WO2017101717A1 - Use of mitochondrial targeting contrast agent molecule as t2 contrast agent - Google Patents

Abstract

Provided are a mitochondrial targeting contrast agent molecule and use thereof as a T ₂ contrast agent. The mitochondrial targeting contrast agent molecule comprises a phosphonium cation of formula -P ⁺(X ₁)(X ₂)(X ₃) for binding to cell mitochondria, and a superparamagnetic metal complex,which is a contrast unit for enhencing contrast during magnetic resonance imaging. Also provided is a mitochondrial targeting contrast agent molecule, in which one or more cations of -P ⁺(X ₁)(X ₂)(X ₃) are bound with 1 to 8 superparamagnetic metal complex molecules through branch or linear molecules, while linker and spacer are used to improve the spatial structure between the cation -P ⁺(X ₁)(X ₂)(X ₃) and the branched or linear molecule and that between the branched or linear molecule and the superparamagnetic metal complex. Meanwhile, provided are magnetic labeled cells labeled with the contrast molecules, a combination of the magnetically labeled cells and scaffold materials, a method for preparing the contrast agent molecule, and a method of magnetic resonance imaging tracing in vivo by using the above materials.

Description

Use of a contrast agent molecule targeting mitochondria as a T2 contrast agent

This application claims priority to December 17, 2015 filed Chinese Patent Application No. 201510946966.9 name invention as "a mitochondrial targeting contrast agent molecule as T ₂ contrast agents of use" Chinese Patent Application, the entire The content is incorporated herein by reference.

Technical field

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.

Background technique

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. In the process of stem cell regenerative medicine, 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. In practical applications, when the image contrast of different tissues is close, 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. The use of magnetic resonance imaging techniques to trace stem cells in vivo first requires magnetic labeling of stem cells to distinguish them from other tissue cells surrounding them.

The introduction of magnetic resonance contrast agents usually accelerates both the longitudinal relaxation rate and the transverse relaxation rate of water protons in the tissue in which they are located, but different magnetic resonance contrast agents accelerate the relative magnitudes of the two relaxation rates. This difference results in some MRI contrast agents suitable for MRI signal enhancement, called T ₁ contrast agents for MRI signals attenuate some suitable, known as T ₂ contrast agents. For example, in general, 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. In addition, 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. However, 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. At present, 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. However, 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.

In vivo magnetic resonance imaging tracing using stem cells labeled with ruthenium complex is also an alternative technical solution. However, in the existing methods, the problems encountered with the use of small molecule ruthenium complexes or targeted contrast agents for cell membrane-bound receptors, in addition to the low longitudinal relaxation rate, the time it takes to stay in the cells is too short. Overcoming problems; when using macromolecules or nanoparticles loaded with ruthenium complexes, the same problems as SPIO nanoparticles will be encountered, including slow removal in the body and possible uptake by other cells leading to interference with image results.

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. When 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.

In summary, there is a need in the art for such a magnetic resonance contrast agent for cell or graft labeling, which not only provides a visual representation of the viability of the cells in vivo and the migration or migration of the cells or transplants. Information such as nests and differentiation can also meet the needs of long-term observation, and can solve the toxicity problems caused by large doses.

Summary of the invention

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 inventors have further found that, after the labeled cells were transplanted into the body of the present application, 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, wherein 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. 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.

In a preferred embodiment of the present application, the targeting unit is a triphenylphosphonium cation or a derivative thereof.

In a preferred embodiment of the present application, 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), 钕 (Nd), 钷 (Pm), 钐 (Sm), 铕 (Eu), 钆 (Gd), 铽 (Tb), 镝 (Dy), 钬 (Ho), 铒(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, DTPA-BMEA, BOPTA, EOB-DTPA or derivatives thereof And any combination of them.

In a preferred embodiment of the present application, 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.

In a preferred embodiment of the present application, the linker is a linear amino acid, preferably lysine; the spacer is a linear amino acid, preferably NH ₂ (CH ₂ ) _p COOH or NH ₂ ( CH ₂ CH ₂ O) _q CH ₂ COOH, wherein p is an integer from 0 to 12, q is an integer from 0 to 4, and when p=0 or q=0, there is no spacer.

In a preferred embodiment of the present application, 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.

In a preferred embodiment of the present application, 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.

Another object of the present application is to provide 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. an aryl group, 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.

In a preferred embodiment of the present application, the linker is selected from a linear amino acid, preferably a lysine; the spacer is selected from a linear amino acid, preferably NH ₂ (CH ₂ ) _p COOH or NH ₂ (CH ₂ CH ₂ O) _q CH ₂ COOH, wherein p is an integer from 0 to 12, q is an integer from 0 to 4, and when p=0 or q=0, there is no spacer.

In a preferred embodiment of the present application, the targeting unit is linked to the 1-8 imaging unit by the dendritic or linear molecule.

In a preferred embodiment of the present application, a plurality of targeting units, preferably 2 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. Attached to a dendritic or linear molecule; 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, and the amino group of the superparamagnetic metal complex is selected from ethylamino group, propylamino group or butylamino group.

In a preferred embodiment of the present application, 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.

In a preferred embodiment of the present application, 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. 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 groundbreaking discovery. On the one hand, there is no effective way to clearly trace the survival rate of cells transplanted into the body and the physiological processes such as migration and homing through live imaging technology. On the other hand, it is because of the long-term 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. More importantly, 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.

