CN106890345A

CN106890345A - A kind of contrast agent molecule of targetted mitochondria is used as T2The purposes of contrast agent

Info

Publication number: CN106890345A
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: 2017-06-27
Anticipated expiration: 2035-12-17
Also published as: CN106890345B; WO2017101717A1

Abstract

The invention discloses a kind of contrast agent molecule of targetted mitochondria as T₂The purposes of contrast agent, the contrast agent molecule of the targetted mitochondria is included for combination cell mitochondrial with-P⁺(X₁)(X₂)(X₃) general structure phosphonium cations and the radiography unit superparamagnetic metals complex compound for strengthening magnetic resonance imaging contrast's degree.The invention also discloses a kind of contrast agent molecule of targetted mitochondria and preparation method thereof, wherein one or more-P⁺(X₁)(X₂)(X₃) cation passes through dendritic or linear molecule combines 1~8 superparamagnetic metals complex molecules, while using connexon and introns improvement-P⁺(X₁)(X₂)(X₃) space structure between cation and dendritic or linear molecule and dendritic or linear molecule and superparamagnetic metals complex compound.The present invention further discloses the Magnetic labeled cells that a kind of contrast agent molecule with the targetted mitochondria is marked, and Magnetic labeled cells and timbering material combination, and nuclear magnetic resonance image vivo tracking method is carried out with them.

Description

A kind of contrast agent molecule of targetted mitochondria is used as T2The purposes of contrast agent

Technical field

The present invention relates to Medical Imaging, more particularly to a kind of contrast agent molecule of targetted mitochondria is used as magnetic Resonance T₂The purposes of contrast agent, the invention further relates to a kind of contrast agent molecule of targetted mitochondria, is marked with it Magnetic labeled cells, and Magnetic labeled cells and timbering material combination, and carry out magnetic resonance shadow with them As (Magnetic Resonance Imaging, MRI) vivo tracking method.

Background technology

Stem cell regenerating medical treatment is an emerging biomedical sector, and its basic ideas is by inducing transplantation Internal stem cell directional differentiation, realizes to damaged tissues and the Regeneration and Repair of organ.In stem cell regenerating doctor During treatment, it is necessary to survival after in real-time tracking stem cell transplantation body, migrate and go back to the nest, directed differentiation etc. Physiological behavior, carries out accurate tissue biological's credit cloth spike to stem cell, and exogenous stem cells in distinguishing, The functioning cell that the stem cell and differentiation that self-renewing is produced produce, so that deeply understanding stem cell is in vivo The physiology courses such as migration, breeding, division and differentiation, and this is for stem cell biology basic research, and The assessment of observation of curative effect and functional rehabilitation is clinically carried out, is all had very important significance.

Nuclear magnetic resonance image is used as with high spatial resolution and soft tissue contrast and without the non-of ionising radiation risk Live body image technology is damaged, is that the most potential technology with atomization is maintained in tracking stem cell body.Magnetic Resonance image is the ordered arrangement process in uniform magnetic field of the spin magnetic moment based on different Water Proton protons After the magnetic moment of formation is subject to specific microwave-excitation, its longitudinal relaxation speed (1/T₁) or transverse relaxation rate (1/T₂) difference is there may be, cause the different contrast difference realities formed in image of signal intensity of echo The 26S Proteasome Structure and Function imaging of cell, tissue and organ now to organism.In practical application, working as different groups When the image contrast for knitting is close, particular organization's such as tumour can also be changed by introducing magnetic resonance contrast agent The relaxation rate of the water proton in tissue, realizes the imaging to the particular organization.Using resonance imaging techniques Vivo tracking stem cell is also required to carry out magnetic marker to stem cell first, so as to by them from other around it Made a distinction in histocyte.

Being introduced into for magnetic resonance contrast agent would generally be while accelerate the longitudinal relaxation speed of water proton in its residing tissue And transverse relaxation rate, but different magnetic resonance contrast agent accelerate two relative amplitudes of relaxation rate exist compared with Big difference.This species diversity causes the magnetic resonance contrast agent having to be suitable for MRI signal to be strengthened, referred to as T₁ Contrast agent, the MRI signal that is suitable for having weakens, referred to as T₂Contrast agent.For example, it is generally the case that gadolinium The acceleration effect that complex compound compares its transverse relaxation rate to the acceleration effect of water proton longitudinal relaxation speed shows Write, be conducive in T₁Weighting produces bright signal as lower, strengthens T₁The contrast of picture is weighted, therefore often by conduct T₁Contrast agent is used.Acceleration of superparamagnetic iron oxide (SPIO) nano-particle to water proton transverse relaxation rate Effect compares the acceleration effect of its longitudinal relaxation speed significantly much, in T₂Dark letter is produced under weighted imaging pattern Number, strengthen T₂The contrast of picture is weighted, is preferable T₂Contrast agent.In addition, identical contrast agent distribution exists On different bioelectric interfaces, its relative amplitude for accelerating two relaxation rates also likely to be present larger difference. As gadolinium complex is distributed in cytoplasm with free state or form of vesicles or is combined with mitochondria in cytoplasm When, there is significant difference in the influence of its vertical and horizontal relaxation rate to cell water proton.

T with SPIO nano-particles as representative₂Contrast agent has transverse relaxation rate very high, therefore, dry Extensive research and application have been obtained in cytokines image.But this cell mark based on SPIO nano-particles SPIO nano-particles migration information in vivo is provided on note technological essence, this migration is received with carrying The mother cell of rice corpuscles occurs, or the nano-particle dissociated after mother cell death is migrated in itself, or be by The migration occurred with macrophage after other cells such as macrophage phagocytic, present image method cannot be given Clear and definite conclusion, as the significant challenge that image information analysis faces.And, this cell marking cannot be straight Tell that the cell of people's mark is still living cells after entering in vivo with seeing, or dead or part is dead Die.

Gadolinium complex is used as T₁Contrast agent has been obtained for being widely applied in clinical medicine, clinical at present to answer Gadolinium complex contrast agent can be divided into two classes：One class is the DOTA and its derivative for having cyclic structure, One class is DTPA and its derivative without ring structure.This small molecule contrast preparation is clearly steady due to chemical constitution It is fixed, can precise control and targeted molecular coupling chemically process and result, therefore as targeting The construction unit of contrast agent has reliability very high.But, the key that gadolinium complex contrast agent faces Problem is that its relaxation rate is far below T₂The relaxation rate of type SPIO nano-particles, therefore in order to obtain enough groups Contrast is knitted, it is necessary to larger dosage, causes to bring metal gadolinium ion in vivo toxicity etc. to ask safely The concern of topic.

It is also an optional technical side to carry out live body nuclear magnetic resonance image spike using gadolinium complex labeled stem cells Case.But in existing method, made using the targeting of small molecule gadolinium complex or Cell-oriented membrane-bound receptor In addition to longitudinal relaxation rate is relatively low, it is also one that the time that it is detained in cell is too short to the problem that shadow agent runs into The problem that needs overcome；During using macromolecular or nanometer particle load gadolinium complex, will run into again and be received with SPIO The same problem of rice corpuscles, including removing speed is slow in vivo and may be caused to image by other cellular uptakes The interference of result.

Patent US20090214437A1 and US20130142735A1 disclose a kind of energy and cell mitochondrial With reference to magnetic resonance contrast agent, the magnetic resonance contrast agent intravenous administration in vivo after, can be lived in mitochondria It is enriched with the tumor tissues of jump, tumor tissues is strengthened in T using it₁Magnetic resonance signal under weighted imaging pattern (bright signal is presented), so as to improve T₁Weight the contrast of picture.This contrast agent is used as T₁Contrast agent application When the live body image of cell transplantation body, it is impossible to clearly determine the survival in vivo of mark cell and migration shape Condition, the need for signal enhancing effect duration can not meet long-term observation.

In sum, such a is needed badly in the art for cell or the magnetic resonance contrast agent of transplanting body tag, Can not only intuitively provide cell existing state and the cell or transplant in vivo migration, The information such as go back to the nest and break up, moreover it is possible to the need for meeting long-term observation, and can solve the toxicity that heavy dose is brought Problem.

