KR-20260062628-A - NANOPARTICLES FOR TISSURE TARGETING AND MANUFACTURING METHOD THREOF

Abstract

The present invention provides a method for producing tissue-targeting nanoparticles comprising the steps of producing phospholipid nanoparticles, producing cell membrane nanoparticles derived from mesenchymal stem cells, and fusing the produced phospholipid nanoparticles and cell membrane nanoparticles so as to produce tissue-targeting nanoparticles, and a tissue-targeting nanoparticle produced by the method comprising phospholipid nanoparticles containing bisphosphoate and cell membrane nanoparticles derived from mesenchymal stem cells.

Inventors

• 이수홍
• 아라이요시에
• 조웅진
• 안진성
• 김덕일
• 차경엽

Assignees

• 동국대학교 산학협력단

Dates

Publication Date

20260507

Application Date

20241029

Claims (20)

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 Article 1, The above organization is, Method for manufacturing tissue-targeting nanoparticles including bone tissue.
3. In Article 1, The above phospholipid nanoparticles are, A method for preparing tissue-targeting nanoparticles selected from the group consisting of 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 peptides ((Asp) 14 , (AspSerSer) 6 ), and CH 6 aptamers.
4. In Article 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 Article 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 Article 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 Article 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 Article 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 Article 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 Article 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 Article 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 Article 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 Article 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 Article 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.

Description

Tissue-targeting nanoparticles and method for manufacturing the same 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. 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. Since the COVID-19 pandemic, the use of such nanotechnology in the pharmaceutical and biotech 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 crucial 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 are being studied in areas such as disease diagnosis, drug delivery, and molecular medical imaging. For instance, nanoparticles incorporating diverse forms of nanotechnology—such as liposomes, proteins, polymers, micelles, emulsions, nanocapsules, dendrimers, and nanoparticles—are being researched and developed. Currently, some nanoparticles have received FDA approval and are being applied in the market and clinical settings; these include encapsulated mRNA (siRNA) or DNA (in gene therapy), inorganic metals and metal complexes, or chemotherapy agents with pharmacological capabilities. 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). 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. FIG. 1a is a flowchart of a method for manufacturing tissue-targeting nanoparticles according to one embodiment of the present invention. 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. 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. 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. 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. FIG. 2 is a schematic diagram of a tissue-targeting nanoparticle according to one embodiment of the present invention. Figure 3 shows the results regarding the size and distribution of tissue-targeting nanoparticles according to one embodiment of the present invention. Figure 4 is a result of confirming the fusion of tissue-targeting nanoparticles according to one embodiment of the present invention. FIGS. 5a to 5d are the results of verifying ex vivo tissue targeting of tissue-tar