DRAWINGS

In order to more clearly illustrate the embodiments of the present application and the technical solutions of the prior art, the following description of the embodiments and the drawings used in the prior art will be briefly introduced. Obviously, the drawings in the following description are only Some embodiments of the application may also be used to obtain other figures from those of ordinary skill in the art without departing from the scope of the invention.

FIG. 1 is a schematic diagram of a magnetic labeled cell for magnetic resonance imaging in vivo tracer provided by the present application.

2 is a schematic view showing the structure of a contrast agent molecule targeting mitochondria provided by the present application.

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.

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. The structural unit of the dendritic or linear molecule is lysine, and the number k of structural units may be an integer from 0 to 7; the number of contrast units DOTA is m+n=k+1.

6 is a 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, and 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. The structural unit of the dendrimer is lysine, the number of structural units is k=3, and the linker is Lysine, wherein the length of one spacer is the case of p=0 of NH ₂ (CH ₂ ) _p COOH, and the structure of the other spacer is NH ₂ (CH ₂ ) ₅ COOH, ie aminohexanoic acid (Acp), The length is the case where p = 5 of NH ₂ (CH ₂ ) _p COOH.

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. ₄ -linker-TPP, the contrast unit is Gd-DOTA, and the targeting units TPP and Gd-DOTA are connected to the dendrimer by carboxyl group. The structural unit of the dendrimer is lysine, and the number of structural units is k=3. Wherein the linker is aminohexanoyl lysine, which comprises the spacer aminocaproic acid, wherein the length of one spacer is the case of p = 0 of NH ₂ (CH ₂ ) _p COOH, and the structure of the other spacer is NH ₂ (CH ₂ ) ₅ COOH, the length is the case where p ₂ of NH ₂ (CH ₂ ) _p COOH.

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. ₄ -linker-TPP, the imaging unit is Gd-DOTA, and the targeting units TPP and Gd-DOTA are connected to the linear molecule through the carboxyl group. The structural unit of the linear molecule is lysine, and the number of structural units is k=3. Wherein the linker is aminohexanoyl lysine comprising a spacer aminocaproic acid, wherein the length of one spacer is the case of p = 0 as described for NH ₂ (CH ₂ ) _p COOH, and the structure of the other spacer It is a case where NH ₂ (CH ₂ ) ₅ COOH has a length of p = 5 of NH ₂ (CH ₂ ) _p COOH.

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. The structural unit of the dendrimer is lysine, and the number of structural units is k=3. Wherein the linker is aminohexanoyl lysine, which comprises the spacer aminocaproic acid, wherein the length of one spacer is the case of p = 0 of NH ₂ (CH ₂ ) _p COOH, and the structure of the other spacer is NH ₂ (CH ₂ ) ₅ COOH, the length is the case where p ₂ of NH ₂ (CH ₂ ) _p COOH.

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. 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. The structural unit of the dendrimer is lysine, and the number of structural units is k=3. Wherein the linker is aminohexanoyl lysine, which comprises the spacer aminocaproic acid, wherein the length of one spacer is the case of p = 0 of NH ₂ (CH ₂ ) _p COOH, and the structure of the other spacer is NH ₂ (CH ₂ ) ₅ COOH, the length is the case where p ₂ of NH ₂ (CH ₂ ) _p COOH.

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, The structural unit of the type molecule is lysine, and the number of structural units is k=3, where the linker is aminohexanoyl lysine, which contains the spacer aminocaproic acid, wherein the length of one spacer is NH ₂ (CH _{2 )} Where p 0 of _p COOH, the structure of the other spacer is NH ₂ (CH ₂ ) ₅ COOH, and the length is _{p 2} of NH ₂ (CH ₂ ) _p COOH.

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. ]-NH ₂ , abbreviated as (Gd-DOTA) ₄ -linker-TPP ₂ , the imaging unit is 4 Gd-DOTA molecules, ₄ DOTAs are attached to the ε-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 structural unit of the linear molecule is lysine, and the number of structural units is k= 3, where the linker is aminohexanoyl lysine, which comprises a spacer aminocaproic acid, wherein the length of one spacer is the case of p = 0 as described for NH ₂ (CH ₂ ) _p COOH, and the other spacer 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.

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.

Figure 17 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.

Figure 18 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.

19 is an in vitro T ₂ -weight MRI image signal intensity obtained by using (Gd-DOTA) ₄ -TPP ₂ as a targeting mitochondria-targeted molecularly labeled mesenchymal stem cells after different time propagation according to the cell reproduction time. The change was compared with the results of Part 10 of Example 10.

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.

detailed description

In order to make the objects, technical solutions, and advantages of the present application more comprehensible, the present application will be further described in detail below with reference to the accompanying drawings. It is apparent that the described embodiments are only a part of the embodiments of the present application, and not all of them. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present application without departing from the inventive scope are the scope of the present application.

The present application provides 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, wherein 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.

In the embodiment of the present application, 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.

In the embodiment of the present application, 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. A derivative obtained by butylamino group or the like.

In the embodiments of the present application, 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. Converting the carboxyl group of the complexing ligand of the contrast unit and the carboxyl group of the triphenylsulfonium or its derivative cation into an NHS active ester which is linked to the amino group of the branched or linear molecule, and after removing the protective group and the superparamagnetic metal Ion complexation results in the described contrast agent molecules that target mitochondria. 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.