The content of the invention

The present invention is based on it was recognized by the inventor that gadolinium complex is distributed on different bioelectric interfaces, and it is to cell There is significant difference in the influence of the vertical and horizontal relaxation rate of water proton, particularly when gadolinium complex is thin After being combined with mitochondria in kytoplasm, the ability of the longitudinal relaxation speed of its acceleration cell water proton is significantly reduced, It is unfavorable for that magnetic resonance signal strengthens, magnetic resonance signal decrease is advantageous on the contrary, therefore more suitable in T₂Plus Dark signal is produced under power imaging pattern.Inventor by mark cell of the invention it has furthermore been found that be transplanted to body After interior, the mark cell can partly discharge contrast agent molecule, and the contrast agent molecule of release can move cell Implant periphery is organized in magnetic resonance T₂Weight as bright signal is presented under pattern, such that it is able to make cell transplantation body More clearly made a distinction with its perienchyma.

It is an object of the invention to provide a kind of contrast agent molecule of targetted mitochondria as T₂The use of contrast agent On the way, the contrast agent molecule of the targetted mitochondria includes targeting unit and radiography unit, wherein, the targeting Unit is that have-P⁺(X₁)(X₂)(X₃) general structure phosphonium cations, wherein X₁、X₂、X₃Represent without taking Generation or the C replaced through one or more substitution bases_1-12Alkyl, C_1-12Alkenyl or C_6-10Aryl, it is described to take Dai Ji includes 1,2 or 3 halogen atoms, C_1-12Alkyl, C_6-10Aryl, hydroxyl, C_1-12Alkoxy, halogen Generation-C_1-12Alkoxy；Wherein X₁、X₂、X₃Can be identical group, or different groups；Institute It is superparamagnetic metals complex compound to state radiography unit.Line in the contrast agent molecule and cell of the targetted mitochondria Plastochondria is presented significant magnetic resonance signal and weakens effectiveness after combining so that the Magnetic labeled cells are in vitro and body Interior magnetic resonance T₂Weighting under pattern as being all presented dark signal；The targeting unit and multiple magnetic resonance radiography lists Unit combines and during for cell marking, stronger magnetic resonance signal is presented and weakens effectiveness, and when can be lasting longer Between.

In a preferred embodiment of the present invention, it is described targeting unit be triphenyl phosphonium cation or its spread out It is biological.

In a preferred embodiment of the present invention, the superparamagnetic metals complex compound by superparamagnetic metals and Complexing agent is formed, wherein：The superparamagnetic metals are the metals with superparamagnetic characteristic, including but not limited to Lanthanide series metal 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 series metal Chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), yttrium (Y), niobium (Nb) Deng；The complexing agent is selected from DOTA, HP-DO3A, DO3A-butrol, DTPA-BMA, DTPA, DTPA-BMEA, BOPTA, EOB-DTPA or derivatives thereof and its any combination.

In a preferred embodiment of the present invention, the targeting unit is directly or by connexon (linker) It is connected with dendritic or linear molecule, the dendritic or linear molecule are directly or by introns (spacer) Be connected with radiography unit, wherein the construction unit of the dendritic or linear molecule be it is any can homopolymerization or copolymerization Form monomer, more preferably preferred amino acid, the lysine of dendritic or linear macromolecule.

In a preferred embodiment of the present invention, described connexon is straight-chain amino acid, preferably relies Propylhomoserin；The introns are straight-chain amino acid, preferably NH₂(CH₂)_pCOOH or NH₂(CH₂CH₂O)_qCH₂COOH, wherein p are 0~12 integers, and q is 0~4 integer, works as p=0 Or q=0 interval scales do not have introns.

In a preferred embodiment of the present invention, described targeting unit is by the dendritic or line style Molecule is connected with 1-8 radiography unit.- P described in each⁺(X₁)(X₂)(X₃) cation pass through dendritic or line Type molecule is combined with multiple superparamagnetic metals complex compounds, and its magnetic resonance signal decrease effect is more notable, in T₂Plus The time that dark signal is presented when being imaged under power pattern can be with longer.

In a preferred embodiment of the present invention, using multiple targeting unit, preferably 2 targeting units Be connected with 1-8 radiography unit by the dendritic or linear molecule, can increase the contrast agent molecule with The intensity that mitochondria is combined, further extends it in T₂The time of dark signal is presented when being imaged under weighting pattern.

It is another object of the present invention to provide a kind of contrast agent molecule of targetted mitochondria, it includes targeting Unit and radiography unit, wherein：The targeting unit is that have-P⁺(X₁)(X₂)(X₃) general structure Phosphonium sun from Son, wherein X₁、X₂、X₃The C that representative is unsubstituted or replaces through one or more substitution bases_1-12Alkyl, C_1-12Alkenyl or C_6-10Aryl, the substitution base includes 1,2 or 3 halogen atoms, C_1-12Alkyl, C_6-10Aryl, hydroxyl, C_1-12Alkoxy, halo-C_1-12Alkoxy；Wherein X₁、X₂、X₃Can be identical Group, or different groups；The radiography unit is superparamagnetic metals complex compound；The targeting Unit is connected directly or by connexon with dendritic or linear molecule, and the dendritic or linear molecule are direct Or be connected with radiography unit by introns, wherein the construction unit of the dendritic or linear molecule is any Can homopolymerization or copolymerization form monomer, more preferably preferred amino acid, the lysine of dendritic or linear macromolecule.

In a preferred embodiment of the present invention, described connexon is selected from straight-chain amino acid, preferably Lysine；The introns are selected from straight-chain amino acid, preferably NH₂(CH₂)_pCOOH or NH₂(CH₂CH₂O)_qCH₂COOH, wherein p are 0~12 integers, and q is 0~4 integer, works as p=0 Or q=0 interval scales do not have introns.

In a preferred embodiment of the present invention, described targeting unit is by the dendritic or line style Molecule is connected with 1-8 radiography unit.

In a preferred embodiment of the present invention, using multiple targeting unit, preferably 2 targeting units It is connected with 1-8 radiography unit by the dendritic or linear molecule.

The present invention also provides a kind of method of the contrast agent molecule for preparing foregoing targetted mitochondria, and it includes：Often Individual described-P⁺(X₁)(X₂)(X₃) cation and halogenated carboxylic acid or halogenated amine reaction generation have carboxyl or amino - the P of functional group⁺(X₁)(X₂)(X₃) the cation ,-P⁺(X₁)(X₂)(X₃) cation by obtain carboxyl or ammonia Base is connected with dendritic or linear molecule；The halogenated carboxylic acid is chloro, bromo or iodo aliphatic acid or fragrance Acid；The superparamagnetic metals complex compound is connected by its carboxyl or amino with dendritic or linear molecule；It is described The carboxyl of superparamagnetic metals complex compound is selected from second carboxyl, the third carboxyl or fourth carboxyl, the superparamagnetic metals complexing The amino of thing is selected from ethylamino, the third amino or fourth amino.

In the preferred embodiment of the present invention, the contrast agent molecule of targetted mitochondria is by the following method Synthesis：Solid phase synthesis process using cross protection deprotection strategy synthesizes with or without connexon successively Or the dendritic or linear molecule and the radiography unit of with or without introns of introns, then connect -P⁺(X₁)(X₂)(X₃) cation and radiography unit.

In the preferred embodiment of the present invention, will-the P⁺(X₁)(X₂)(X₃) cation, radiography unit And/or a converting carboxylate groups for unit in dendritic or linear molecule into active ester or through after overactivation with The amino of another unit, sulfydryl or hydroxyl are coupled；Or by click chemistry (click chemistry) Connection targeting unit, dendritic or linear molecule and radiography unit.

The present invention also provides the Magnetic labeled cells that a kind of contrast agent molecule with described targetted mitochondria is marked, The Magnetic labeled cells are to can be used for cell transplantation by any of contrast agent molecule mark of targetted mitochondria The cell for the treatment of, selected from mescenchymal stem cell, NSC, Cardiac Stem Cells, embryonic stem cell, lures Lead multipotential stem cell.

The present invention a kind of method of cell marking is also provided, by the contrast agent molecule of targetted mitochondria insert containing In the nutrient solution of the cell, using the endocytosis or pinocytosis function of cell by the contrast agent of the targetted mitochondria Molecule introduces intracellular, and the contrast agent molecule of described targetted mitochondria can be combined with the mitochondria of intracellular, can Effectively to extend its holdup time in the mark cell.

The method that the present invention also provides another cell marking, it is characterised in that：By the radiography of targetted mitochondria Agent molecule is inserted in the nutrient solution containing the cell, electrotransfection buffer solution or physiological saline, using pulse electricity The contrast agent molecule of described targetted mitochondria is introduced intracellular by the method for perforation.

