CN106890345B

CN106890345B - Contrast agent molecule targeting mitochondria as T2Use of contrast agents

Info

Publication number: CN106890345B
Application number: CN201510946966.9A
Authority: CN
Inventors: 邓宗武; 谭波; 张艳辉; 张宏岩; 张海禄
Original assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Current assignee: Suzhou Institute of Nano Tech and Nano Bionics of CAS
Priority date: 2015-12-17
Filing date: 2015-12-17
Publication date: 2020-09-01
Anticipated expiration: 2035-12-17
Also published as: CN106890345A; WO2017101717A1

Abstract

The invention discloses a contrast agent molecule of a targeted mitochondrion as T₂Use of contrast agents, the mitochondria-targeted contrast agent molecules comprising a peptide having-P for binding to cellular mitochondria⁺(X₁)(X₂)(X₃) Phosphonium cations of the general structural formula and a contrast unit superparamagnetic metal complex for enhancing the contrast of magnetic resonance imaging. The invention also discloses a contrast agent molecule targeting mitochondria and a preparation method thereof, wherein one or more-P⁺(X₁)(X₂)(X₃) Cation is combined with 1-8 superparamagnetic metal complex molecules through dendritic or linear molecules, and a linker and a spacer are used for improving-P⁺(X₁)(X₂)(X₃) Yang (Yang)Ions and dendrimeric or linear molecules and steric structure between dendrimeric or linear molecules and superparamagnetic metal complexes. The invention further discloses a magnetic labeling cell labeled by the mitochondrion-targeted contrast agent molecule, a combination of the magnetic labeling cell and a bracket material, and a magnetic resonance imaging in vivo tracing method by using the magnetic labeling cell and the combination.

Description

Contrast agent molecule targeting mitochondria as T2Use of contrast agents

Technical Field

The invention relates to the field of medical imaging, in particular to a magnetic resonance T using a contrast agent molecule targeting mitochondria₂The invention also relates to a contrast agent molecule targeting mitochondria, Magnetic labeled cells labeled by the contrast agent molecule, a combination of the Magnetic labeled cells and a bracket material, and a Magnetic Resonance Imaging (MRI) in vivo tracing method by using the contrast agent molecule and the Magnetic labeled cells.

Background

Stem cell regeneration medicine is a new biomedical field, and the basic idea is to realize regeneration and repair of damaged tissues and organs by inducing the directional differentiation of stem cells in a transplanted body. In the stem cell regeneration medical treatment process, the physiological behaviors of survival, migration, homing, directional differentiation and the like of stem cells transplanted into a body need to be tracked in real time, accurate tissue biological distribution tracing is carried out on the stem cells, and the internal and external stem cells, the stem cells generated by self-renewal and the functional cells generated by differentiation are distinguished, so that the physiological processes of migration, propagation, division, differentiation and the like of the stem cells in the body are deeply known, and the method has very important significance for basic research of stem cell biology and clinical evaluation of curative effect observation and function recovery.

Magnetic resonance imaging as a soft group with high spatial resolutionThe non-invasive in vivo imaging technique with tissue contrast and no risk of ionizing radiation is the most potential technique for tracing the maintenance and differentiation process of stem cells in vivo. The magnetic resonance image is based on the longitudinal relaxation rate (1/T) of the magnetic moment formed in the process that the spin magnetic moments of water protons in different biological tissues are orderly arranged in a uniform magnetic field after being excited by specific microwaves₁) Or transverse relaxation rate (1/T)₂) There may be differences in contrast resulting in differences in signal intensity of the echoes formed in the image enabling structural and functional imaging of cells, tissues and organs of the organism. In practical applications, when the contrast of images of different tissues is close, the relaxation rate of water protons in a specific tissue, such as tumor tissue, can be changed by introducing a magnetic resonance contrast agent, so as to realize imaging of the specific tissue. The in vivo tracking of stem cells using magnetic resonance imaging techniques also requires first magnetic labeling of the stem cells to distinguish them from other tissue cells surrounding them.

The introduction of a magnetic resonance contrast agent will generally increase both the longitudinal and transverse relaxation rates of water protons in the tissue in which it is located, but there is a large difference in the relative magnitudes of the two relaxation rates that different magnetic resonance contrast agents increase. This difference results in the suitability of some magnetic resonance contrast agents for MRI signal enhancement, called T₁Contrast agents, some of which are suitable for MRI signal reduction, are known as T₂A contrast agent. For example, in general, the gadolinium complex has a greater acceleration effect on the longitudinal relaxation rate of water protons than on the transverse relaxation rate, favoring the acceleration at T₁Weighted under-image generation of bright signals, enhancing T₁The contrast of the weighted image is therefore often referred to as T₁Contrast agents are used. The acceleration effect of superparamagnetic iron oxide (SPIO) nanoparticles on transverse relaxation rate of water protons is much more significant than that of longitudinal relaxation rate thereof, at T₂Dark signal generation in weighted imaging mode to enhance T₂The contrast of the weighted image is ideal T₂A contrast agent. In addition, the same contrast agent is distributed over different biological interfaces, and there may also be a large difference in the relative amplitudes at which it accelerates the two relaxation rates. Such as gadolinium complexes in the free state or in the capsuleThe distribution of the vacuoles in the cytoplasm or binding to mitochondria in the cytoplasm differ significantly in their effect on the longitudinal and transverse relaxation rates of cellular water protons.

T represented by SPIO nanoparticles₂The contrast agent has high transverse relaxation rate, so that the contrast agent is widely researched and applied to stem cell in vivo images. However, the cell labeling technology based on the SPIO nanoparticles essentially provides information on the migration of the SPIO nanoparticles in vivo, the migration is caused by the mother cells carrying the nanoparticles, the free nanoparticles migrate after the death of the mother cells, or the migration caused by macrophages after being phagocytosed by other cells such as macrophages, and the current imaging method cannot give a clear conclusion and becomes a main challenge for analyzing the image information. Moreover, such cell markers do not intuitively tell one that the marked cells remain viable after entry into the body, or have died or partially died.

Gadolinium complexes as T₁Contrast agents have been widely used in clinical medicine, and gadolinium complex contrast agents currently used in clinical medicine can be classified into two types: one is DOTA with a ring structure and derivatives thereof, and the other is DTPA with an acyclic structure and derivatives thereof. The small molecule contrast agent has definite and stable chemical structure, and can precisely control the process and the result of coupling with the targeting molecule through a chemical method, so that the small molecule contrast agent has high reliability as a structural unit of the targeting contrast agent. However, a key problem faced by gadolinium complex contrast agents is their relaxivity well below T₂The relaxation rate of the SPIO type nanoparticles requires a large dose to obtain a sufficient tissue contrast, which leads to concern about safety problems such as toxicity of metal gadolinium ions in vivo.

The gadolinium complex labeled stem cells are also an optional technical scheme for in vivo magnetic resonance image tracing. However, in the existing method, the problems encountered by using small-molecule gadolinium complex or targeted contrast agent facing to cell membrane bound receptor are that besides the low longitudinal relaxation rate, the short residence time in the cell is a problem to be overcome; the same problems encountered with gadolinium complexes loaded with macromolecules or nanoparticles as with SPIO nanoparticles include slow clearance in vivo and possible interference with imaging results due to uptake by other cells.

US20090214437A1 and US20130142735A1 disclose magnetic resonance contrast agents which bind to the mitochondria of cells and which, after intravenous injection into the body, can be enriched in the tumor tissue where the mitochondria are active and can be used to enhance the T-cell activation of the tumor tissue₁Weighting the magnetic resonance signal (presenting a bright signal) in the imaging mode, thereby increasing T₁The contrast of the image is weighted. This contrast agent is referred to as T₁When the contrast agent is applied to the living body image of the cell transplant, the survival and migration conditions of the labeled cells in the body cannot be clearly determined, and the duration of the signal enhancement effect cannot meet the requirement of long-term observation.

In view of the above, there is a need in the art for a magnetic resonance contrast agent for labeling cells or transplants, which not only intuitively provides information about the survival status of the cells in vivo and the migration, homing, and differentiation of the cells or transplants, but also satisfies the need for long-term observation, and solves the problem of toxicity caused by a large dose.

Disclosure of Invention

The invention is based on the recognition by the inventor that the gadolinium complex is distributed on different biological interfaces, the influence of the gadolinium complex on the longitudinal relaxation rate and the transverse relaxation rate of the water protons of the cells is remarkably different, and particularly after the gadolinium complex is combined with mitochondria in cytoplasm, the gadolinium complex has remarkably reduced capability of accelerating the longitudinal relaxation rate of the water protons of the cells, is not beneficial to magnetic resonance signal enhancement, but is beneficial to magnetic resonance signal reduction, and is more suitable for T₂Dark signals are generated in the weighted imaging mode. The inventors have further discovered that after the labeled cells of the invention are transplanted into a body, the labeled cells partially release the contrast agent molecules, and the released contrast agent molecules cause the tissues surrounding the cell transplant to undergo magnetic resonance T₂The weighted image mode presents a bright signal, which allows the cell transplant to be more clearly distinguished from its surrounding tissue.

Hair brushIt is an object of the present invention to provide a mitochondrial-targeted contrast agent molecule as T₂Use of a contrast agent, the mitochondrially targeted contrast agent molecule comprising a targeting unit and a contrast unit, wherein the targeting unit is of the formula-P⁺(X₁)(X₂)(X₃) Phosphonium cations of the general structural formula wherein X₁、X₂、X₃Represents C unsubstituted or substituted by one or more substituents_1-12Alkyl radical, C_1-12Alkenyl, or C_6-10Aryl, said substituents comprising 1, 2 or 3 halogen atoms, C_1-12Alkyl radical, C_6-10Aryl, hydroxy, C_1-12Alkoxy, halo-C_1-12An alkoxy group; wherein X₁、X₂、X₃May be the same group or different groups; the contrast unit is a superparamagnetic metal complex. The mitochondria-targeted contrast agent molecule exhibits significant magnetic resonance signal attenuation effects after binding to mitochondria in cells, allowing magnetic resonance T of the magnetically labeled cells in vitro and in vivo₂Dark signals are presented in the weighted image mode; the targeting unit, when combined with a plurality of magnetic resonance imaging units and used for cell marking, exhibits stronger magnetic resonance signal attenuation effect and can last for a longer time.

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

In a preferred embodiment of the present invention, the superparamagnetic metal complex is formed of a superparamagnetic metal and a complexing agent, wherein: the superparamagnetic metals are metals having superparamagnetic properties, including, but not limited to, lanthanide metals praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), and non-lanthanide metals chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), yttrium (Y), niobium (Nb), etc.; the complexing agent is selected from DOTA, HP-DO3A, DO3A-butrol, DTPA-BMA, DTPA, DTPA-BMEA, BOPTA, EOB-DTPA or derivatives thereof and any combination thereof.