In one embodiment of the present application, 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. The number k of structural units of the dendritic or linear molecule may be an integer from 0 to 7; the number of the DOTA of the contrast unit is m+n=k+1, the linker is lysine, and the structure of the spacer molecule is NH ₂ ( CH ₂ ) _p COOH, wherein p is an integer from 0 to 12.

In one embodiment of the present application, 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.

In one embodiment of the present application, Gd-DOTA is used as a contrast unit, and the targeting unit triphenylsulfonium molecule and Gd-DOTA are each linked to a dendrimer through a carboxyl group, and the structural unit of the dendrimer is lysine, a structural unit. When the number k=3, n=2, and m=2, 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.

In one embodiment of the present application, Gd-DOTA is used as a contrast unit, and the targeting unit triphenylsulfonium molecule and Gd-DOTA are each linked to a dendrimer through a carboxyl group, and the structural unit of the dendrimer is lysine, a structural unit. When the number k=3, n=2, and m=2, 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.

In one embodiment of the present application, Gd-DOTA is used as a contrast unit, and 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. When the number k=3, m=1, n=3, 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.

In one embodiment of the present application, 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. When the number k=3, n=2, and m=2, 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.

In one embodiment of the present application, 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. When the number k=3, n=2, and m=2, 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.

In one embodiment of the present application, 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 structural unit of the dendrimer adopts lysine, the number of structural units is k=3, n=2, and when m=2, the linker is aminohexanoyl lysine, which contains spacer aminocaproic acid, one of which is separated. 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.

In one embodiment of the present application, 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. When the number of structural units is k=3, m=1, and n=3, 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 .

In one embodiment of the present application, there is provided a cell labeled with a magnetic resonance contrast agent molecule targeting mitochondria as described herein, and a magnetic resonance contrast agent utilizing mitochondria targeting mitochondria by pulse electroporation Molecular marker mesenchymal stem cell method.

In one embodiment of the present application, there is provided a method of binding observed mitochondria in vitro magnetic resonance imaging contrast agents 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.

In one embodiment of the present application, there is provided a method of transplanting mitochondrial magnetic resonance contrast agent molecularly labeled mesenchymal stem cells into a mouse by site-directed surgical transplantation/injection using 11.7 T in vivo magnetic resonance T ₂ weighted imaging, observe the change of image contrast of cell graft and its surrounding tissues at different time points after cell transplantation.

In one embodiment of the present application, there is provided 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.

The specific implementation is as follows:

Example 1: Synthesis of contrast agent molecule Gd-DOTA-TPP targeting mitochondria (Fig. 6)

1. Synthesis of Bu ^t ₃ DOTA (1,4,7-tris(tert-butoxycarbonylmethyl)-10-(acetic acid)-1,4,7,10-tetraazacyclododecane). The synthesis was started from 1,4,7,10-tetraazacyclododecane (Cyclen) as follows:

a) 10.0 g of Cyclen and 29.3 g of NaHCO ₃ were weighed into a 1 L three-necked flask and 50 mL of acetonitrile was added. 37.4 g of t-butyl bromoacetate was weighed in a fume hood, and 20 mL of acetonitrile was added, mixed uniformly, and placed in a dropping funnel. An acetonitrile solution of t-butyl bromoacetate was slowly added dropwise to the reaction mixture in a three-necked flask under ice bath and N ₂ . After the dropwise addition was completed, stirring was continued for 30 hours at room temperature. The solid was filtered off, acetonitrile was removed by rotary evaporation, recrystallized twice from toluene to give a white solid 16g Bu ^t ₃ DO3A (1,4,7- tris (tert-butoxycarbonyl methyl) -1,4,7,10-tetraazacyclododecane Azacyclododecane).

b) Weigh 1.38 g of K ₂ CO ₃ and 2.57 g of Bu ^t ₃ DO3A in a 250 mL three-necked flask, add 50 mL of acetonitrile, and add 1.0 g of ethyl bromoacetate in 5 mL of acetonitrile under N ₂ protection and stirring at room temperature. Raise to 70 ° C for 12-24 hours. Cool to room temperature, filter and remove the solvent by rotary evaporation. Column chromatography using CH ₂ Cl ₂ /MeOH (20:1) as a solvent afforded 2.5 g of pale yellow viscous foamy product 1,4,7-tris (tert-butoxycarbonylmethyl)-10 -(ethoxycarbonylmethyl) 1,4,7,10-tetraazacyclododecane (Bu ^t ₃ Et-DOTA).

c) 1.5 g of Bu ^t ₃ Et-DOTA was weighed into a 250 mL three-necked flask, dissolved in 50 mL of dioxane, and 25 mL of 1.2 M aqueous NaOH solution was added under N ₂ protection, and stirred at 50-70 ° C for 4 hours. The dioxane was removed by rotary evaporation and extracted three times with dichloromethane (25 mL). Solvent was removed by rotary evaporation, is separated by column chromatography with eluent _{CH 2 Cl 2 / MeOH (20:1} ), to give 1.0g product as a pale yellow viscous foam Bu ^t ₃ DOTA.

