WO2017101717A1 - Utilisation d'une molécule d'agent de contraste de ciblage mitochondrial comme agent de contraste t2 - Google Patents

Utilisation d'une molécule d'agent de contraste de ciblage mitochondrial comme agent de contraste t2 Download PDF

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WO2017101717A1
WO2017101717A1 PCT/CN2016/108801 CN2016108801W WO2017101717A1 WO 2017101717 A1 WO2017101717 A1 WO 2017101717A1 CN 2016108801 W CN2016108801 W CN 2016108801W WO 2017101717 A1 WO2017101717 A1 WO 2017101717A1
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targeting
group
molecule
contrast agent
contrast
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Chinese (zh)
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邓宗武
谭波
张艳辉
张宏岩
张海禄
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中国科学院苏州纳米技术与纳米仿生研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/12—Macromolecular compounds
    • A61K49/124—Macromolecular compounds dendrimers, dendrons, hyperbranched compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A61K49/146—Peptides, e.g. proteins the peptide being a polyamino acid, e.g. poly-lysine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0623—Stem cells
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells

Definitions

  • the present application relates to the field of medical imaging, and in particular to the use of a contrast agent molecule targeting mitochondria as a magnetic resonance T 2 contrast agent, and the present application also relates to a contrast agent molecule targeting mitochondria, labeled magnetic labeled cells, And a combination of magnetically labeled cells and scaffold materials, and magnetic resonance imaging (MRI) in vivo tracing methods.
  • MRI magnetic resonance imaging
  • Stem cell regenerative medicine is an emerging field of biomedicine.
  • the basic idea is to achieve the regeneration and repair of damaged tissues and organs by inducing the differentiation of stem cells in the transplanted body.
  • it is necessary to track the physiological behaviors such as survival, migration and homing, and directed differentiation after stem cell transplantation in vivo, and accurately trace the tissue biology of stem cells, and distinguish between internal and external stem cells and self-renewal.
  • Stem cells and functional cells produced by differentiation so as to deeply understand the physiological processes of stem cell migration, reproduction, division and differentiation in vivo, and this has the basic research on stem cell biology and the evaluation of clinical observation and functional recovery. Very important meaning.
  • Magnetic resonance imaging as a non-invasive bioimaging technique with high spatial resolution and soft tissue contrast and no risk of ionizing radiation, is the most promising technique for tracing stem cell maintenance and differentiation in vivo.
  • Magnetic resonance imaging is based on the spine magnetic moment of water protons in different biological tissues. The magnetic moment formed by the ordered arrangement of the magnetic field in a uniform magnetic field is excited by a specific microwave, and its longitudinal relaxation rate (1/T 1 ) or transverse relaxation. The rate (1/T 2 ) may be different, resulting in different signal intensities of the echoes. The difference in contrast formed in the image enables structural and functional imaging of cells, tissues and organs of the organism.
  • the imaging of the specific tissue can also be achieved by introducing a magnetic resonance contrast agent to change the relaxation rate of water protons in a specific tissue such as tumor tissue.
  • a magnetic resonance contrast agent to change the relaxation rate of water protons in a specific tissue such as tumor tissue.
  • T 1 contrast agents for MRI signals attenuate some suitable, known as T 2 contrast agents.
  • T 2 contrast agents for MRI signals attenuate some suitable, known as T 1 contrast agents.
  • the acceleration effect of the ruthenium complex on the longitudinal relaxation rate of water protons is significantly higher than the acceleration effect on the transverse relaxation rate, which is beneficial for generating bright signals under T 1 -weighted images and enhancing T 1 -weighted images. Contrast is therefore often used as a T 1 contrast agent.
  • the accelerated effect of superparamagnetic iron oxide (SPIO) nanoparticles on the transverse relaxation rate of water protons is much more pronounced than the acceleration of their longitudinal relaxation rate, producing dark signals and enhancing T 2 -weighted images in T 2 -weight imaging mode.
  • the contrast is ideal for T 2 contrast agents.
  • the same contrast agent is distributed at different biological interfaces, and there may be a large difference in the relative amplitude of the two relaxation rates. If the ruthenium complex is distributed in the cytoplasm in the form of free or vesicles or when it binds to mitochondria in the cytoplasm, there is a significant difference in the effects of the longitudinal and transverse relaxation rates of the water protons.
  • the T 2 contrast agent represented by SPIO nanoparticles has a high transverse relaxation rate and, therefore, has been widely studied and applied in the living cell image of stem cells.
  • this SPIO nanoparticle-based cell labeling technology essentially provides information on the migration of SPIO nanoparticles in vivo. This migration occurs with the parent cells carrying the nanoparticles, or the migration of the free nanoparticles themselves after the death of the mother cells. Or the migration of macrophages after phagocytosis by other cells such as macrophages, the current imaging methods can not give clear conclusions, and become the main challenge of image information analysis. Moreover, such cell markers do not intuitively tell people that labeled cells are still living cells after entering the body, or have died or partially died.
  • the ruthenium complex has been widely used as a T 1 contrast agent in clinical medicine.
  • the clinical application of ruthenium complex contrast agents can be divided into two categories: one is a cyclic structure of DOTA and its derivatives.
  • One class is DTPA and its derivatives with acyclic structure. Due to the clear and stable chemical structure of the small molecule contrast agent, the process and the result of coupling with the targeting molecule can be precisely controlled by chemical methods, and thus the structural unit as a targeted contrast agent has high reliability.
  • a key problem faced by ruthenium complex contrast agents is that their relaxation rate is much lower than that of T 2 -type SPIO nanoparticles, so in order to obtain sufficient tissue contrast, a larger dose is required, resulting in a metal ruthenium. Ions in the body may pose concerns about safety issues such as toxicity.
  • the patent publication US20090214437A1 and US20130142735A1 a magnetic resonance contrast agent which binds to the mitochondria
  • the MRI contrast agent is injected intravenously into the body, it may be enriched in active mitochondria of tumor tissue, the tumor tissue to enhance its use in T 1 the magnetic resonance signal (light signal rendering) of weighted imaging mode, thereby improving the contrast ratio of T 1 weighted images.
  • the contrast agent is applied as a T 1 contrast agent to a living body image of a cell transplant, the survival and migration of the labeled cells in the body cannot be clearly determined, and the duration of the signal enhancement effect cannot meet the needs of long-term observation.
  • the present application is based on the inventors' recognition that iridium complexes are distributed at different biological interfaces, and that their effects on the longitudinal and transverse relaxation rates of cellular water protons are significantly different, especially when the ruthenium complex is in the cytoplasm.
  • After mitochondria binding its ability to accelerate the longitudinal relaxation rate of cell water progeny is significantly reduced, which is not conducive to the enhancement of magnetic resonance signals, but is conducive to the attenuation of magnetic resonance signals, so it is more suitable for generating dark signals in T 2 weighted imaging mode.