In a preferred embodiment of the invention, the targeting unit is linked to a dendritic or linear molecule directly or via a linker (linker) and the dendritic or linear molecule is linked to the contrast unit directly or via a spacer (spacer), wherein the building blocks of the dendritic or linear molecule are any monomers, preferably amino acids, more preferably lysine, which can be homopolymerized or copolymerized to form a dendritic or linear macromolecule.

In a preferred embodiment of the present invention, the linker is a linear amino acid, preferably lysine; the spacer is a linear amino acid, preferably NH₂(CH₂)_pCOOH or NH₂(CH₂CH₂O)_qCH₂COOH, wherein p is an integer of 0 to 12, q is an integer of 0 to 4, and when p or q is 0, it represents no spacer.

In a preferred embodiment of the invention said targeting unit is linked to 1-8 contrast units via said dendritic or linear molecule. Each of said-P⁺(X₁)(X₂)(X₃) The cations are combined with a plurality of superparamagnetic metal complexes through dendritic or linear molecules, the magnetic resonance signal weakening effect is more remarkable, and the effect is shown in T₂The dark signal may be presented for a longer time when imaging in the weighting mode.

In a preferred embodiment of the invention, the strength of the binding of the contrast agent molecule to mitochondria can be increased and further extended at T by using a plurality of targeting units, preferably 2 targeting units, linked to 1-8 contrast units via the dendritic or linear molecule₂The time when the dark signal is present when imaging in the weighting mode.

It is another object of the present invention to provide a mitochondrial-targeted contrast agent molecule comprising a targeting unit and a contrast unit, wherein: the targeting unit is of-P⁺(X₁)(X₂)(X₃) Phosphonium cations of the general structural formula wherein X₁、X₂、X₃Represents C unsubstituted or substituted by one or more substituents_1-12Alkyl radical, C_1-12Alkenyl, or C_6-10Aryl, said substituents comprising 1, 2 or 3 halogen atoms, C_1-12Alkyl radical, C_6-10Aryl, hydroxy, C_1-12Alkoxy, halo-C_1-12An alkoxy group; wherein X₁、X₂、X₃May be the same group or different groups; 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 is linked to a 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, that can be homopolymerized or copolymerized to form a dendritic or linear macromolecule.

In a preferred embodiment of the present invention, the linker is selected from the group consisting of linear amino acids, preferably lysine; the spacer is selected from linear amino acids, preferably NH₂(CH₂)_pCOOH or NH₂(CH₂CH₂O)_qCH₂COOH, wherein p is an integer of 0 to 12, q is an integer of 0 to 4, and when p or q is 0, it represents no spacer.

In a preferred embodiment of the invention said targeting unit is linked to 1-8 contrast units via said dendritic or linear molecule.

In a preferred embodiment of the invention, a plurality of targeting units, preferably 2 targeting units, are used linked to 1-8 contrast units via said dendritic or linear molecule.

The invention also provides a method for preparing the mitochondrially targeted contrast agent molecule, which comprises the following steps: each of said-P⁺(X₁)(X₂)(X₃) Reaction of cations with halocarboxylic acids or haloamines to form-P having carboxyl or amino functions⁺(X₁)(X₂)(X₃) A cation of formula-P⁺(X₁)(X₂)(X₃) The cation is connected with the branch type or linear molecule through the obtained carboxyl or amino; the halogenated carboxylic acid is chloro, bromo or iodo fatty acid or aromatic acid; the superparamagnetic metal complex is linked to the dendritic or linear molecule through a carboxyl or amino group thereof; the superparamagnetic metal complexThe carboxyl of the compound is selected from ethylcarboxyl, propylcarboxyl or butylcarboxyl, and the amino of the superparamagnetic metal complex is selected from ethylamino, propylamino or butylamino.

In a preferred embodiment of the invention, the mitochondrial-targeted contrast agent molecule is synthesized by: solid phase synthesis using cross-protection deprotection strategy for sequential synthesis of dendritic or linear molecules with or without linker or spacer and imaging units with or without spacer followed by ligation of-P⁺(X₁)(X₂)(X₃) A cation and a contrast unit.

In a preferred embodiment of the present invention, said-P is⁺(X₁)(X₂)(X₃) The carboxyl of one unit of cation, contrast unit and/or dendritic or linear molecule is converted into active ester or coupled with the amino, sulfydryl or hydroxyl of another unit after being activated; or by click chemistry (click chemistry) to link targeting units, dendritic or linear molecules and contrast units.

The invention also provides a magnetic labeling cell marked by the contrast agent molecule targeting mitochondria, which is any cell marked by the contrast agent molecule targeting mitochondria and can be used for cell transplantation therapy and is selected from mesenchymal stem cells, neural stem cells, myocardial stem cells, embryonic stem cells and induced pluripotent stem cells.

The invention also provides a cell marking method, which is characterized in that the contrast agent molecules targeting mitochondria are put into the culture solution containing the cells, and are introduced into the cells by utilizing the endocytosis or pinocytosis function of the cells, and the contrast agent molecules targeting mitochondria can be combined with the mitochondria in the cells, so that the retention time of the contrast agent molecules in the marked cells can be effectively prolonged.

The present invention also provides another method for cell labeling, which is characterized in that: and (2) putting the contrast agent molecules targeting mitochondria into a culture solution, an electrotransfection buffer solution or physiological saline containing the cells, and introducing the contrast agent molecules targeting mitochondria into the cells by using a pulse electroporation method.

The invention also provides a combination of the magnetic labeled cells and a scaffold material, wherein the scaffold material is any medical material capable of forming a combination with the cells and is selected from collagen, various synthetic macromolecules or inorganic scaffold materials; the scaffold material may or may not contain various trophic factors that support cell survival and growth.

The invention also provides a magnetic resonance image living body tracing method, which comprises the following steps: injecting the magnetic labeled cells or the combination of the magnetic labeled cells and the scaffold material into a human or animal body through site-specific surgery transplantation/vein injection; placing the human or animal in magnetic resonance imaging equipment, and performing magnetic resonance T₂Imaging in a weighted mode.

The invention provides a contrast agent molecule targeting mitochondria as T₂The contrast agent can be widely applied to the survival rate of cells transplanted into a living body in cell therapy and physiological processes of migration, homing and the like of the cells. This is a pioneering finding, on the one hand, because there is no effective method for clearly tracing the survival rate of cells transplanted into the body and physiological processes such as migration and homing thereof by using in vivo imaging technology, and on the other hand, because the application of gadolinium complex contrast agents has been focused only on the signal enhancement effect thereof for a long time, only in T₁Imaging in the weighting mode has unsatisfactory image contrast and shows irregular change. The invention is based on that after the contrast agent molecule of the targeted mitochondria is combined with the mitochondria in the cell, the magnetic resonance signal of the magnetic marked cell can be greatly reduced, and the stable and regular signal attenuation effect is presented for a long time, so the invention is used in T₂The effect of weakening the magnetic resonance signal presented by the gadolinium complex after being combined with mitochondria is fully utilized in a weighting mode. More importantly, the invention discovers that after the cells marked by the invention are transplanted into a body, the marked cells release contrast agent molecules targeting mitochondria, and the released contrast agent molecules targeting mitochondria can cause tissues around the cell transplant to be subjected to magnetic resonance T₂The weighted image mode presents a bright signal, which allows the cell transplant to be more clearly distinguished from its surrounding tissue.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.

Fig. 1 is a schematic diagram of a method for magnetic resonance imaging in vivo tracking of magnetically labeled cells according to the present invention.

Fig. 2 is a schematic structural diagram of a mitochondrial-targeted contrast agent molecule according to the present invention.

FIG. 3 is a structural general diagram of a phosphonium cation, which is a targeting unit of a mitochondrial contrast agent molecule provided by the present invention, and Triphenylphosphonium (TPP) cation and its derivatives ((p-tolyl) which are targeting units used in the examples of the present invention₃P) structural drawing of the cation.

FIG. 4 is a structural diagram of a superparamagnetic metal complex of a contrast unit according to the present invention, where M represents a metal with superparamagnetic properties, which can be used to enhance contrast in magnetic resonance imaging.

Fig. 5 is a schematic molecular structure diagram of a mitochondrion-targeted contrast agent provided by the present invention, wherein the contrast unit Gd-DOTA and the targeting unit triphenyl phosphonium cation are both connected to a dendritic or linear molecule through a carboxyl group. The structural unit of the dendritic or linear molecule is lysine, and the number k of the structural units can be an integer of 0-7; the number of contrast units DOTA is m + n ═ k + 1.

Fig. 6 shows a mitochondrion-targeted contrast agent molecule TPP-K (Gd-DOTA) -OH, which is abbreviated as Gd-DOTA-TPP, the contrast unit is Gd-DOTA, and both the targeting units TPP and Gd-DOTA are connected to two amino groups of lysine (abbreviated as K) through carboxyl groups in embodiment 1 of the present invention.

FIG. 7 shows a mitochondrion-targeted contrast agent molecule [ Gd-DOTA-Acp-K (Gd-DOTA) according to example 2 of the present invention]₂K (TPP) -OH, abbreviated (Gd-DOTA)₄TPP, contrast mediumThe unit is Gd-DOTA, the targeting units TPP and Gd-DOTA are connected with the dendritic molecule through carboxyl, the structural unit of the dendritic molecule is lysine, the number of the structural units is k ═ 3, the linker is lysine, the length of one spacer is NH₂(CH₂)_pIn the case where p of COOH is 0, the other spacer has a structure of NH₂(CH₂)₅COOH, aminocaproic acid (Acp), length NH₂(CH₂)_pAnd p in COOH is 5.

FIG. 8 shows a mitochondrion-targeted contrast agent molecule [ Gd-DOTA-Acp-K (Gd-DOTA) according to example 3 of the present invention]₂linker-K (TPP) -OH, abbreviated (Gd-DOTA)₄linker-TPP, the contrast unit is Gd-DOTA, both targeting units TPP and Gd-DOTA are linked to the dendrimer via carboxyl groups, the building unit of the dendrimer is lysine, the number of building units is k ═ 3, where the linker is aminocaproyllysine, which contains the spacer aminocaproic acid, where the length of one spacer is NH₂(CH₂)_pIn the case where p of COOH is 0, the other spacer has a structure of NH₂(CH₂)₅COOH, length NH₂(CH₂)_pAnd p in COOH is 5.

