WO2023068362A1 - レドックスナノ粒子の細胞処理への使用 - Google Patents

レドックスナノ粒子の細胞処理への使用 Download PDF

Info

Publication number
WO2023068362A1
WO2023068362A1 PCT/JP2022/039300 JP2022039300W WO2023068362A1 WO 2023068362 A1 WO2023068362 A1 WO 2023068362A1 JP 2022039300 W JP2022039300 W JP 2022039300W WO 2023068362 A1 WO2023068362 A1 WO 2023068362A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
formula
residue
nanoparticles
represented
Prior art date
Application number
PCT/JP2022/039300
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
愛樹 丸島
浩二 平田
幸夫 長崎
祐司 松丸
博 石川
寛樹 武川
アルネラ ムヤギチ
晃弘 大山
順子 豊村
英明 松村
暁 平山
淞湖 文
Original Assignee
ＣｒｅｓｔｅｃＢｉｏ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ＣｒｅｓｔｅｃＢｉｏ株式会社 filed Critical ＣｒｅｓｔｅｃＢｉｏ株式会社
Priority to JP2023554758A priority Critical patent/JPWO2023068362A1/ja
Publication of WO2023068362A1 publication Critical patent/WO2023068362A1/ja

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present invention relates to the use of redox-active copolymer-based nanoparticles for the treatment or modification of mammalian cells, more specifically poly(ethylene glycol) (PEG) segments and cyclic nitroxide radicals. Novel application or use of nanoparticles of amphiphilic copolymers containing polymer chain segments having as pendants for cell processing.
  • PEG poly(ethylene glycol)
  • Non-Patent Document 1 Korean Cells
  • Non-Patent Document 3 Non-Patent Document 3
  • redox nanoparticles in particular block copolymers containing hydrophobic polymer segments with cyclic nitroxyl radicals in their side chains and poly(ethylene glycol) (PEG) segments, self-assemble from an aqueous solution.
  • Polymer micellized nanoparticles are known as a preparation capable of scavenging free radicals generated by ischemia and inflammation (WO 2009/133647 A (hereinafter abbreviated as Patent Document 1), WO 2016 /052463 A (hereinafter abbreviated as Patent Document 2)).
  • amphiphilic copolymers comprising polymer chain segments having PEG segments and cyclic nitroxide radicals as pendant (or side chains), specifically copolymers of maleic anhydride and styrene, wherein maleic anhydride Nanoparticles based on copolymers, to which PEG chains are grafted via units and covalently attached pendant residues of agents such as cyclic nitroxide radicals, have also been investigated in vitro or in vitro. It is known to exhibit a redox action caused by a cyclic nitroxide radical in vivo (Japanese Patent Application Laid-Open No. 2019-123773 (hereinafter abbreviated as Patent Document 3)).
  • Non-Patent Document 5 it has been shown that when the former nanoparticles are administered to a mouse model of transient cerebral ischemia through the carotid artery, they are incorporated into the cytoplasm of neurons in cerebral ischemic lesions and exhibit neuroprotective effects (Mujagic A. , et al. Brain research, 2020; 1743: 146922, doi: 1016/j.braineres. , 2017, doi: 10. 1161/STROKAHA. 116. 016356 (hereinafter abbreviated as Non-Patent Document 5)).
  • the combined use of cells and redox nanoparticles before transplantation can correct or improve the above-mentioned disadvantages of cell transplantation technology, it can be applied to a wide range of applications including regenerative medicine. It will contribute to the development of technical fields using such cells.
  • an object of the present invention is to provide mammalian cells (hereinafter also referred to as transplanted cells or simply cells) for treating or preserving them or stabilizing them in a place or area where they may reside.
  • the purpose of the present invention is to obtain a means that can be handled easily and efficiently, and can be administered directly to a predetermined site or region where the cell transplantation or the like is desired, although this is not a limitation.
  • the present inventors have conducted research and found that specific redox nanoparticles based on the copolymers disclosed in Patent Document 1 or 2 or Patent Document 3 and mammalian cells or When combined or used together with cells for transplantation, the cells are modified, and stress such as oxidative stress in the external environment to the cells is suppressed under preserving or culturing conditions of the cells before transplantation. It has been found that the stress-induced cell damage caused by the stress is reversed. It has been found that cells modified in this way can significantly suppress oxidative stress caused by free radicals and reactive oxygen species on the cells even in a predetermined place or region where transplantation is desired after transplantation. Furthermore, it is understood that the redox nanoparticles are not limited to cells for transplantation, and similarly act or effect on a wide range of cells.
  • A represents unsubstituted or substituted C 1 -C 12 alkyl and the substituent, if substituted, represents a formyl group, a group of formula R′R ′′ CH—, where R ′ and R ′′ are independently and C 1 -C 4 alkoxy or R ′ and R ′′ together represent —OCH 2 CH 2 O—, —O(CH 2 ) 3 O— or —O(CH 2 ) 4 O—, L 1 represents a direct bond or a divalent linking group, L 2 —R 1 is a group in which L 2 is —(CH 2 ) a —NH—(CH 2 ) a — or —(CH 2 ) a —O—(CH 2 ) a —, and R 1 is represented by the formula any of the cyclic nitroxide radical residues represented by R2 is chloro, bromo or hydroxyl; In the above, the repeating units in the polymer backbone with L 2
  • R 1 or R 2 is a1: the following formula , where TEMPO is the following formula any of the cyclic nitroxide radical residues represented by any residue represented by a2: the following formula A residue represented by either a3: the following formula is represented by wherein R 3 is a C 1-3 alkyl group and r is an integer from 2 to 6, the residue A residue selected from the group consisting of the other is OH, or (b) R 1 and R 2 together represent —O— and form a cyclic anhydride residue, or (c) R 1 and R 2 are each OH represents However, in the repeating unit marked with x, (i) either one of R 1 or R 2 in (a) comprises the residue of a1, or (ii) either one of R 1 or R 2 in (