The present invention also provides the combination of a kind of Magnetic labeled cells and timbering material, and the timbering material is Any medical material that combination can be formed with cell, selected from collagen, various synthesis macromolecules or nothing Machine support material；The various trophic factors that the timbering material includes or survived not comprising sertoli cell and grown.

The present invention also provides a kind of nuclear magnetic resonance image vivo tracking method, and it includes：By the Magnetic labeled cells Or the Magnetic labeled cells arrive human or animal with the combination of timbering material by pinpointing operation transplantation/intravenous injection In vivo；Above-mentioned human or animal is placed in nuclear magnetic resonance image equipment, in magnetic resonance T₂It is imaged under weighting pattern.

The contrast agent molecule of the targetted mitochondria that the present invention is provided is used as T₂The purposes of contrast agent, can answer extensively The survival rate and its migration and the physiology mistake such as go back to the nest of internal cell are transplanted to for vivo tracking in cell therapy Journey.This is an initiative discovery, on the one hand because can be by live body there is presently no effective method The survival rate and its migration and the physiology course such as go back to the nest of internal cell are transplanted in the clear and definite spike of image technology, separately On the one hand because its signal enhancing effect is only absorbed in the application of gadolinium complex contrast agent for a long time, therefore Only in T₁It is imaged under weighting pattern, image contrast is undesirable, and irregular change is presented.The present invention is base After the contrast agent molecule of described targetted mitochondria is combined with intracellular mitochondria, institute can be greatly lowered The magnetic resonance signal of Magnetic labeled cells is stated, is presented stablize regular signal decrease effect for a long time, therefore this Invention is in T₂The magnetic resonance signal presented after making full use of gadolinium complex to be combined with mitochondria under weighting pattern subtracts Weak effect.More importantly it is a discovery of the invention that using the present invention mark cell be transplanted in vivo after, it is described Mark cell can discharge the contrast agent molecule of targetted mitochondria, the contrast agent molecule of the targetted mitochondria of release Cell transplantation body periphery can be made is organized in magnetic resonance T₂Weight as bright signal is presented under pattern, such that it is able to make Cell transplantation body more clearly makes a distinction with its perienchyma.

Brief description of the drawings

In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to implementing Example or the accompanying drawing to be used needed for description of the prior art are briefly described.It should be evident that description below In accompanying drawing be only some embodiments of the present invention, for those of ordinary skill in the art, do not paying On the premise of going out creative work, other accompanying drawings can also be obtained according to these accompanying drawings.

A kind of Magnetic labeled cells showing for the method for nuclear magnetic resonance image vivo tracking that Fig. 1 is provided for the present invention It is intended to.

A kind of structural representation of the contrast agent molecule of targetted mitochondria that Fig. 2 is provided for the present invention.

The targeting unit i.e. knot of phosphonium cation of the contrast agent molecule of the targetted mitochondria that Fig. 3 is provided for the present invention Targeting unit triphenyl phosphonium (TPP) cation and its derivative used in structure formula figure and the embodiment of the present invention Thing ((p-methylphenyl)₃P) the structure chart of cation.

The radiography unit superparamagnetic metals that can be used to strengthen magnetic resonance imaging contrast's degree that Fig. 4 is provided for the present invention The structure chart of complex compound, wherein M represent the metal with superparamagnetic characteristic.

A kind of contrast agent molecule structural representation of targetted mitochondria that Fig. 5 is provided for the present invention, wherein radiography Unit Gd-DOTA, targeting unit triphenyl phosphonium cation are connected by carboxyl with dendritic or linear molecule. The construction unit of dendritic or linear molecule is lysine, and construction unit number k can be 0~7 integer； The number of radiography cells D OTA is m+n=k+1.

Fig. 6 is a kind of contrast agent molecule of targetted mitochondria that the embodiment of the present invention 1 is provided TPP-K (Gd-DOTA)-OH, is abbreviated as Gd-DOTA-TPP, and radiography unit is Gd-DOTA, and targeting is single First TPP and Gd-DOTA are connected by carboxyl with two amino of lysine (being abbreviated as K).

Fig. 7 is a kind of contrast agent molecule of targetted mitochondria that the embodiment of the present invention 2 is provided [Gd-DOTA-Acp-K(Gd-DOTA)]₂- K (TPP)-OH, is abbreviated as (Gd-DOTA)₄- TPP, radiography unit It is Gd-DOTA, targeting unit TPP and Gd-DOTA are connected by carboxyl with dendritic molecule, branch The construction unit of type molecule is lysine, and construction unit number is k=3, and connexon is lysine, one of them The length of introns is NH₂(CH₂)_pThe situation of the p=0 of COOH, the structure of another introns is NH₂(CH₂)₅COOH, i.e. aminocaproic acid (Acp), length is NH₂(CH₂)_pThe situation of the p=5 of COOH.

Fig. 8 is a kind of contrast agent molecule of targetted mitochondria that the embodiment of the present invention 3 is provided [Gd-DOTA-Acp-K(Gd-DOTA)]₂- linker-K (TPP)-OH, is abbreviated as (Gd-DOTA)₄-linker- TPP, radiography unit is Gd-DOTA, and targeting unit TPP and Gd-DOTA pass through carboxyl and dendritic Molecule is connected, and the construction unit of dendritic molecule is lysine, and construction unit number is k=3, here connexon It is aminohexanoyl lysine, wherein comprising introns aminocaproic acid, the length of one of introns is NH₂(CH₂)_pThe situation of the p=0 of COOH, the structure of another introns is NH₂(CH₂)₅COOH, it is long Degree is NH₂(CH₂)_pThe situation of the p=5 of COOH.

Fig. 9 is a kind of contrast agent molecule of targetted mitochondria that the embodiment of the present invention 4 is provided [Gd-DOTA-Acp-K(Gd-DOTA)]₂- linker-K (TPP)-OH, is abbreviated as (Gd-DOTA)₄-linker- TPP, radiography unit is Gd-DOTA, and targeting unit TPP and Gd-DOTA are by carboxyl and line style point Son is connected, and the construction unit of linear molecule is lysine, and construction unit number is k=3, and connexon is ammonia here Base hexanoyl lysine, wherein comprising introns aminocaproic acid, the length of one of introns is NH₂(CH₂)_pThe situation of the p=0 described in COOH, the structure of another introns is NH₂(CH₂)₅COOH, Length is NH₂(CH₂)_pThe situation of the p=5 of COOH.

Figure 10 is a kind of contrast agent molecule of targetted mitochondria that the embodiment of the present invention 5 is provided [Dy-DOTA-Acp-K(Gd-DOTA)]₂- linker-K (TPP)-OH, is abbreviated as (Dy-DOTA)₄-linker- TPP, radiography unit is Dy-DOTA, and targeting unit TPP and Dy-DOTA pass through carboxyl and dendritic Molecule is connected, and the construction unit of dendritic molecule is lysine, and construction unit number is k=3, here connexon It is aminohexanoyl lysine, wherein comprising introns aminocaproic acid, the length of one of introns is NH₂(CH₂)_pThe situation of the p=0 of COOH, the structure of another introns is NH₂(CH₂)₅COOH, it is long Degree is NH₂(CH₂)_pThe situation of the p=5 of COOH.

Figure 11 is a kind of contrast agent molecule of targetted mitochondria that the embodiment of the present invention 6 is provided [Gd-DTPA-Acp-K(Gd-DTPA)]₂- linker-K (TPP)-OH, is abbreviated as (Gd-DTPA)₄-linker- TPP, radiography unit is Gd-DTPA, and targeting unit TPP and Gd-DTPA are by carboxyl and dendritic point Son is connected, and the construction unit of dendritic molecule is lysine, and construction unit number is k=3, and connexon is here Aminohexanoyl lysine, wherein comprising introns aminocaproic acid, the length of one of introns is NH₂(CH₂)_pThe situation of the p=0 of COOH, the structure of another introns is NH₂(CH₂)₅COOH, it is long Degree is NH₂(CH₂)_pThe situation of the p=5 of COOH.