FIG. 9 shows a mitochondrion-targeted contrast agent molecule [ Gd-DOTA-Acp-K (Gd-DOTA) according to example 4 of the present invention]₂linker-K (TPP) -OH, abbreviated (Gd-DOTA)₄linker-TPP, the contrast unit is Gd-DOTA, the targeting units TPP and Gd-DOTA are both linked via carboxyl groups to a linear molecule, the structural unit of the linear molecule is lysine, the number of structural units is k ═ 3, where the linker is aminocaproyl lysine, which comprises the spacer aminocaproic acid, where one spacer is NH in length₂(CH₂)_pIn the case where p is 0 in COOH, the other spacer has a structure of NH₂(CH₂)₅COOH, length NH₂(CH₂)_pAnd p in COOH is 5.

FIG. 10 shows a mitochondrion-targeted contrast agent molecule [ Dy-DOTA-Acp-K (Gd-DOTA) according to example 5 of the present invention]₂-linker-K(TPP)-OH，Is abbreviated as (Dy-DOTA)₄linker-TPP, the contrast unit Dy-DOTA, both targeting units TPP and Dy-DOTA being linked to the dendrimer via a carboxyl group, the building block of the dendrimer being lysine, the number of building blocks being k ═ 3, where the linker is aminocaproyllysine comprising the spacer aminocaproic acid, wherein one spacer has the length NH₂(CH₂)_pIn the case where p of COOH is 0, the other spacer has a structure of NH₂(CH₂)₅COOH, length NH₂(CH₂)_pAnd p in COOH is 5.

FIG. 11 shows a mitochondrion-targeted contrast agent molecule [ Gd-DTPA-Acp-K (Gd-DTPA) according to example 6 of the present invention]₂-linker-K (TPP) -OH, abbreviated (Gd-DTPA)₄linker-TPP, the contrast unit is Gd-DTPA, the targeting units TPP and Gd-DTPA are both linked to the dendrimer via carboxyl groups, the building block of the dendrimer is lysine, the number of building blocks is k ═ 3, where the linker is aminocaproyl lysine, which contains the spacer aminocaproic acid, where the length of one spacer is NH₂(CH₂)_pIn the case where p of COOH is 0, the other spacer has a structure of NH₂(CH₂)₅COOH, length NH₂(CH₂)_pAnd p in COOH is 5.

FIG. 12 shows a mitochondrion-targeted contrast agent molecule [ Gd-DOTA-Acp-K (Gd-DOTA) according to example 7 of the present invention]₂-linker-K ((p-tolyl)₃P) -OH, abbreviated (Gd-DOTA)₄-linker- (p-tolyl)₃P, contrast unit is Gd-DOTA, targeting unit (P-tolyl)₃P and Gd-DOTA are both connected with the dendritic molecule through carboxyl, the structural unit of the dendritic molecule is lysine, the number of the structural units is k ═ 3, the linker is aminocaproyl lysine, the linker comprises a spacer aminocaproic acid, and the length of one spacer is NH₂(CH₂)_pIn the case where p of COOH is 0, the other spacer has a structure of NH₂(CH₂)₅COOH, length NH₂(CH₂)_pAnd p in COOH is 5.

FIG. 13 shows a mitochondrial targeting contrast molecule TPP-Lys (TPP) -Lys [ DOTA-Acp-Lys (DOTA) ((DOTA))]-NH₂Abbreviated as (Gd-DOTA)₄-linker-TPP₂The contrast unit is 4 Gd-DOTA molecules, and the 4 DOTA molecules are connected on the-amino (side chain) of the linear polypeptide Lys and the amino at the N end of the polypeptide to form linear arrangement; the targeting unit is that two TPP molecules are connected at the same end of a linear molecule, and the carboxyl of Lys at the end is amidated; the structural unit of the linear molecule is lysine, the number of the structural units is k ═ 3, and the linker is aminocaproyl lysine which comprises a spacer aminocaproic acid, wherein the length of one spacer is NH₂(CH₂)_pIn the case where p is 0 in COOH, the other spacer has a structure of NH₂(CH₂)₅COOH, length NH₂(CH₂)_pAnd p in COOH is 5.

FIG. 14 shows the use of (Gd-DOTA) in example 10 of the present invention_1,4In vitro magnetic resonance T of mesenchymal stem cells labelled with TPP as a mitochondrial-targeting contrast agent molecule₁Weighted sum T₂Weighting the image effect map. The first row of images are T of Gd-DOTA labeling mesenchymal stem cells at different cell propagation time nodes₁And T₂The image is weighted. The second, third and fourth rows of images are respectively the T of 5, 10, 20mMGd-DOTA-TPP marking mesenchymal stem cells at different cell propagation time nodes₁And T₂The image is weighted. The fifth row image was 20mM (Gd-DOTA)₄TPP labeling of T of mesenchymal stem cells at different cell propagation time nodes₁And T₂The image is weighted. The numbers below the images are the time nodes (days) at which the images were taken during the incubation period following cell labeling.

FIG. 15 is an in vitro T cell obtained by propagating labeled cells provided in example 10 of the present invention at different times₁The intensity of the weighted MRI image signal as a function of cell proliferation time.

FIG. 16 is an in vitro T cell obtained by propagating labeled cells provided in example 10 of the present invention at different times₂Weighting MRI image signal intensity as a function of cell proliferation timeAnd (4) changing.

FIG. 17 example 11 of the present invention uses 20mM (Gd-DOTA)₄TPP is contrast agent molecular marker mesenchymal stem cells targeting mitochondria, transplanted into the cranium of a mouse through site-specific surgery injection, and 11.7T magnetic resonance T collected at different time nodes₂Weighting the image effect map.

FIG. 18 example 12 of the present invention used 20mM (Gd-DOTA)₄TPP is contrast agent molecule marking mesenchymal stem cells targeting mitochondria, and 3T magnetic resonance T collected from rat foreleg muscle is transplanted through site-specific injection₂Weighting the image effect map.

FIG. 19 shows the use of (Gd-DOTA) in example 13 of the present invention₄-TPP₂In-vitro T obtained by propagating mesenchymal stem cells labeled by contrast agent molecules serving as targeted mitochondria at different times₂Comparison of the weighted MRI image signal intensity as a function of cell proliferation time with the results of example 10.

FIG. 20 is an in vitro T cell obtained by propagating labeled cells provided in examples 10 and 13 of the present invention for different periods of time₂The intensity of the weighted MRI image signal is related to the change of Gd content in the cells.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a contrast agent molecule targeting mitochondria as T₂Use of a contrast agent, the mitochondrially targeted contrast agent molecule comprising a targeting unit and a contrast unit, wherein the targeting unit is of the formula-P⁺(X₁)(X₂)(X₃) Phosphonium cations of the general structural formula wherein X₁、X₂、X₃Represents C unsubstituted or substituted by one or more substituents_1-12Alkyl radical, C_1-12Alkenyl, or C_6-10Aryl, said substituents comprising 1, 2 or 3 halogen atoms, C_1-12Alkyl radical, C_6-10Aryl, hydroxy, C_1-12Alkoxy, halo-C_1-12An alkoxy group; wherein X₁、X₂、X₃May be the same group or different groups; the contrast unit is a superparamagnetic metal complex.

In the embodiment of the invention, the structure of the molecule of the targeting unit triphenylphosphonium or the derivative thereof can be any one shown in fig. 3, and can also be other structural derivatives with the structural general formula shown in fig. 3.

In the embodiment of the present invention, the structure of the contrast unit superparamagnetic metal complex may be any one of the complexes shown in fig. 4, or may be a derivative of any one of the complexes shown in fig. 4, for example, a derivative obtained by replacing acetyl groups connected to dendritic or linear molecules in a complexing ligand of a superparamagnetic metal complex with propionyl groups or butyryl groups, or replacing ethylcarbonyl groups with ethylamino groups, propylamino groups, butylamino groups, or the like.

In the embodiment of the invention, the mitochondrion-targeted contrast agent molecule can be directly prepared by a polypeptide solid phase synthesis technology; it is also possible to synthesize each unit separately and then convert the carboxyl group of one unit to an NHS active ester or to link it to another unit after activation, or to link it by click chemistry. For example, the complex ligand (with protecting group) of the contrast unit superparamagnetic metal complex, the triphenyl phosphonium cation or the derivative cation thereof and the dendritic or linear molecule with amino are synthesized, the carboxyl of the complex ligand of the contrast unit and the carboxyl of the triphenyl phosphonium cation or the derivative cation thereof are converted into NHS active ester to be connected with the amino of the dendritic or linear molecule, and the carboxyl and the amino are complexed with superparamagnetic metal ions after the protecting group is removed to obtain the contrast agent molecule targeting mitochondria. The dendritic or linear molecule may provide for the attachment of amino groups, thiol groups, hydroxyl groups, etc., and may also provide for the attachment of carboxyl groups; accordingly, the superparamagnetic metal complex and the triphenylphosphonium or the derivative cation thereof may be linked to the dendritic or linear molecule through a carboxyl group, or may be linked to the dendritic or linear molecule through an amino group, a mercapto group, a hydroxyl group, or the like; or click chemistry azide-alkynyl cycloaddition.

In one embodiment of the invention, Gd-DOTA is used as a contrast unit, a targeting unit triphenyl phosphonium molecule and Gd-DOTA are connected with a dendritic or linear molecule through carboxyl, a structural unit of the dendritic or linear molecule adopts lysine, and the structure of the synthesized triphenyl phosphonium magnetic resonance targeted mitochondrial contrast agent molecule is shown in figure 5. The number k of structural units of the dendritic or linear molecule can be an integer of 0-7; the number of contrast units DOTA is m + n ═ k +1, the linker is lysine, and the spacer molecular structure adopts NH₂(CH₂)_pCOOH, wherein p is an integer of 0 to 12.

In one embodiment of the invention, Gd-DOTA is used as a contrast unit, triphenyl phosphonium molecules and Gd-DOTA of a targeting unit are connected with two amino groups of lysine through carboxyl, and the structure of the synthesized triphenyl phosphonium targeted mitochondrial contrast agent molecules is shown in figure 6 and corresponds to probe molecules Gd-DOTA-TPP.