2. Synthesis of Ph ₃ P(Br)(CH ₂ ) ₄ COOH [(4-carboxybutyltriphenylphosphonium bromide)]

7.42 g of triphenylsulfonium and 5.01 g of 5-bromopentanoic acid were weighed and placed in a 100 mL round bottom flask, and 35 mL of xylene was added thereto, followed by stirring to dissolve. Heat to 140 ° C and condense for 4 hours. The mixture was cooled to 40 ° C to 50 ° C, and 30 mL of dimethyl ether was slowly added dropwise with a separating funnel under vigorous stirring to obtain white crystals. Filtering, white The color crystals were washed twice with a small amount of diethyl ether to give product (br.

3. Synthesize DOTA-TPP by solid phase synthesis:

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.

After condensing Ph ₃ P(Br)(CH ₂ ) ₄ COOH, 50 mL of 1% TFA in dichloromethane was added to remove the protecting group Mtt and drained. Wash three times with DMF, methanol and dichloromethane, respectively. Add 1.72g Bu ^t ₃ DOTA, 50mL DMF solvent, 0.96g TBTU, 0.41g HOBt condensing agent and 2.5mL base DIPEA, react at 25 ° C, judge the coloration of carboxyl group and amino group after ninhydrin color development, and then drain the solvent. Wash three times with 50 mL of methanol, DMF, methanol and dichloromethane. 50 mL of 50% TFA in dichloromethane was added and the reaction was carried out at 25 ° C for 40 minutes. Filtration, the filtrate was taken, the pH of the filtrate was adjusted to neutral with triethylamine and concentrated to dryness. Diethyl ether was added to precipitate a white solid to give a crude product.

The crude product was further purified by HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5μm 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.

4. The deprotected DOTA-TPP is complexed with Gd ^{3+ to} obtain 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. The apparatus was rotated overnight, and then the pH was carefully adjusted to 7-8 with 1.0 M aqueous ammonia and 1.0 M hydrochloric acid, and the clear aqueous solution was freeze-dried to obtain a white powder 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 5μm 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.

Example 2: Synthesis of (Gd-DOTA) ₄ -TPP targeting mitochondrial contrast agent molecules (Figure 7)

1, as described in Synthesis Example 1 and Bu ^t ₃ DOTA _{Ph 3 P (Br) (CH} 2) 4 COOH.

2. Synthesis of DOTA ₄ -TPP by solid phase synthesis: The steps are as follows: synthesized on a solid phase synthesizer according to the traditional Fmoc method, and the amino acids are 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.

After condensing Ph ₃ P(Br)(CH ₂ ) ₄ COOH, 50 mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt of the lysine side chain, and the solvent was drained. Wash three times with DMF, methanol and dichloromethane, respectively. The Fmoc protected amino acid was then added for condensation (2.0 g Fmoc-Lys(Fmoc)-OH, 4.0 g Fmoc-Lys(Mtt)-OH, 2.12 g Fmoc-ε-Acp-OH). 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. Remove the conditions of the previous Fmoc before adding the next Fmoc protected amino acid: 25mL 20% piperidine / DMF reaction at room temperature for 0.5 hours, drain, add 25mL 20% piperidine / DMF reaction for 0.5 hours, drain, use DMF respectively Methanol and dichloromethane were washed three times each.

After finally condensing Fmoc-ε-Acp-OH and removing Fmoc, 100 mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt, and drained. Wash three times with DMF, methanol and dichloromethane, respectively. Add 7g Bu ^t ₃ DOTA, 200mL DMF solvent, 4g TBTU, 1.64g HOBt condensing agent and 10mL base DIPEA, react at 25 ° C, judge the carboxy group and amino group condensation after ninhydrin color development, then drain the solvent, respectively, use 50mL Methanol, washed three times with DMF, methanol and dichloromethane. 50 mL of 50% TFA in dichloromethane was added and the reaction was carried out at 25 ° C for 40 minutes. Filtration, the filtrate was taken, the pH of the filtrate was adjusted to neutral with triethylamine and concentrated to dryness. Diethyl ether was added to precipitate a white solid to give a crude product.

The crude product was further purified by HPLC, Waters 2535_2707_2998_WFC, XBridge Pre C18 5 μm 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.

3. The deprotected DOTA ₄ -TPP is complexed with Gd ^{3+ to} obtain a contrast agent molecule targeting (Gd-DOTA) ₄ -TPP targeting mitochondria. Specifically, as follows: about 0.5 mL of an 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 -TPP for 3 hours, and the pH was adjusted to about 6 with 1.0 M aqueous ammonia, and static at room temperature. The mixer was rotated overnight, then the pH was carefully adjusted to 7-8 with 1.0 M aqueous ammonia and 1.0 M hydrochloric acid, and the clear aqueous solution was lyophilized to give a white powder (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 5μm 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 3: (Gd-DOTA) _4- linker-TPP targeting mitochondrial synthesis of contrast agent molecules (where spacer is Acp) (Figure 8, dendrimer)

1, according to Example 1 were synthesized Bu ^t ₃ DOTA and _{Ph 3 P (Br) (CH} 2) 4 COOH.

2, DOTA ₄ -linker-TPP synthesized by solid phase synthesis:

The steps are briefly described as follows: synthesis is carried out on a solid phase synthesizer according to the conventional Fmoc method, and amino acids are 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.