  • the marker portions cells release the contrast agent molecule, releasing contrast agent molecule causes the surrounding tissue at the magnetic resonance cell transplant T 2 weighted mode A bright signal is presented so that the cell transplant can be more clearly distinguished from its surrounding tissue.
  • the object of the present application is to provide a targeted contrast agent molecules mitochondrial use as T 2 contrast agents
  • the mitochondrial targeting contrast agent comprises a targeting molecule and a contrast unit cell
  • the targeting means is a - P + (X 1 )(X 2 )(X 3 ) a phosphonium cation of the formula wherein X 1 , X 2 , X 3 represents a C 1-12 alkyl group which is unsubstituted or substituted with one or more substituents.
  • a C 1-12 alkenyl group or a C 6-10 aryl group the substituent comprising 1, 2 or 3 halogen atoms, C 1-12 alkyl group, C 6-10 aryl group, hydroxyl group, C 1-12 Alkoxy, halo-C 1-12 alkoxy; wherein X 1 , X 2 , X 3 may be the same group or different groups; the contrast unit is a superparamagnetic metal complex Things.
  • Mitochondria After the contrast agent targeting molecule Mitochondria Mitochondria binding showed significantly reduced the effectiveness of magnetic resonance signals, such that the magnetically labeled cells in vitro and in vivo MRI T 2 weighted images are rendered dark pattern signal; the When the targeting unit is combined with a plurality of magnetic resonance imaging units and used for cell labeling, it exhibits a stronger magnetic resonance signal attenuating effect and can last longer.
  • the targeting unit is a triphenylphosphonium cation or a derivative thereof.
  • the superparamagnetic metal complex is formed of a superparamagnetic metal and a complexing agent, wherein: the superparamagnetic metal is a metal having superparamagnetic properties, including but not Limited to lanthanide metal lanthanum (Pr), ⁇ (Nd), ⁇ (Pm), ⁇ (Sm), ⁇ (Eu), ⁇ (Gd), ⁇ (Tb), ⁇ (Dy), ⁇ (Ho), ⁇ (Er), yttrium (Tm), yttrium (Yb), lanthanum (Lu), and non-lanthanide metal chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), yttrium (Y), ytterbium (Nb), etc.; the complexing agent is selected from the group consisting of DOTA, HP-DO3A, DO3A-butrol, DTPA-BMA, DTPA
  • the targeting unit is linked to a dendritic or linear molecule, either directly or via a linker, directly or through a spacer and contrast.
  • the unit is joined, wherein the structural unit of the dendritic or linear molecule is any monomer, preferably an amino acid, more preferably lysine, which can be homopolymerized or copolymerized to form a dendritic or linear macromolecule.
  • the linker is a linear amino acid, preferably lysine;
  • the targeting unit is linked to the 1-8 imaging unit by the dendritic or linear molecule.
  • Each of said -P + (X 1) (X 2) (X 3) or linear cationic dendrimer molecule by binding a plurality of superparamagnetic metal complex magnetic resonance signals attenuate more pronounced effect, in The time for dark signals to be imaged in the T 2 weighting mode can be longer.
  • the contrast agent molecule and mitochondria can be increased by using a plurality of targeting units, preferably 2 targeting units, by connecting the dendritic or linear molecules to 1-8 contrast units. strength of the bond, to further extend its presentation time when the dark signal is imaged at T 2 weighting pattern.
  • a contrast agent molecule targeting mitochondria comprising a targeting unit and a contrast unit, wherein: the targeting unit has -P + (X 1 )(X 2 ) (X 3 a phosphonium cation of the general formula wherein X 1 , X 2 , X 3 represents a C 1-12 alkyl group, a C 1-12 alkenyl group, or a C 6-10 which is unsubstituted or substituted with one or more substituents.
  • the substituent includes 1, 2 or 3 halogen atoms, C 1-12 alkyl, C 6-10 aryl, hydroxy, C 1-12 alkoxy, halo alkoxy -C 1-12 a group; wherein X 1 , X 2 , X 3 may be the same group or a different group;
  • the contrast unit is a superparamagnetic metal complex;
  • the targeting unit is directly or through a linker a dendritic or linear molecule linked, the dendritic or linear molecule being attached to the contrast unit directly or via a spacer, wherein the structural unit of the dendritic or linear molecule is any homopolymerizable or copolymerizable to form a dendrimer or line A monomer, preferably an amino acid, of a macromolecule, more preferably lysine.
  • the linker is selected from a linear amino acid, preferably a lysine;
  • the targeting unit is linked to the 1-8 imaging unit by the dendritic or linear molecule.
  • a plurality of targeting units are employed by the dendritic or linear molecules to be coupled to 1-8 contrasting units.
  • the present application also provides a method of preparing the aforementioned contrast agent molecule targeting mitochondria, comprising: each of said -P + (X 1 )(X 2 )(X 3 ) cations with a halogenated carboxylic acid or a halogenated
  • the amine reacts to form a -P + (X 1 )(X 2 )(X 3 ) cation having a carboxyl or amino functional group, and the -P + (X 1 )(X 2 )(X 3 ) cation passes through the obtained carboxyl group or amino group.
  • the halocarboxylic acid is a chloro, bromo or iodo fatty acid or an aromatic acid
  • the superparamagnetic metal complex passes through its carboxyl or amino group with a dendritic or linear molecule
  • the carboxyl group of the superparamagnetic metal complex is selected from the group consisting of ethyl carboxyl group, propyl carboxyl group or butyl carboxyl group
  • the amino group of the superparamagnetic metal complex is selected from ethylamino group, propylamino group or butylamino group.
  • a mitochondrial-targeting contrast agent molecule is synthesized by a solid phase synthesis method using a cross-protection deprotection strategy to sequentially synthesize a dendrimer with or without a linker or spacer. Or a linear molecule, and a contrast unit with or without a spacer, then a -P + (X 1 )(X 2 )(X 3 ) cation and a contrast unit are attached.
  • the carboxyl group of one of the -P + (X 1 )(X 2 )(X 3 ) cation, the contrast unit and/or the dendritic or linear molecule is converted into
  • the active ester is either coupled to the amino, sulfhydryl or hydroxyl group of another unit after activation; or the targeting unit, the dendrimer or linear molecule and the contrast unit are linked by click chemistry.
  • the present application also provides a magnetically labeled cell labeled with the targeting mitochondrial contrast agent molecule, which is any cell that can be used for cell transplantation therapy by targeting a mitochondrial contrast agent molecule.
  • the targeting mitochondrial contrast agent molecule is any cell that can be used for cell transplantation therapy by targeting a mitochondrial contrast agent molecule. From mesenchymal stem cells, neural stem cells, cardiac stem cells, embryonic stem cells, induced pluripotent stem cells.