In one embodiment of the invention, Gd-DOTA is used as a contrast unit, the targeting units triphenyl phosphonium molecule and Gd-DOTA are both connected with the dendritic molecule through carboxyl, lysine is adopted as the structural unit of the dendritic molecule, when the number of the structural units is k-3, n-2 and m-2, the linker is lysine, the length of one spacer is 0, and the structure of the other spacer is NH₂(CH₂)₅COOH, the structure of the synthesized triphenylphosphonium mitochondrion-targeted contrast agent molecule is shown in FIG. 7, corresponding to the probe molecule (Gd-DOTA)₄-TPP。

In one embodiment of the invention, Gd-DOTA is used as a contrast unit, the targeting units, namely triphenyl phosphonium molecule and Gd-DOTA, are connected with the dendritic molecule through carboxyl, lysine is adopted as the structural unit of the dendritic molecule, when the number of the structural units is k & lt3 & gt, n & lt2 & gt, and m & lt2 & gt, the linker is aminocaproyl lysine which comprises spacers aminocaproic acid, wherein the length of one spacer is 0, and the structure of the other spacer is NH₂(CH₂)₅COOH, toThe structure of the contrast agent molecule targeting the mitochondria of the formed triphenyl phosphonium is shown in figure 8, and the contrast agent molecule corresponds to a probe molecule (Gd-DOTA)₄-linker-TPP。

In one embodiment of the invention, Gd-DOTA is used as a contrast unit, the targeting units, namely triphenyl phosphonium molecule and Gd-DOTA, are connected with a linear molecule through carboxyl, lysine is adopted as a structural unit of the linear molecule, the number k of the structural units is 3, m is 1, and when n is 3, a linker is aminocaproyl lysine which comprises spacers of aminocaproic acid, wherein the length of one spacer is 0, and the structure of the other spacer is NH₂(CH₂)₅COOH, the structure of the synthesized triphenylphosphonium mitochondrion-targeted contrast agent molecule is shown in FIG. 9, corresponding to the probe molecule (Gd-DOTA)₄-linker-TPP。

In one embodiment of the invention, Dy-DOTA is used as a contrast unit, the targeting units triphenyl phosphonium cation and Dy-DOTA are connected with dendritic molecules through carboxyl, lysine is used as a structural unit of the dendritic molecules, when the number of the structural units is k & lt3 & gt, n & lt2 & gt and m & lt2 & gt, the linker is aminocaproyl lysine containing spacers aminocaproic acid, wherein the length of one spacer is 0, and the structure of the other spacer is NH₂(CH₂)₅COOH, the structure of the synthesized triphenylphosphonium mitochondrion-targeted contrast agent molecule is shown in FIG. 10, which corresponds to a probe molecule (Dy-DOTA)₄-TPP。

In one embodiment of the invention, Gd-DTPA is used as a contrast unit, the targeting units, namely triphenyl phosphonium molecule and Gd-DTPA, are connected with dendritic molecules through carboxyl, lysine is adopted as a structural unit of the dendritic molecules, when the number of the structural units is k & lt3 & gt, n & lt2 & gt, and m & lt2 & gt, the linker is aminocaproyl lysine which comprises spacers aminocaproic acid, wherein the length of one spacer is 0, and the structure of the other spacer is NH₂(CH₂)₅COOH, the structure of the synthesized triphenylphosphonium mitochondrion-targeted contrast agent molecule is shown in FIG. 11, corresponding to probe molecule (Gd-DTPA)₄-linker-TPP。

In one embodiment of the invention, Gd-DOTA is used as the contrast unit and the targeting unit is III(p-methylphenyl) phosphonium molecule ((p-tolyl)₃P) and Gd-DOTA are both connected with the dendrimer through carboxyl, the structural unit of the dendrimer adopts lysine, the number of the structural units is k & lt3 & gt, n & lt2 & gt, and when m & lt2 & gt, the linker is aminocaproyl lysine, the linker comprises spacer aminocaproic acid, the length of one spacer is 0, and the structure of the other spacer is NH₂(CH₂)₅COOH, the structure of the synthesized triphenylphosphonium mitochondrion-targeted contrast agent molecule is shown in FIG. 12, corresponding to probe molecule (Gd-DOTA)₄-linker- (p-tolyl)₃P。

In one embodiment of the invention, Gd-DOTA is used as a contrast unit, 2 targeting units, namely triphenylphosphonium molecules and Gd-DOTA, are connected with linear molecules through carboxyl, lysine is adopted as a structural unit of the linear molecules, when the number of the structural units is k-3, m-1 and n-3, a linker is aminocaproyl lysine which comprises spacers aminocaproic acid, wherein the length of one spacer is 0, and the structure of the other spacer is NH₂(CH₂)₅COOH, the structure of the synthesized triphenylphosphonium mitochondrion-targeted contrast agent molecule is shown in FIG. 13, corresponding to probe molecule (Gd-DOTA)₄-linker-TPP₂。

In one embodiment of the invention, a cell labeled with the mitochondrial-targeted magnetic resonance contrast agent molecule of the invention is provided, as well as a method for labeling a mesenchymal stem cell with a mitochondrial-targeted magnetic resonance contrast agent molecule bound to the cell's mitochondria by pulsed electroporation.

In one embodiment of the invention, a method is provided for visualizing contrast agent molecularly labeled mesenchymal stem cells bound to the mitochondria of cells by in vitro magnetic resonance imaging, at different time nodes after cell labeling, their T₁Weighted imaging sum T₂The course of the change in imaging contrast is weighted.

In one embodiment of the invention, a method is provided for transplanting mesenchymal stem cells labeled by mitochondria-targeted magnetic resonance contrast agent molecules into mice by site-directed surgical transplantation/injection, and utilizing 11.7T living body magnetic resonance T₂And (4) weighted imaging, namely observing the change process of the image contrast of the cell transplant and the peripheral tissues thereof at different time nodes after the cell transplantation.

In one embodiment of the invention, a method is provided for transplanting mesenchymal stem cells marked by mitochondria-targeted magnetic resonance contrast agent molecules into a rat body by site-directed surgical transplantation/injection, and utilizing 3T living body magnetic resonance T₂And (4) weighted imaging, and observing the image contrast of the cell transplant and the peripheral tissues thereof.

The specific embodiment is as follows:

example 1: synthesis of mitochondrial-targeted contrast molecule Gd-DOTA-TPP (FIG. 6)

1、Bu^t ₃Synthesis of 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) according to the following procedure:

a) 10.0g of Cyclen and 29.3g of NaHCO are weighed out₃Put into a 1L three-necked flask, and 50mL of acetonitrile was added. 37.4g of tert-butyl bromoacetate was weighed in a fume hood, added with 20mL of acetonitrile, mixed well and placed in a dropping funnel. In an ice bath and N₂Under protection, a solution of tert-butyl bromoacetate in acetonitrile was slowly added dropwise to the reaction mixture in a three-necked flask. After the addition was complete, stirring was continued at room temperature for 30 hours. The solid was filtered off, acetonitrile was removed by rotary evaporation, and recrystallization from toluene was carried out 2 times to give 16g of Bu as a white solid^t ₃DO3A (1,4, 7-tris (tert-butoxycarbonylmethyl) -1,4,7, 10-tetraazacyclododecane).

b) Weighing 1.38g K₂CO₃And 2.57g Bu^t ₃DO3A was placed in a 250mL three-necked flask and 50mL acetonitrile was added under N₂Under protection and stirring at room temperature, 1.0g of ethyl bromoacetate in 5mL of acetonitrile is added dropwise, and the temperature is raised to 70 ℃ for reaction for 12-24 hours. Cooled to room temperature, filtered and the solvent removed by rotary evaporation. By CH₂Cl₂Column chromatography with/MeOH (20:1) as developing solvent gave 2.5g of 1,4, 7-tris (tert-butoxycarbonylmethyl) -10- (ethoxycarbonylmethyl) 1,4,7, 10-tetraazacyclododecane (Bu) as a pale yellow viscous foam product^t ₃Et-DOTA)。

c) 1.5g of Bu are weighed^t ₃Et-DOTA into a 250mL three-necked flask, 50mL dioxane was added to dissolve, N₂Under protection, 25mL of 1.2M NaOH aqueous solution was added, and the mixture was stirred at 50-70 ℃ for 4 hours. The dioxane was removed by rotary evaporation, extracted three times with dichloromethane (25 mL each), and the combined extracts were dried over anhydrous sodium sulfate. Removing the solvent by rotary evaporation, using CH₂Cl₂The column chromatography separation is carried out by taking/MeOH (20:1) as a developing agent to obtain 1.0g of a light yellow viscous foam-like product Bu^t ₃DOTA。

2、Ph₃P(Br)(CH₂)₄COOH [ bromide (4-carboxybutyltriphenylphosphonium)]Synthesis of (2)

7.42g of triphenylphosphonium and 5.01g of 5-bromovaleric acid were weighed into a 100mL round-bottomed flask, and 35mL of xylene was added and dissolved with stirring. Heated to 140 ℃ and condensed at reflux for 4 hours. Cooling to 40-50 deg.c, and dropping 30mL dimethyl ether via separating funnel while stirring to obtain white crystal. The white crystals were filtered off with suction and washed twice with a small amount of diethyl ether to give the product, (4-carboxybutyltriphenylphosphonium) bromide, 8.51 g, in 68% yield.

3. Synthesis of DOTA-TPP by solid phase synthesis:

the steps are briefly described as follows: the synthesis was carried out by the conventional Fmoc (fluorenylmethyl chloroformate) method on a solid phase synthesizer, and the amino acids were coupled in sequence from the C-terminus to the N-terminus according to the structure shown in FIG. 6. First, 1g of solid phase carrier 2-chlorotrityl resin was sequentially added with 2.0g of Fmoc-Lys (Mtt) -OH, 1.77g of Ph₃P(Br)(CH₂)₄COOH is condensed, the condensation condition of carboxyl and amino in each step is that 50mL of DMF is taken as a solvent, 0.96g of TBTU, 0.41g of HOBt condensing agent and 2.5mL of alkali DIPEA are added, the reaction is carried out for about 24 hours at 25 ℃, and whether the condensation of carboxyl and amino is finished or not is judged according to the color development of ninhydrin in specific time; after the condensation has ended, the solvent is drained off and washed three times with 50mL of methanol, DMF, methanol and dichloromethane respectively. Adding Ph₃P(Br)(CH₂)₄Conditions for Fmoc removal before COOH: 25mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, which was then drained, and 25mL of 20% piperidine/DMF was added and reacted for 0.5 hour, which was drained, and the reaction solution was then washed with water and driedWashed three times with each of DMF, methanol, dichloromethane.

Condensed Ph₃P(Br)(CH₂)₄After COOH, the protecting group Mtt was removed by adding 50mL of 1% TFA/dichloromethane and suction dried. Washed three times with DMF, methanol, dichloromethane, respectively. 1.72g of Bu were added^t ₃DOTA, 50mL of DMF solvent, 0.96g of TBTU, 0.41g of HOBt condensing agent and 2.5mL of base DIPEA were reacted at 25 ℃ and after the end of the condensation of the carboxyl and amino groups, as judged by ninhydrin color development, the solvent was drained and washed three times with 50mL of methanol, DMF, methanol and dichloromethane, respectively. 50mL of 50% TFA/dichloromethane was added and the reaction was carried out at 25 ℃ for 40 minutes. Filtering, taking the filtrate, adjusting the pH of the filtrate to be neutral by triethylamine, and concentrating to be dry. Adding ether to separate out white solid to obtain crude product.