Figure 12 is a kind of contrast agent molecule of targetted mitochondria that the embodiment of the present invention 7 is provided [Gd-DOTA-Acp-K(Gd-DOTA)]₂- linker-K ((p-methylphenyl)₃P)-OH, is abbreviated as (Gd-DOTA)₄- Linker- (p-methylphenyl)₃P, radiography unit is Gd-DOTA, targeting unit (p-methylphenyl)₃P and Gd-DOTA It is connected with dendritic molecule by carboxyl, the construction unit of dendritic molecule is lysine, construction unit Number is k=3, and connexon is aminohexanoyl lysine here, wherein comprising introns aminocaproic acid, one of them The length of introns is NH₂(CH₂)_pThe situation of the p=0 of COOH, the structure of another introns is NH₂(CH₂)₅COOH, length is NH₂(CH₂)_pThe situation of the p=5 of COOH.

Figure 13 is a kind of contrast agent molecule of targetted mitochondria that the embodiment of the present invention 8 is provided TPP-Lys(TPP)-Lys[DOTA-Acp-Lys(DOTA)-Acp-Lys(DOTA)-Acp-Lys(DOTA)]-NH₂, it is abbreviated as (Gd-DOTA)₄-linker-TPP₂, radiography unit is 4 Gd-DOTA molecules, 4 DOTA Linear arrangement on amino with polypeptide N-terminal is connected on the epsilon-amino of linear polypeptide Lys (side chain)；Targeting Unit is same one end that two TPP molecules are connected on linear molecule, and the carboxyl of the Lys of this one end is amidated； The construction unit of linear molecule is lysine, and construction unit number is k=3, and connexon is that aminohexanoyl relies here Propylhomoserin, wherein comprising introns aminocaproic acid, the length of one of introns is NH₂(CH₂)_pCOOH institutes The situation of the p=0 for stating, the structure of another introns is NH₂(CH₂)₅COOH, length is NH₂(CH₂)_pThe situation of the p=5 of COOH.

Figure 14 is that the embodiment of the present invention 10 is used (Gd-DOTA)_1,4- TPP as targetted mitochondria contrast agent The external magnetic resonance T of molecular labeling mescenchymal stem cell₁Weighted sum T₂Weighting image effect figure.First row image It is T of the Gd-DOTA mark mescenchymal stem cells in different cell generation time nodes₁And T₂Weighted image. Second and third, four row's images be respectively 5,10,20mM Gd-DOTA-TPP mark mescenchymal stem cell exist The T of different cell generation time nodes₁And T₂Weighted image.5th row's image is 20mM (Gd-DOTA)₄- TPP marks mescenchymal stem cell in the T of different cell generation time nodes₁And T₂Weighted graph Picture.Numeral below image is during continuing after cell marking to be incubated, to gather the timing node of image (my god).

Figure 15 is the external T obtained after the mark cell that the embodiment of the present invention 10 is provided is bred through different time₁ Weighted mri signal of video signal intensity with cell generation time change.

Figure 16 is the external T obtained after the mark cell that the embodiment of the present invention 10 is provided is bred through different time₂ Weighted mri signal of video signal intensity with cell generation time change.

Figure 17 embodiment of the present invention 11 uses 20mM (Gd-DOTA)₄- TPP is the contrast agent of targetted mitochondria Molecular labeling mescenchymal stem cell, by pinpointing operation injection transplantation to mouse intracranial, in different time node The 11.7T magnetic resonance T of collection₂Weighting image effect figure.

Figure 18 embodiment of the present invention 12 uses 20mM (Gd-DOTA)₄- TPP is the contrast agent of targetted mitochondria Molecular labeling mescenchymal stem cell, by the 3T magnetic resonance gathered in leg muscle before fixed-point injection transplanting rat T₂Weighting image effect figure.

Figure 19 is that the embodiment of the present invention 13 is used (Gd-DOTA)₄-TPP₂As the contrast agent of targetted mitochondria The external T that molecular labeling mescenchymal stem cell is obtained after being bred through different time₂Weighted mri signal of video signal intensity With the change and the contrast of the partial results of embodiment 10 of cell generation time.

Figure 20 is the body obtained after the mark cell that the embodiment of the present invention 10 and 13 is provided is bred through different time Outer T₂The variation relation of Gd contents in weighted mri signal of video signal intensity and cell.

Specific embodiment

Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clearly Chu, it is fully described by.Obviously, described embodiment is only a part of embodiment of the invention, rather than Whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creation Property work under the premise of the every other embodiment that is obtained, belong to the scope of protection of the invention.

The present invention provides a kind of contrast agent molecule of targetted mitochondria as T₂The purposes of contrast agent, the targeting Mitochondrial contrast agent molecule includes targeting unit and radiography unit, wherein, the targeting unit is that have -P⁺(X₁)(X₂)(X₃) general structure phosphonium cations, wherein X₁、X₂、X₃Representative is unsubstituted or through one The C that individual or multiple substitution bases replace_1-12Alkyl, C_1-12Alkenyl or C_6-10Aryl, the substitution base include 1, 2 or 3 halogen atoms, C_1-12Alkyl, C_6-10Aryl, hydroxyl, C_1-12Alkoxy, halo-C_1-12Alcoxyl Base；Wherein X₁、X₂、X₃Can be identical group, or different groups；The radiography unit It is superparamagnetic metals complex compound.

In the embodiment of the present invention, the structure of described targeting unit triphenyl phosphonium or derivatives thereof molecule can be Any one shown in Fig. 3, or the other structures derivative with general structure shown in Fig. 3.

In the embodiment of the present invention, the structure of described radiography unit superparamagnetic metals complex compound can be institute in Fig. 4 The derivative of any one complex compound shown in any one complex compound, or Fig. 4 for showing, such as by superparamagnetic The acetyl group being connected with dendritic or linear molecule in the complexing ligand of metal complex replaces with propiono or fourth Acyl group, it is also possible to which B carbonyl is replaced with the derivative that ethylamino, the third amino, fourth amino etc. are obtained.

In the embodiment of the present invention, the contrast agent molecule of the targetted mitochondria can be by Solid-phase synthesis peptides skill Art is directly prepared；Unit can also be respectively synthesized, then the converting carboxylate groups of a certain unit are lived into NHS Sprinkle ester or be attached with another unit after activating, or connected by click chemistry.Such as first synthesize The complexing ligand (with protection group) of described radiography unit superparamagnetic metals complex compound, described triphenyl phosphonium sun Ion or derivatives thereof cation and dendritic or linear molecule with amino, match somebody with somebody the complexing of radiography unit The converting carboxylate groups of the carboxyl and triphenyl phosphonium of body or derivatives thereof cation into the active esters of NHS and its dendritic or The amino of linear molecule is attached, and sloughs after protection group and to obtain described with superparamagnetic metals ion complexation The contrast agent molecule of targetted mitochondria.Described dendritic or linear molecule can provide amino, sulfydryl, hydroxyl Base etc. is connected, it is also possible to provide carboxyl connection；Correspondingly, described superparamagnetic metals complex compound and triphen Ji Phosphonium or derivatives thereof cation can be connected by carboxyl with dendritic or linear molecule, it is also possible to by ammonia Base, sulfydryl, hydroxyl etc. are connected with dendritic or linear molecule；Or nitrine-the alkynyl of click chemistry Cycloaddition reaction.

In one embodiment of the invention, by the use of Gd-DOTA as radiography unit, unit triphenyl is targetted Phosphonium molecule is all connected by carboxyl with Gd-DOTA with dendritic or linear molecule, dendritic or linear molecule Construction unit uses lysine, the structure of the contrast agent molecule of the triphenyl phosphonium magnetic resonance targetted mitochondria of synthesis As shown in Figure 5.The construction unit number k of dendritic or linear molecule can be 0~7 integer；Radiography unit The number of DOTA is m+n=k+1, and connexon is lysine, and spacer molecule structure is used NH₂(CH₂)_pCOOH, wherein p are 0~12 integers.

In one embodiment of the invention, by the use of Gd-DOTA as radiography unit, unit triphenyl is targetted Phosphonium molecule and Gd-DOTA are connected by carboxyl with two amino of lysine, the triphenyl phosphonium targeting of synthesis The structure of mitochondrial contrast agent molecule is as shown in fig. 6, correspondence probe molecule Gd-DOTA-TPP.