  • the prepared mammalian cells may be damaged in advance by stress in an external environment, and when the stress is applied, the modification is caused by the stress. while the modification protects the cell from the stress when not subjected to the stress.
  • the in vitro combining the nanoparticles and the mammalian cells comprises mixing the nanoparticles and the mammalian cells in a medium for culturing the mammalian cells.
  • nanoparticles based on the copolymer represented by formula (I) or formula (II) can effectively modify mammalian cells.
  • the nanoparticles can protect mammalian cells from stress in the external environment, including oxidative stress, or can recover mammalian cells from damage caused by the stress, thus facilitating the handling of the cells.
  • the mammalian cell formulation that is combined in vitro with the nanoparticles is also free in vivo, where the modified mammalian cells can exist in a predetermined place or region in the living body in which they have been transplanted. Since oxidative stress caused by radicals and reactive oxygen species can be suppressed, cell survival and engraftment rate can be kept high even after transplantation. Therefore, the present invention facilitates in vitro handling of mammalian cells, enables effective use of mammalian cells, and can contribute to, for example, the development of regenerative medicine.
  • FIG. 1 is a diagram showing the results of fluorescent immunostaining in 1-3-1 of Example 1.
  • FIG. 1 is a diagram and graph showing the results of RT-PCR and real-time PCR in 1-3-2 of Example 1.
  • FIG. 2 is a graph showing the results of cell viability assay in 2-2 of Example 2.
  • FIG. 2 is a graph and photographs showing the evaluation results of apoptosis in 2-3 of Example 2.
  • FIG. 2 is a graph and photographs showing evaluation results of superoxide in 2-3 of Example 2.
  • FIG. 2 is a graph showing evaluation results of inflammatory cytokines in 2-4 of Example 2.
  • FIG. 3 is a photograph of an electron spin resonance spectrum and fluorescence immunostaining showing the evaluation results of dynamics of redox nanoparticles in a culture environment in Example 3.
  • FIG. 4 is a graph showing the results of transplantation of cells and redox nanoparticles for determining the optimum concentration of redox nanoparticles to be used in direct intracerebral transplantation of nervous system cells into the cerebral infarction mice of Example 5.
  • FIG. 10 is a graph showing mouse behavior evaluation results in 5-2 of Example 5.
  • FIG. 10 is a graph showing the results of a test of uptake of redox nanoparticles of Example 6 into nervous system cells.
  • Fig. 10 is a graph showing the results of a test of uptake of the redox nanoparticles of Example 7 (related to the copolymer of formula (II)) into nervous system cells.
  • FIG. 11 is a conceptual diagram related to the content of an experiment in Example 8(1);
  • FIG. 11 is a conceptual diagram related to the content of an experiment in Example 8(2);
  • FIG. 11 is a conceptual diagram related to the experimental content of Example 9(1).
  • FIG. 10 is a conceptual diagram relating to the content of an experiment in Example 9(2); It is a fluorescence photographed image (the original is a color image) in Example 8 (1).
  • FIG. 10 is a graph showing the results of intracellular RNP quantification in Example 8(1).
  • Fig. 10 is a graph showing the measurement results of the intracellular residual amount of RNP in Example 8(1).
  • Fig. 10 is a graph showing the measurement results of intracellular RNP 8 hours after OGD treatment in Example 8(2).
  • FIG. 10 is a graphical representation of molar concentration-converted values of intracellular RNP measurement results in Example 8(2).
  • FIG. Fig. 10 is a graph showing the measurement results of RNP-positive cells before and after OGD treatment in Example 9(1). It is a fluorescence photographed image (original is a color image) in Example 9 (1).
  • Fig. 10 is a graph showing the measurement results of RNP-positive cells before and after OGD loading in Example 9 (1).
  • FIG. 10 is a graph showing the measurement results of viable cells before and after OGD loading in Example 9(2).
  • Fig. 10 is a graph showing the measurement results of viable cells before and after RNP and OGD treatment in Example 9(2).
  • Fig. 10 is a graph showing the measurement results of viable cells before and after OGD loading (fold change) in Example 9(2).
  • FIG. 10 is a graph showing the results of calculating the cell-level response to RNP administration in an experimental example preceding Example 8, based only on the average value of fluorescence intensity.
  • Copolymer-based nanoparticles comprising poly(ethylene glycol) segments and polymer chain segments having pendant cyclic nitroxides of formula (I) or formula (II) disclosed herein are As long as the object of the present invention is met, not only nanoparticles composed only of these copolymers, but also copolymers represented by the formula (I), wherein L 2 is —(CH 2 ) a —NH—(CH 2 ) a —, interaction with polyanionic polymers such as poly(meth)acrylic acid, polysulfonic acid, etc., as well as through self-assembly of the copolymer.
  • the block copolymer represented by formula (I) and nanoparticles thereof can be obtained by the methods disclosed in Patent Documents 1 and 2. Specifically, a plurality of block copolymers obtained by the methods described in these patent documents are added to an aqueous medium (containing water, if necessary, a phosphate buffer and / or salt, a water-soluble organic solvent can be contained.), the copolymer can self-assemble or associate to form the nanoparticles.
  • an aqueous medium containing water, if necessary, a phosphate buffer and / or salt, a water-soluble organic solvent can be contained.