  • the present application also provides a method for cell labeling, placing a mitochondrial-targeting contrast agent molecule into a culture solution containing the cell, and introducing the targeting mitochondrial contrast agent molecule by utilizing cell endocytosis or pinocytosis function within the cell, the targeting mitochondrial contrast agent molecule binds to intracellular mitochondria and can effectively prolong its residence time in the labeled cells.
  • the present application also provides another method for cell labeling, characterized in that a contrast agent molecule targeting mitochondria is placed in a culture solution containing the cells, an electrotransfection buffer or a physiological saline, and a pulse electroporation method is used.
  • the mitochondrial targeting contrast agent molecule is introduced into the cell.
  • the present application also provides a combination of the magnetic labeled cells and a scaffold material, wherein the scaffold material is any medical material that can form a combination with cells, and is selected from the group consisting of collagen, various synthetic polymers or inorganic scaffold materials;
  • the scaffolding material contains or does not contain various trophic factors that support cell survival and growth.
  • the present application also provides a magnetic resonance imaging living body tracing method, comprising: transferring the magnetic labeled cells or the combination of the magnetic labeled cells and the scaffold material through a fixed-point surgical transplantation/intravenous injection into a human or an animal; a human or animal is placed in the MRI device, a magnetic resonance imaging at T 2 weighting pattern.
  • the mitochondrial-targeted contrast agent molecule provided by the present application can be widely used as a T 2 contrast agent in a cell treatment to trace the survival rate of cells transplanted into the body and the physiological processes such as migration and homing.
  • This is a technological discovery.
  • the application of iridium complex contrast agent only focuses on its signal enhancement effect, so it is only imaged in T 1 weighted mode, the image contrast is not ideal, and it shows irregular changes.
  • the present application is based on the fact that the contrast agent molecule targeting the mitochondria binds to the mitochondria in the cell, and the magnetic resonance signal of the magnetic labeled cell is greatly reduced, and the stable and regular signal attenuation effect is exhibited for a long time, so the present application
  • the magnetic resonance signal attenuation effect exhibited by the combination of the ruthenium complex and the mitochondria is fully utilized in the T 2 weighting mode.
  • the present application finds that after transplantation of cells labeled with the present application into the body, the labeled cells release a contrast agent molecule that targets the mitochondria, and the release of the targeting mitochondrial contrast agent molecule will cause the periphery of the cell transplant.
  • tissue at the magnetic resonance signal is rendered bright T 2 weighted images mode, the cells can be more clearly their transplant tissue surrounding separate region.
  • FIG. 1 is a schematic diagram of a magnetic labeled cell for magnetic resonance imaging in vivo tracer provided by the present application.
  • FIG. 2 is a schematic view showing the structure of a contrast agent molecule targeting mitochondria provided by the present application.
  • FIG. 3 is a structural diagram of a targeting unit of a mitochondria-targeting contrast agent molecule, that is, a phosphonium cation provided by the present application, and a targeting unit triphenylsulfonium (TPP) cation and a derivative thereof used in the examples of the present application. (Strong structure of (p-tolyl) 3 P) cation.
  • FIG. 4 is a structural diagram of a contrast unit superparamagnetic metal complex that can be used to enhance magnetic resonance imaging contrast provided by the present application, wherein M represents a metal having superparamagnetic properties.
  • FIG. 5 is a schematic diagram showing the molecular structure of a contrast agent targeting mitochondria provided by the present application, wherein the contrast unit Gd-DOTA and the targeting unit triphenylphosphonium cation pass through a carboxyl group and a dendritic or linear molecule. connection.
  • Gd-DOTA-TPP mitochondrial-targeting contrast agent molecule TPP-K(Gd-DOTA)-OH provided by Example 1 of the present application, abbreviated as Gd-DOTA-TPP
  • the imaging unit is Gd-DOTA
  • the targeting unit TPP and Gd-DOTA is linked via a carboxyl group to two amino groups of lysine (abbreviated as K).
  • Figure 7 is a contrast agent molecule [Gd-DOTA-Acp-K(Gd-DOTA)] 2 -K(TPP)-OH targeting mitochondria provided in Example 2 of the present application, abbreviated as (Gd-DOTA) 4 -TPP, the contrast unit is Gd-DOTA, and the targeting units TPP and Gd-DOTA are connected to the dendrimer via a carboxyl group.
  • Figure 8 is a mitochondrial targeting agent molecule [Gd-DOTA-Acp-K(Gd-DOTA)] 2 -linker-K(TPP)-OH, abbreviated as (Gd-DOTA) provided in Example 3 of the present application.
  • Gd-DOTA mitochondrial targeting agent molecule
  • 4 -linker-TPP the contrast unit is Gd-DOTA
  • the targeting units TPP and Gd-DOTA are connected to the dendrimer by carboxyl group.
  • Figure 9 is a mitochondrial targeting agent molecule [Gd-DOTA-Acp-K(Gd-DOTA)] 2 -linker-K(TPP)-OH, abbreviated as (Gd-DOTA) provided in Example 4 of the present application.
  • the imaging unit is Gd-DOTA
  • the targeting units TPP and Gd-DOTA are connected to the linear molecule through the carboxyl group.
  • Figure 10 is a contrast agent molecule [Dy-DOTA-Acp-K(Gd-DOTA)] 2 -linker-K(TPP)-OH targeting mitochondria provided in Example 5 of the present application, abbreviated as (Dy-DOTA) 4 -linker-TPP, the imaging unit is Dy-DOTA, and the targeting units TPP and Dy-DOTA are connected to the dendrimer via a carboxyl group.
  • Figure 11 is a mitochondrial targeting agent molecule [Gd-DTPA-Acp-K(Gd-DTPA)] 2 -linker-K(TPP)-OH, abbreviated as (Gd-DTPA) provided in Example 6 of the present application.
  • Gd-DTPA mitochondrial targeting agent molecule
  • 4 -linker-TPP the imaging unit is Gd-DTPA, and the targeting units TPP and Gd-DTPA are connected to the dendrimer by carboxyl group.
  • Figure 12 is a mitochondrial targeting agent molecule [Gd-DOTA-Acp-K(Gd-DOTA)] 2 -linker-K((p-tolyl) 3 P)-OH, which is provided in Example 7 of the present application, Abbreviated as (Gd-DOTA) 4- linker-(p-tolyl) 3 P, the contrast unit is Gd-DOTA, and the targeting unit (p-tolyl) 3 P and Gd-DOTA are both linked to the dendrimer via a carboxyl group,
  • Figure 13 is a mitochondrial targeting agent molecule TPP-Lys(TPP)-Lys[DOTA-Acp-Lys(DOTA)-Acp-Lys(DOTA)-Acp-Lys(DOTA) provided in Example 8 of the present application.