In one embodiment of the invention, by the use of Gd-DOTA as radiography unit, unit triphenyl is targetted Phosphonium molecule and Gd-DOTA are connected by carboxyl with dendritic molecule, and the construction unit of dendritic molecule is used Lysine, construction unit number k=3, n=2, during m=2, connexon is lysine, one of introns Length be 0, the structure of another introns is NH₂(CH₂)₅COOH, the triphenyl phosphonium targeting line grain of synthesis The structure of the contrast agent molecule of body is as shown in fig. 7, correspondence probe molecule (Gd-DOTA)₄-TPP。

In one embodiment of the invention, by the use of Gd-DOTA as radiography unit, unit triphenyl is targetted Phosphonium molecule and Gd-DOTA are connected by carboxyl with dendritic molecule, and the construction unit of dendritic molecule is used Lysine, construction unit number k=3, n=2, during m=2, connexon is aminohexanoyl lysine, wherein wrapping Aminocaproic acid containing introns, the length of one of introns is 0, and the structure of another introns is NH₂(CH₂)₅COOH, the structure of the contrast agent molecule of the triphenyl phosphonium targetted mitochondria of synthesis as shown in figure 8, Correspondence probe molecule (Gd-DOTA)₄-linker-TPP。

In one embodiment of the invention, by the use of Gd-DOTA as radiography unit, unit triphenyl is targetted Phosphonium molecule and Gd-DOTA are connected by carboxyl with linear molecule, and the construction unit of linear molecule is using bad ammonia Acid, construction unit number k=3, m=1, during n=3, connexon is aminohexanoyl lysine, wherein comprising Every sub- aminocaproic acid, the length of one of introns is 0, and the structure of another introns is NH₂(CH₂)₅COOH, the structure of the contrast agent molecule of the triphenyl phosphonium targetted mitochondria of synthesis as shown in figure 9, Correspondence probe molecule (Gd-DOTA)₄-linker-TPP。

In one embodiment of the invention, by the use of Dy-DOTA as radiography unit, unit triphenyl is targetted Phosphonium cation and Dy-DOTA are connected by carboxyl with dendritic molecule, and the construction unit of dendritic molecule is adopted With lysine, construction unit number k=3, n=2, during m=2, connexon is aminohexanoyl lysine, wherein Comprising introns aminocaproic acid, the length of one of introns is 0, and the structure of another introns is NH₂(CH₂)₅COOH, the structure of the contrast agent molecule of the triphenyl phosphonium targetted mitochondria of synthesis is as shown in Figure 10, Correspondence probe molecule (Dy-DOTA)₄-TPP。

In one embodiment of the invention, by the use of Gd-DTPA as radiography unit, unit triphenyl is targetted Phosphonium molecule and Gd-DTPA are connected by carboxyl with dendritic molecule, and the construction unit of dendritic molecule is used Lysine, construction unit number k=3, n=2, during m=2, connexon is aminohexanoyl lysine, wherein wrapping Aminocaproic acid containing introns, the length of one of introns is 0, and the structure of another introns is NH₂(CH₂)₅COOH, the structure of the contrast agent molecule of the triphenyl phosphonium targetted mitochondria of synthesis is as shown in figure 11, Correspondence probe molecule (Gd-DTPA)₄-linker-TPP。

In one embodiment of the invention, by the use of Gd-DOTA as radiography unit, targeting unit three is (right Aminomethyl phenyl) Phosphonium molecule ((p-methylphenyl)₃P) it is connected with dendritic molecule by carboxyl with Gd-DOTA, The construction unit of dendritic molecule uses lysine, construction unit number k=3, n=2, during m=2, connexon It is aminohexanoyl lysine, wherein comprising introns aminocaproic acid, the length of one of introns is 0, separately The structure of one introns is NH₂(CH₂)₅COOH, the contrast agent point of the triphenyl phosphonium targetted mitochondria of synthesis The structure of son is as shown in figure 12, correspondence probe molecule (Gd-DOTA)₄- linker- (p-methylphenyl)₃P。

In one embodiment of the invention, by the use of Gd-DOTA as radiography unit, 2 target units three Ben Ji Phosphonium molecules and Gd-DOTA are connected by carboxyl with linear molecule, and the construction unit of linear molecule is used Lysine, construction unit number k=3, m=1, during n=3, connexon is aminohexanoyl lysine, wherein wrapping Aminocaproic acid containing introns, the length of one of introns is 0, and the structure of another introns is NH₂(CH₂)₅COOH, structure such as Figure 13 institutes of the contrast agent molecule of the triphenyl phosphonium targetted mitochondria of synthesis Show, correspondence probe molecule (Gd-DOTA)₄-linker-TPP₂。

In one embodiment of the invention, there is provided a kind of utilization targetted mitochondria of the present invention The cell of magnetic resonance contrast agent molecular labeling, and by pulse electroporation in conjunction with cell mitochondrial target To mitochondrial magnetic resonance contrast agent molecular labeling mescenchymal stem cell method.

In one embodiment of the invention, there is provided a kind of method observes knot by external nuclear magnetic resonance image The mescenchymal stem cell of the contrast agent molecule mark of conjunction cell mitochondrial, the different time node after cell marking, Its T₁Weighted imaging and T₂The change procedure of weighted imaging contrast.

In one embodiment of the invention, there is provided a kind of method, will by pinpointing operation transplantation/injection The mesenchymal stem cell transplantation of the magnetic resonance contrast agent molecular labeling of targetted mitochondria is utilized in Mice Body 11.7T live body magnetic resonance T₂The image contrast of weighted imaging, observation of cell transplant and its perienchyma is thin The change procedure of different time node after born of the same parents' transplanting.

In one embodiment of the invention, there is provided a kind of method, will by pinpointing operation transplantation/injection The mesenchymal stem cell transplantation of the magnetic resonance contrast agent molecular labeling of targetted mitochondria in rat body, using 3T Live body magnetic resonance T₂The image contrast of weighted imaging, observation of cell transplant and its perienchyma.

Specific embodiment is as follows：

Embodiment 1：The synthesis (Fig. 6) of the contrast agent molecule Gd-DOTA-TPP of targetted mitochondria

1、Bu^t ₃DOTA (1,4,7-three (tert-butoxycarbonylmethyl)-10- (acetic acid)-1,4,7,10-tetraazacyclododecanes 12 Alkane) synthesis.Synthesize since 1,4,7,10-tetraazacyclododecanand (Cyclen) according to the following steps：

A) 10.0g Cyclen and 29.3g NaHCO are weighed₃It is placed in the there-necked flask of 1L, adds 50mL second Nitrile.It is well mixed after 37.4g bromo-acetic acid tert-butyls, plus 20mL acetonitriles are weighed in fume hood, is put into dropping liquid In funnel.In ice bath and N₂The acetonitrile solution of bromo-acetic acid tert-butyl is slowly added dropwise into there-necked flask under protection In reactant mixture.After completion of dropping, continue to stir 30 hours at room temperature.Solid is filtered, rotary evaporation is removed Acetonitrile is removed, with re crystallization from toluene 2 times, 16g white solids Bu is obtained^t ₃DO3A (1,4,7-three (tertiary butyloxycarbonyl first Base)-1,4,7,10-tetraazacyclododecanand).

B) 1.38g K are weighed₂CO₃With 2.57g Bu^t ₃DO3A is placed in the there-necked flask of 250mL, is added 50mL acetonitriles, in N₂The 5mL acetonitrile solutions of lower dropwise addition 1.0g bromoacetates are protected and are stirred at room temperature, temperature Degree rises to 70 DEG C and reacts 12-24 hours.Room temperature is cooled to, is filtered, rotary evaporation removes solvent.With CH₂Cl₂/MeOH(20:1) for solvent carries out column chromatogram chromatography separation, the faint yellow thick bubbles of 2.5g are obtained Foam shape product 1,4,7-three (tert-butoxycarbonylmethyl)-10- (ethoxycarbonymetyl) 1,4,7,10-tetraazacyclododecanand (Bu^t ₃Et-DOTA)。

C) 1.5g Bu are weighed^t ₃Et-DOTA adds the dissolving of 50mL dioxane in the there-necked flask of 250mL, N₂Protection is lower to add the 25mL 1.2M NaOH aqueous solution, and 50-70 DEG C is stirred 4 hours.Rotary evaporation removes dioxy Six rings, three times (each 25mL) is extracted with dichloromethane, and combining extraction liquid uses anhydrous sodium sulfate drying.Rotation Turn evaporation of solvent, use CH₂Cl₂/MeOH(20:1) for solvent carries out column chromatogram chromatography separation, obtain The faint yellow foam like consistency shape product Bu of 1.0g^t ₃DOTA。

2、Ph₃P(Br)(CH₂)₄The synthesis of COOH [bromination (4- carboxylic Ding bases triphenyl phosphonium)]

Weigh 7.42g triphenyl phosphoniums and 5.01g 5- bromine valeric acids are placed in 100mL round-bottomed flasks, add 35mL Dimethylbenzene, stirring and dissolving.It is heated to 140 DEG C, condensing reflux 4 hours.40 DEG C~50 DEG C are cooled to, strong 30mL dimethyl ethers are slowly added dropwise with separatory funnel under strong stirring, white crystal is obtained.Suction filtration, will be white brilliant Body is washed twice with a small amount of Anaesthetie Ether, is obtained 8.51 grams of product brominated (4- carboxylic Ding bases triphenyl phosphonium), yield 68%.