The crude product was further purified by HPLC using Waters 2535-2707-2998-WFC, Xbridge Pre C185 μm19 × 150mm as mobile phase solvent A (0.1% TFA, water), solvent B (0.1% TFA, CH) as mobile phase₃CN), solvent a decreased from 50% to 25% in 25 minutes; the flow rate was 10 mL/min. About 200mg of DOTA-TPP product is obtained, and the purity is more than 95%.

4. Deprotected DOTA-TPP with Gd³⁺Complexing to obtain Gd-DOTA-TPP targeted mitochondrial contrast agent molecules. The method comprises the following specific steps: will contain 35.7mg of GdCl₃·6H₂Approximately 1.0mL of aqueous O solution was added dropwise to 100mg of the above aqueous DOTA-TPP solution and mixed for 3 hours, carefully adjusted to pH 6 with 1.0M ammonia, the static mixer was rotated overnight at room temperature, then carefully adjusted to pH 7-8 with 1.0M ammonia and 1.0M hydrochloric acid, and the clear aqueous solution was freeze-dried to give white powder Gd-DOTA-TPP mitochondrial targeting contrast agent molecules₃CN, 20% water), solvent B rose from 22% to 42% in 20 minutes; the flow rate was 1.0 mL/min. The purity is more than 95 percent.

Example 2: (Gd-DOTA)₄Synthesis of TPP-Mitochondrially Targeted contrast agent molecule (FIG. 7)

1. Bu Synthesis according to example 1^t ₃DOTA and Ph₃P(Br)(CH₂)₄COOH。

2. Synthesis of DOTA by solid phase Synthesis₄-TPP: the steps are briefly described as follows: the amino acids were coupled sequentially from C-terminus to N-terminus according to the structure shown in FIG. 7, as synthesized by the conventional Fmoc method on a solid phase synthesizer. First, 1g of solid-phase carrier 2-chlorotrityl resin was sequentially added with 2.0g of Fmoc-Lys (Mtt) -OH and 1.77g of Ph₃P(Br)(CH₂)₄COOH is condensed, the condensation condition of carboxyl and amino in each step is that 50mL of DMF is taken as a solvent, 0.96g of TBTU, 0.41g of HOBt condensing agent and 2.5mL of alkali DIPEA are added, the reaction is carried out for about 24 hours at 25 ℃, and whether the condensation of carboxyl and amino is finished or not is judged according to the color development of ninhydrin in specific time; after the condensation has ended, the solvent is drained off and washed three times with 50mL of methanol, DMF, methanol and dichloromethane respectively. Adding Ph₃P(Br)(CH₂)₄Conditions for Fmoc removal before COOH: 25mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, which was then dried by suction, and 25mL of 20% piperidine/DMF was added and reacted for 0.5 hour, which was then dried by suction and washed with DMF, methanol and dichloromethane three times, respectively.

Condensed Ph₃P(Br)(CH₂)₄After COOH, 50mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt from the lysine side chain, and the solvent was drained. Washed three times with DMF, methanol, dichloromethane, respectively. Fmoc-protected amino acids were then added sequentially for condensation (2.0g Fmoc-Lys (Fmoc) -OH, 4.0g Fmoc-Lys (Mtt) -OH, 2.12g Fmoc-Acp-OH). The condensation condition of carboxyl and amino in each step is that 50mL of DMF is taken as a solvent, 1.92g of TBTU, 0.82g of HOBt condensing agent and 5mL of alkali DIPEA are added, the reaction is carried out for about 24 hours at 25 ℃, and whether the condensation of carboxyl and amino is finished or not is judged according to the color development of ninhydrin in specific time; after the condensation has ended, the solvent is drained off and washed three times with 50mL of methanol, DMF, methanol and dichloromethane respectively. Conditions for removal of the previous Fmoc before addition of the next Fmoc-protected amino acid: 25mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, which was then dried by suction, and 25mL of 20% piperidine/DMF was added and reacted for 0.5 hour, which was then dried by suction and washed with DMF, methanol and dichloromethane three times, respectively.

Fmoc-Acp-OH is finally condensed and eliminatedAfter Fmoc, the protecting group Mtt was removed by addition of 100mL of 1% TFA/dichloromethane and drained. Washed three times with DMF, methanol, dichloromethane, respectively. 7g of Bu were added^t ₃DOTA, 200mL of DMF solvent, 4g of TBTU, 1.64g of HOBt condensing agent and 10mL of base DIPEA were reacted at 25 ℃, and after the condensation of carboxyl and amino groups was completed as judged by ninhydrin color development, the solvent was drained and washed three times with 50mL of methanol, DMF, methanol and methylene chloride, respectively. 50mL of 50% TFA/dichloromethane was added and the reaction was carried out at 25 ℃ for 40 minutes. Filtering, taking the filtrate, adjusting the pH of the filtrate to be neutral by triethylamine, and concentrating to be dry. Adding ether to separate out white solid to obtain crude product.

The crude product was further purified by HPLC using Waters 2535-2707-2998-WFC, Xbridge Pre C185 μm19 × 150mm as mobile phase solvent A (0.1% TFA, water), solvent B (0.1% TFA, CH) as mobile phase₃CN), solvent a15 decreased from 80% to 65% in minutes; the flow rate was 10 mL/min. About 200mg of product (DOTA) is obtained₄TPP, purity above 95%.

3. Deprotected DOTA₄TPP with Gd³⁺Complexing to obtain (Gd-DOTA)₄TPP targeting mitochondrial contrast agent molecules. The method comprises the following specific steps: will contain 12.8mg of GdCl₃·6H₂About 0.5mL of an aqueous solution of O was added dropwise to 100mg of DOTA above₄Mixing TPP in water for 3 hours, carefully adjusting pH to about 6 with 1.0M ammonia, rotating the static mixer overnight at room temperature, carefully adjusting pH to 7-8 with 1.0M ammonia and 1.0M hydrochloric acid, and freeze-drying the clear water solution to obtain white powder (Gd-DOTA)₄TPP targeting mitochondrial contrast agent molecules. Detection by HPLC (Gd-DOTA)₄HPLC conditions, Waters 2535-2707-2998, Sapphire C185 μm4.6 × 250mm, mobile phase solvent A (0.1% TFA, water), solvent B (0.1% TFA, 80% CH)₃CN, 20% water), solvent B rose from 24% to 44% in 20 minutes; the flow rate was 1.0 mL/min. The purity is more than 95 percent.

Example 3: (Gd-DOTA)₄Synthesis of linker-TPP mitochondrially targeted contrast agent molecule (where spacer is Acp) (FIG. 8, dendrimer)

1. Respective Bu was synthesized according to the method of example 1^t ₃DOTA and Ph₃P(Br)(CH₂)₄COOH。

2. Synthesis of DOTA by solid phase Synthesis₄-linker-TPP：

The steps are briefly described as follows: the amino acids were coupled sequentially from C-terminus to N-terminus according to the structure shown in FIG. 8, as synthesized by the conventional Fmoc method on a solid phase synthesizer. First, 1g of solid-phase carrier 2-chlorotrityl resin was sequentially added with 2.0g of Fmoc-Lys (Mtt) -OH and 1.77g of Ph₃P(Br)(CH₂)₄COOH is condensed, the condensation condition of carboxyl and amino in each step is that 50mL of DMF is taken as a solvent, 0.96g of TBTU, 0.41g of HOBt condensing agent and 2.5mL of alkali DIPEA are added, the reaction is carried out for about 24 hours at 25 ℃, and whether the condensation of carboxyl and amino is finished or not is judged according to the color development of ninhydrin in specific time; after the condensation has ended, the solvent is drained off and washed three times with 50mL of methanol, DMF, methanol and dichloromethane respectively. Adding Ph₃P(Br)(CH₂)₄Conditions for Fmoc removal before COOH: 25mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, which was then dried by suction, and 25mL of 20% piperidine/DMF was added and reacted for 0.5 hour, which was then dried by suction and washed with DMF, methanol and dichloromethane three times, respectively.

Condensed Ph₃P(Br)(CH₂)₄After COOH, 50mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt from the lysine side chain, and the solvent was drained. Washed three times with DMF, methanol, dichloromethane, respectively. Fmoc protected amino acids were then added sequentially for condensation (1.06g Fmoc- -Acp-OH, 2.0g Fmoc-Lys (Fmoc) - -OH, 4.0g Fmoc-Lys (Mtt) - -OH, 2.12g Fmoc- -Acp-OH). The condensation condition of carboxyl and amino in each step is that 50mL of DMF is taken as a solvent, 1.92g of TBTU, 0.82g of HOBt condensing agent and 5mL of alkali DIPEA are added, the reaction is carried out for about 24 hours at 25 ℃, and whether the condensation of carboxyl and amino is finished or not is judged according to the color development of ninhydrin in specific time; after the condensation has ended, the solvent is drained off and washed three times with 50mL of methanol, DMF, methanol and dichloromethane respectively. Conditions for removal of the previous Fmoc before addition of the next Fmoc-protected amino acid: 25mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, which was then dried by suction, and 25mL of 20% piperidine/DMF was added and reacted for 0.5 hour, which was then dried by suction and washed with DMF, methanol and dichloromethane three times, respectively.

After final condensation of Fmoc- -Acp- -OH and removal of Fmoc, the protecting group Mtt was removed by addition of 100mL of 1% TFA/dichloromethane and drained. Washed three times with DMF, methanol, dichloromethane, respectively. 7g of Bu were added^t ₃DOTA, 200mL of DMF solvent, 4g of TBTU, 1.64g of HOBt condensing agent and 10mL of base DIPEA were reacted at 25 ℃, and after the condensation of carboxyl and amino groups was completed as judged by ninhydrin color development, the solvent was drained and washed three times with 50mL of methanol, DMF, methanol and methylene chloride, respectively. 50mL of 50% TFA/dichloromethane was added and the reaction was carried out at 25 ℃ for 40 minutes. Filtering, taking the filtrate, adjusting the pH of the filtrate to be neutral by triethylamine, and concentrating to be dry. Adding ether to separate out white solid to obtain crude product.

The crude product was further purified by HPLC using Waters 2535-2707-2998-WFC, Xbridge Pre C185 μm19 × 150mm as mobile phase solvent A (0.1% TFA, water), solvent B (0.1% TFA, CH) as mobile phase₃CN), solvent a decreased from 80% to 65% in 15 minutes; the flow rate was 10 mL/min. About 200mg of product (DOTA) is obtained₄linker-TPP with purity of more than 95%.