  • the imaging unit is 4 Gd-DOTA molecules, 4 DOTAs are attached to the ⁇ -amino group (side chain) of the linear polypeptide Lys and the N-terminus of the polypeptide
  • the amino group is linearly arranged;
  • the targeting unit is the same end of the two TPP molecules connected to the linear molecule, the carboxyl group of Lys at this end is amidated;
  • the structure is NH 2 (CH 2 ) 5 COOH, and the length is the case where p 2 of NH 2 (CH 2 ) p COOH.
  • Figure 14 is a graph showing the in vitro magnetic resonance T 1 -weighted and T 2 -weighted images of contrast agent molecularly labeled mesenchymal stem cells using (Gd-DOTA) 1,4 -TPP as targeting mitochondria in Example 10 of the present application.
  • the first row of images is a T 1 and T 2 weighted image of Gd-DOTA labeled mesenchymal stem cells at different cell reproduction time nodes.
  • the second, third, and fourth rows of images are T 1 and T 2 weighted images of 5, 10, 20 mM Gd-DOTA-TPP labeled mesenchymal stem cells at different cell reproduction time nodes, respectively.
  • the fifth row of images is a T 1 and T 2 weighted image of 20 mM (Gd-DOTA) 4 -TPP labeled mesenchymal stem cells at different cell reproduction time nodes.
  • the number below the image is the time node (days) at which the image was acquired during the incubation period after cell labeling.
  • Example 15 is a graph showing changes in the intensity of an in vitro T 1 -weight MRI image signal obtained by the labeled cells provided in Example 10 of the present application after propagation over time.
  • FIG 16 is an in vitro T 2 weighted MRI imaging signal intensity of the labeled cells obtained in Example 10 provided after the propagation time via different application of the present embodiment changes over time in cell proliferation.
  • Example 11 of the present application uses 20 mM (Gd-DOTA) 4 -TPP as a target molecule for targeting mitochondria to label mesenchymal stem cells, which are transplanted into the mouse brain by site-directed surgery, and 11.7T collected at different time points. Magnetic resonance T 2 weighted image renderings.
  • Example 3 of the present application uses 20 mM (Gd-DOTA) 4 -TPP as a targeting mitochondrial contrast molecule labeled mesenchymal stem cells, and 3T magnetic resonance T 2 weighted images acquired by transplanting the forebrain muscles of the rat by site injection. Effect chart.
  • Figure 20 is a graph showing the relationship between the in vitro T 2 -weight MRI image signal intensity and the Gd content in cells obtained by the labeled cells provided in Examples 10 and 13 of the present application after being propagated at different times.
  • the mitochondrial targeting contrast agent comprises a targeting molecule and a contrast unit cell
  • the targeting means is a -P + ( X 1 )(X 2 )(X 3 ) a phosphonium cation of the general formula wherein X 1 , X 2 , X 3 represents a C 1-12 alkyl group which is unsubstituted or substituted with one or more substituents, C 1 a -12 alkenyl group, or a C 6-10 aryl group, the substituent comprising 1, 2 or 3 halogen atoms, a C 1-12 alkyl group, a C 6-10 aryl group, a hydroxyl group, a C 1-12 alkoxy group And a halogenated-C 1-12 alkoxy group; wherein X 1 , X 2 , X 3 may be the same group or a different group; the contrast unit is a superparamagnetic metal complex.
  • the structure of the targeting unit triphenylsulfonium or a derivative thereof may be any of the structures shown in FIG. 3 or other structures having the structural formula shown in FIG. 3. derivative.
  • the structure of the contrast unit superparamagnetic metal complex may be any one of the complexes shown in FIG. 4, or may be any of the complexes shown in FIG. a derivative, for example, a acetyl group attached to a dendritic or linear molecule in a complex ligand of a superparamagnetic metal complex is replaced by a propionyl or butyryl group, and an ethyl carbonyl group may be replaced with an ethylamino group or a propylamino group.
  • the targeting mitochondrial contrast agent molecule can be directly prepared by a polypeptide solid phase synthesis technology; each unit can also be separately synthesized, and then the carboxyl group of one unit is converted into an NHS active ester or activated and then The unit is connected or connected by clicking on the chemical. For example, synthesizing the complexing ligand (protecting group) of the superparamagnetic metal complex of the contrast unit, the cation of the triphenylsulfonium cation or its derivative, and the dendritic or linear molecule with an amino group.
  • the dendritic or linear molecule may provide a linkage of an amino group, a thiol group, a hydroxyl group or the like, and may also provide a carboxyl linkage; accordingly, the superparamagnetic metal complex and the triphenylphosphonium or a derivative thereof may be
  • the carboxyl group is linked to a dendritic or linear molecule, and may also be attached to a dendritic or linear molecule via an amino group, a thiol group, a hydroxyl group or the like; or a cycloaddition reaction of a chemical azide-alkynyl group.
  • Gd-DOTA is used as a contrast unit, and the targeting unit triphenylsulfonium molecule and Gd-DOTA are both linked to a dendritic or linear molecule through a carboxyl group, and the structural unit of the dendritic or linear molecule is adopted.
  • the structure of the lysine, synthetic triphenylsulfonium magnetic resonance targeting mitochondrial contrast agent molecule is shown in Figure 5.
  • Gd-DOTA is used as a contrast unit, and the targeting unit triphenylsulfonium molecule and Gd-DOTA are both linked to the two amino groups of lysine via a carboxyl group, and the synthesized triphenylsulfonium is targeted to mitochondria.
  • the structure of the contrast agent molecule is shown in Figure 6, corresponding to the probe molecule Gd-DOTA-TPP.
  • Gd-DOTA is used as a contrast unit
  • the targeting unit triphenylsulfonium molecule and Gd-DOTA are each linked to a dendrimer through a carboxyl group
  • the structural unit of the dendrimer is lysine, a structural unit.
  • the linker is lysine, wherein one spacer has a length of 0, and the other spacer has a structure of NH 2 (CH 2 ) 5 COOH, and the synthesized three
  • the structure of the contrast agent molecule of phenylhydrazine targeting mitochondria is shown in Figure 7, corresponding to the probe molecule (Gd-DOTA) 4- TPP.
  • Gd-DOTA is used as a contrast unit
  • the targeting unit triphenylsulfonium molecule and Gd-DOTA are each linked to a dendrimer through a carboxyl group
  • the structural unit of the dendrimer is lysine, a structural unit.
  • the linker is aminohexanoyl lysine, which contains the spacer aminocaproic acid, wherein one spacer has a length of 0 and the other spacer has a structure of NH. 2 (CH 2 ) 5 COOH
  • the structure of the contrast agent molecule of the synthesized triphenylsulfonium targeting mitochondria is shown in Fig. 8, corresponding to the probe molecule (Gd-DOTA) 4- linker-TPP.
  • Gd-DOTA is used as a contrast unit
  • the targeting unit triphenylsulfonium molecule and Gd-DOTA are both linked to a linear molecule through a carboxyl group, and the structural unit of the linear molecule is lysine, a structural unit.