3rd, DOTA-TPP is synthesized with the method for synthesis in solid state：

Step is summarized as follows：Synthesize on solid phase synthetic instrument by traditional Fmoc (chloro-carbonic acid fluorenes methyl esters) method, Structure as shown in Figure 6, from C-terminal to N-terminal successively coupling amino acid.Solid phase carrier 2- chlorine trityl tree first Fat 1g, sequentially adds 2.0g Fmoc-Lys (Mtt)-OH, 1.77g Ph₃P(Br)(CH₂)₄COOH is condensed, The condition of carboxyl and the amino condensation for often walking be the DMF with 50mL as solvent, add 0.96g TBTU, 0.41g The alkali DIPEA of HOBt condensing agents and 2.5mL, 25 DEG C are reacted about 24 hours, and the specific time is aobvious with ninhydrin Color is defined to judge whether carboxyl and amino condensation terminate；Condensation drains solvent after terminating, and 50mL is used respectively Methyl alcohol, is washed three times with DMF, methyl alcohol and dichloromethane.Add Ph₃P(Br)(CH₂)₄Fmoc is removed before COOH Condition：20% piperidines of 25mL/DMF room temperature reactions 0.5 hour, drains, and adds the piperidines of 25mL 20% / DMF reacts 0.5 hour, drains, and is respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.

Condensation Ph₃P(Br)(CH₂)₄After COOH, 50mL 1%TFA/ dichloromethane is added to slough blocking group Mtt, drains.Respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.Add 1.72g Bu^t ₃DOTA, The DMF solvent of 50mL, the alkali DIPEA of 0.96g TBTU, 0.41g HOBt condensing agents and 2.5mL, 25 DEG C Reaction, judges that carboxyl and amino condensation drain solvent after terminating with ninhydrin colour developing, and 50mL methyl alcohol is used respectively, Washed three times with DMF, methyl alcohol and dichloromethane.Add 50mL 50%TFA/ dichloromethane, 25 DEG C of reactions 40 Minute.Filtering, takes filtrate, and filtrate pH is transferred into neutrality with triethylamine, is concentrated to dryness.Add ether, analysis Go out white solid, obtain crude product.

Crude product is further purified with HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5 μm of 19 × 150mm, mobile phase is：Solvent orange 2 A (0.1%TFA, water), solvent B (0.1%TFA, CH₃CN), Solvent orange 2 A dropped to 25% in 25 minutes from 50%；Flow velocity 10mL/ minutes.Obtain about 200mg products DOTA-TPP, purity more than 95%.

4th, by the DOTA-TPP and Gd of deprotection³⁺Complexing, obtains making for Gd-DOTA-TPP targetted mitochondrias Shadow agent molecule.It is specific as follows：By the GdCl containing 35.7mg₃·6H₂The aqueous solution of O about 1.0mL is added drop-wise to Mix 3 hours in the aqueous solution of the above-mentioned DOTA-TPP of 100mg, careful 1.0M ammoniacal liquor adjusts pH value to 6 or so, Static mixer is rotated overnight at room temperature, then carefully adjusts pH value to 7-8 with 1.0M ammoniacal liquor and 1.0M hydrochloric acid, clear The contrast agent molecule of white powder Gd-DOTA-TPP targetted mitochondrias is obtained after clear water solution freeze-drying.With HPLC detects the purity of Gd-DOTA-TPP.HPLC conditions, Waters2535_2707_2998, Sapphire 5 μm of 4.6 × 250mm of C18, mobile phase is：Solvent orange 2 A (0.1%TFA, water), solvent B (0.1%TFA, 80%CH₃CN, 20% water), solvent B rose to 42% in 20 minutes from 22%；Flow velocity 1.0mL/ minutes.It is pure Degree more than 95%.

Embodiment 2：(Gd-DOTA)₄The synthesis (Fig. 7) of the contrast agent molecule of-TPP targetted mitochondrias

1st, Bu is synthesized according to embodiment 1^t ₃DOTA and Ph₃P(Br)(CH₂)₄COOH。

2nd, DOTA is synthesized with the method for synthesis in solid state₄-TPP：Step is summarized as follows：Exist by traditional Fmoc methods Synthesize on solid phase synthetic instrument, structure as shown in Figure 7, from C-terminal to N-terminal successively coupling amino acid.Solid phase first Carrier 2- chlorine trityl resin 1g, sequentially add 2.0g Fmoc-Lys (Mtt)-OH, 1.77g Ph₃P(Br)(CH₂)₄COOH is condensed, and the condition of carboxyl and the amino condensation for often walking is with the DMF of 50mL It is solvent, adds the alkali DIPEA of 0.96g TBTU, 0.41g HOBt condensing agents and 2.5mL, 25 DEG C of reactions is big About 24 hours, the specific time was defined to judge whether carboxyl and amino condensation terminate by ninhydrin colour developing；Condensation Solvent is drained after end, 50mL methyl alcohol is used respectively, washed three times with DMF, methyl alcohol and dichloromethane.Add Ph₃P(Br)(CH₂)₄The condition of Fmoc is removed before COOH：20% piperidines of 25mL/DMF room temperature reactions 0.5 are small When, drain, add 20% piperidines of 25mL/DMF and react 0.5 hour, drain, respectively with DMF, methyl alcohol, Dichloromethane respectively washing three times.

Condensation Ph₃P(Br)(CH₂)₄After COOH, 50mL 1%TFA/ dichloromethane is added to slough lysine side The blocking group Mtt of chain, drains solvent.Respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.Then The amino acid for sequentially adding Fmoc protections is condensed (2.0g Fmoc-Lys (Fmoc)-OH, 4.0g Fmoc-Lys (Mtt)-OH, 2.12g Fmoc- ε-Acp-OH).Often walk carboxyl and amino condensation condition be with The DMF of 50mL is solvent, adds the alkali DIPEA of 1.92g TBTU, 0.82g HOBt condensing agents and 5mL, 25 DEG C react about 24 hours, the specific time by ninhydrin colour developing be defined judge carboxyl and amino condensation whether Terminate；Condensation drains solvent after terminating, and 50mL methyl alcohol is used respectively, is washed with DMF, methyl alcohol and dichloromethane Three times.A condition of Fmoc is removed before the amino acid for adding next Fmoc protections：25mL 20% Piperidines/DMF room temperature reactions 0.5 hour, drains, and adds 20% piperidines of 25mL/DMF and reacts 0.5 hour, takes out It is dry, respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.

After being finally condensed Fmoc- ε-Acp-OH and taking off Fmoc, 100mL 1%TFA/ dichloromethanes are added Alkane sloughs blocking group Mtt, drains.Respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.Add 7g Bu^t ₃The DMF solvent of DOTA, 200mL, the alkali of 4g TBTU, 1.64g HOBt condensing agents and 10mL DIPEA, 25 DEG C of reactions judge that carboxyl and amino condensation drain solvent after terminating with ninhydrin colour developing, point 50mL methyl alcohol is not used, is washed three times with DMF, methyl alcohol and dichloromethane.Add 50mL 50%TFA/ dichloromethanes Alkane, 25 DEG C are reacted 40 minutes.Filtering, takes filtrate, and filtrate pH is transferred into neutrality with triethylamine, is concentrated to dryness. Ether is added, white solid is separated out, crude product is obtained.

Crude product is further purified with HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5 μm of 19 × 150mm, mobile phase is：Solvent orange 2 A (0.1%TFA, water), solvent B (0.1%TFA, CH₃CN), Solvent orange 2 A drops to 65% in 15 minutes from 80%；Flow velocity 10mL/ minutes.Obtain about 200mg products (DOTA)₄- TPP, purity more than 95%.