  • such molecular assemblies are shelled with poly(ethylene glycol) (PEG) segments that are highly soluble and highly mobile in aqueous media, and consist primarily of L 2 -R 1 and It is understood to form core-shell nanoparticles with a region (core) formed from hydrophobic polymer segments with repeating units of R 3 .
  • Nanoparticles are on the order of nanometers in size, and are contemplated to have sizes in the range of, but not limited to, 5 nm to 500 nm, 10 nm to 300 nm, 10 nm to 100 nm, or 10 nm to 60 nm. .
  • the nanosize of nanoparticles means the average particle size that can be determined in an aqueous solution or homogeneous aqueous dispersion containing them by dynamic light scattering (DLS) analysis. Nano-sized particles formed in an aqueous medium can be obtained as a solid by, for example, freeze-drying, centrifugation, and the like. Such nanoparticles may be abbreviated herein as redox nanoparticles.
  • the divalent linking group defined for L 1 is a poly(ethylene glycol) (hereinafter sometimes abbreviated as PEG) segment and a cyclic nitroxide radical as a side chain may not adversely affect the functionality of the attached poly(methylstyrene) segments, such as the aforementioned nanoparticle-forming ability, redox functionality due to cyclic nitroxide radicals.
  • PEG poly(ethylene glycol)
  • non-limiting divalent linking groups generally refer to groups containing up to 34, preferably 18, more preferably up to 10 carbons and optionally oxygen and nitrogen atoms. do. Specific examples of such a linking group include the following groups:
  • “In vitro” means combinations, preparations, formulations of mammalian cells and said nanoparticles that are treated or prepared ex vivo, and in vivo combination or exclude anything that is prepared or formulated. Specifically, after the redox nanoparticles are administered in vivo, the particles are formed in vivo with cells, organs containing cells, organs or tissues, or parts thereof, or combined. are outside the scope of said preparations and formulations.
  • Modification of mammalian cells means that the cells are changed or modified so as to meet the purpose of the present invention, specifically, suppressing stress under the external environment to mammalian cells , or to alter the cell so that it can be protected from the external environment.
  • stress in the external environment means an action that may have some adverse effect on the functions originally maintained by cells in an environment in which cells can exist in vitro,
  • the causative agent may be oxygen, which may be present in the air.
  • redox nanoparticles when cells are cultured, it may be an action based on reactive oxygen species or free radicals that may be generated in the culture environment, which may adversely affect the survival of the cells.
  • redox nanoparticles When redox nanoparticles are used together with cells for transplantation for cell transplantation or cell therapy, such effects may be due to the in vivo transplantation area or administration site of the transplanted cells or the lesion or lesion requiring treatment. Or it may be an effect caused by reactive oxygen species or free radicals in the vicinity thereof.
  • Environmental stress may be oxidative stress, and also includes stress resulting from oxidation or, in some cases, reduction, where the aforementioned adverse effects on cells are primarily based on reactive oxygen species or free radicals, etc. can.
  • “Protecting stressed cells in an external environment” includes suppressing the decrease in cell viability due to the aforementioned stress, and in some cases, improving cell viability.
  • “recovering damage to mammalian cells caused by the stress” means that when mammalian cells are damaged in advance by the stress, the nanoparticles recover the damage afterwards. means to let Thus, a method comprising a step of combining redox nanoparticles and mammalian cells, a preparation comprising redox nanoparticles as an active ingredient can be used for the preservation of mammalian cells, and when the cells are transplanted or administered in vivo.
  • the nanoparticles that may be contained in a medium in which animal cells can be cultured and the mammalian cells that may be contained in a diluent or in a medium for culturing mammalian cells are combined into one. means. These components are not limited to being integrally contained in a single container or one element of a kit, and even if they are contained in separate containers, they are also included in the configuration of the combination.
  • the diluent may be an aqueous medium commonly used in the art (distilled water, deionized water, phosphate buffered saline (PBS), physiological saline, etc.), and the medium in which mammalian cells can be cultured is , known per se in the art and may be commercially available media.
  • a specific example of such a combination is, for example, the formulation of aspect 6, wherein said nanoparticles and said mammalian cells are in coexistence in a medium in which the mammal is cultured. Further, in the combined form, the nanoparticles and the mammalian cells coexist in a culture medium for culturing a mammal, and the mammalian cells are permeated with the nanoparticles. can be mentioned.
  • an “active ingredient” is an ingredient that helps exhibit the aforementioned actions or functions.
  • a “preparation” is an entity made to perform such an action or function.
  • “Mammalian cells” include, but are not limited to, embryonic stem cells, induced pluripotent stem cells, pluripotent stem cells, somatic (tissue) stem cells, hematopoietic stem cells, mesenchymal stem cells, and neural stem cells. , and nerve cells and glial cells (astrocytes, microglia, oligodendrocytes) differentiated from these cells, or brain, spinal cord, peripheral nerves, heart, liver, kidney, pancreas, lung, intestine, blood, blood vessels , Bone, muscle, and other target tissues, organs, and all progenitor cells and cells induced to differentiate into organs, for example, mammalian cells including human cells.