  • the linker is aminohexanoyl lysine, which contains the spacer aminocaproic acid, wherein one spacer has a length of 0 and the other spacer has a structure of NH. 2 (CH 2 ) 5 COOH
  • the structure of the contrast agent molecule of the synthesized triphenylsulfonium targeting mitochondria is shown in Figure 9, corresponding to the probe molecule (Gd-DOTA) 4- linker-TPP.
  • Dy-DOTA is used as a contrast unit, and the targeting unit triphenylphosphonium cation and Dy-DOTA are each linked to a dendrimer through a carboxyl group, and the structural unit of the dendrimer is lysine, a structural unit.
  • the linker is aminohexanoyl lysine, which contains the spacer aminocaproic acid, wherein one spacer has a length of 0 and the other spacer has a structure of NH. 2 (CH 2 ) 5 COOH, the structure of the contrast agent molecule of the synthesized triphenylsulfonium targeting mitochondria is shown in Figure 10, corresponding to the probe molecule (Dy-DOTA) 4 -TPP.
  • Gd-DTPA is used as a contrast unit, and the targeting unit triphenylsulfonium molecule and Gd-DTPA are both linked to the dendrimer via a carboxyl group, and the structural unit of the dendrimer is lysine.
  • the linker is aminohexanoyl lysine, which contains the spacer aminocaproic acid, wherein one spacer has a length of 0 and the other spacer has a structure of NH. 2 (CH 2) 5 COOH, molecular structure of the contrast agent is synthesized mitochondrial targeting triphenylphosphonium As shown, the corresponding probe molecules (Gd-DTPA) 4 -linker- TPP 11.
  • Gd-DOTA is used as a contrast unit, and the targeting unit tris(p-methylphenyl)fluorene molecule ((p-tolyl) 3 P) and Gd-DOTA are each linked to a dendrimer via a carboxyl group.
  • the length of the other is 0, the structure of the other spacer is NH 2 (CH 2 ) 5 COOH, and the structure of the contrast molecule of the synthesized triphenylsulfonium targeting mitochondria is shown in Figure 12, corresponding to the probe molecule (Gd- DOTA) 4- linker-(p-tolyl) 3 P.
  • Gd-DOTA is used as a contrast unit, and two targeting units, a triphenylphosphonium molecule and a Gd-DOTA, are each linked to a linear molecule through a carboxyl group, and a structural unit of the linear molecule is lysine.
  • the linker is aminohexanoyl lysine, which contains a spacer aminocaproic acid, wherein the length of one spacer is 0, and the structure of the other spacer It is NH 2 (CH 2 ) 5 COOH, and the structure of the contrast agent molecule of the synthesized triphenylsulfonium targeting mitochondria is as shown in FIG. 13 , corresponding to the probe molecule (Gd-DOTA) 4 -linker-TPP 2 .
  • a method of binding observed mitochondria in vitro magnetic resonance imaging contrast agents by inter-molecular markers of mesenchymal stem cells by inter-molecular markers of mesenchymal stem cells, the cells at different times after the node flag which T 1 and T 2 are weighted imaging The process of weighting the contrast of the imaging contrast.
  • a method of transplanting mitochondrial magnetic resonance contrast agent molecularly labeled mesenchymal stem cells into a rat by site-directed surgical transplantation/injection using 3T in vivo magnetic resonance T 2 Weighted imaging was performed to observe the image contrast of the cell graft and its surrounding tissues.
  • Example 1 Synthesis of contrast agent molecule Gd-DOTA-TPP targeting mitochondria (Fig. 6)
  • the steps are briefly described as follows: The conventional Fmoc (methyl chloroformate) method was synthesized on a solid phase synthesizer, and the amino acid was sequentially coupled from the C terminal to the N terminal according to the structure shown in FIG. First, 1 g of the solid support 2-chlorotrityl resin, followed by the addition of 2.0 g of Fmoc-Lys(Mtt)-OH, 1.77 g of Ph 3 P(Br)(CH 2 ) 4 COOH for condensation, each step of carboxyl and amino groups
  • the condensation conditions were as follows: 50 mL of DMF was used as a solvent, 0.96 g of TBTU, 0.41 g of HOBt condensing agent and 2.5 mL of alkali DIPEA were added, and the reaction was carried out at 25 ° C for about 24 hours.
  • the specific time was determined by the color of ninhydrin to determine the carboxyl group and the amino group. Whether or not the condensation was completed; after the end of the condensation, the solvent was drained, washed with 50 mL of methanol and then three times with DMF, methanol and dichloromethane.
  • Conditions for removing Fmoc before adding Ph 3 P(Br)(CH 2 ) 4 COOH 25 mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, drained, and then added with 25 mL of 20% piperidine/DMF for 0.5 hour, drained. They were washed three times with DMF, methanol and dichloromethane, respectively.
  • the crude product was further purified by HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5 ⁇ m 19 ⁇ 150mm, mobile phase: Solvent A (0.1% TFA, aqueous), solvent B (0.1% TFA, CH 3 CN), solvent A from within 25 minutes 50% dropped to 25%; flow rate 10 mL/min. Approximately 200 mg of product DOTA-TPP was obtained with a purity of 95% or more.
  • the deprotected DOTA-TPP is complexed with Gd 3+ to obtain a contrast agent molecule targeting Gd-DOTA-TPP targeting mitochondria.
  • a contrast agent molecule targeting Gd-DOTA-TPP targeting mitochondria Specifically, as follows: about 1.0 mL of an aqueous solution containing 35.7 mg of GdCl 3 ⁇ 6H 2 O was added dropwise to 100 mg of the above aqueous solution of DOTA-TPP and mixed for 3 hours, and the pH was adjusted to about 6 with 1.0 M aqueous ammonia, and statically mixed at room temperature.
  • Gd-DOTA-TPP targeting mitochondrial contrast agent molecule.
  • the purity of Gd-DOTA-TPP was measured by HPLC. HPLC conditions, Waters 2535_2707_2998, Sapphire C18 5 ⁇ m 4.6 ⁇ 250mm, mobile phase: solvent A (0.1% TFA, water), solvent B (0.1% TFA, 80% CH 3 CN, 20% water), solvent B within 20 minutes From 22% to 42%; flow rate 1.0 mL / min. The purity is 95% or more.
  • the conditions for the condensation of the carboxyl group and the amino group in each step are as follows: 50 mL of DMF is used as a solvent, 1.92 g of TBTU, 0.82 g of HOBt condensing agent and 5 mL of alkali DIPEA are added, and the reaction is carried out at 25 ° C for about 24 hours, and the specific time is determined by ninhydrin color development. It was judged whether or not the condensation of the carboxyl group and the amino group was completed; after the end of the condensation, the solvent was drained, and washed with 50 ml of methanol, three times with DMF, methanol and dichloromethane.
