[0079] Hereinafter, with reference to FIGS. 1a to 2, a tissue-targeting nanoparticle according to one embodiment of the present invention and a method for manufacturing the same will be described.

[0080] FIG. 1a is a flowchart of a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention. For convenience of explanation, the description will be made with reference to FIG. 1b to 1e.

[0081] Referring to FIG. 1a, a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention comprises the steps of generating phospholipid nanoparticles (S110), generating cell membrane nanoparticles derived from mesenchymal stem cells (S120), and fusing the generated phospholipid nanoparticles and cell membrane nanoparticles (S130).

[0082] At this time, the nanoparticles in the step (S110) of generating phospholipid nanoparticles may include bisphosphonate (BP), and accordingly, can target bone tissue. Meanwhile, the phospholipid nanoparticles in the method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention may include not only BP but also various tissue-targeting substances such as small molecules, proteins, antibodies, peptides, and aptamers, specifically Tetracyclines, CXC chemokine receptor type 4 (CXCR4), Ephrin type B receptor 4 (EphB4), Dentin Matrix Protein 1 (DMP1), Anti-Sclerostin antibody, Anti-Type I collagen antibody, Acidic oligopeptide, TRAP binding peptide, Ser-Asp-Ser-Ser-Asp (SDSSD) peptide, (DSS)6 peptide, Asp-rich peptide ((Asp) 14 It may include , (AspSerSer)6), CH6 aptamers, etc., but preferably may be BP, but is not limited thereto.

[0083] In this regard, referring to FIG. 1b, a flowchart of a method for generating phospholipid nanoparticles in a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention is illustrated, wherein the step of generating phospholipid nanoparticles (S110) comprises mixing a first solution containing DSPE-PEG-MAL (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)2000]) and a second solution containing Thiol-BP (S210), dissolving a mixture containing DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, DSPE-PEG, and the generated DSPE-PEG-BP in a non-polar solvent (S220), and inducing self-assembly of the obtained lipid membrane (S230). Accordingly, phospholipid nanoparticles containing BP can be generated.

[0084] More specifically, the mixing step (S210) is a step for producing DSPE-PEG-BP, comprising the steps of: mixing a first solution containing DSPE-PEG-MAL and a second solution to obtain a first reaction solution; dialyzing the obtained first reaction solution to obtain a purified second reaction solution so as to remove residual thiol-BP in the first reaction solution; and freeze-drying the second reaction solution to produce DSPE-PEG-BP.

[0085] At this time, the first solution may be a solution containing 30 to 35 mg / ml of DSPE-PEG-MAL, and the solvent may be DPBS (Dulbecco's phosphate buffered saline), but is not limited thereto, and various media and solvents used for cell culture and experiments may be used.

[0086] The second solution may be a solution containing 35 to 45 mg / ml of Thiol-BP, wherein the solvent may be the same solvent as the first solution described above.

[0087] For example, the step of obtaining a first reaction solution may include preparing a first solution by dissolving about 50 mg of DSPE-PEG-MAL in about 1.5 ml of DPBS, preparing a second solution by dissolving about 20.1 mg of BP having a thiol functional group (Thiol-BP) in about 0.5 ml of DPBS, mixing the prepared first solution and second solution and reacting (click-reaction) through a stirred incubator for about 60 minutes. Accordingly, a first reaction solution containing DSPE-PEG-BP can be obtained from the first solution and the second solution.

[0088] Meanwhile, the first reaction solution may contain residual DSPE-PEG-MAL or Thiol-BP substances that have not completely reacted. Accordingly, the step of generating DSPE-PEG-BP includes, after the step of obtaining the first reaction solution, the step of obtaining a purified second reaction solution by dialyzing the first reaction solution to remove insoluble substances within the first reaction solution.

[0089] For example, the step of obtaining a second reaction solution can be performed through a dialysis method using a dialysis filtration membrane. More specifically, a first dialysis can be performed by introducing a first reaction solution and a dialysis solution containing 50 mM sodium chloride into a device including a dialysis filtration membrane, followed by stirring for about 4 hours, and this first dialysis process can be performed two or more times. Then, a second dialysis can be performed by introducing additional distilled water into the device including a dialysis filtration membrane, followed by stirring for about 4 hours, and this second dialysis process can be performed three or more times. Accordingly, a second reaction solution purified from the first reaction solution can be obtained.

[0090] At this time, sodium chloride and distilled water may be used as dialysis fluids, but are not limited thereto, and any various dialysis fluids capable of inducing the diffusion of insoluble substances may be used.

[0091] The dissolving step (S220) is a step of generating a lipid membrane from a mixture, wherein the mixture may contain DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, DSPE-PEG, and the generated DSPE-PEG-BP in a ratio of 65 to 75 : 15 to 25 : 3 to 7 : 3 to 7 mol %, respectively, and the solvent in which the mixture is dissolved may be chloroform, but is not limited thereto, and any non-polar solvent capable of dissolving the aforementioned mixture may be used. For example, DSPC, cholesterol, DSPE-PEG, and the generated DSPE-PEG-BP may be dissolved in about 300 μl of chloroform in amounts of about 5.94 mg, about 0.83 mg, about 1.50 mg, and about 1.74 mg, respectively.

[0092] Furthermore, the dissolving step (S220) can be performed with stirring for 40 to 56 hours, and for example, the dissolving step (S220) can be performed for about 48 hours using a rotary evaporator. Accordingly, a lipid membrane can be formed from the mixture.

[0093] The step of inducing self-assembly (S230) is a step of generating phospholipid nanoparticles containing BP from the generated lipid membrane, and can be performed by a physical method. More specifically, the step of inducing self-assembly (S230) includes a step of ultrasonically treating the generated lipid membrane and a step of extruding the ultrasonically treated lipid membrane. At this time, the ultrasonically treating step can be performed after treating the generated lipid membrane with a medium such as DPBS. Ultimately, self-assembly of the lipid membrane is induced by physical methods such as the aforementioned ultrasonography and extrusion, and phospholipid nanoparticles containing BP can be generated.