After condensing Ph ₃ P(Br)(CH ₂ ) ₄ COOH, 50 mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt of the lysine side chain, and the solvent was drained. Wash three times with DMF, methanol and dichloromethane, respectively. The Fmoc protected amino acid was then added for condensation (1.06 g Fmoc-ε-Acp-OH, 2.0 g Fmoc-Lys(Fmoc)-OH, 4.0 g Fmoc-Lys(Mtt)-OH, 2.12 g Fmoc-ε-Acp-OH ). 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. Remove the conditions of the previous Fmoc before adding the next Fmoc protected amino acid: 25mL 20% piperidine / DMF reaction at room temperature for 0.5 hours, drain, add 25mL 20% piperidine / DMF reaction for 0.5 hours, drain, use DMF respectively Methanol and dichloromethane were washed three times each.

After finally condensing Fmoc-ε-Acp-OH and removing Fmoc, 100 mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt, and drained. Wash three times with DMF, methanol and dichloromethane, respectively. Add 7g Bu ^t ₃ DOTA, 200mL DMF solvent, 4g TBTU, 1.64g HOBt condensing agent and 10mL base DIPEA, react at 25 ° C, judge the carboxyl group and amino group after the condensation of ninhydrin color development, then drain the solvent, respectively, use 50mL Methanol, washed three times with DMF, methanol and dichloromethane. 50 mL of 50% TFA in dichloromethane was added and the reaction was carried out at 25 ° C for 40 minutes. Filtration, the filtrate was taken, the pH of the filtrate was adjusted to neutral with triethylamine and concentrated to dryness. Diethyl ether was added to precipitate a white solid to give a crude product.

The crude product was further purified by HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5μm 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.

3. 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. Specifically, as follows: about 0.5 mL of an 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 lower static mixer was rotated overnight, then the pH was adjusted to 7-8 with 1.0 M ammonia and 1.0 M hydrochloric acid, and the clear aqueous solution was freeze-dried to obtain a white powder (Gd-DOTA). ₄ -linker-TPP targeting mitochondria contrast agent molecules . The purity of (Gd-DOTA) _4- linker-TPP was determined by HPLC. HPLC conditions, Waters 2535_2707_2998, Sapphire C18 5μm 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 4: (Gd-DOTA) _4- linker-TPP targeting mitochondrial synthesis of contrast agent molecules (where spacer is Acp) (Figure 9, linear molecule)

1, the synthesis Bu ^t ₃ DOTA and _{Ph 3 P (Br) (CH} 2) 4 COOH The procedure of Example 1.

2. According to the method of synthesizing DOTA ₄ -linker-TPP according to Example 3, a linear molecule-linked DOTA ₄ -linker-TPP was synthesized by a solid phase synthesis method according to the structure shown in FIG.

3. 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. Specifically, as follows: about 0.5 mL of an 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 lower static mixer was rotated overnight, then the pH was adjusted to 7-8 with 1.0 M ammonia and 1.0 M hydrochloric acid, and the clear aqueous solution was freeze-dried to obtain a white powder (Gd-DOTA). ₄ -linker-TPP targeting mitochondria contrast agent molecules . The purity of (Gd-DOTA) _4- linker-TPP was determined by HPLC. HPLC conditions, Waters 2535_2707_2998, Sapphire C18 5μm 4.6×250mm, mobile phase: solvent A (0.1% TFA water), solvent B (0.1% TFA, 80% CH ₃ CN, 20% water), solvent B in 20 minutes 24% rose to 44%; flow rate 1.0 mL / min. The purity is 95% or more.

Example 5: (Dy-DOTA) _4- linker-TPP targeting mitochondrial synthesis of contrast agent molecules (linker=Acp here) (Figure 10)

1, the synthesis Bu ^t ₃ DOTA and _{Ph 3 P (Br) (CH} 2) 4 COOH The procedure of Example 1.

2. Synthesize DOTA ₄ -linker-TPP according to the method of Example 3:

3. The deprotected DOTA ₄ -linker-TPP is complexed with Dy ^{3+ to} obtain (Dy-DOTA) _4- linker-TPP targeting mitochondrial contrast agent molecules. Specifically, as follows: 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 lower static mixer was rotated overnight, then carefully adjusted to pH 7-8 with 1.0 M ammonia and 1.0 M hydrochloric acid, and the clear aqueous solution was freeze-dried to obtain a white powder (Dy-DOTA). ₄ -linker-TPP targeting mitochondria contrast agent molecules . The purity of (Dy-DOTA) _4- linker-TPP was determined by HPLC. HPLC conditions, Waters 2535_2707_2998, Sapphire C18 5μm 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 6: (Gd-DTPA) _4- linker-TPP targeting mitochondrial synthesis of contrast agent molecules (linker=Acp here) (Figure 11)

1. Synthesis of Ph ₃ P(Br)(CH ₂ ) ₄ COOH according to the method of Example 1.

2. Synthesis of DTPA ₄ -linker-TPP by solid phase synthesis:

The steps are briefly described as follows: synthesis is carried out on a solid phase synthesizer according to the conventional Fmoc method, and amino acids are 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.