3rd, by the DOTA of deprotection₄- TPP and Gd³⁺Complexing, obtains (Gd-DOTA)₄- TPP targetted mitochondrias Contrast agent molecule.It is specific as follows：By the GdCl containing 12.8mg₃·6H₂The aqueous solution of O about 0.5mL is added drop-wise to The above-mentioned DOTA of 100mg₄Mix 3 hours in the aqueous solution of-TPP, careful 1.0M ammoniacal liquor adjusts pH value to 6 or so, Static mixer is rotated overnight at room temperature, then carefully adjusts pH value to 7-8 with 1.0M ammoniacal liquor and 1.0M hydrochloric acid, clear White powder (Gd-DOTA) is obtained after clear water solution freeze-drying₄The contrast agent molecule of-TPP targetted mitochondrias. (Gd-DOTA) is detected with HPLC₄The purity of-TPP.HPLC conditions, Waters2535_2707_2998, 5 μm of 4.6 × 250mm of Sapphire C18, mobile phase is：Solvent orange 2 A (0.1%TFA, water), solvent B (0.1%TFA, 80%CH₃CN, 20% water), solvent B rose to 44% in 20 minutes from 24%；Flow velocity 1.0mL/ minutes.It is pure Degree more than 95%.

Embodiment 3：(Gd-DOTA)₄The synthesis of the contrast agent molecule of-linker-TPP targetted mitochondrias is (here Spacer is Acp) (Fig. 8, dendritic molecule)

1st, according to the method synthesis difference Bu of embodiment 1^t ₃DOTA and Ph₃P(Br)(CH₂)₄COOH。

2nd, DOTA is synthesized with the method for synthesis in solid state₄-linker-TPP：

Step is summarized as follows：Synthesize on solid phase synthetic instrument by traditional Fmoc methods, structure as shown in Figure 8, From C-terminal to N-terminal successively coupling amino acid.Solid phase carrier 2- chlorine trityl resins 1g, sequentially adds 2.0g first Fmoc-Lys (Mtt)-OH, 1.77g Ph₃P(Br)(CH₂)₄COOH is condensed, carboxyl and the amino contracting for often walking The condition of conjunction be the DMF with 50mL as solvent, add 0.96g TBTU, 0.41g HOBt condensing agents and 2.5mL Alkali DIPEA, 25 DEG C are reacted about 24 hours, the specific time be defined by ninhydrin colour developing judge carboxyl and Whether amino condensation terminates；Condensation drains solvent after terminating, and 50mL methyl alcohol is used respectively, with DMF, methyl alcohol Washed three times with dichloromethane.Add Ph₃P(Br)(CH₂)₄The condition of Fmoc is removed before COOH：25mL 20% Piperidines/DMF room temperature reactions 0.5 hour, drains, and adds 20% piperidines of 25mL/DMF and reacts 0.5 hour, takes out It is dry, respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.

Condensation Ph₃P(Br)(CH₂)₄After COOH, 50mL 1%TFA/ dichloromethane is added to slough lysine side The blocking group Mtt of chain, drains solvent.Respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.Then The amino acid for sequentially adding Fmoc protections is condensed (1.06g Fmoc- ε-Acp-OH, 2.0g Fmoc-Lys (Fmoc)-OH, 4.0g Fmoc-Lys (Mtt)-OH, 2.12g Fmoc- ε-Acp-OH).Often walk Carboxyl and amino condensation condition be the DMF with 50mL as solvent, add 1.92g TBTU, 0.82g HOBt The alkali DIPEA of condensing agent and 5mL, 25 DEG C are reacted about 24 hours, and the specific time is defined by ninhydrin colour developing Judge whether carboxyl and amino condensation terminate；Condensation drains solvent after terminating, and 50mL methyl alcohol is used respectively, uses DMF, methyl alcohol and dichloromethane are washed three times.One is removed before the amino acid for adding next Fmoc protections The condition of Fmoc：20% piperidines of 25mL/DMF room temperature reactions 0.5 hour, drains, and adds 25mL 20% Piperidines/DMF reacts 0.5 hour, drains, and is respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.

Crude product is further purified with HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5 μm of 19 × 150mm, mobile phase is：Solvent orange 2 A (0.1%TFA, water), solvent B (0.1%TFA, CH₃CN), Solvent orange 2 A dropped to 65% at 15 minutes from 80%；Flow velocity 10mL/ minutes.Obtain about 200mg products (DOTA)₄- linker-TPP, purity more than 95%.

3rd, by the DOTA of deprotection₄- spacer-TPP and Gd³⁺Complexing, obtains (Gd-DOTA)₄-linker-TPP The contrast agent molecule of targetted mitochondria.It is specific as follows：By the GdCl containing 12.8mg₃·6H₂The aqueous solution of O is about 0.5mL is added drop-wise to the above-mentioned DOTA of 100mg₄Mix 3 hours in the aqueous solution of-linker-TPP, carefully use 1.0M ammonia Water adjusts pH value to 6 or so, and static mixer is rotated overnight, then with 1.0M ammoniacal liquor and 1.0M hydrochloric acid at room temperature It is careful to adjust pH value to 7-8, white powder (Gd-DOTA) is obtained after clear aqueous solution freeze-drying₄-linker-TPP The contrast agent molecule of targetted mitochondria.(Gd-DOTA) is detected with HPLC₄The purity of-linker-TPP.HPLC Condition, 5 μm of 4.6 × 250mm of Waters2535_2707_2998, Sapphire C18, mobile phase is：Solvent A (0.1%TFA, water), solvent B (0.1%TFA, 80%CH₃CN, 20% water), solvent B is in 20 minutes 44% is risen to from 24%；Flow velocity 1.0mL/ minutes.Purity more than 95%.

Embodiment 4：(Gd-DOTA)₄The synthesis of the contrast agent molecule of-linker-TPP targetted mitochondrias is (here Spacer is Acp) (Fig. 9, linear molecule)

1st, Bu is synthesized according to the method for embodiment 1^t ₃DOTA and Ph₃P(Br)(CH₂)₄COOH。

2nd, DOTA is synthesized according to embodiment 3₄The method of-linker-TPP, structure as shown in Figure 9 is closed with solid phase Into method synthetic linear molecule connection DOTA₄-linker-TPP。

3rd, by the DOTA of deprotection₄- spacer-TPP and Gd³⁺Complexing, obtains (Gd-DOTA)₄-linker-TPP The contrast agent molecule of targetted mitochondria.It is specific as follows：By the GdCl containing 12.8mg₃·6H₂The aqueous solution of O is about 0.5mL is added drop-wise to the above-mentioned DOTA of 100mg₄Mix 3 hours in the aqueous solution of-linker-TPP, carefully use 1.0M ammonia Water adjusts pH value to 6 or so, and static mixer is rotated overnight, then with 1.0M ammoniacal liquor and 1.0M hydrochloric acid at room temperature It is careful to adjust pH value to 7-8, white powder (Gd-DOTA) is obtained after clear aqueous solution freeze-drying₄-linker-TPP The contrast agent molecule of targetted mitochondria.(Gd-DOTA) is detected with HPLC₄The purity of-linker-TPP.HPLC Condition, 5 μm of 4.6 × 250mm of Waters2535_2707_2998, Sapphire C18, mobile phase is：Solvent A (0.1%TFA water), solvent B (0.1%TFA, 80%CH₃CN, 20% water), solvent B in 20 minutes from 24% rises to 44%；Flow velocity 1.0mL/ minutes.Purity more than 95%.

Embodiment 5：(Dy-DOTA)₄The synthesis of the contrast agent molecule of-linker-TPP targetted mitochondrias is (here Linker=Acp) (Figure 10)

2nd, DOTA is synthesized according to the method for embodiment 3₄-linker-TPP：

3rd, by the DOTA of deprotection₄- linker-TPP and Dy³⁺Complexing, obtains (Dy-DOTA)₄-linker-TPP The contrast agent molecule of targetted mitochondria.It is specific as follows：By the DyCl containing 13.0mg₃·6H₂The aqueous solution of O is about 0.5mL is added drop-wise to the above-mentioned DOTA of 100mg₄Mix 3 hours in the aqueous solution of-linker-TPP, carefully use 1.0M ammonia Water adjusts pH value to 6 or so, and static mixer is rotated overnight, then with 1.0M ammoniacal liquor and 1.0M hydrochloric acid at room temperature It is careful to adjust pH value to 7-8, white powder (Dy-DOTA) is obtained after clear aqueous solution freeze-drying₄-linker-TPP The contrast agent molecule of targetted mitochondria.(Dy-DOTA) is detected with HPLC₄The purity of-linker-TPP.HPLC Condition, 5 μm of 4.6 × 250mm of Waters2535_2707_2998, Sapphire C18, mobile phase is：Solvent A (0.1%TFA, water), solvent B (0.1%TFA, 80%CH₃CN, 20% water), solvent B is in 20 minutes 44% is risen to from 24%；Flow velocity 1.0mL/ minutes.Purity more than 95%.