  • the crude product was further purified by HPLC, Waters 2535_2707_2998_WFC, XBridge Pre C18 5 ⁇ m 19 ⁇ 150 mm, mobile phase: solvent A (0.1% TFA, water), solvent B (0.1% TFA, CH 3 CN), solvent A from 80% within 15 minutes Drop to 65%; flow rate 10mL / min. Approximately 200 mg of product (DOTA) 4- TPP was obtained with a purity of 95% or more.
  • the deprotected DOTA 4 -TPP is complexed with Gd 3+ to obtain a contrast agent molecule targeting (Gd-DOTA) 4 -TPP targeting mitochondria.
  • Gd-DOTA contrast agent molecule targeting
  • (Gd-DOTA) 4- TPP targeting mitochondrial contrast agent molecules.
  • the purity of (Gd-DOTA) 4- TPP was determined by HPLC. HPLC conditions, Waters 2535_2707_2998, Sapphire C18 5 ⁇ m 4.6 ⁇ 250mm, mobile phase: solvent A (0.1% TFA, water), solvent B (0.1% TFA, 80% CH 3 CN, 20% water), solvent B within 20 minutes From 24% to 44%; flow rate 1.0mL / min. The purity is 95% or more.
  • Example 1 1, according to Example 1 were synthesized Bu t 3 DOTA and Ph 3 P (Br) (CH 2) 4 COOH.
  • the specific time was determined by the color of ninhydrin to determine the carboxyl group and the amino group. Whether or not the condensation was completed; after the end of the condensation, the solvent was drained, washed with 50 mL of methanol and then three times with DMF, methanol and dichloromethane.
  • Conditions for removing Fmoc before adding Ph 3 P(Br)(CH 2 ) 4 COOH 25 mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, drained, and then added with 25 mL of 20% piperidine/DMF for 0.5 hour, drained. They were washed three times with DMF, methanol and dichloromethane, respectively.
  • the conditions for the condensation of the carboxyl group and the amino group in each step are as follows: 50 mL of DMF is used as a solvent, 1.92 g of TBTU, 0.82 g of HOBt condensing agent and 5 mL of alkali DIPEA are added, and the reaction is carried out at 25 ° C for about 24 hours, and the specific time is determined by ninhydrin color development. It was judged whether or not the condensation of the carboxyl group and the amino group was completed; after the end of the condensation, the solvent was drained, and washed with 50 ml of methanol, three times with DMF, methanol and dichloromethane.
  • the crude product was further purified by HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5 ⁇ m 19 ⁇ 150mm, mobile phase: Solvent A (0.1% TFA, aqueous), solvent B (0.1% TFA, CH 3 CN), solvent A in 15 minutes 80 % dropped to 65%; flow rate 10 mL / min. Approximately 200 mg of product (DOTA) 4- linker-TPP was obtained with a purity of 95% or more.
  • the deprotected DOTA 4 -spacer-TPP is complexed with Gd 3+ to obtain a contrast agent molecule targeting (Gd-DOTA) 4- linker-TPP targeting mitochondria.
  • Gd-DOTA contrast agent molecule targeting
  • aqueous solution containing 12.8 mg of GdCl 3 ⁇ 6H 2 O was added dropwise to 100 mg of the above aqueous solution of DOTA 4 -linker-TPP for 3 hours, and the pH was adjusted to about 6 with 1.0 M aqueous ammonia, room temperature.
  • the deprotected DOTA 4 -spacer-TPP is complexed with Gd 3+ to obtain a contrast agent molecule targeting (Gd-DOTA) 4- linker-TPP targeting mitochondria.
  • Gd-DOTA contrast agent molecule targeting
  • aqueous solution containing 12.8 mg of GdCl 3 ⁇ 6H 2 O was added dropwise to 100 mg of the above aqueous solution of DOTA 4 -linker-TPP for 3 hours, and the pH was adjusted to about 6 with 1.0 M aqueous ammonia, room temperature.
  • the deprotected DOTA 4 -linker-TPP is complexed with Dy 3+ to obtain (Dy-DOTA) 4- linker-TPP targeting mitochondrial contrast agent molecules.
  • Dy-DOTA 4- linker-TPP targeting mitochondrial contrast agent molecules.
  • about 0.5 mL of an aqueous solution containing 13.0 mg of DyCl 3 ⁇ 6H 2 O was added dropwise to 100 mg of the above aqueous solution of DOTA 4 -linker-TPP for 3 hours, and the pH was adjusted to about 6 with 1.0 M aqueous ammonia, room temperature.
  • the specific time was determined by the color of ninhydrin to determine the carboxyl group and the amino group. Whether or not the condensation was completed; after the end of the condensation, the solvent was drained, washed with 50 mL of methanol and then three times with DMF, methanol and dichloromethane.
  • Conditions for removing Fmoc before adding Ph 3 P(Br)(CH 2 ) 4 COOH 25 mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, drained, and then added with 25 mL of 20% piperidine/DMF for 0.5 hour, drained. They were washed three times with DMF, methanol and dichloromethane, respectively.
  • the conditions for the condensation of the carboxyl group and the amino group in each step are as follows: 50 mL of DMF is used as a solvent, 1.92 g of TBTU, 0.82 g of HOBt condensing agent and 5 mL of alkali DIPEA are added, and the reaction is carried out at 25 ° C for about 24 hours, and the specific time is determined by ninhydrin color development. It was judged whether or not the condensation of the carboxyl group and the amino group was completed; after the end of the condensation, the solvent was drained, and washed with 50 ml of methanol, three times with DMF, methanol and dichloromethane.
  • the crude product was further purified by HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5 ⁇ m 19 ⁇ 150mm, mobile phase: Solvent A (0.1% TFA, aqueous), solvent B (0.1% TFA, CH 3 CN), solvent A from within 15 minutes 80% dropped to 65%; flow rate 10mL/min. Approximately 200 mg of product (DTPA) 4- spacer-TPP was obtained with a purity of 95% or more.
  • the deprotected DTPA 4- linker-TPP is complexed with Gd 3+ to obtain a contrast agent molecule targeting (Gd-DTPA) 4- linker-TPP targeting mitochondria.
  • Gd-DTPA contrast agent molecule targeting 4- linker-TPP targeting mitochondria.
  • aqueous solution containing 12.8 mg of GdCl 3 ⁇ 6H 2 O was added dropwise to 100 mg of the above aqueous solution of DTPA 4 -linker-TPP and mixed for 3 hours, and the pH was adjusted to about 6 with 1.0 M aqueous ammonia, room temperature.
  • the deprotected DOTA 4 -linker-(p-tolyl) 3 P is complexed with Gd 3+ to obtain a contrast agent molecule targeting (Gd-DOTA) 4- linker-(p-tolyl) 3 P targeting mitochondria.
  • Gd-DOTA contrast agent molecule targeting
  • Example 3 about 0.5 mL of an aqueous solution containing 12.6 mg of GdCl 3 ⁇ 6H 2 O was added dropwise to 100 mg of the above aqueous solution of DOTA 4 -linker-(p-tolyl) 3 P, and other raw materials and Conditions and detection steps are unchanged.