[0094] Meanwhile, during the aforementioned physical method process, lipid byproducts for which self-structuring was not induced may be generated. Accordingly, the step of inducing self-assembly (S230) further includes a step of removing byproducts using a filter after the extrusion step. For example, the step of removing byproducts may be performed using a method utilizing tangential flow filtration and a centrifugal filter, but is not limited thereto.

[0095] Ultimately, nanoparticles containing BP can be produced by a process for producing phospholipid nanoparticles comprising the steps of: mixing a first solution containing DSPE-PEG-MAL and a second solution containing Thiol-BP (S210); dissolving a mixture containing DSPC, cholesterol, DSPE-PEG, and the generated DSPE-PEG-BP in a non-polar solvent (S220); and inducing self-assembly of the obtained lipid membrane (S230).

[0096] Meanwhile, the method for generating phospholipid nanoparticles is not limited to the method illustrated in FIG. 1b above, and may include reverse phase evaporation methods including thin film hydration and microfluidic hydrodynamic focusing methods.

[0097] Referring again to FIG. 1a, a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention may perform a step of generating cell membrane nanoparticles derived from mesenchymal stem cells (S120) after a step of generating phospholipid nanoparticles (S110). At this time, the step of generating phospholipid nanoparticles (S110) and the step of generating cell membrane nanoparticles (S120) may be performed in order as described above, but are not limited thereto; they may be performed simultaneously regardless of order, or the step of generating cell membrane nanoparticles (S120) may be performed first.

[0098] More specifically, with reference to FIG. 1c, a flowchart of a method for generating cell membrane nanoparticles in a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention is shown, and the step of generating cell membrane nanoparticles derived from mesenchymal stem cells (S120) includes the step of crushing mesenchymal stem cells to a size of 0.5 to 1.5 μm (S310) and the step of extruding the crushed mesenchymal stem cells (S320).

[0099] The crushing step (S310) can be performed through a freezing and thawing process so that the mesenchymal stem cells are physically crushed. Accordingly, the crushing step (S310) may include the step of preparing a first cell suspension by suspending the mesenchymal stem cells in a culture medium to crush the mesenchymal stem cells, and the step of freezing and thawing the first cell suspension. In this case, the freezing and thawing process may be performed five or more times, and the thawing process may be performed for 30 to 50 minutes. Additionally, the culture medium may be DPBS, but is not limited thereto, and may include all various culture media used in cell experiments.

[0100] For example, to pulverize mesenchymal stem cells, a cell suspension (first cell suspension) is prepared by suspending 1 million mesenchymal stem cells in 1 ml of DPBS, and a tube (container) containing the cell suspension is frozen by immersing it in liquid nitrogen for about 1 minute, followed by a freezing and thawing method at room temperature for about 40 minutes. At this time, the freezing and thawing process may be repeated 5 times.

[0101] The step of generating cell membrane nanoparticles (S120) may include an extrusion step (S320) after the crushing step, and the extrusion step (S320) is a step for inducing self-assembly of crushed mesenchymal stem cells, and can induce self-assembly between the cell membrane of the mesenchymal stem cells and intracellular substances by extruding the crushed mesenchymal stem cells.

[0102] Meanwhile, in the aforementioned process, cell byproducts that have not been induced to self-structure may be generated. Accordingly, the step of generating cell membrane nanoparticles (S120) may further include a step of removing byproducts using a filter after the extrusion step. For example, the step of removing byproducts may be performed using a method utilizing tangential flow filtration and a centrifugal filter, but is not limited thereto.

[0103] Ultimately, cell membrane nanoparticles derived from mesenchymal stem cells can be produced through a step (S120) of producing cell membrane nanoparticles, which includes a step (S310) of crushing the above mesenchymal stem cells to a size of 0.5 to 1.5 μm and a step (S320) of extruding the crushed mesenchymal stem cells.

[0104] Again, referring to FIG. 1a, a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention includes a step (S110) of generating phospholipid nanoparticles and a step (S120) of generating cell membrane nanoparticles, and a step (S130) of fusing the phospholipid nanoparticles and cell membrane nanoparticles.

[0105] More specifically, with reference to FIG. 1d, a flowchart of a method for fusing tissue-targeting nanoparticles in a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention is shown, and the fusing step (S130) includes a step of preparing a first nanoparticle suspension by suspending the generated phospholipid nanoparticles and cell membrane nanoparticles in a culture medium (S410), a step of generating fused particles by freezing and thawing the first nanoparticle suspension (S420), and a step of extruding the generated fused particles (S430).

[0106] First, the step of preparing the first nanoparticle suspension (S410) includes the step of suspending the generated phospholipid nanoparticles and cell membrane nanoparticles in a medium at a 1:1 ratio, wherein the medium may be DPBS, but is not limited thereto, and may include all various media used in cell experiments.

[0107] For example, the step of preparing the first nanoparticle suspension (S410) is 1 billion (1×10⁻⁶). 9 phospholipid nanoparticles (units) and 1 billion (1×10⁻⁶) 9 The method may include a process of suspending cell membrane nanoparticles in 500 μl of DPBS and then combining them to prepare a first nanoparticle suspension of 1 ml.

[0108] Next, in the fusion step (S130), the phospholipid nanoparticles and cell membrane nanoparticles contained in the first nanoparticle suspension can be fused (combined) through a physical method, and the fusion step (S130) may include a step (S420) of generating fused particles by freezing and thawing the first nanoparticle suspension. At this time, the step (S420) of generating fused particles, that is, the freezing and thawing process, may be performed 10 or more times, and the thawing process may be performed for 30 to 50 minutes.

[0109] For example, to pulverize mesenchymal stem cells, a process of freezing a tube (container) containing the first nanoparticle suspension by immersing it in liquid nitrogen for about 1 minute, and then thawing it at room temperature for about 40 minutes (Freezing and thawing method) may be performed. At this time, the freezing and thawing process may be repeated 10 times.