  • the cells are embryonic stem cells, induced pluripotent stem cells, pluripotent stem cells, somatic (tissue) stem cells, hematopoietic stem cells, neural stem cells, intercellular stem cells.
  • Leaf stem cells and cells induced to differentiate from these cells can be used.
  • Formulations comprising said redox nanoparticles and said cells in vitro are also provided, wherein the redox nanoparticles and cells can be present in combination.
  • Combined forms include, but are not limited to, contained independently in the same container or device, or contained together, e.g., to form a culture system, or different It may be contained individually in a container to form, for example, a cell preparation kit or the like.
  • the redox nanoparticles and the cells can be a mixture containing, if necessary, nutrients and the like required for culturing cells known per se.
  • Such a mixture may be in a state in which the redox nanoparticles and the cells are brought into contact or cultured together so that the particles are endocytosed into the cells.
  • the formulation referred to herein may include redox nanoparticles that are incorporated into cells.
  • Such formulations include embryonic stem cells, induced pluripotent stem cells, pluripotent stem cells, somatic (tissue) stem cells, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and induced differentiation from these cells.
  • the target site or region for such transplantation is not limited as long as the object of the present invention is met, but includes brain, spinal cord, peripheral nerve, heart, liver, kidney, lung, pancreas, intestinal tract, blood, blood vessel, It can be part or all of bone, cartilage, muscle, and eye (retina).
  • ischemic bowel disease blood diseases such as leukemia, immune diseases such as autoimmune diseases, Examples include sepsis, severe infections, graft-versus-host disease (GVHD), bone/cartilage diseases, muscle diseases, retinitis pigmentosa, ischemic optic neuropathy, and the like.
  • GVHD graft-versus-host disease
  • bone/cartilage diseases muscle diseases, retinitis pigmentosa, ischemic optic neuropathy, and the like.
  • formulations can be injected or implanted directly into a lesion or lesion, but cells and redox nanoparticles are present individually, or redox nanoparticles are incorporated into cells, and may be administered intravenously, cerebral artery, or Administration, intrathecal administration, subcutaneous implantation, intramuscular administration, and the like can be used.
  • Such formulations can also be prepared by including excipients and diluents commonly used in the art.
  • 2,2,6,6-tetramethylpiperidine-1-oxylamine is sometimes abbreviated as TEMPOL.
  • PEG-b-PMNT was dissolved in dimethylformamide (DMF) at a concentration of 150 mg/mL, and the solution was placed in a dialysis membrane, sealed, and dialyzed against distilled water to prepare redox nanoparticles (RNP). .
  • Distilled water was replaced every 2, 4, 8 and 20 hours from the start of dialysis, and the solution in the dialysis membrane was collected after 24 hours.
  • a 10-fold concentration of PBS was added to the solution in the dialysis membrane so that the final concentration was 1-fold of the PBS concentration.
  • the particle size (Z-Ave) was 26.5 nm
  • the polydispersity index (PDI) was 0.12
  • the particle size distribution was uniform. .
  • Minced pieces were seeded into 6 cm dishes with 3 ml of growth medium. After the spindle-shaped cells grown from the slices reached 80% confluence, they were subcultured at a ratio of 1:3 with 0.1% trypsin containing 0.2% EDTA. 1 ⁇ 10 4 cells were plated in growth medium in 10 cm dishes.
  • the largest colony was recovered by colonial cloning using the filter paper method, and cultured in the same growth medium.
  • the separated cells were dental pulp stem cells, and the material used for the medium was purchased from Thermo Fisher Science Co. Ltd.
  • ⁇ 10 5 dental pulp stem cells were cultured in a 6 cm dish in the aforementioned growth medium. After the cells reached 80% confluence, they were cultured in a neural differentiation medium.
  • the neural differentiation induction medium used had the following composition (DMEM/F12, 5% FBS supplemented with 15nM all-trans-retinoic acid (ATRA), 20nM progesterone, 20nM estradiol, 20nM NGF-1, 10ng/ml thyroxine, 10 nM dexamethasone, 50 ⁇ M ascorbic acid, and 20 ng/ml IGF-1).
  • the neural differentiation induction medium was exchanged twice a week.
  • 1-3 Evaluation of cultured cells 1-3-1. Fluorescent Immunostaining 2 ⁇ 10 5 dental pulp stem cells and 2 ⁇ 10 5 neural cells were cultured on a cover glass placed in a 35 mm dish. Cells were fixed with 99.8% methanol at ⁇ 30° C. for 15 minutes, washed with PBS three times, and treated with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) for 10 minutes at room temperature. Cells were subjected to antigen-antibody reaction using the following primary antibodies overnight at 4°C, and then reacted with appropriate secondary antibodies for 60 minutes at room temperature in a dark room. After washing with PBS three times, the cells were mounted using a mounting medium containing DAPI (SCR-038448, dianova).
  • DAPI SCR-038448, dianova
  • Antibodies used rabbit anti-Nestin antibody (1:200; cat. No., N5413; Sigma-Aldrich), rabbit anti-Doublecortin (DCX) antibody (1:1000; cat. No., ab18723; Abcam), mouse anti-MAP2 Antibody (1:500; cat. No., M4403; Sigma-Aldrich), rabbit anti-GFAP antibody (1:1000; cat. No., ab7260; Abcam).
  • RNA was extracted from cultured cells using Trizol reagent (Cat. No., 15596-018; Invitrogen). The specimens used were dental pulp stem cells induced from the pulp of another 3 individuals (n 3) and nervous system cells induced therefrom. cDNA was prepared from the extracted total RNA using a reverse transcription kit (Applied Biosystems, Cheshire, UK). For RT-PCR, cDNA was reacted using the following primers and a thermal cycler.