After condensing Ph ₃ P(Br)(CH ₂ ) ₄ COOH, 50 mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt of the lysine side chain, and the solvent was drained. Wash three times with DMF, methanol and dichloromethane, respectively. The Fmoc protected amino acid was then added for condensation (1.06 g Fmoc-ε-Acp-OH, 2.0 g Fmoc-Lys(Fmoc)-OH, 4.0 g Fmoc-Lys(Mtt)-OH, 2.12 g Fmoc-ε-Acp-OH ). 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. Remove the conditions of the previous Fmoc before adding the next Fmoc protected amino acid: 25mL 20% piperidine / DMF reaction at room temperature for 0.5 hours, drain, add 25mL 20% piperidine / DMF reaction for 0.5 hours, drain, use DMF respectively Methanol and dichloromethane were washed three times each.

After finally condensing Fmoc-ε-Acp-OH and removing Fmoc, 100 mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt, and drained. Wash three times with DMF, methanol and dichloromethane, respectively. Add 4.0 g of DTPAA (diethylenetriamine pentaacetic anhydride), 200 mL of DMF solvent and 10 mL of alkali DIPEA, react at 25 ° C, judge the carboxyl group and the amino group after the condensation of ninhydrin, and then drain the solvent, respectively, using 50 mL of methanol. Wash three times with DMF, methanol and dichloromethane. 50 mL of 50% TFA in dichloromethane was added and the reaction was carried out at 25 ° C for 40 minutes. Filtration, the filtrate was taken, the pH of the filtrate was adjusted to neutral with triethylamine and concentrated to dryness. Diethyl ether was added to precipitate a white solid to give a crude product.

The crude product was further purified by HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5μm 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.

3. 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. Specifically, as follows: about 0.5 mL of an 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 lower static mixer was rotated overnight, then the pH was adjusted to 7-8 with 1.0 M ammonia and 1.0 M hydrochloric acid, and the clear aqueous solution was freeze-dried to obtain a white powder (Gd-DTPA). ₄ -linker-TPP targeting mitochondria contrast agent molecules . The purity of (Gd-DTPA) _4- linker-TPP was determined by HPLC. HPLC conditions, Waters 2535_2707_2998, Sapphire C18 5μm 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 7: Synthesis of (Gd-DOTA) _4- linker-(p-tolyl) ₃ P targeting mitochondrial contrast agent molecules (linker=Acp here) (Fig. 12)

1, as in Example 1 Synthesis Bu ^t ₃ DOTA.

2. Synthesis of (p-tolyl) ₃ P(Br)(CH ₂ ) ₄ COOH [bromination (4-carboxybutyltris(p-methylphenyl)phosphonium)]

The procedure for synthesizing Ph ₃ P(Br)(CH ₂ ) ₄ COOH according to Example 1 was carried out, and 7.42 g of the triphenylphosphine was replaced with 8.61 g of tri-p-tolylphosphine, and the other materials and conditions were unchanged. The product obtained was brominated (4-carboxybutyltri-p-tolylhydrazine) 8.0 g, yield 58%.

3. Synthesis of DOTA ₄ -linker-(p-tolyl) ₃ P by solid phase synthesis:

According to the synthesis procedure of Example 3, 1.77 g of Ph ₃ P(Br)(CH ₂ ) ₄ COOH was exchanged for 1.94 g of (p-tolyl) ₃ P(Br)(CH ₂ ) ₄ COOH, other materials and conditions. And the detection steps are unchanged.

4. 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. According to the complexing step of 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)

1, the synthesis Bu ^t ₃ DOTA and _{Ph 3 P (Br) (CH} 2) 4 COOH The procedure of Example 1.

2. The linear molecule-linked DOTA ₄ -linker-TPP _{2 was} synthesized by solid phase synthesis according to the structure shown in FIG. In this embodiment, 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 steps are briefly described as follows: synthesis is carried out on a solid phase synthesizer according to the conventional Fmoc method, and amino acids are sequentially coupled from the C terminal to the N terminal according to the structure shown in FIG. First, 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. 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.

After condensing Ph ₃ P(Br)(CH ₂ ) ₄ COOH, 50 mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt of the lysine side chain, and the solvent was drained. Wash three times with DMF, methanol and dichloromethane, respectively. Then, 1.06 g of Fmoc-ε-Acp-OH, 2.0 g of Mtt-Lys(Fmoc)-OH, and 1.72 g of Bu ^t ₃ DOTA were successively added for condensation. 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. Add the conditions of the previous Fmoc before adding the next molecule condensation: 25mL 20% piperidine / DMF reaction at room temperature for 0.5 hours, drain, add 25mL 20% piperidine / DMF reaction for 0.5 hours, drain, use DMF, methanol respectively The dichloromethane was washed three times each. This completes the linear connection of a contrast unit complexing agent DOTA. This process was repeated twice more to complete the linear connection of the three contrast unit complexes DOTA; the linear connection of the last contrast unit complex agent DOTA was the same as the above procedure, but the added condensation molecules were only added to 1.06 g of Fmoc-ε-Acp. -OH, 1.72 g Bu ^t ₃ DOTA was condensed without the addition of Mtt-Lys(Fmoc)-OH molecules.

After total condensation, 50 mL of 50% TFA in dichloromethane was added and the reaction was carried out at 25 ° C for 40 minutes. Filtration, the filtrate was taken, the pH of the filtrate was adjusted to neutral with triethylamine and concentrated to dryness. Diethyl ether was added to precipitate a white solid to give a crude product.