3. Deprotected DOTA₄-spacer-TPP with Gd³⁺Complexing to obtain (Gd-DOTA)₄linker-TPP mitochondrial-targeted contrast agent molecules. The method comprises the following specific steps: will contain 12.8mg of GdCl₃·6H₂About 0.5mL of an aqueous solution of O was added dropwise to 100mg of DOTA above₄-linker-TPP in water for 3 hours, carefully adjusting the pH to about 6 with 1.0M ammonia, rotating the static mixer overnight at room temperature, carefully adjusting the pH to 7-8 with 1.0M ammonia and 1.0M hydrochloric acid, and freeze-drying the clear aqueous solution to obtain a white powder (Gd-DOTA)₄linker-TPP mitochondrial-targeted contrast agent molecules. Detection by HPLC (Gd-DOTA)₄HPLC conditions, Waters 2535-2707-2998, Sapphire C185 μm4.6 × 250mm, solvent A (0.1% TFA, water), solvent B (0.1% TFA, 80% CH) as mobile phase₃CN, 20% water), solvent B rose from 24% to 44% in 20 minutes; the flow rate was 1.0 mL/min. The purity is more than 95 percent.

Example 4: (Gd-DOTA)₄Synthesis of linker-TPP mitochondrially targeted contrast agent molecule (where spacer is Acp) (FIG. 9, lineType molecule)

1. Bu Synthesis according to the procedure of example 1^t ₃DOTA and Ph₃P(Br)(CH₂)₄COOH。

2. Synthesis of DOTA according to example 3₄linker-TPP method, Synthesis of Linear molecularly bound DOTA by solid phase Synthesis according to the Structure shown in FIG. 9₄-linker-TPP。

3. Deprotected DOTA₄-spacer-TPP with Gd³⁺Complexing to obtain (Gd-DOTA)₄linker-TPP mitochondrial-targeted contrast agent molecules. The method comprises the following specific steps: will contain 12.8mg of GdCl₃·6H₂About 0.5mL of an aqueous solution of O was added dropwise to 100mg of DOTA above₄-linker-TPP in water for 3 hours, carefully adjusting the pH to about 6 with 1.0M ammonia, rotating the static mixer overnight at room temperature, carefully adjusting the pH to 7-8 with 1.0M ammonia and 1.0M hydrochloric acid, and freeze-drying the clear aqueous solution to obtain a white powder (Gd-DOTA)₄linker-TPP mitochondrial-targeted contrast agent molecules. Detection by HPLC (Gd-DOTA)₄HPLC conditions, Waters 2535-2707-2998, Sapphire C185 μm4.6 × 250mm, solvent A (0.1% TFA water), solvent B (0.1% TFA, 80% CH) as mobile phase₃CN, 20% water), solvent B rose from 24% to 44% in 20 minutes; the flow rate was 1.0 mL/min. The purity is more than 95 percent.

Example 5: (Dy-DOTA)₄Synthesis of linker-TPP mitochondrially targeted contrast agent molecules (herein linker ═ Acp) (fig. 10)

2. DOTA Synthesis according to example 3₄-linker-TPP：

3. Deprotected DOTA₄linker-TPP with Dy³⁺Complexing to obtain (Dy-DOTA)₄linker-TPP mitochondrial-targeted contrast agent molecules. The method comprises the following specific steps: will contain 13.0mg of DyCl₃·6H₂About 0.5mL of an aqueous solution of O was added dropwise to 100mg of DOTA above₄-linker-TPP in water for 3 hours, carefully using 1Adjusting pH to about 6 with 0M ammonia water, rotating the static mixer overnight at room temperature, carefully adjusting pH to 7-8 with 1.0M ammonia water and 1.0M hydrochloric acid, and freeze drying the clear aqueous solution to obtain white powder (Dy-DOTA)₄linker-TPP mitochondrial-targeted contrast agent molecules. Detection by HPLC (Dy-DOTA)₄HPLC conditions, Waters 2535-2707-2998, Sapphire C185 μm4.6 × 250mm, solvent A (0.1% TFA, water), solvent B (0.1% TFA, 80% CH) as mobile phase₃CN, 20% water), solvent B rose from 24% to 44% in 20 minutes; the flow rate was 1.0 mL/min. The purity is more than 95 percent.

Example 6: (Gd-DTPA)₄Synthesis of linker-TPP mitochondrially targeted contrast agent molecules (herein linker ═ Acp) (fig. 11)

1. Ph was synthesized according to the method of example 1₃P(Br)(CH₂)₄COOH

2. Synthesis of DTPA by solid phase Synthesis₄-linker-TPP：

The steps are briefly described as follows: the amino acids were synthesized by conventional Fmoc method on a solid phase synthesizer, and coupled in sequence from the C-terminus to the N-terminus according to the structure shown in FIG. 10. First, 1g of solid-phase carrier 2-chlorotrityl resin was sequentially added with 2.0g of Fmoc-Lys (Mtt) -OH and 1.77g of Ph₃P(Br)(CH₂)₄COOH is condensed, the condensation condition of carboxyl and amino in each step is that 50mL of DMF is taken as a solvent, 0.96g of TBTU, 0.41g of HOBt condensing agent and 2.5mL of alkali DIPEA are added, the reaction is carried out for about 24 hours at 25 ℃, and whether the condensation of carboxyl and amino is finished or not is judged according to the color development of ninhydrin in specific time; after the condensation has ended, the solvent is drained off and washed three times with 50mL of methanol, DMF, methanol and dichloromethane respectively. Adding Ph₃P(Br)(CH₂)₄Conditions for Fmoc removal before COOH: 25mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, which was then dried by suction, and 25mL of 20% piperidine/DMF was added and reacted for 0.5 hour, which was then dried by suction and washed with DMF, methanol and dichloromethane three times, respectively.

Condensed Ph₃P(Br)(CH₂)₄After COOH, 50mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt from the lysine side chain, and the solvent was drained. Respectively using DMF, methanol and dichloroThe methane was washed three times each. Fmoc protected amino acids were then added sequentially for condensation (1.06g Fmoc- -Acp-OH, 2.0g Fmoc-Lys (Fmoc) - -OH, 4.0g Fmoc-Lys (Mtt) - -OH, 2.12g Fmoc- -Acp-OH). The condensation condition of carboxyl and amino in each step is that 50mL of DMF is taken as a solvent, 1.92g of TBTU, 0.82g of HOBt condensing agent and 5mL of alkali DIPEA are added, the reaction is carried out for about 24 hours at 25 ℃, and whether the condensation of carboxyl and amino is finished or not is judged according to the color development of ninhydrin in specific time; after the condensation has ended, the solvent is drained off and washed three times with 50mL of methanol, DMF, methanol and dichloromethane respectively. Conditions for removal of the previous Fmoc before addition of the next Fmoc-protected amino acid: 25mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, which was then dried by suction, and 25mL of 20% piperidine/DMF was added and reacted for 0.5 hour, which was then dried by suction and washed with DMF, methanol and dichloromethane three times, respectively.

After final condensation of Fmoc- -Acp- -OH and removal of Fmoc, the protecting group Mtt was removed by addition of 100mL of 1% TFA/dichloromethane and drained. Washed three times with DMF, methanol, dichloromethane, respectively. 4.0g DTPAA (diethylenetriaminepentaacetic anhydride), 200mL DMF solvent and 10mL base DIPEA were added, reacted at 25 ℃, and after the condensation of carboxyl and amino groups was completed as judged by ninhydrin color development, the solvent was drained and washed three times with 50mL methanol, DMF, methanol and dichloromethane, respectively. 50mL of 50% TFA/dichloromethane was added and the reaction was carried out at 25 ℃ for 40 minutes. Filtering, taking the filtrate, adjusting the pH of the filtrate to be neutral by triethylamine, and concentrating to be dry. Adding ether to separate out white solid to obtain crude product.

The crude product was further purified by HPLC using Waters 2535-2707-2998-WFC, Xbridge Pre C185 μm19 × 150mm as mobile phase solvent A (0.1% TFA, water), solvent B (0.1% TFA, CH) as mobile phase₃CN), solvent a decreased from 80% to 65% in 15 minutes; the flow rate was 10 mL/min. About 200mg of product (DTPA) are obtained₄-spacer-TPP with a purity of more than 95%.

3. Deprotected DTPA₄linker-TPP with Gd³⁺Complexing to obtain (Gd-DTPA)₄linker-TPP mitochondrial-targeted contrast agent molecules. The method comprises the following specific steps: will contain 12.8mg of GdCl₃·6H₂About 0.5mL of an aqueous solution of O was added dropwise to 100mg of the above DTPA₄-linker-TPP in aqueous solutionAfter 3 hours, carefully adjust the pH to about 6 with 1.0M ammonia, rotate the static mixer overnight at room temperature, carefully adjust the pH to 7-8 with 1.0M ammonia and 1.0M hydrochloric acid, freeze-dry the clear aqueous solution to give a white powder (Gd-DTPA)₄linker-TPP mitochondrial-targeted contrast agent molecules. Detection by HPLC (Gd-DTPA)₄HPLC conditions, Waters 2535-2707-2998, Sapphire C185 μm4.6 × 250mm, mobile phase solvent A (0.1%, TFA water), solvent B (0.1% TFA, 80% CH. RTM. water)₃CN, 20% water), solvent B rose from 24% to 44% in 20 minutes; the flow rate was 1.0 mL/min. The purity is more than 95 percent.

Example 7: (Gd-DOTA)₄-linker- (p-tolyl)₃Synthesis of P-targeted mitochondrial contrast agent molecules (herein linker ═ Acp) (fig. 12)

1. Bu Synthesis according to example 1^t ₃DOTA。

2. (p-tolyl)₃P(Br)(CH₂)₄COOH [ 4-carboxybutyltris (p-methylphenyl) phosphonium bromide)]Synthesis of (2)

Synthesis of Ph as in example 1₃P(Br)(CH₂)₄COOH step, 7.42g of triphenylphosphine as a starting material was changed to 8.61g of tri-p-tolylphosphine, and other starting materials and conditions were not changed. The product, (4-carboxybutyltri-p-tolylphosphonium) bromide, was obtained in 8.0 g, 58% yield.

3. Synthesis of DOTA by solid phase Synthesis₄-linker- (p-tolyl)₃P：

Following the synthetic procedure of example 3, starting with 1.77g of Ph₃P(Br)(CH₂)₄COOH was changed to 1.94g (p-tolyl)₃P(Br)(CH₂)₄COOH, other raw materials and conditions and the detection procedure were unchanged.

4. Deprotected DOTA₄-linker- (p-tolyl)₃P and Gd³⁺Complexing to obtain (Gd-DOTA)₄-linker- (p-tolyl)₃P is a mitochondrial-targeted contrast agent molecule. Following the complexation procedure of example 3, a solution containing 12.6mg of GdCl₃·6H₂About 0.5mL of an aqueous solution of O was added dropwise to 100mg of DOTA above₄-linker- (p-tolyl)₃P in an aqueous solution, and other raw materials and conditions and the detection step were not changed.