[0110] Next, the extrusion step (S430) is a step that induces self-assembly of the generated fusion particles, and the fusion particles can be extruded to produce final tissue-targeting nanoparticles.

[0111] Meanwhile, in the aforementioned process, cell byproducts for which self-structuring was not induced may be generated. Accordingly, the fusion step (S130) may further include a step of removing byproducts using a filter after the extrusion step. For example, the step of removing byproducts may be performed using a method utilizing tangential flow filtration and a centrifugal filter, but is not limited thereto.

[0112] Ultimately, tissue-targeting nanoparticles can be produced through a fusion step (S130) comprising the steps of: suspending the above-mentioned phospholipid nanoparticles and cell membrane nanoparticles in a culture medium to prepare a first nanoparticle suspension (S410); freezing and thawing the first nanoparticle suspension to produce fused particles (S420); and extruding the produced fused particles (S430).

[0113] Meanwhile, tissue-targeting nanoparticles according to one embodiment of the present invention can be utilized as a drug delivery system (DDS) by carrying a drug containing genetic material. More specifically, a tissue-targeting nanoparticle carrying a drug containing genetic material can be produced by loading a drug containing genetic material onto phospholipid nanoparticles, which are constituent components of the tissue-targeting nanoparticles according to one embodiment of the present invention, and fusing them with cell membrane nanoparticles derived from mesenchymal stem cells.

[0114] Accordingly, the step of generating phospholipid nanoparticles (S110) further includes the step of loading a drug containing genetic material onto the generated phospholipid nanoparticles. More specifically, with reference to FIG. 1e, a flowchart of a method for loading a drug containing genetic material onto phospholipid nanoparticles in a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention is shown. When the loaded drug is genetic material, the loading step includes the step of preparing a second nanoparticle suspension by suspending phospholipid nanoparticles in an organic solvent (S510), the step of preparing a genetic material suspension by suspending the genetic material in a buffer solution (S520), and the step of dual-injecting the second nanoparticle suspension and the genetic material suspension into a single container (S530).

[0115] At this time, the genetic material may include mRNA and plasmid DNA, but is not limited thereto, and may include all genetic material that can be loaded onto phospholipid nanoparticles.

[0116] The organic solvent used in the step (S510) of preparing the second nanoparticle suspension may be ethanol (ethyl alcohol, EtOH), but is not limited thereto, and may include any organic solvent capable of diluting phospholipid nanoparticles. Furthermore, the pH of the organic solvent may be 3.5 to 4.5, but preferably 4.0.

[0117] The phospholipid nanoparticles used in the step (S510) of preparing the second nanoparticle suspension may be phospholipid nanoparticles produced through the process of FIG. 1b described above, and in the step (S510) of preparing the second nanoparticle suspension, they may be used after freeze-drying and then suspended in an organic solvent.

[0118] For example, the step (S510) of preparing the second nanoparticle suspension is 1 × 10 produced through the process of FIG. 1b described above. 7The method may include the step of freeze-drying phospholipid nanoparticles and then suspending the freeze-dried phospholipid nanoparticles in about 250 μl of pH 4.0 ethanol to prepare a second nanoparticle suspension.

[0119] Next, the step of preparing a genetic material suspension (S520) is a step of preparing a suspension containing genetic material to be loaded. For example, the step of preparing a genetic material suspension (S520) may include the step of preparing a genetic material suspension by suspending about 1 μg of nucleic acid mRNA or plasmid DNA in about 250 μl of pH 4.0 citrate buffer solution.

[0120] The buffer solution used in the step (S520) of preparing a genetic material suspension may be a citrate buffer, but is not limited thereto, and may include any solvent or buffer solution capable of diluting the genetic material. Furthermore, the pH of the buffer solution may be 3.5 to 4.5, but preferably 4.0.

[0121] The parallel injection step (S530) may include a method in which equal amounts of the second nanoparticle suspension and the dielectric material suspension are injected simultaneously within a single container. For example, the parallel injection step (S530) may include a parallel injection method in which the second nanoparticle suspension and the dielectric material suspension are each prepared by being placed into a syringe and then dropped simultaneously into a single microtube, thereby allowing the dielectric material to be loaded onto the phospholipid nanoparticles.

[0122] Meanwhile, during the aforementioned process, genetic material byproducts that are not loaded onto the phospholipid nanoparticles may be generated. Accordingly, the loading step may further include a step of removing the byproducts using a filter after the parallel injection step. More specifically, the loading step may be performed using tangential flow filtration and a centrifugal filter, but is not limited thereto. Furthermore, after the step of removing these byproducts, the phospholipid nanoparticles containing the genetic material from which the byproducts have been removed are stored in suspension in DPBS and can be used to generate tissue-targeting nanoparticles.

[0123] Meanwhile, in Fig. 1e, a parallel injection method was described as a method of loading genetic material onto phospholipid nanoparticles, but it is not limited thereto, and various drug loading technologies including genetic material such as microfluidic chips, electroporation, and extrusion technology can be applied.

[0124] The phospholipid nanoparticles loaded with genetic material generated through the process of FIG. 1e described above can be used to generate tissue-targeting nanoparticles through the same process of FIG. 1d described above. For example, the generated phospholipid nanoparticles loaded with genetic material and cell membrane nanoparticles are each 5×10 10 A single nanoparticle suspension can be prepared by suspending (mixing) each nanoparticle in 400 ml of DPBS, and then a fused particle can be produced by using a physical method such as freezing and thawing the prepared nanoparticle suspension, and then extruding the produced fused particle to produce a tissue-targeting nanoparticle carrying genetic material.

[0125] Ultimately, in accordance with the process described above, the tissue-targeting nanoparticles according to one embodiment of the present invention can be utilized as a drug delivery system (DDS). For example, if the tissue-targeting nanoparticles according to one embodiment of the present invention carry genetic material (nucleic acid) as described above, they can be utilized as a gene therapy agent. Furthermore, since the tissue-targeting nanoparticles according to one embodiment of the present invention can carry various drugs, they can be applied to a wide range of diseases.