The crude product was further purified by HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5μm 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.

3. 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. Specifically, as follows: about 0.5 mL of an 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. The clear aqueous solution was lyophilized to give a white powder (Gd-DOTA). _4- linker-TPP ₂ targeted mitochondrial angiography Agent molecule. The purity of (Gd-DOTA) _4- linker-TPP _{2 was determined} by HPLC. HPLC conditions, Waters 2535_2707_2998, Sapphire C18 5μm 4.6×250mm, mobile phase: solvent A (0.1% TFA water), solvent B (0.1% TFA, 80% CH ₃ CN, 20% water), solvent B in 20 minutes 24% rose to 44%; flow rate 1.0 mL / min. The purity is 95% or more.

Example 9: Concentration of triphenylsulfonium targeting mitochondria molecular molecule labeled mesenchymal stem cells

1. Human bone marrow mesenchymal stem cells (hMSCs) 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. Add 3 mL of complete medium in a centrifuge tube, blow up the cell pellet, take out the cell suspension, place in a 100×20 mm culture dish, continue to add 5 mL of complete medium, gently shake the dish to evenly disperse the cells in the complete medium. in. It was then placed in an incubator at 37 ° C in a 5% carbon dioxide solution. On the second day of cell resuscitation, the medium was changed and the culture was continued. When the adherent cell density reaches 80% to 90%, the medium is aspirated. Gently wash the cell-filled culture dish with 2 mL of sterile PBS solution, add 1 mL of PBS and 1 mL of trypsin, and observe the cell morphology under the microscope. After the cells are completely digested, 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.

2, 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.

3. Electroporation labeled cells

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 μL of the sample solution in the cell pellet (100 μL 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 μs, 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

1. 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.

2. 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.

3, the capillary cells are T ₁ and T ₂ weighted weighted imaging in a magnetic resonance spectrometer 11.7T. The T ₁ weighted image uses a saturation recovery sequence, TE = 5.2 ms, TR = 500 ms, FOV = 12 × 12 mm ² , matrix = 96 × 96, slice thickness = 0.8 mm, slice interval = 0.2 mm, cumulative number = 4; The T ₂ weighted image uses multi-layer echo, TR=3000ms, TE=80ms, 20 echoes, FOV=12×12mm ² , matrix=96×96, layer thickness=0.8mm, slice interval=0.2mm, The cumulative number of times = 1.

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. Can be seen that: (1) 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; (2) With the proliferation of magnetic labeled cells, 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. (2) Magnetically labeled cells In the T ₂ -weighted MRI image, 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

1. The method of the method of Example 9, using (Gd-DOTA) ₄ -TPP targeting mitochondrial contrast agent molecules labeled mesenchymal stem cells by pulse electroporation, targeting mitochondria contrast agent molecule concentration of 20 mM;

2. About 3×10 ⁵ magnetically labeled cells were transplanted into the mouse brain by site-directed injection, and 11.7T magnetic resonance T ₂ weighting was performed on the cell transplantation site at different time points after cell transplantation (D0～D10). Imaging (S is the slice number of the image). Use a bird cage coil with a diameter of 38mm, using RARE sequence parameter settings: TE = 7ms, TR = 125, 300, 500, 750, 1000, 1500, 3000, 5000ms, FOV = 20 × 20mm ² , matrix = 128 × 128, Layer thickness = 0.5 mm, average = 4. The obtained image effect is shown in Fig. 17: 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, and then (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). After one week, 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. This is a significant feature of the magnetic resonance targeting mitochondrial contrast agent molecules and imaging methods provided by the present application. In practical applications, 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

1. 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;

2. About 1×10 ⁷ magnetically labeled cells were transplanted into the forelimb muscle of the mice by site-directed injection, and 3T magnetic resonance T ₂ weighted imaging was performed on the magnetically labeled cell transplantation site after magnetic labeling cell transplantation. The image effect is shown in Fig. 18. The cell transplant body exhibits a significant dark signal, indicating that the mitochondrial targeting agent molecule provided by the present application, the magnetic labeled cell labeled by the same, and the magnetic resonance imaging live tracer method are on the clinical imaging device. It also has application feasibility.

Example 13: In vitro MRI image of contrast agent molecularly labeled mesenchymal stem cells targeting bistriphenylguanidine targeting mitochondria

Double triphenylphosphonium mitochondrial targeting contrast agent molecules prepared in Example 8, between the markers according to Example 9 of the mesenchymal stem cells were magnetically labeled cells in vitro T ₂ weighted MRI imaging method according to Example 10.

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.

The above is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc., which are made within the spirit and principles of the present application, should be included in the present application. Within the scope of protection.