Example 8: (Gd-DOTA)₄-linker-TPP₂Synthesis of contrast agent molecules targeting mitochondria (FIG. 13, where linker is Lys (Acp) -NH₂The contrast unit Gd-DOTA is linked to the linear polypeptide molecule, the two targeting units TPP are linked to the same end of the linear molecule, the carboxyl of Lys is amidated)

2. Synthesis of Linear molecularly bonded DOTA by solid phase Synthesis according to the Structure shown in FIG. 13₄-linker-TPP₂. In this example, the carboxyl group of the linker lysine was converted to an amide group (the carboxyl group of the linker lysine of all the mitochondrial contrast agent molecules of the present invention can be converted to an amide group and have the same contrast effect).

The steps are briefly described as follows: the amino acids were synthesized by conventional Fmoc method on a solid phase synthesizer, and coupled in sequence from the C-terminus to the N-terminus according to the structure shown in FIG. 10. First, a solid phase carrier is prepared by using a Resin for producing a C-terminal Amide-terminated polypeptide such as RinkAmide AM Resin or Rink Amide MBHA Resin/Knorr Resin 1g, and sequentially adding 2.0g of Fmoc-Lys (Mtt) -OH, 2.0g of Fmoc-Lys (Fmoc) -OH, 3.54g of Ph₃P(Br)(CH₂)₄COOH is condensed. The conditions of the condensation of carboxyl and amino groups in each step, the judgment standard of whether the condensation of carboxyl and amino groups is finished, the post-treatment after the condensation is finished, and the addition of Ph₃P(Br)(CH₂)₄The conditions for Fmoc removal before COOH, etc. were the same as in example 3.

Condensed Ph₃P(Br)(CH₂)₄After COOH, 50mL of 1% TFA/dichloromethane was added to remove the protecting group Mtt from the lysine side chain, and the solvent was drained. Washed three times with DMF, methanol, dichloromethane, respectively. Then 1.06g of Fmoc-Acp-OH, 2.0g of Mtt-Lys (Fmoc) -OH,1.72g of Bu were added in that order^t ₃DOTA condensation was carried out. The condensation of the carboxyl and amino groups in each step being carried out under the conditionsAdding 1.92g of TBTU, 0.82g of HOBt condensing agent and 5mL of alkali DIPEA into 50mL of DMF as a solvent, and reacting at 25 ℃ for about 24 hours, wherein the specific time is based on ninhydrin color development to judge whether the condensation of carboxyl and amino is finished; after the condensation has ended, the solvent is drained off and washed three times with 50mL of methanol, DMF, methanol and dichloromethane respectively. The conditions for removing the last Fmoc prior to adding the next molecule for condensation: 25mL of 20% piperidine/DMF was reacted at room temperature for 0.5 hour, which was then dried by suction, and 25mL of 20% piperidine/DMF was added and reacted for 0.5 hour, which was then dried by suction and washed with DMF, methanol and dichloromethane three times, respectively. This completes a linear ligation of contrast element complexing agent DOTA. Repeating the process twice to complete the linear connection of three contrast unit complexing agents DOTA; the linear ligation of the last contrast unit complexing agent DOTA was identical to the procedure described above, but only 1.06g of Fmoc- -Acp- -OH,1.72g of Bu were added to the condensation molecule added^t ₃DOTA was condensed without the addition of Mtt-Lys (Fmoc) -OH molecules.

After all condensation, 50mL of 50% TFA/dichloromethane was added and the reaction was carried out at 25 ℃ for 40 minutes. Filtering, taking the filtrate, adjusting the pH of the filtrate to be neutral by triethylamine, and concentrating to be dry. Adding ether to separate out white solid to obtain crude product.

3. Deprotected DOTA₄-linker-TPP₂With Gd³⁺Complexing to obtain (Gd-DOTA)₄-linker-TPP₂A contrast agent molecule targeted to mitochondria. The method comprises the following specific steps: will contain 12.8mg of GdCl₃·6H₂About 0.5mL of an aqueous solution of O was added dropwise to 100mg of DOTA above₄-linker-TPP₂The aqueous solution of (A) was mixed for 3 hours, the pH value was carefully adjusted to about 6 with 1.0M ammonia, the static mixer was rotated overnight at room temperature, the pH value was carefully adjusted to 7-8 with 1.0M ammonia and 1.0M hydrochloric acid, and the clear aqueous solution was freeze-dried to give a white powder (Gd-DOTA)₄-linker-TPP₂A contrast agent molecule targeted to mitochondria. Detection by HPLC (Gd-DOTA)₄-linker-TPP₂HPLC conditions, Waters 2535-2707-2998, Sapphire C185 μm4.6 × 250mm, mobile phase solvent A (0.1% TFA water), solvent B (0.1% TFA, 80% CH 4.6 mm₃CN, 20% water), solvent B rose from 24% to 44% in 20 minutes; the flow rate was 1.0 mL/min. The purity is more than 95 percent.

Example 9: preparation method of triphenyl phosphonium targeted mitochondrial contrast agent molecule-labeled mesenchymal stem cells

1. Human mesenchymal stem cells (hMSCs) frozen in liquid nitrogen were removed and rapidly thawed in a 37 ℃ water bath. In a clean bench, the frozen stock of thawed cells was removed with a 1mL pipette and placed in a 10mL sterile centrifuge tube, while 2mL of complete medium was added: (Basic culture mediumDMEM-F1280% -90%, Australia fetal calf serum 10% -20%, double antibody 1%), centrifuging for 5 minutes at 1000 rpm, sucking out the culture medium, adding 3mL of complete culture medium in a centrifuge tube, blowing out cell precipitates, taking out cell suspension, placing the cell suspension in a 100 × 20mm culture dish, continuously adding 5mL of complete culture medium, slightly shaking the culture dish to uniformly disperse the cells in the complete culture medium, placing the cell suspension in an incubator at 37 ℃ and 5% carbon dioxide for culture, recovering the cells for the second day, replacing the culture medium, continuously culturing, sucking out the culture medium when the density of adherent cells reaches 80% -90%, slightly washing the culture dish with 2mL of sterile solution, then adding 1mL of PBS and 1mL of trypsin, observing the cell morphology under a microscope in time, sucking out the trypsin after the cells are completely digested, blowing out the cells with the complete culture medium containing 4m, transferring the cells into two culture dishes with a size of 100 × 20mm, and respectively adding 6mL of the complete culture medium to continuously culture the cells for 6-9 generations of experiments.

2. Cell treatment: when the density of adherent cells reaches 80% -90%, gently washing the culture dish full of cells with 2mL of sterile PBS solution, then adding 1mL of PBS and 1mL of trypsin, observing the cell morphology under a microscope in time, and after the cells are completely digested, absorbing the trypsin. The cells were blown up with 4mL of complete medium, the cell suspension was transferred to a 10mL sterile centrifuge tube, centrifuged at 1000 rpm for 5 minutes and the medium was aspirated off to obtain a cell pellet. The same resuspension procedure was followed with 4mL PBS.

3. Electroporation labelling of cells

The triphenylphosphonium-targeted mitochondrial contrast agent molecules prepared by the method are dissolved in physiological saline to prepare concentration series of 1, 2, 5, 10, 20 and 40 mM. 100 mu L of sample solution is placed in a cell sediment (100 mu L of the sample solution contains 100-200 ten thousand cells), the cells are blown off, the blown off cells are placed in a 96-well plate, an electro-transfection instrument with one reaching voltage of 120V and pulse width of 100 mu s is adopted, the electric shock experiment condition is repeated for 6 times at intervals of 1000ms, the contrast agent molecules targeting mitochondria are introduced into cytoplasm, and when a certain electric pulse is applied to carry out the experiment, the cells in the 96-well plate are ensured to be uniformly dispersed in physiological saline instead of being deposited at the bottom of the 96-well plate.

Example 10: in-vitro MRI (magnetic resonance imaging) imaging method for mesenchymal stem cells labeled by triphenyl phosphonium targeted mitochondrial contrast agent molecules

1. Mesenchymal stem cells were labeled according to the method of example 9 using the triphenylphosphonium-targeted mitochondrial contrast agent molecules prepared in examples 1 and 2, half of the cells labeled for each probe concentration were transferred to a 10mL centrifuge tube, and the petri dish was washed with 3mL PBS and likewise transferred to the centrifuge tube. Centrifuge for 5 minutes at 1200 rpm and remove PBS. The cells were transferred to a capillary with an inner diameter of 1.5mm and a closed end using a capillary with an outer diameter of 1.3mm, centrifuged at 1500 rpm for 10 minutes to pack the cells close together at the bottom of the capillary for in vitro MRI imaging.

2. The other half of the cells were cultured until the number of cells doubled, and then half of the cells were packed close to the bottom of the capillary tube for in vitro MRI imaging. The other half of the cells were cultured until the number of cells doubled. This was done until there was no difference in the results of in vitro cellular MRI imaging experiments.

3. Subjecting the cells in the capillary to T in an 11.7T magnetic resonance spectrometer₁Weighted sum T₂And (4) weighted imaging. T is₁The weighted image is a saturation recovery sequence, TE 5.2ms, TR 500ms, FOV 12 × 12mm²96 × 96, 0.8mm layer thickness, 0.2mm layer spacing, 4 times, T₂The weighted image uses multi-slice echoes, TR 3000ms, TE 80ms, 20 echoes, FOV 12 × 12mm²The matrix is 96 × 96, the slice thickness is 0.8mm, the slice spacing is 0.2mm, and the cumulative count is 1.

FIG. 14 shows in vitro T-cell markers obtained by labeling mesenchymal stem cells with triphenylphosphonium-containing mitochondrial-targeting contrast agent molecules of different structures at different probe concentrations, and propagating the labeled cells for different periods of time₁Weighted sum T₂Weighting the MRI image results. The examples also include the use of Gd-DOTA labeled cells without cell binding capacity as a reference contrast for in vitro MRI imaging experiments.

FIG. 15 is an in vitro T-cell population of magnetically labeled cells obtained in this example at various times₁The intensity of the weighted MRI image signal as a function of cell proliferation time. It can be seen that: (1) Gd-DOTA labels T of cells as soon as they are labeled₁The weighted MRI image signal enhancement effect is obvious, and T of magnetically marked cells is marked by triphenylphosphonium-containing mitochondrial-targeted contrast agent molecules₁The weighted MRI image signal enhancement effect is not obvious, even the signal attenuation effect is presented; (2) t of all magnetically labeled cells accompanying the propagation of magnetically labeled cells₁The signal intensity of the weighted MRI images is rapidly recovered (within 1-2 days) to the signal intensity level of the unmarked cells, which indicates that the magnetic resonance T is₁The limitation of long-term tracking of transplanted cell bodies in a weighted mode.