[0126] FIG. 2 is a schematic diagram of a tissue-targeting nanoparticle according to one embodiment of the present invention.

[0127] Referring to FIG. 2, a tissue-targeting nanoparticle according to one embodiment of the present invention is a nanoparticle produced by fusing a phospholipid nanoparticle (BP-Lipid nanoparticle) containing bisphosphonate (BP) and a cell membrane nanoparticle (CM-NV) derived from a mesenchymal stem cell (MSC).

[0128] At this time, the phospholipid nanoparticles may include phospholipids and sterol lipids. For example, in a method for preparing tissue-targeting nanoparticles according to one embodiment of the present invention, the phospholipid nanoparticles may be formed by a mixture containing DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, DSPE-PEG, and the generated DSPE-PEG-BP in a ratio of 65 to 75 : 15 to 25 : 3 to 7 : 3 to 7 mol %, respectively, and thus, the tissue-targeting nanoparticles according to one embodiment of the present invention may include phospholipids and sterol lipids.At this time, the phospholipids are DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000]), DSPE-PEG-MAL(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)2000]), DOPE(1,2-diacyl-sn-glycero-3-phosphoethanolamine), DOPE-mal(dioleoyl-sn-glycero-3-phosphoethanolamine-N-(maleimidomethyl)), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine), DMPC(1,2-dipalmitoyl-sn- It may include at least one of glycero-3-phosphocholine, DMPG (1,2-dipalmitoyl-sn-glycero-3-Phospho-rac-(1-glycerol)), DLPC (1,2-dilauroylsn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), but is not limited thereto; preferably, it may include DSPC and DSPE-PEG, as in the method for preparing tissue-targeting nanoparticles according to one embodiment of the present invention described above.

[0129] Bisphosphoate (BP) may be in a form bound to a phospholipid, namely DSPE-PEG, but is not limited thereto.

[0130] BP can bind to Ca ions. Accordingly, tissue-targeting nanoparticles according to one embodiment of the present invention can target tissues containing Ca, and for example, since bisphosphonate (BP) can bind to hydroxyapatite (Ca5(PO4)3) of bone tissue (surface), tissue-targeting nanoparticles according to one embodiment of the present invention can target bone tissue.

[0131] In the method for preparing tissue-targeting nanoparticles according to one embodiment of the present invention, the phospholipid nanoparticles are shown to contain cholesterol, but are not limited thereto, and sterol lipids such as sitosterol, stigmasterol, campesterol, and ergosterol may be used. Accordingly, the tissue-targeting nanoparticles according to one embodiment of the present invention may include cholesterol, sitosterol, stigmasterol, campesterol, and ergosterol as sterol lipids.

[0132] Phospholipid nanoparticles and cell membrane nanoparticles of tissue-targeting nanoparticles according to one embodiment of the present invention may be included in a 1:1 ratio, but are not limited thereto. In order to maximize the production efficiency of tissue-targeting nanoparticles, a 1:1 ratio for each of the phospholipid nanoparticles and cell membrane nanoparticles of tissue-targeting nanoparticles according to one embodiment of the present invention may be most preferable.

[0133] The phospholipid nanoparticles and cell membrane nanoparticles of the tissue-targeting nanoparticles according to one embodiment of the present invention can be fused by a physical method, for example, the phospholipid nanoparticles and cell membrane nanoparticles of the tissue-targeting nanoparticles according to one embodiment of the present invention can be fused based on a freezing and thawing method and a mechanical extrusion method, but are not limited thereto, and may include all various methods by which the phospholipid nanoparticles and cell membrane nanoparticles can be fused.

[0134] The size, i.e., the diameter, of the tissue-targeting nanoparticles according to one embodiment of the present invention may be 1 to 200 nm, but is not limited thereto. For example, the tissue-targeting nanoparticles according to one embodiment of the present invention may include nanoparticles having a diameter of 1 to 200 nm and may have an average size of about 180 nm.

[0135] Since the tissue-targeting nanoparticles according to one embodiment of the present invention can selectively target tissues, they can be used as a drug delivery system (DDS) by carrying a drug. Accordingly, the tissue-targeting nanoparticles according to one embodiment of the present invention may further include at least one drug among genetic material drugs, ionic drugs, and protein drugs.

[0136] For example, the genetic material of a genetic material drug may include at least one of mRNA and plasmid DNA, wherein, if the loaded genetic material drug is mRNA, the mRNA is a tissue-targeting nanoparticle 1×10 10 16 ng can be loaded per unit. In addition, when the genetic material drug loaded onto the tissue-targeting nanoparticle according to one embodiment of the present invention is plasmid DNA, the plasmid DNA is 1×10 of the tissue-targeting nanoparticle. 10 More than 29 ng can be loaded per piece.

[0137] Confirmation of the characteristics of tissue-targeting nanoparticles according to one embodiment of the present invention

[0138] Hereinafter, with reference to FIGS. 3 and 4, the characteristics of tissue-targeting nanoparticles according to one embodiment of the present invention will be identified.

[0139] Figure 3 shows the results regarding the size and distribution of tissue-targeting nanoparticles according to one embodiment of the present invention. In this case, Comparative Example 1, Comparative Example 2, and Example 1 are nanoparticles produced by the method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention, where Comparative Example 1 is a cell membrane nanoparticle derived from mesenchymal stem cells (CM-NV), Comparative Example 2 is a phospholipid nanoparticle containing BP (BP-LNP), and Example 1 is a tissue-targeting nanoparticle (BP-Hybrid) according to one embodiment of the present invention. Furthermore, the size and distribution of the nanoparticles were confirmed through dynamic light scattering (DLS).

[0140] First, referring to FIG. 3(a), Example 1, Comparative Example 1, and Comparative Example 2 all contain particles with a size of 100 to 200 nm.

[0141] Next, referring to FIG. 3(b), Example 1, Comparative Example 1, and Comparative Example 2 are shown to have average sizes of 160.2 nm, 135.8 nm, and 182.4 nm, respectively.