  • Example 8 (Gd-DOTA) 4- linker-TPP 2 targeting mitochondrial synthesis of contrast agent molecules (Fig. 13, here the linker is Lys(Acp)-NH 2 , and the contrast unit Gd-DOTA is linked to the linear polypeptide molecule Above, two targeting units TPP are linked to the same end of the linear molecule, and the carboxyl group of the Lys is amidated)
  • the linear molecule-linked DOTA 4 -linker-TPP 2 was synthesized by solid phase synthesis according to the structure shown in FIG.
  • the carboxyl group of the linker lysine is converted into an amide group (all of the carboxyl groups of the linker lysine of the contrast agent molecule targeting the mitochondria described herein can be converted into an amide group and have the same Contrast effect).
  • the solid phase carrier uses a resin for producing a C-terminal amide-terminated polypeptide such as Rink Amide AM Resin or Rink Amide MBHA Resin/Knorr Resin 1 g, followed by 2.0 g of Fmoc-Lys(Mtt)-OH, 2.0 g of Fmoc-Lys ( Fmoc)-OH, 3.54 g of Ph 3 P(Br)(CH 2 ) 4 COOH was condensed.
  • a resin for producing a C-terminal amide-terminated polypeptide such as Rink Amide AM Resin or Rink Amide MBHA Resin/Knorr Resin 1 g
  • a resin for producing a C-terminal amide-terminated polypeptide such as Rink Amide AM Resin or Rink Amide MBHA Resin/Knorr Resin 1 g
  • 2.0 g of Fmoc-Lys(Mtt)-OH 2.0 g of Fmoc-Ly
  • the conditions for the condensation of the carboxyl group and the amino group in each step, the criterion for determining whether or not the condensation of the carboxyl group and the amino group is completed, the post-treatment after completion of the condensation, the conditions for removing the Fmoc before the addition of Ph 3 P(Br)(CH 2 ) 4 COOH, and the like are the same as the examples. 3 is the same.
  • the conditions for the condensation of the carboxyl group and the amino group in each step are as follows: 50 mL of DMF is used as a solvent, 1.92 g of TBTU, 0.82 g of HOBt condensing agent and 5 mL of alkali DIPEA are added, and the reaction is carried out at 25 ° C for about 24 hours, and the specific time is determined by ninhydrin color development. It was judged whether or not the condensation of the carboxyl group and the amino group was completed; after the end of the condensation, the solvent was drained, and washed with 50 ml of methanol, three times with DMF, methanol and dichloromethane.
  • the crude product was further purified by HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5 ⁇ m 19 ⁇ 150mm, mobile phase: Solvent A (0.1% TFA, aqueous), solvent B (0.1% TFA, CH 3 CN), solvent A from within 15 minutes 80% dropped to 65%; flow rate 10mL/min. Approximately 200 mg of product (DTPA) 4- spacer-TPP was obtained with a purity of 95% or more.
  • the deprotected DOTA 4 -linker-TPP 2 is complexed with Gd 3+ to obtain a contrast agent molecule targeting (Gd-DOTA) 4- linker-TPP 2 targeting mitochondria.
  • Gd-DOTA contrast agent molecule targeting
  • aqueous solution containing 12.8 mg of GdCl 3 ⁇ 6H 2 O was added dropwise to 100 mg of the above aqueous solution of DOTA 4 -linker-TPP 2 and mixed for 3 hours, and the pH was adjusted to about 6 with 1.0 M aqueous ammonia.
  • the static mixer was rotated overnight at room temperature, then carefully adjusted to pH 7-8 with 1.0 M ammonia and 1.0 M hydrochloric acid.
  • Example 9 Concentration of triphenylsulfonium targeting mitochondria molecular molecule labeled mesenchymal stem cells
  • hMSCs Human bone marrow mesenchymal stem cells frozen in liquid nitrogen were taken out and thawed rapidly in a 37 ° C water bath. In a clean bench, remove the frozen solution of the thawed cells with a 1 mL pipette and place in a 10 mL sterile centrifuge tube while adding 2 mL of complete medium (basal medium DMEM-F1280% to 90%, Australian fetal calf Serum 10% to 20%, double antibody 1%), centrifuged at 1000 rpm for 5 minutes per minute, and the medium was aspirated.
  • complete medium basic medium DMEM-F1280% to 90%, Australian fetal calf Serum 10% to 20%, double antibody 1%
  • trypsin is removed and blown with 4 m complete medium.
  • the cells were transferred to two 100 ⁇ 20 mm culture dishes in two divided portions, and then further cultured by adding 6 mL of complete medium. The experiment used 6-9 generation cells.
  • cell treatment When the density of adherent cells reaches 80% to 90%, gently wash the cells with long cells with 2ml of sterile PBS solution, then add 1mL PBS and 1mL trypsin, observe the cell morphology in time. After the cells are completely digested, trypsin is aspirated. The cells were pipetted with 4 mL of complete medium, the cell suspension was transferred to a 10 mL sterile centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the medium was aspirated to obtain a cell pellet. The same resuspension operation was carried out with 4 mL of PBS.
  • the contrast agent molecules of triphenylsulfonium-targeted mitochondria prepared according to the method of the present application are dissolved in physiological saline to prepare a concentration series of 1, 2, 5, 10, 20, 40 mM. Take 100 ⁇ L of the sample solution in the cell pellet (100 ⁇ L contains 1 million to 2 million cells), blow off the cells, place the blown cells in a 96-well plate, and use a transfection instrument with a voltage of 120V. The pulse width is 100 ⁇ s, the interval is 1000 ms, and the electrophoresis test conditions are repeated 6 times.
  • the contrast agent molecules targeting the mitochondria are introduced into the cytoplasm, and when the experiment is performed by applying a certain electric pulse, it is necessary to ensure that the cells in the 96-well plate are uniformly dispersed. In saline, instead of having been deposited on the bottom of a 96-well plate.
  • Example 10 In vitro MRI imaging method of triphenylsulfonium targeting mitochondria contrast agent molecularly labeled mesenchymal stem cells
  • the contrast agent molecules of the mitochondria targeted by triphenylsulfonium prepared in Examples 1 and 2, according to the examples The method of 9 labeled mesenchymal stem cells, and half of the cells labeled with each probe concentration were transferred to a 10 mL centrifuge tube, and the culture dishes were washed with 3 mL of PBS, and the tubes were also transferred to a centrifuge tube. Centrifuge for 5 minutes at 1200 rpm and remove PBS. The cells were transferred to a capped capillary having an inner diameter of 1.5 mm using a capillary having an outer diameter of 1.3 mm, centrifuged at 1500 rpm for 10 minutes, and the cells were densely packed at the bottom of the capillary for in vitro MRI imaging.