  • cDNA was mixed with qPCR master mix (Cat. No., A15297; Applied Biosystems) and TaqMan probes (Nestin, DCX, MAP2, GFAP, Olig2) and reacted with QuantStudio5 (Thermo Fisher Scientific).
  • PRL13A Hs03043887_gH was used as a control. Data were compared using the comparative C T ( ⁇ CT) method.
  • Example 2 Examination of neuroprotective effect of redox nanoparticles under hypoxic culture + reperfusion environment 2-1.
  • Method of hypoxic culture + reperfusion culture A hypoxic culture + reperfusion model was used in vitro as a pseudo model of peri-infarct foci (Abramov AY, et al., J Neurosci. 2007;27(5):1129- 38.).
  • Hypoxic culture 1% oxygen, 37°C
  • A TEMPOL
  • B redox nanoparticles
  • C TUNEL staining Red indicates TUNEL staining
  • blue indicates cell nuclei
  • A TEMPOL
  • B redox nanoparticles
  • C MitoSOX Red indicates superoxide that reacted with MitoSOX
  • blue indicates cell nuclei
  • Control ii
  • TEMPOL iii
  • Example 3 Evaluation of dynamics of redox nanoparticles in a culture environment (electron spin resonance method and fluorescence immunostaining) Rhodamine-labeled redox nanoparticles were used to assess the dynamics of redox nanoparticles in neurons (Hosoo H, et al., Stroke. 2017;48(8):2238-47). Electron spin resonance method was used to measure the structure and quantity of free radicals (Yoshitomi T, et al., Bioconjugate chemistry. 2009;20(9):1792-8, Vong LB, et al., Biomaterials. 2015 ;55:54-63).
  • Nervous system cells were prepared by normal culture for 9 hours and hypoxia culture + reperfusion for 1 hour, and TEMPOL and rhodamine-labeled redox nanoparticles were administered to 200 ⁇ M as described above.
  • Cell supernatants and cell suspensions were prepared for evaluation by electron spin resonance. The cell suspension was washed three times with Hanks solution to remove TEMPOL- or rhodamine-labeled redox nanoparticles adhering to the outside of the cells. The cell suspension was adjusted to 6.0 ⁇ 10 6 cells/400 ⁇ l, and 100 ⁇ l was measured by the electron spin resonance method.
  • reduced radicals in the culture supernatant and cell suspension were measured by reoxidation using potassium hexacyanoferrate(III) (10 mM; Kanto Chemical Co., Japan).
  • the ESR measurement conditions were Magnetic Field 335.5+/-7.5mT, Gain x790, Modulation Width 0.2mT, Time constant 1sec., Sweep time 2min.
  • nervous system cells were prepared by normal culture for 9 hours and hypoxia culture + reperfusion for 1 hour, and rhodamine-labeled redox nanoparticles were administered to 200 ⁇ M. After fixing the cells with 4% paraformaldehyde, the cells were mounted with a DAPI-containing mounting medium and observed under a fluorescence microscope.
  • the redox nanoparticles were disintegrated in the cells and existed in a polymer state, indicating that redox reactions occurred ((Yoshitomi T, et al., Biomacromolecules. 2009;10( 3):596-601.) Also, in the examination of the culture supernatant (Fig. 7.C), a sharp triplet signal was detected in the TEMPOL group, while a broad signal was detected in the rhodamine-labeled redox nanoparticle group. (See above.) Furthermore, even when potassium hexacyanoferrate(III) was added to the culture supernatant, a sharp triplet signal was detected in the TEMPOL group.
  • FIG. 7. A, B Fluorescent immunostaining showed that rhodamine-labeled redox nanoparticles were in the cytoplasm both after normal culture and hypoxic culture + reperfusion. confirmed (Fig. 7. A, B).
  • A electron spin resonance method and fluorescence immunostaining of normal culture
  • B electron spin resonance method and fluorescence immunostaining of hypoxic culture + reperfusion
  • C electron spin of culture supernatant of hypoxic culture + reperfusion Resonance method Red indicates rhodamine and blue indicates cell nuclei.
  • Example 4 Creation of cerebral infarction model mice due to distal middle cerebral artery occlusion The animal experiment plan was approved by the University of Tsukuba Life Science Animal Resource Center (approval number: #20-132). All experiments were conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals.”
  • mice Male, 7-8 weeks old, body weight 20-25 g were purchased from CLEA, Japan and used.
  • a mouse distal middle cerebral artery occlusion model was prepared by the method reported by Taguchi et al. (See Taguchi A, et al., The Journal of clinical investigation. 2004;114(3):330-8.). Briefly, mice were anesthetized by intraperitoneal injection of a mixed solution of ketamine (70 mg/kg) and xylazine (14 mg/kg). A skin incision was made on the temporal region of the mouse and a small craniotomy was made in the temporal bone with a dental drill.
  • Example 5 Method of direct intracerebral transplantation of nervous system cells into mice with cerebral infarction Four weeks after transplantation of cells and redox nanoparticles (0, 200, 500, 1000 ⁇ M) to determine the optimal concentration of redox nanoparticles in advance The mouse brain was taken out and the number of surviving transplanted cells was confirmed. A cell suspension of nervous system cells was prepared in advance and adjusted so that 1.0 ⁇ 10 5 cells would be transplanted. In addition, the redox nanoparticles were mixed with the cell suspension before cell transplantation to form a mixed solution. 500, 1000 ⁇ M showed more survival than 0, 200, and no difference was observed at 500, 1000 ⁇ M. See FIG.
  • the transplanted cells or substances were divided into 4 groups: PBS, redox nanoparticles, cells + PBS, and cells + redox nanoparticles.
  • PBS head fixator
  • redox nanoparticles cells + PBS
  • cells + redox nanoparticles Two days after the cerebral infarction treatment, the mice were fixed in a head fixator (NARISHIGE, Japan) after general anesthesia as in 3-6. A skin incision was made in the midline of the head, and Hamilton Syringe (Cat. No., 4025-11701, GLSciences) was used at the periinfarct site 1 mm anteriorly and 1.5 mm laterally from bregma to a depth of 2 mm from the surface of the brain. was injected into the brain by pointing the needle to The injection was performed slowly over 10 minutes, and the needle was slowly withdrawn after waiting 5 minutes after injection.