[0142] In addition, regarding the polydispersity index, Example 1, Comparative Example 1, and Comparative Example 2 were found to have indices of 0.150, 0.229, and 0.158, respectively. Since Example 1 was found to have the lowest polydispersity index, it may mean that the tissue-targeting nanoparticles according to one embodiment of the present invention have the lowest standard deviation of the molecular weight distribution.

[0143] Figure 4 shows the results of confirming the fusion of tissue-targeting nanoparticles according to one embodiment of the present invention. In this case, the confirmation of the fusion of tissue-targeting nanoparticles according to one embodiment of the present invention was performed using a fluorescence resonance energy transfer assay (FRET assay). The conditions for the nanoparticles of Comparative Examples 1 to 5 are as shown in Table 1 below.

[0144] [Table 1]

[0145]

[0146] Referring to FIG. 4d(a), Examples 1 and 2 are shown to have the same wavelength pattern as the Comparative Example. Furthermore, Examples 3 to 5 are shown to have different wavelength patterns from the Comparative Example, and the wavelength patterns among them are similar. That is, it may mean that fusion between nanoparticles can be formed when the number of phospholipid nanoparticles relative to cell membrane nanoparticles is 0.5 times or more.

[0147] Referring to Fig. 4d (b), Example 4 shows the highest relative intensity for wavelengths of 530 nm and 590 nm. This implies that the highest fusion efficiency is achieved when cell membrane nanoparticles and phospholipid nanoparticles are fused in equal numbers.

[0148] Accordingly, in the tissue-targeting nanoparticles and the method for manufacturing the same according to one embodiment of the present invention, the ratio of phospholipid nanoparticles to fused cell membrane nanoparticles may be 0.5 to 10, but most preferably 1, and specifically, the ratio of phospholipid nanoparticles to cell membrane nanoparticles in the tissue-targeting nanoparticles and the method for manufacturing the same according to one embodiment of the present invention may be 1:1.

[0149] Confirmation and verification of tissue targeting of tissue-targeting nanoparticles according to one embodiment of the present invention

[0150] Hereinafter, with reference to FIGS. 5a to 7, the tissue targeting efficiency of tissue targeting nanoparticles according to one embodiment of the present invention will be confirmed and verified.

[0151] FIGS. 5a to 5d are the results of ex vivo tissue targeting verification of tissue-targeting nanoparticles according to one embodiment of the present invention with and without BP.

[0152] First, referring to FIG. 5a, to verify the tissue targeting of nanoparticles based on the presence or absence of bisphosphonate (BP), a tissue targeting substance, a bone tissue-like scaffold prepared with hydroxyapatide and collagen in a 30:70 wt ratio (%) and mouse-derived tissues (heart, liver, lung, spleen, kidney, muscle, and femur) were used. Comparative Example 1, Comparative Example 2, and the Example were treated on the bone tissue-like scaffold and mouse-derived tissues, and then fluorescence microscopy observation was performed. Furthermore, the conditions for Comparative Example 1, Comparative Example 2, and the Example are as shown in Table 2 below, and Comparative Example 2 and the Example were loaded with a fluorescent substance (DiO).

[0153] [Table 2]

[0154]

[0155] Bisphosphonate (BP) can bind to hydroxyapatite (Ca5(PO4)3) in bone tissue, and accordingly, nanoparticles containing BP can target bone tissue. Accordingly, tissue-targeting nanoparticles according to one embodiment of the present invention can selectively target bone tissue by including phospholipid nanoparticles containing BP.

[0156] Accordingly, referring to FIG. 5b, the fluorescence expression results for tissue-targeting nanoparticles according to one embodiment of the present invention on a bone-like scaffold are shown, and the fluorescence intensity of the example is about 50, which is statistically significantly higher than that of Comparative Example 1 and Comparative Example 2 (p<0.0001). That is, since the tissue-targeting nanoparticles according to one embodiment of the present invention contain BP, it may mean that targeting of calcium-containing tissues is possible.

[0157] Referring to FIG. 5c, the fluorescence expression results for tissue-targeting nanoparticles according to one embodiment of the present invention in a mouse-derived femoral bone fragment are shown, and the fluorescence intensity of the example is about 50, which is statistically significantly higher than that of Comparative Example 1 and Comparative Example 2 (p<0.0001). That is, since the tissue-targeting nanoparticles according to one embodiment of the present invention contain BP, it may mean that targeting of bone tissue in vivo is possible.

[0158] Referring to FIG. 5d, the ex vivo fluorescence expression results for tissue-targeting nanoparticles according to one embodiment of the present invention in mouse-derived tissues (heart, liver, lung, spleen, kidney, muscle, femur) are shown, and the fluorescence intensity in the femur (bone) is 30 or higher, which is statistically significantly higher than that of tissues in other organs (p<0.0001). That is, this implies that the tissue-targeting nanoparticles according to one embodiment of the present invention can specifically achieve high targeting in bone tissue. Accordingly, since the tissue-targeting nanoparticles according to one embodiment of the present invention can selectively target only bone tissue, they can have a higher pharmacological delivery effect in relation to bone tissue and diseases thereof.

[0159] Figures 6a to 6c show the results of in vivo tissue targeting verification of tissue-targeting nanoparticles according to one embodiment of the present invention.

[0160] First, referring to FIG. 6a, to verify the targeting of tissue-targeting nanoparticles according to one embodiment of the present invention on in vivo tissues, Comparative Example 1, Comparative Example 2, and Example were administered every two days for one week via retro-orbital injection, and Comparative Example 1, Comparative Example 2, and Example were administered at a dose of 1 mg per 1 kg of mouse body weight (mg / kg). Organ extraction of the liver, lung, heart, kidney, spleen, brain, femur, and tibia from mice was performed on the 7th day of administration, and the fluorescence expression of Comparative Example 1, Comparative Example 2, and Example in the extracted organs was confirmed using an In-vivo Optical Imaging System (IVIS). Furthermore, the conditions for Comparative Example 1, Comparative Example 2, and Example are as shown in Table 3 below, and Comparative Example 2 and Example carried a fluorescent substance (DiO).