Claims (15)

1. A use of a mitochondrial-targeting contrast agent molecule as a T ₂ contrast agent, wherein the mitochondrial-targeting contrast agent molecule comprises a targeting unit and a contrast unit, wherein

   The targeting unit is a phosphonium cation having a structural formula of -P ⁺ (X ₁ )(X ₂ )(X ₃ ), wherein X ₁ , X ₂ , X ₃ represents unsubstituted or substituted by one or more substituents Substituted C _1-12 alkyl, C _1-12 alkenyl, or C _6-10 aryl, the substituent comprising 1, 2 or 3 halogen atoms, C _1-12 alkyl, C _6-10 aryl a group, a hydroxyl group, a C _1-12 alkoxy group, a halogeno-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.
2. The use according to claim 1, wherein the targeting unit is a triphenylphosphonium cation or a derivative thereof.
3. The use according to claim 1, wherein the superparamagnetic metal complex is formed of a superparamagnetic metal and a complexing agent, wherein:

   The superparamagnetic metal is selected from the group consisting of lanthanide metals Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and non-lanthanide metals Cr, Mn, Fe, Co, Ni, Cu, Y, Nb;

   The complexing agent is selected from the group consisting of DOTA, HP-DO3A, DO3A-butrol, DTPA-BMA, DTPA, DTPA-BMEA, BOPTA, EOB-DTPA or derivatives thereof, and any combination thereof.
4. The use according to claim 1, wherein the targeting unit is linked to a dendritic or linear molecule directly or via a linker, the dendritic or linear molecule being linked to the contrast unit directly or via a spacer, wherein the branch The structural unit of a type 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.
5. The use according to claim 4, wherein said linker is a linear amino acid, preferably lysine; said spacer is a linear amino acid, preferably NH ₂ (CH ₂ ) _p COOH or NH ₂ (CH) ₂ CH ₂ O) _q CH ₂ COOH, wherein p is an integer from 0 to 12, q is an integer from 0 to 4, and when p=0 or q=0, there is no spacer.
6. The use according to any one of claims 4 to 5, wherein the dendritic or linear molecule is linked to 1 to 2 targeting units and 1 to 8 contrasting units, preferably to 2 targeting units and 4 Contrast unit connection.
7. The use according to any one of claims 4 to 6, wherein when the dendritic or linear molecule is linked to two targeting units, the binding positions of the targeting units may be adjacent, preferably in the presence of linear molecules. The same end; may not be adjacent, preferably at both ends of the linear molecule.
8. A contrast agent molecule targeting mitochondria, comprising a targeting unit and a contrast unit, wherein:

   The targeting unit is a phosphonium cation having a structural formula of -P ⁺ (X ₁ )(X ₂ )(X ₃ ), wherein X ₁ , X ₂ , X ₃ represents unsubstituted or substituted by one or more substituents Substituted C _1-12 alkyl, C _1-12 alkenyl, or C _6-10 aryl, the substituent comprising 1, 2 or 3 halogen atoms, C _1-12 alkyl, C _6-10 aryl a group, a hydroxyl group, a C _1-12 alkoxy group, a halogeno-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 targeting unit is linked to a dendritic or linear molecule directly or via a linker, the dendritic or linear molecule being linked to the contrast unit directly or via a spacer, 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.
9. The mitochondrial-targeting contrast agent molecule according to claim 8, wherein the linker is selected from a linear amino acid, preferably lysine; the spacer is selected from a linear amino acid, preferably NH ₂ (CH _{2 ) p} COOH or NH ₂ (CH ₂ CH ₂ O) _q CH ₂ COOH, wherein p is an integer from 0 to 12, q is an integer from 0 to 4, and when p=0 or q=0, there is no spacer.
10. The mitochondrial-targeting contrast agent molecule according to claim 8 or 9, wherein the dendritic or linear molecule is linked to 1 to 2 targeting units and 1-8 imaging units, preferably to 2 targeting units Connected to 4 contrast units.
11. The mitochondrial-targeting contrast agent molecule according to any of claims 8-10, wherein when the dendritic or linear molecule is linked to two targeting units, the binding positions of the targeting units may be adjacent, preferably in a line. The same end of the type of molecule; or may not be adjacent, preferably at both ends of the linear molecule.
12. A method of preparing a mitochondrial-targeting contrast agent molecule of claims 8-11, comprising:

    Each of said -P ⁺ (X ₁ )(X ₂ )(X ₃ ) cations is reacted with a halogenated carboxylic acid or a halogenated amine to form -P ⁺ (X ₁ )(X ₂ ) having a carboxyl group or an amino functional group ( X ₃ ) a cation in which the -P ⁺ (X ₁ )(X ₂ )(X ₃ ) cation is bonded to a dendritic or linear molecule through a obtained carboxyl group or amino group; the halogenated carboxylic acid is chloro, bromo Or an iodo fatty acid or an aromatic acid;

    The superparamagnetic metal complex is linked to a dendritic or linear molecule via its carboxyl group or amino group; 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 superparamagnetic The amino group of the metal complex is selected from the group consisting of ethylamino, propylamino or butylamino.
13. A magnetically labeled cell labeled with a contrast agent molecule targeted to mitochondria according to claims 8-11, the magnetically labeled cell being any cell that can be used for cell transplantation therapy by targeting a mitochondrial contrast agent molecule , selected from the group consisting of mesenchymal stem cells, neural stem cells, cardiac stem cells, embryonic stem cells, and induced pluripotent stem cells.
14. A combination of magnetic labeling cells and scaffold materials according to claim 13, wherein the scaffold material is any medical material that can form a combination with cells, 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.
15. A magnetic resonance imaging living body tracing method, comprising:

    The magnetic labeled cell of claim 13 or the combination of the magnetic labeled cell of claim 14 and the scaffold material by site-directed surgery/intravenous injection into a human or animal body;

    The above human or animal was placed in a magnetic resonance imaging apparatus and imaged in a magnetic resonance T ₂ weighting mode.

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