  • mice transplanted with PBS and redox nanoparticles were compared. Two days after the cerebral infarction treatment, mice were given general anesthesia as described above, and PBS and redox nanoparticles were implanted into the peri-infarct foci. One hour after transplantation, 27 mg/kg of dihydroethidium (Cat. No., D1168, Invitrogen) (200 ⁇ l in total) was intraperitoneally administered twice at 30-minute intervals (Hu D, et al., The Journal of Neuroscience. 2006; 26(15):3933-41).
  • dihydroethidium Cat. No., D1168, Invitrogen
  • mice were placed in a transparent plastic cylinder with a diameter of 8 cm and a height of 12 cm, and the number of times they used their forelimbs was measured.
  • the rate of forelimb use was calculated using the following formula [paralyzed forelimb/(paralyzed forelimb + non-paralyzed forelimb + both sides)] x 100 (Craft TK, et al., Stroke. 2005;36(9):2006- 11.).
  • FIG. 10 The results are shown in FIG. From FIG. 10, it was confirmed that cell therapy using cells + redox nanoparticles according to the present invention improved neurological symptoms in cerebral infarction model mice. Specifically, it is as follows. Adhesive removal test (Fig. 10. A) and Cylinder test (Fig. 10. B) behavioral evaluation of mice showed improvement in all four groups. Furthermore, the groups containing cells (cells + PBS and cells + redox nanoparticles group) showed better improvement in behavioral assessment from 1 week after transplantation compared to the groups without cells (PBS, redox nanoparticles group). rice field.
  • mice were perfusion-fixed with 4% paraformaldehyde one week and six weeks after transplantation, and the mouse brains were excised and cryosections were prepared.
  • Antigen-antibody treatment was performed overnight at 4°C using the following primary antibodies, followed by 60 minutes at room temperature in the dark using appropriate secondary antibodies.
  • Mounting was performed using DAPI-containing mounting medium (SCR-038448, dianova). Observations were made using a fluorescence microscope (Leica Microsystems Wetzlar, Germany).
  • the primary antibodies used were mouse anti-STEM121 antibody (1:1000, cat.
  • FIG. 11.A One week after transplantation (Fig. 11.A), there was no difference in the survival rate of transplanted cells between the PBS and redox nanoparticle groups. However, at 6 weeks after transplantation (Fig. 11.B), the survival rate of transplanted cells was higher in the redox nanoparticles group than in the PBS group (P ⁇ 0.05). Fluorescent immunostaining showed positive cells for both Doublecortin, MAP2, and GFAP in both the PBS group (Fig. 11. A-i) and the redox nanoparticle group (Fig. 11. A-ii) one week after transplantation. Six weeks after transplantation, most of the cells in the PBS group (Fig. 11.A).
  • Example 6 Uptake of Redox Nanoparticles (Related to the Copolymer of Formula (I)) into Neural Cells
  • Neural cells were cultured in a 24-well plate at 6 ⁇ 10 4 cells/well.
  • Rhodamine-labeled redox nanoparticles were separately administered to the culture solution at 100 ⁇ M and 500 ⁇ M, and normal culture was performed. After 15 minutes, 1 hour, 3 hours, 6 hours and 24 hours after administration, observation was performed using a fluorescence microscope. After that, each well was washed twice with Hanks, and the cells were fixed with 4% paraformaldehyde for 15 minutes.
  • antigen-antibody reaction was performed overnight at 4°C using the following primary antibody, and then reaction was allowed to proceed for 60 minutes at room temperature in a dark room using an appropriate secondary antibody. After washing with Hanks, they were mounted using DAPI-containing mounting medium (SCR-038448, dianova). Observation was performed at the same time using a fluorescence microscope, fluorescence intensity was measured using ImageJ, and average values were compared.
  • Antibody used mouse anti-rhodamine antibody (1:250; Cat. No., ab9093; Abcam).
  • Example 7 Uptake of Redox Nanoparticles (Related to the Copolymer of Formula (II)) into Neural Cells
  • Neural cells were cultured in a 24-well plate at 3.2 ⁇ 10 4 cells/well.
  • Cy5-labeled nanoparticles prepared according to "Production Example 17: Preparation of nanoparticles of SMAPo TN (N564)" in Patent Document 3 (Japanese Patent Application Laid-Open No. 2019-123773) were administered into the culture medium at a concentration of 3.2 mM. Then, normal culture was performed. Observation was performed using a fluorescence microscope 1 hour, 3 hours, 6 hours, and 24 hours after administration. Fluorescence intensity was measured using ImageJ and average values were compared.
  • Example 8 Verification of cell permeability of RNP (1) Establishment of RNP penetration time In order to evaluate the permeability of RNP over time by treatment time and establish the most effective concentration and treatment time, Based on 100 ⁇ M, which was determined to be present, the intracellular RNP amount was measured by fluorescence observation of Rhodamine in RNP for each treatment time.
  • FIG. 14 shows a conceptual diagram of the contents of the experiment. Cells were seeded at a density of 10,000 cells/well on a 2.5 v/v % Matrigel-coated 24-well plate. NB27 medium, which is a nerve cell medium, was used as the medium. RNP treatment of the cells was carried out by normal culture at 37° C.
  • the treatment time was set to 15 minutes, 1 hour, 3 hours, 6 hours and 12 hours, respectively, and untreated cells were cultured for the same time as a control group.
  • each cell was washed twice with PBS(-), the medium was returned to the normal neuronal cell medium NB27 medium, and all-in-one fluorescence was used by Keyence. Photographs were taken with a microscope (BZ-X800). Photographs were taken immediately after each RNP treatment and after washing, and normal culture was performed for 48 hours after all conditions were completed.