[0161] [Table 3]

[0162]

[0163] Referring to FIG. 6b, the fluorescence expression images of the excised mouse organs are shown, and the fluorescence expression of the example for liver, lung, heart, kidney, spleen, and brain tissue samples appears to be similar to that of Comparative Example 2, but the fluorescence expression for the femur and tibia bone tissues shows that the example has a very high fluorescence expression intensity.

[0164] More specifically, referring to FIG. 6c, the graph results for the fluorescence intensity for the aforementioned FIG. 6b are shown, and it is indicated that the fluorescence intensity of the example has a statistically significantly higher value only in bone tissue. That is, it can be said that the tissue-targeting nanoparticles according to one embodiment of the present invention can accurately and specifically target bone tissue even in vivo.

[0165] Figure 7 shows the results of an analysis of the intracellular delivery efficiency of tissue-targeting nanoparticles according to one embodiment of the present invention. In order to confirm the intracellular delivery efficiency of tissue-targeting nanoparticles according to one embodiment of the present invention, Examples and Comparative Examples 1 to 3 were treated with mesenchymal stem cells (MSC), osteoblasts (OB), and macrophages, respectively. The conditions for Examples and Comparative Examples 1 to 3 are as shown in Table 4 below, and a fluorescent substance (PKH67) was loaded for fluorescence analysis.

[0166] [Table 4]

[0167]

[0168] Referring to FIG. 7, the example is shown to have a higher fluorescence expression intensity in mesenchymal stem cells (MSC) and osteoblasts (OB) compared to Comparative Examples 1 and 3, together with Comparative Example 2, which may mean that the forms of the example and Comparative Example 2 can have high efficiency in intracellular delivery to target cells.

[0169] On the other hand, in macrophages, the example was shown to have a lower fluorescence intensity than Comparative Example 3, which may mean that the form of the example may have low efficiency in intracellular delivery to phagocytes.

[0170] Ultimately, the tissue-targeting nanoparticles according to one embodiment of the present invention can actively induce intracellular delivery to target cells, namely mesenchymal stem cells and osteoblasts, and reduce intracellular delivery to macrophages, thereby increasing the in vivo circulation time and allowing for the expectation of enhanced tissue regeneration efficacy.

[0171] Confirmation and verification of drug delivery effects for tissue-targeting nanoparticles according to one embodiment of the present invention

[0172] Hereinafter, with reference to FIGS. 8 to 11, the drug delivery effect of tissue-targeting nanoparticles according to one embodiment of the present invention will be verified.

[0173] Figure 8 shows the results regarding the loading efficiency of genetic material (mRNA, plasmid DNA) of tissue-targeting nanoparticles according to one embodiment of the present invention. In this case, Example 1 and Example 2 are nanoparticles loaded with genetic material produced through the process of Figure 1e described above, Example 1 is a tissue-targeting nanoparticle loaded with mRNA, and Example 2 is a tissue-targeting nanoparticle loaded with plasmid DNA. Furthermore, BP-LNP is a comparative example, which is a phospholipid nanoparticle that is a component of the tissue-targeting nanoparticle according to one embodiment of the present invention.

[0174] Referring to FIG. 8, the nucleic acid loading efficiency for phospholipid nanoparticles (BP-LNP), which are components of tissue-targeting nanoparticles according to one embodiment of the present invention, is shown to be 50.64%.

[0175] Furthermore, Example 1, produced based on these phospholipid nanoparticles, comprises nanoparticles 1×10 10 It appears that about 16 ng of mRNA is loaded per particle.

[0176] In addition, Example 2, produced based on phospholipid nanoparticles, consists of nanoparticles ×10 10It is found that approximately 29 ng of plasmid DNA (pDNA) is loaded per particle.

[0177] Figure 9 shows the results regarding the gene delivery efficiency of tissue-targeting nanoparticles loaded with mRNA according to one embodiment of the present invention. In this case, to verify the gene delivery efficiency of tissue-targeting nanoparticles loaded with mRNA according to one embodiment of the present invention, RUNX2 gene mRNA was loaded onto tissue-targeting nanoparticles according to one embodiment of the present invention, and osteoblasts were used as target cells. The conditions for Comparative Example 1, Comparative Example 2, and the example are as shown in Table 5 below. More specifically, 40,000 osteoblasts were seeded into each well of a 12-well plate, and after approximately 24 hours, Comparative Example 1, Comparative Example 2, and the example were 1×10 10 Each was treated individually, and the gene expression of the cells was confirmed via qPCR 48 hours after treatment of Comparative Example 1, Comparative Example 2, and Example.

[0178] [Table 5]

[0179]

[0180] Referring to Fig. 9, the RUNX2 gene expression (mRNA expression) in osteoblasts was found to be statistically significantly highest in the example (p<0.05), which may mean that the tissue-targeting nanoparticles according to one embodiment of the present invention can deliver mRNA-based drugs loaded on the target cells with high efficiency.

[0181] Furthermore, since the present invention is derived from phospholipid nanoparticles containing BP, it can accurately deliver drugs to bone tissue and associated bone cells (including osteoblasts).

[0182] Figure 10 shows the results regarding the gene delivery efficiency of tissue-targeting nanoparticles loaded with plasmid DNA according to one embodiment of the present invention. In order to verify the gene delivery efficiency of tissue-targeting nanoparticles loaded with plasmid DNA according to one embodiment of the present invention, RUNX2 gene plasmid DNA was loaded onto tissue-targeting nanoparticles according to one embodiment of the present invention, and osteoblasts were used as target cells. The conditions for Comparative Example 1, Comparative Example 2, and the example are as shown in Table 6 below. More specifically, 40,000 osteoblasts were dispensed into each well of a 12-well plate, and Comparative Example 1, Comparative Example 2, and the example were treated after approximately 24 hours had elapsed. Comparative Example 2 is a mixture prepared by mixing 0.1 μl of Lipofectamine, which is mainly used for gene transduction, and 29 ng of RUNX2 gene plasmid DNA, and the mixture was treated on osteoblasts 15 minutes after the mixture was prepared. In the case of the example, tissue-targeting nanoparticles according to one embodiment of the present invention carrying 29 ng of plasmid DNA are 1×10 10 Each was treated individually, and the gene expression of the cells was confirmed via qPCR 48 hours after treatment of Comparative Example 1, Comparative Example 2, and Example.