FIG. 16 is an in vitro T-cell population of magnetically labeled cells obtained in this example at various times₂The intensity of the weighted MRI image signal as a function of cell proliferation time. It can be seen that: (1) Gd-DOTA-labeled magnetically labeled cell T just after the cells were labeled₂The weighted MRI image signal presents obvious enhancement effect, and T of the magnetically marked cells is marked by triphenylphosphonium-containing contrast agent molecules targeting mitochondria₂The weighted MRI image signal presents a remarkable signal attenuation effect, and the signal attenuation degree is more remarkable along with the increase of the probe concentration during cell marking, and even can reach or even be lower than the noise level; (2) accompanied byPropagation of magnetically labeled cells, T of magnetically labeled cells labeled with triphenylphosphonium-containing mitochondrial-targeted contrast agent molecules₂The weighted MRI image signal intensity recovery speed is obviously slower than the T₁The recovery rate of the enhanced effect of the weighted MRI image signal is still at the noise level within about 5 days, and still exhibits a significant contrast difference (dark signal) with the unlabeled cells within about 10 days, requiring about 16 days to reach the signal intensity level of the unlabeled cells, indicating that magnetic resonance T₂Viability of transplanted cell bodies was traced for long periods of time in a weighted mode.

Example 11: 11.7T living body magnetic resonance T in vivo of mesenchymal stem cell transplantation mouse labeled by triphenylphosphonium targeted mitochondrial contrast agent molecules₂Weighted image

1. Following the procedure of example 9, using (Gd-DOTA)₄TPP mitochondrial targeted contrast agent molecules label mesenchymal stem cells by a pulsed electroporation method, with a mitochondrial targeted contrast agent molecule concentration of 20 mM;

2. about 3 × 10⁵Magnetically labeled cells are transplanted into the intracranial of mice by a site-specific injection method, and 11.7T magnetic resonance T is carried out on the cell transplantation positions at different time points (D0-D10) after the cell transplantation₂Weighted imaging (S is the slice number of the image) using a 38mm diameter birdcage coil, RARE sequence parameters were set such that TE is 7ms, TR is 125, 300, 500, 750, 1000, 1500, 3000, 5000ms, and FOV is 20 × 20mm²The matrix 128 × 128, with a layer thickness of 0.5mm and an average number of 4, is shown in fig. 17, where the transplanted cells are partially located in the brain and exhibit significant dark signals (white arrows indicating positions) for a long period of time (D0-D10), partially located in the ventricle, and rapidly migrating and exhibiting significant dark signals in the ventricle immediately after transplantation (D0), and then (D1-D4) the cell transplant begins to release mitochondrial-targeted contrast agent molecules and causes significant light signals (gray arrows indicating positions) in the peripheral tissues where the cells are locatedThe magnetic resonance mitochondrion-targeted contrast agent molecule and the imaging method provided by the invention have the remarkable characteristic that in practical application, a cell transplant can be clearly distinguished from peripheral tissues.

Example 12: 3T living body magnetic resonance T in vivo of rat transplanted with mesenchymal stem cells labeled by triphenylphosphonium targeted mitochondrial contrast agent molecules₂Weighted image

2. will be about 1 × 10⁷Transplanting the magnetically marked cells into the muscle of the foreleg of the mouse by a fixed-point injection method, and carrying out 3T magnetic resonance T on the transplanting position of the magnetically marked cells after the magnetically marked cells are transplanted₂The weighted imaging results show that the obtained image effect is shown in fig. 18, and the cell transplant presents a significant dark signal, which shows that the mitochondrial targeting contrast agent molecule, the magnetic labeling cell labeled by the mitochondrial targeting contrast agent molecule and the magnetic resonance image living body tracing method provided by the invention have application feasibility on clinical imaging equipment.

Example 13: in vitro MRI (magnetic resonance imaging) image of mesenchymal stem cells marked by contrast agent molecules of bi-triphenyl phosphonium targeted mitochondria

Using the bis-triphenylphosphonium-targeted mitochondrial contrast agent molecule prepared in example 8, mesenchymal stem cells were labeled according to the method of example 9, and in vitro T-labeling of cells was performed according to the method of example 10₂The MRI images are weighted.

FIG. 19 is an in vitro T-cell population of magnetically labeled cells obtained in this example at various times₂Comparison of the intensity of weighted MRI image signals as a function of cell proliferation time with some of the results in example 10. It can be seen that: magnetically labeling T of cells via molecular labeling of mitochondrially targeted contrast agents containing bis-triphenylphosphonium₂The recovery speed of the weighted MRI image signal intensity is obviously slower than that of the magnetic labeled cells labeled by the triphenylphosphonium-containing mitochondrion-targeted contrast agent molecules, which shows that the weighted MRI image signal intensity is in magnetic resonance T₂Can be longer in the weighting modeThe transplanted cell bodies are traced in vivo over a time frame.

FIG. 20 is an in vitro T-cell population of magnetically labeled cells obtained in this example at various times₂The relationship between the intensity of the weighted MRI image signal and the Gd content change in the cells indicates that the magnetic labeled cells are required to be subjected to T₂The minimum cellular Gd content required to weight the MRI image signal intensity down to noise levels can be as low as 5 × 10⁹Gd/cell.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. Preparation of T-shaped contrast agent molecule targeting mitochondria₂Use in an agent for a contrast agent, the molecule of the contrast agent targeting mitochondria comprising a targeting unit and a contrast unit, wherein,

the targeting unit is triphenyl phosphonium cation or one or more C thereof_6-10Aryl-substituted triphenyl phosphonium cations of (a);

the targeting unit is connected with a dendritic or linear molecule through a linker, the dendritic or linear molecule is connected with the contrast unit through a spacer, and the structural unit of the dendritic or linear molecule is any monomer which can be homopolymerized or copolymerized to form a dendritic or linear macromolecule; the linker is a linear amino acid; the spacer is a linear amino acid;

the contrast unit is a superparamagnetic metal complex; the superparamagnetic metal complex is formed from a superparamagnetic metal and a complexing agent, wherein: the superparamagnetic metal is Gd; the complexing agent is selected from DOTA, HP-DO3A, DO3A-butrol, DTPA-BMA, DTPA, DTPA-BMEA, BOPTA, EOB-DTPA and any combination thereof.

2. Use according to claim 1, the building blocks of said dendritic or linear molecule being amino acids.

3. Use according to claim 2, the building block of said dendritic or linear molecule being lysine.

4. The use of claim 1, wherein the linker is lysine; the spacer is NH₂(CH₂)_pCOOH or NH₂(CH₂CH₂O)_qCH₂COOH, wherein p is an integer of 0 to 12, q is an integer of 0 to 4, and represents no spacer when p =0 or q = 0.

5. Use according to any one of claims 2 to 4, wherein said dendritic or linear molecule is linked to 1 to 2 targeting units and 1 to 8 contrast units.

6. Use according to claim 5, said dendritic or linear molecule being linked to 2 targeting units and 4 contrast units.

7. Use according to any of claims 2 to 4, wherein the dendritic or linear molecule is linked to 2 targeting units, said targeting units being located adjacent to each other at the same end of the linear molecule.

8. Use according to any of claims 2 to 4, wherein the dendritic or linear molecule is linked to 2 targeting units, wherein the binding sites of the targeting units are not adjacent and are located at either end of the linear molecule.

9. A contrast agent molecule targeted to mitochondria comprising a targeting unit and a contrast unit, wherein:

the contrast unit is a superparamagnetic metal complex; the superparamagnetic metal complex is formed from a superparamagnetic metal and a complexing agent, wherein: the superparamagnetic metal is Gd; the complexing agent is selected from DOTA, HP-DO3A, DO3A-butrol, DTPA-BMA, DTPA, DTPA-BMEA, BOPTA, EOB-DTPA and any combination thereof;

the targeting unit is connected with a dendritic or linear molecule through a linker, the dendritic or linear molecule is connected with the contrast unit through a spacer, and the structural unit of the dendritic or linear molecule is any monomer which can be homopolymerized or copolymerized to form a dendritic or linear macromolecule; the linker is a linear amino acid; the spacer is a linear amino acid.

10. The mitochondrially targeted contrast agent molecule according to claim 9, wherein the structural units of the dendritic or linear molecule are amino acids.

11. The mitochondrion-targeted contrast agent molecule of claim 10, wherein the structural unit of the dendritic or linear molecule is lysine.

12. The mitochondrion-targeted contrast agent molecule of claim 9, wherein the linker is lysine; the spacer is NH₂(CH₂)_pCOOH or NH₂(CH₂CH₂O)_qCH₂COOH, wherein p is an integer of 0 to 12, q is an integer of 0 to 4, and represents no spacer when p =0 or q = 0.

13. The mitochondrial targeting contrast agent molecule of any of claims 9-12, wherein the dendritic or linear molecule is linked to 1-2 targeting units and 1-8 contrast units.

14. The mitochondrion-targeted contrast agent molecule of claim 13, wherein the dendritic or linear molecule is linked to 2 targeting units and 4 contrast units.

15. The mitochondrion-targeted contrast agent molecule of claim 14 where the dendritic or linear molecule is linked to 2 targeting units that bind adjacent to each other at the same end of the linear molecule.

16. The mitochondrion-targeted contrast agent molecule of claim 14, wherein when the dendritic or linear molecule is linked to 2 targeting units, the binding sites of the targeting units are not adjacent and are located at the two ends of the linear molecule.

17. A method of making the mitochondrial-targeted contrast agent molecule of any of claims 9-16, comprising:

each of said triphenylphosphonium cations or salts thereof having one or more C_6-10With a halogen-substituted carboxylic acid or a halogen-substituted amine to form a triphenyl phosphonium cation having carboxyl or amino functionality or via one or more C_6-10Aryl-substituted triphenylphosphonium cation of (a), the triphenylphosphonium cation or a salt thereof having one or more C groups_6-10The aryl-substituted triphenylphosphonium cation of (a) is linked to a dendritic or linear molecule through the carboxyl or amino group obtained; the halogenated carboxylic acid is chloro, bromo or iodo fatty acid or aromatic acid;

the superparamagnetic metal complex is linked to the dendritic or linear molecule through a carboxyl or amino group thereof; the carboxyl of the superparamagnetic metal complex is selected from an ethylcarboxyl group, a propylcarboxyl group or a butylcarboxyl group, and the amino of the superparamagnetic metal complex is selected from an ethylamino group, a propylamino group or a butylamino group.

18. A magnetically labeled cell labeled with the mitochondrial targeted contrast agent molecule according to any one of claims 9-16, which is any cell labeled with the mitochondrial targeted contrast agent molecule that can be used for cell transplantation therapy, selected from the group consisting of mesenchymal stem cells, neural stem cells, cardiac stem cells, embryonic stem cells, induced pluripotent stem cells.

19. A combination of magnetically labeled cells as claimed in claim 18 and a scaffold material, wherein the scaffold material is any medical material capable of forming a combination with cells, selected from collagen, various synthetic polymers or inorganic scaffold materials; the scaffold material may or may not contain various trophic factors that support cell survival and growth.