  • imageJ software (Ver.1.53q) was used to determine the total area of positive Rhodamine signals from fluorescence images and the total area of cells from phase contrast images. changed.
  • the threshold for positive signal calculation was determined according to the ISO Data parameter of the software.
  • FIG. 15 shows a conceptual diagram of the contents of the experiment.
  • Cells were seeded at a density of 10,000 cells/well on a 2.5 v/v % Matrigel-coated 24-well plate.
  • NB27 medium which is a nerve cell medium, was used as the medium.
  • RNP treatment of the cells was carried out by normal culture at 37° C. under 5% CO 2 environment for 48 hours after seeding, and then the medium was changed to RNP:DMEM/LG medium adjusted to each concentration.
  • the OGD load was applied immediately after the medium exchange, and culture was performed for 8 hours. After completion of OGD, each cell was washed twice with PBS(-), the medium was returned to normal neuronal cell medium NB27 medium, and photographed with a Keyence All-in-one microscope (BZ-X800). Imaging was performed after washing immediately after OGD.
  • imageJ software (Ver.1.53q) was used to determine the total area of Rhodamine signal-positive from the fluorescence image and the total area of the entire cell from the phase contrast image, and quantify the positive area divided by the cell area. . Threshold for positive signal calculation was performed according to the ISOData parameter of the software.
  • Example 9 Verification of antioxidant effect of RNP (1) Verification of antioxidant capacity of RNP against reperfusion damage
  • RNP was applied to cells before and after OGD loading. It was set to show preventive and therapeutic effects, respectively.
  • FIG. 16 shows a conceptual diagram of the contents of the experiment. Cells were seeded at a density of 10,000 cells/well on a 2.5 v/v % Matrigel-coated 24-well plate. NB27 medium, which is a nerve cell medium, was used as the medium. Cells were treated with RNP after seeding and normal culture at 37°C in a 5% CO2 environment for 48 hours. rice field.
  • Samples (PreRNP) for prophylactic effect observation were cultured normally for 48 hours, then the medium was changed to RNP medium, and OGD was applied for 8 hours at 37°C in a 1% O 2 environment. After OGD, the medium was removed by aspiration, the cells were washed twice with PBS(-), and the neuron medium was replaced with NB27 medium, and simulated reperfusion damage was given at 37°C and 5% CO 2 for 48 hours. rice field.
  • Samples (PostRNP) for observation of therapeutic effects were cultured for 48 hours, washed twice with PBS(-), replaced with DMEM/LG medium, and placed under OGD at 37°C and 1% O2 for 8 hours. processed. After OGD, the medium was replaced with RNP:DMEM/LG medium with the RNP concentration adjusted as described above, and cultured at 37°C under 5% CO 2 environment for 12 hours. After RNP treatment, the medium was removed by aspiration, the cells were washed twice with PBS(-), and the neuron medium was replaced with NB27 medium, and simulated reperfusion damage was given at 37°C in a 5% CO 2 environment. .
  • FIG. 17 shows a conceptual diagram of the contents of the experiment.
  • RNP was administered to the cells before and after OGD loading, and the prophylactic and therapeutic effects of each were determined.
  • Cells were seeded at a density of 5,000 cells/well on 2.5 v/v % matrigel-coated 96-well plates.
  • NB27 medium which is a nerve cell medium, was used as the medium.
  • RNP treatment of the cells was carried out using DMEM/LG medium adjusted to RNP concentrations of 10, 50, 100 and 500 ⁇ M, respectively, after normal culture at 37° C. and 5% CO 2 for 48 hours after seeding.
  • cell viability was measured using a 1:1 mixture of CellTiter Glo2.0 (Promega) reagent and DMEM/LG.
  • the medium was removed, the cells were washed twice with PBS(-), replaced with CellTiter Glo2.0:DMEM/LG solution, the well plate was gently agitated for 2 minutes, and allowed to stand at room temperature for 10 minutes for reaction. After the reaction, the plate was set in a Varioskan LUX microplate reader (Thermo) and the yellow luminescence of Luciferin reacted with ATP in living cells was quantified by luminometric measurement.
  • FIG. 23 shows the results converted to . From FIG. 23, it can be seen that there was no significant difference between 50 ⁇ M and 100 ⁇ M, but the RNP permeability increased at 500 ⁇ M when administered after OGD loading.
  • FIG. 24 shows the results of photographing the cells in each experiment taken with a fluorescence microscope after the completion of each experiment in Example 9(1). Qualitative analysis of cell damage was performed by qualitatively comparing axonal damage (yellow circles) with dendrite and cell body damage (white circles) in FIG.
  • the amount of RNP also increased in a concentration-dependent manner, but the efficiency was similarly high at the dose concentration-to-signal ratio of 50 ⁇ M and 100 ⁇ M, and the signal was the highest at 500 ⁇ M, but the efficiency was 100 ⁇ M. was about 40% lower than the From this result, while 100 ⁇ M obtained in the above experimental example seems to be an appropriate concentration, it was necessary to verify whether not only the penetration rate but also the therapeutic effect showed the same efficiency.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Developmental Biology & Embryology (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Rheumatology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Toxicology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Hematology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/JP2022/039300 2021-10-22 2022-10-21 レドックスナノ粒子の細胞処理への使用 WO2023068362A1 (ja)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023554758A JPWO2023068362A1 (enrdf_load_stackoverflow) 2021-10-22 2022-10-21