[0183] [Table 6]

[0184]

[0185] Referring to Fig. 10, the RUNX2 gene expression (mRNA expression) in osteoblasts is statistically significantly highest in the example (p<0.0001), which may mean that the tissue-targeting nanoparticles according to one embodiment of the present invention can deliver a plasmid DNA-based drug loaded on the target cells with high efficiency.

[0186] Ultimately, according to the results of 9 and 10, tissue-targeting nanoparticles according to one embodiment of the present invention, which carry a drug containing genetic material, are shown to have excellent drug delivery and gene transduction efficiency and can be used as next-generation disease-specific gene delivery vehicles.

[0187] Figure 11 shows the results regarding the osteogenic differentiation efficiency of tissue-targeting nanoparticles loaded with mRNA according to one embodiment of the present invention. In this case, mesenchymal stem cells were used to verify the osteogenic differentiation efficiency; 100,000 mesenchymal stem cells were seeded into each well of a 12-well plate, and after approximately 24 hours, the comparative example and the example were tested at a rate of 1 × 10⁶ 10 Each was treated individually, and the expression levels of bone marker genes (COL1, OPN) were confirmed via qPCR 7 days after treatment. The conditions for the comparative example and the example are as shown in Table 7 below.

[0188] [Table 7]

[0189]

[0190] Referring to Fig. 11, the expression levels of mRNA for the bone marker genes COL1 (Type 1 collagen) and OPN (Osteopontin) are shown to be statistically significantly higher in the example than in the comparative example (p<0.0001, p=0.0147).

[0191] This means that tissue-targeting nanoparticles according to one embodiment of the present invention can deliver and express bone marker genes carried within stem cells, and accordingly, tissue-targeting nanoparticles according to one embodiment of the present invention can improve bone differentiation ability in an individual.

[0192] The present invention will be explained in more detail below through examples. However, since these examples are merely illustrative of the present invention, the scope of the present invention should not be interpreted as being limited by these examples.

[0193]

[0194] [National R&D projects that supported this invention]

[0195] [Project ID] 1711190102

[0196] [Assignment No.] 2022R1A2C3004850

[0197] [Ministry Name] Ministry of Science and ICT

[0198] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0199] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0200] [Project Title] Development of Technology to Enhance Therapeutic Efficacy for Inflammatory Skeletal Diseases through Mass Production and Modification of Patient-Specific Stem Cell-Derived Multifunctional Exosomes Using a 3D Fusion System

[0201] [Name of Project Performing Organization] Dongguk University

[0202] [Research Period] 20230301 ~ 20240229

[0203]

[0204] [National R&D projects that supported this invention]

[0205] [Project ID] 2710018148

[0206] [Assignment No.] 00405381

[0207] [Ministry Name] Ministry of Science and ICT

[0208] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0209] [Research Project Name] Basic Research Laboratory Pioneer Type

[0210] [Project Title] Basic Research Laboratory for the Development of Multimodal Induced Regeneration Biomaterials

[0211] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0212] [Research Period] 20240801 ~ 20270430

Claims

1. Step of generating phospholipid nanoparticles; Steps for generating cell membrane nanoparticles derived from mesenchymal stem cells, and A method for manufacturing tissue-targeting nanoparticles, comprising the step of fusing the generated phospholipid nanoparticles and the cell membrane nanoparticles so as to generate tissue-targeting nanoparticles.

2. In Paragraph 1, The above organization is, Method for manufacturing tissue-targeting nanoparticles including bone tissue.

3. In Paragraph 1, The above phospholipid nanoparticles are, Bisphosphonate (BP), Tetracyclines, CXC chemokine receptor type 4 (CXCR4), Ephrin type B receptor 4 (EphB4), Dentin Matrix Protein 1 (DMP1), Anti-Sclerostin antibody, Anti-Type I collagen antibody, Acidic oligopeptide, TRAP binding peptide, Ser-Asp-Ser-Ser-Asp (SDSSD) peptide, (DSS)6 peptide, Asp-rich peptide ((Asp) 14 A method for preparing tissue-targeting nanoparticles selected from the group consisting of (AspSerSer)6) and CH6 aptamers.

4. In Paragraph 1, The step of generating the above phospholipid nanoparticles is, A step of mixing a first solution containing DSPE-PEG-MAL(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)2000]) and a second solution containing Thiol-BP to produce DSPE-PEG-BP; A step of producing a lipid membrane by dissolving a mixture comprising DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, DSPE-PEG, and the generated DSPE-PEG-BP in a non-polar solvent, and A method for manufacturing tissue-targeting nanoparticles, comprising the step of inducing self-assembly of the generated lipid membrane to produce the phospholipid nanoparticles.

5. In Paragraph 4, The step of generating the above DSPE-PEG-BP is A step of obtaining a first reaction solution by mixing the first solution and the second solution; A step of obtaining a purified second reaction solution by dialyzing the obtained first reaction solution so as to remove residual thiol-BP in the first reaction solution, and A method for preparing tissue-targeting nanoparticles, comprising the step of freeze-drying the second reaction solution to produce DSPE-PEG-BP.

6. In Paragraph 5, The step of obtaining the first reaction solution above is, A method for preparing tissue-targeting nanoparticles, performed for 50 to 70 minutes.

7. In Paragraph 4, The above first solution is, Contains 30 to 35 mg / ml of DSPE-PEG-MAL, The above second solution is, A method for preparing tissue-targeting nanoparticles comprising 35 to 45 mg / ml of Thiol-BP.