  • the other half of the cells continue to culture until the number of cells doubles, and then half of the cells are densely packed at the bottom of the capillary for in vitro MRI imaging experiments.
  • the other half of the cells continue to grow until the number of cells doubles. Therefore, there was no difference in the results of in vitro cell MRI imaging experiments.
  • the capillary cells are T 1 and T 2 weighted weighted imaging in a magnetic resonance spectrometer 11.7T.
  • Figure 14 is a diagram showing the in vitro T 1 weighting of the mesenchymal stem cells labeled with different structures of triphenylsulfonium-containing targeting mitochondria at different probe concentrations in the present embodiment. And T 2 weighted MRI image results. Gd-DOTA labeled cells with no cell binding ability were also included in the examples as a reference comparison for in vitro MRI imaging experiments.
  • Fig. 15 is a graph showing the change of the in vitro T 1 -weight MRI image signal intensity obtained by the magnetic labeled cells obtained in the present example with different cell proliferation time.
  • the cell has just been marked, a T-weighted MRI enhancement effect of the video signal Gd-DOTA labeled cells was significantly, mitochondria targeted contrast agent molecules through triphenylphosphonium containing magnetically labeled cells labeled T 1
  • the weighted MRI image signal enhancement effect is not significant, and even shows signal attenuation effect
  • the T 1 weighted MRI image signal intensity of all magnetic labeled cells recovers rapidly (within 1 to 2 days) to
  • the signal intensity level of unlabeled cells indicates the limitation of long-term tracer transplantation in the magnetic resonance T 1 -weighted model.
  • FIG 16 is an in vitro T 2 weighted MRI imaging signal intensity of magnetically labeled cells obtained in this embodiment is obtained via different propagation times after the change over time in cell proliferation. It can be seen that: (1) the cell has just been marked, T 2 weighted MRI image signal magnetically labeled cells labeled Gd-DOTA showed a significant enhancement effect by containing triphenylphosphonium mitochondria targeted contrast agent molecules labeled The T 2 -weighted MRI image signal of magnetically labeled cells showed a significant signal-attenuating effect. The signal attenuation increased with the increase of probe concentration when the cells were labeled, and even reached or even below the noise level.
  • the intensity of the T 2 -weighted MRI image of the magnetically labeled cells labeled with the target molecule of the mitochondria containing triphenylsulfonate is significantly slower than that of the T 1 -weighted MRI image enhancement effect, within about 5 days. Still at the noise level, it still shows significant contrast difference (dark signal) with unlabeled cells within about 10 days, and it takes about 16 days to reach the signal intensity level of unlabeled cells, indicating long-term tracer transplantation in magnetic resonance T 2 -weighted mode. The feasibility of the body.
  • Example 11 triphenylphosphonium between mitochondrial targeting contrast agent of molecular markers of mesenchymal stem cells in mice in vivo MR 11.7T T 2 weighted images
  • a part of the transplanted cells is located in the cranium, and a significant dark signal (white arrow indicates the position) is displayed for a long time (D0 to D10); a part is located in the ventricle, and the part of the cells is on the day after transplantation.
  • D0 rapidly migrates in the ventricle and presents a significant dark signal
  • D1 ⁇ D4 the cell transplant begins to release the contrast agent molecules targeting the mitochondria and present a prominent bright signal to the surrounding tissue (gray arrow Indicate the location).
  • the cells began to die, and the mitochondrial-targeting contrast molecules released after the cell membrane of the dead cells ruptured also showed a significant bright signal (the black arrow indicates the position) of the surrounding tissue.
  • the cell transplant body can be clearly distinguished from the surrounding tissue.
  • Example 12 triphenylphosphonium between mitochondrial targeting contrast agent of molecular markers in vivo mesenchymal stem 3T MRI T 2 weighted images rats transplanted cells
  • Example 9 The method of Example 9 was used to label the mesenchymal stem cells by pulse electroporation using a contrast agent molecule targeting (Gd-DOTA) 4 -TPP targeting mitochondria, and the concentration of the contrast agent targeting the mitochondria was 20 mM;
  • Example 13 In vitro MRI image of contrast agent molecularly labeled mesenchymal stem cells targeting bistriphenylguanidine targeting mitochondria
  • Example 19 is a comparison of the in vitro T 2 -weighted MRI image signal intensity obtained by the magnetic labeled cells obtained in the present example with the cell reproduction time and the partial results in Example 10. It can be seen that the intensity of T 2 -weighted MRI image signal recovery of magnetically labeled cells labeled with contrast agent molecules containing bistriphenylphosphonium-targeted mitochondria is significantly slower than that of targeted mitochondria containing a triphenylsulfonium.
  • the molecularly labeled magnetically labeled cells indicate that the former can trace the transplanted cells in vivo over a longer time frame in the magnetic resonance T 2 weighting mode.
  • Figure 20 is a graph showing the relationship between the in vitro T 2 -weighted MRI image signal intensity and the change of Gd content in cells obtained by multiplying magnetic labeled cells obtained in the present embodiment, indicating that the intensity of the magnetically labeled cells in the T 2 -weight MRI image is lowered.
  • the minimum cellular Gd content required to reach noise levels can be as low as 5 x 10 9 Gd/cell.

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Abstract

L'invention concerne une molécule d'agent de contraste de ciblage mitochondrial et son utilisation comme agent de contraste T 2. La molécule d'agent de contraste de ciblage mitochondrial comprend un cation phosphonium de formule -P +(X 1)(X 2)(X 3) pour la liaison à des mitochondries de cellule, et un complexe métallique superparamagnétique, qui est une unité de contraste pouvant améliorer le contraste en imagerie par résonance magnétique. L'invention concerne également une molécule d'agent de contraste de ciblage mitochondrial, un ou plusieurs cations de -P +(X 1)(X 2)(X 3) étant liés avec 1 à 8 molécules de complexe métallique supraparamagnétique par le biais de molécules linéaires ou ramifiées, un lieur et un espaceur étant utilisés pour améliorer la structure spatiale entre le cation -P +(X 1)(X 2)(X 3) et la molécule linéaire ou ramifiée et celle entre la molécule linéaire ou ramifiée et le complexe métallique supraparamagnétique. Par ailleurs, l'invention concerne des cellules marquées magnétiquement marquées avec les molécules de contraste, une combinaison des cellules marquées magnétiquement et de matériaux d'échafaudage, un procédé de préparation de la molécule d'agent de contraste et un procédé d'imagerie par résonance magnétique in vivo utilisant les matériaux ci-dessus.
PCT/CN2016/108801 2015-12-17 2016-12-07 Utilisation d'une molécule d'agent de contraste de ciblage mitochondrial comme agent de contraste t2 WO2017101717A1 (fr)

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CN201510946966.9A CN106890345B (zh) 2015-12-17 2015-12-17 一种靶向线粒体的造影剂分子作为t2造影剂的用途

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