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021172795 2021-10-22
JP2021-172795 2021-10-22

Publications (1)

Publication Number Publication Date
WO2023068362A1 true WO2023068362A1 (ja) 2023-04-27

Family

ID=86058332

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/039300 WO2023068362A1 (ja) 2021-10-22 2022-10-21 レドックスナノ粒子の細胞処理への使用

Country Status (2)

Country Link
JP (1) JPWO2023068362A1 (enrdf_load_stackoverflow)
WO (1) WO2023068362A1 (enrdf_load_stackoverflow)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025074996A1 (ja) * 2023-10-02 2025-04-10 国立大学法人筑波大学 外科手術又は移植手術における虚血再灌流障害を抑制するための製剤

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019123773A (ja) * 2018-01-12 2019-07-25 国立大学法人 筑波大学 修飾されたスチレン−無水マレイン酸共重合体及びその使用
JP2020186181A (ja) * 2019-05-10 2020-11-19 国立大学法人 筑波大学 運動能力向上用製剤

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019123773A (ja) * 2018-01-12 2019-07-25 国立大学法人 筑波大学 修飾されたスチレン−無水マレイン酸共重合体及びその使用
JP2020186181A (ja) * 2019-05-10 2020-11-19 国立大学法人 筑波大学 運動能力向上用製剤

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025074996A1 (ja) * 2023-10-02 2025-04-10 国立大学法人筑波大学 外科手術又は移植手術における虚血再灌流障害を抑制するための製剤

Also Published As

Publication number Publication date
JPWO2023068362A1 (enrdf_load_stackoverflow) 2023-04-27

Similar Documents

Publication Publication Date Title
Itosaka et al. Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering
JP5529021B2 (ja) 臍帯血由来の間葉系幹細胞を含むＩＬ−８またはＧＲＯ−α発現細胞に関連する疾患の診断、予防または治療用の組成物
Guan et al. Photo-triggered gadofullerene: enhanced cancer therapy by combining tumor vascular disruption and stimulation of anti-tumor immune responses
KR102081666B1 (ko) 암 치료용 약학 조성물
Tan et al. Integrating hydrogels manipulate ECM deposition after spinal cord injury for specific neural reconnections via neuronal relays
CN104105493A (zh) 哺乳动物胎儿肺细胞以及其治疗用途
CN113975397B (zh) 一种基因/小分子化合物纳米递药系统及其制备方法与应用
WO2023068362A1 (ja) レドックスナノ粒子の細胞処理への使用
Xiong et al. Transplantation of hematopoietic stem cells promotes functional improvement associated with NT-3-MEK-1 activation in spinal cord-transected rats
Yan et al. Adhesive and conductive hydrogel-based therapy simultaneously targeting neuroinflammation and neurofunctional damage after brain injury
Ko et al. Transplantation of neuron‐inducing grafts embedding positively charged gold nanoparticles for the treatment of spinal cord injury
Zeng et al. Injectable hydrogel microspheres encapsulating extracellular vesicles derived from melatonin-stimulated NSCs promote neurogenesis and alleviate inflammation in spinal cord injury
Francipane et al. The lymph node as a new site for kidney organogenesis
Gui et al. Nanoscale coordination polymer Fe-DMY downregulating Poldip2-Nox4-H2O2 pathway and alleviating diabetic retinopathy
Liu et al. AN‐Cadherin Nano‐Antagonist Hydrogel Enhances Recovery From Spinal Cord Injury by Impeding Glial Scarring
Yan et al. A mussel-inspired, antibacterial, antioxidant, injectable composite hydrogel for the sustain delivery of salvianolic acid B for the treatment of frozen shoulder
CN116059400A (zh) 调节髓核氧代谢平衡的水凝胶微球的制备方法及其应用
Wei et al. Highly aligned electroactive ultrafine fibers promote the differentiation of mesenchymal stem cells into Schwann-like cells for nerve regeneration
Au et al. An injectable subcutaneous colon-specific immune niche for the treatment of ulcerative colitis
KR101635148B1 (ko) 신경세포 및 엘라스틴 유사 폴리펩타이드를 포함하는 파킨슨병 치료 촉진용 약학적 조성물
US20180280446A1 (en) Agent for Treating Urinary Incontinence Including Stem Cells Derived from Amniotic Fluid
Sun et al. Dual sustained-release BMP7-nanoparticle hydrogel scaffolds for enhanced BMSC neuronal differentiation and spinal cord injury repair
CN113499318A (zh) 红细胞膜包被载药纳米粒/探针及其在脑胶质母细胞瘤诊疗中的应用
CN116421565B (zh) 一种生物钟调节水凝胶微球及其制备方法与应用
CN116473991B (zh) miR-143-3p在制备治疗周围神经损伤药物或材料中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22879639

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023554758

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22879639

Country of ref document: EP

Kind code of ref document: A1