8. In Paragraph 4, The step of generating the above lipid membrane is, A method for preparing tissue-targeting nanoparticles, performed for 40 to 56 hours.

9. In Paragraph 4, The above mixture is, Regarding DSPC, cholesterol, DSPE-PEG, and the generated DSPE-PEG-BP, A method for preparing tissue-targeting nanoparticles comprising, respectively, in a ratio of 65 to 75 : 15 to 25 : 3 to 7 : 3 to 7 mol %.

10. In Paragraph 4, The step of generating the above phospholipid nanoparticles is, A step of ultrasonically treating the generated lipid membrane; A step of extruding the ultrasonically treated lipid membrane, and, A method for manufacturing tissue-targeting nanoparticles, comprising the step of removing byproducts using a filter.

11. In Paragraph 1, The step of generating the above cell membrane nanoparticles is, A step of crushing mesenchymal stem cells to a size of 0.5 to 1.5 μm, and A step of extruding the pulverized mesenchymal stem cells to induce self-assembly of the pulverized mesenchymal stem cells, and A method for manufacturing tissue-targeting nanoparticles, comprising the step of removing byproducts using a filter.

12. In Paragraph 11, The above grinding step is, A step of preparing a first cell suspension by suspending the above mesenchymal stem cells in a culture medium, and A method for manufacturing tissue-targeting nanoparticles, comprising the step of freezing and thawing the first cell suspension so that the mesenchymal stem cells are physically ruptured.

13. In Paragraph 12, The step of freezing and thawing the first cell suspension is, It is performed 5 or more times, and The above thawing is, A method for preparing tissue-targeting nanoparticles, performed for 30 to 50 minutes.

14. In Paragraph 1, The above-mentioned fusing step is, A step of preparing a first nanoparticle suspension by suspending the generated phospholipid nanoparticles and cell membrane nanoparticles in a culture medium; A step of generating fused particles by freezing and thawing the first nanoparticle suspension, and A method for manufacturing tissue-targeting nanoparticles, comprising the step of extruding the generated fusion particles to generate final tissue-targeting nanoparticles from the fusion particles.

15. In Paragraph 14, The step of preparing the first nanoparticle suspension described above is, A method for preparing tissue-targeting nanoparticles, comprising the step of suspending the generated phospholipid nanoparticles and the cell membrane nanoparticles in a culture medium at a 1:1 ratio.

16. In Paragraph 14, The step of generating the above-mentioned fusion particles is, A method for manufacturing tissue-targeting nanoparticles performed 10 or more times.

17. In Paragraph 14, The above thawing is, A method for preparing tissue-targeting nanoparticles, performed for 30 to 50 minutes.

18. In Paragraph 14, The above-mentioned fusing step is, A method for manufacturing tissue-targeting nanoparticles, further comprising the step of removing byproducts using a filter after the extrusion step described above.

19. In Paragraph 1, The step of generating the above phospholipid nanoparticles is, A method for manufacturing tissue-targeting nanoparticles, further comprising the step of loading a drug containing genetic material onto the generated phospholipid nanoparticles.

20. In Paragraph 19, The above-mentioned supporting step is, A step of preparing a second nanoparticle suspension by suspending the above phospholipid nanoparticles in an organic solvent; A step of preparing a genetic material suspension by suspending the above genetic material in a buffer solution, and A method for manufacturing tissue-targeting nanoparticles, comprising the step of dual-injecting the second nanoparticle suspension and the genetic material suspension into a single container.

21. In Paragraph 19, The above genetic material is, Method for preparing tissue-targeting nanoparticles comprising mRNA and plasmid DNA.

22. Tissue-targeting nanoparticles prepared by the method of any one of claims 1 to 21, wherein Phospholipid nanoparticles containing bisphosphonate (BP) and Tissue-targeting nanoparticles fused from cell membrane nanoparticles derived from mesenchymal stem cells.

23. In Paragraph 22, The above phospholipid nanoparticles are, Tissue-targeting nanoparticles containing phospholipids and sterol lipids.

24. In Paragraph 23, The above phospholipid is, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE-PEG(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)2000]), DSPE-PEG-MAL(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)2000]), DOPE(1,2-diacyl-sn-glycero-3-phosphoethanolamine), DOPE-mal(dioleoyl-sn-glycero-3-phosphoethanolamine-N-(maleimidomethyl)), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine), DMPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPG( 1,2-dipalmitoyl-sn-glycero-3-Phospho-rac-(1-glycerol)), Tissue-targeting nanoparticles comprising at least one of DLPC (1,2-dilauroylsn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).

25. In Paragraph 24, The above BP is, Tissue-targeting nanoparticles in a form bound to the above DSPE-PEG.

26. In Paragraph 23, The above-mentioned sterol lipids are, Tissue-targeting nanoparticles comprising at least one of cholesterol, sitosterol, stigmasterol, campesterol, and ergosterol.

27. In Paragraph 22, The above tissue-targeting nanoparticles are, Tissue-targeting nanoparticles having a diameter of 1 to 200 nm.

28. In Paragraph 22, The above phospholipid nanoparticles and cell membrane nanoparticles are, Tissue-targeting nanoparticles with a 1:1 ratio.

29. In Paragraph 22, The above tissue-targeting nanoparticles are, Tissue-targeting nanoparticles that target tissues containing Ca.

30. In Paragraph 22, The above tissue-targeting nanoparticles are, Tissue-targeting nanoparticles comprising at least one drug among genetic material drugs, ionic drugs, and protein drugs.

31. In Paragraph 30, The above genetic material is, Tissue-targeting nanoparticles comprising at least one of mRNA and plasmid DNA.

32. In Paragraph 31, The above mRNA is The above tissue-targeting nanoparticles 1×10 10 Tissue-targeting nanoparticles loaded with 16 ng or more per particle.

33. In Paragraph 31, The above plasmid DNA is The above tissue-targeting nanoparticles 1×10 10 Tissue-targeting nanoparticles loaded with more than 29 ng per particle.

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