Embodiment 6：(Gd-DTPA)₄The synthesis of the contrast agent molecule of-linker-TPP targetted mitochondrias is (here Linker=Acp) (Figure 11)

1st, Ph is synthesized according to the method for embodiment 1₃P(Br)(CH₂)₄COOH

2nd, DTPA is synthesized with the method for synthesis in solid state₄-linker-TPP：

Step is summarized as follows：Synthesize on solid phase synthetic instrument by traditional Fmoc methods, structure as shown in Figure 10, From C-terminal to N-terminal successively coupling amino acid.Solid phase carrier 2- chlorine trityl resins 1g, sequentially adds 2.0g first Fmoc-Lys (Mtt)-OH, 1.77g Ph₃P(Br)(CH₂)₄COOH is condensed, carboxyl and the amino contracting for often walking The condition of conjunction be the DMF with 50mL as solvent, add 0.96g TBTU, 0.41g HOBt condensing agents and 2.5mL Alkali DIPEA, 25 DEG C are reacted about 24 hours, the specific time be defined by ninhydrin colour developing judge carboxyl and Whether amino condensation terminates；Condensation drains solvent after terminating, and 50mL methyl alcohol is used respectively, with DMF, methyl alcohol Washed three times with dichloromethane.Add Ph₃P(Br)(CH₂)₄The condition of Fmoc is removed before COOH：25mL 20% Piperidines/DMF room temperature reactions 0.5 hour, drains, and adds 20% piperidines of 25mL/DMF and reacts 0.5 hour, takes out It is dry, respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.

After being finally condensed Fmoc- ε-Acp-OH and taking off Fmoc, 100mL 1%TFA/ dichloromethanes are added Alkane sloughs blocking group Mtt, drains.Respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.Add 4.0g The alkali DIPEA of the DMF solvent of DTPAA (diethylenetriamine pentaacetic acid acid anhydrides), 200mL and 10mL, 25 DEG C Reaction, judges that carboxyl and amino condensation drain solvent after terminating with ninhydrin colour developing, and 50mL methyl alcohol is used respectively, Washed three times with DMF, methyl alcohol and dichloromethane.Add 50mL 50%TFA/ dichloromethane, 25 DEG C of reactions 40 Minute.Filtering, takes filtrate, and filtrate pH is transferred into neutrality with triethylamine, is concentrated to dryness.Add ether, analysis Go out white solid, obtain crude product.

Crude product is further purified with HPLC, Waters2535_2707_2998_WFC, XBridge Pre C18 5 μm of 19 × 150mm, mobile phase is：Solvent orange 2 A (0.1%TFA, water), solvent B (0.1%TFA, CH₃CN), Solvent orange 2 A dropped to 65% in 15 minutes from 80%；Flow velocity 10mL/ minutes.Obtain about 200mg products (DTPA)₄- spacer-TPP, purity more than 95%.

3rd, by the DTPA of deprotection₄- linker-TPP and Gd³⁺Complexing, obtains (Gd-DTPA)₄-linker-TPP The contrast agent molecule of targetted mitochondria.It is specific as follows：By the GdCl containing 12.8mg₃·6H₂The aqueous solution of O is about 0.5mL is added drop-wise to the above-mentioned DTPA of 100mg₄Mix 3 hours in the aqueous solution of-linker-TPP, carefully use 1.0M ammonia Water adjusts pH value to 6 or so, and static mixer is rotated overnight, then with 1.0M ammoniacal liquor and 1.0M hydrochloric acid at room temperature It is careful to adjust pH value to 7-8, white powder (Gd-DTPA) is obtained after clear aqueous solution freeze-drying₄-linker-TPP The contrast agent molecule of targetted mitochondria.(Gd-DTPA) is detected with HPLC₄The purity of-linker-TPP.HPLC Condition, 5 μm of 4.6 × 250mm of Waters2535_2707_2998, Sapphire C18, mobile phase is：Solvent A (0.1%, TFA water), solvent B (0.1%TFA, 80%CH₃CN, 20% water), solvent B is in 20 minutes 44% is risen to from 24%；Flow velocity 1.0mL/ minutes.Purity more than 95%.

Embodiment 7：(Gd-DOTA)₄- linker- (p-methylphenyl)₃The synthesis of the contrast agent molecule of P targetted mitochondrias (linker=Acp here) (Figure 12)

1st, Bu is synthesized according to embodiment 1^t ₃DOTA。

2nd, (p-methylphenyl)₃P(Br)(CH₂)₄The conjunction of COOH [bromination (4- carboxylics butyl three (p-methylphenyl) Phosphonium)] Into

Synthesize Ph according to embodiment 1₃P(Br)(CH₂)₄The step of COOH, the triphenylphosphine of raw material 7.42g Change the three p-methylphenyl phosphines of 8.61g into, other raw materials and condition are all constant.Obtain product brominated (4- carboxylics butyl three pairs Jia Ben Ji Phosphonium) 8.0 grams, yield 58%.

3rd, DOTA is synthesized with the method for synthesis in solid state₄- linker- (p-methylphenyl)₃P：

According to the synthesis step of embodiment 3, raw material 1.77g Ph₃P(Br)(CH₂)₄COOH changes the (right of 1.94g into Tolyl)₃P(Br)(CH₂)₄COOH, other raw materials and condition and detecting step are all constant.

4th, by the DOTA of deprotection₄- linker- (p-methylphenyl)₃P and Gd³⁺Complexing, obtains (Gd-DOTA)₄- linker- (p-methylphenyl)₃The contrast agent molecule of P targetted mitochondrias.According to the complexing of embodiment 3 Step, by the GdCl containing 12.6mg₃·6H₂It is above-mentioned that the aqueous solution of O about 0.5mL is added drop-wise to 100mg DOTA₄- linker- (p-methylphenyl)₃Mix in the aqueous solution of P, other raw materials and condition and detecting step are not Become.

Embodiment 8：(Gd-DOTA)₄-linker-TPP₂The contrast agent molecule of targetted mitochondria synthesis (Figure 13, Here linker is Lys (Acp)-NH₂, radiography unit Gd-DOTA is connected on line style peptide molecule, two Targeting unit TPP is connected to same one end of linear molecule, and the carboxyl of the Lys is amidated)

2nd, the method synthetic linear molecule of the structure synthesis in solid state as shown in Figure 13 is connected DOTA₄-linker-TPP₂.In the present embodiment, the carboxyl of connexon lysine is converted to amide groups (this hair The carboxyl of the connexon lysine of the contrast agent molecule of bright described all targetted mitochondrias may be converted into acyl Amido, and with same contrasting effects).

Step is summarized as follows：Synthesize on solid phase synthetic instrument by traditional Fmoc methods, structure as shown in Figure 10, From C-terminal to N-terminal successively coupling amino acid.Solid phase carrier is used for producing the polypeptide that C-terminal acid amides ends up first Resin such as Rink Amide AM Resin or Rink Amide MBHA Resin/Knorr Resin 1g, according to Secondary addition 2.0g Fmoc-Lys (Mtt)-OH, 2.0g Fmoc-Lys (Fmoc)-OH, 3.54g Ph₃P(Br)(CH₂)₄COOH is condensed.Condition, carboxyl and amino contracting that the carboxyl and amino for often walking are condensed Close the criterion for whether terminating, the post processing being condensed after terminating, add Ph₃P(Br)(CH₂)₄COOH it The condition of preceding removing Fmoc etc. is same as Example 3.

Condensation Ph₃P(Br)(CH₂)₄After COOH, 50mL 1%TFA/ dichloromethane is added to slough lysine side The blocking group Mtt of chain, drains solvent.Respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.Then Sequentially add 1.06g Fmoc- ε-Acp-OH, 2.0g Mtt-Lys (Fmoc)-OH, 1.72g Bu^t ₃DOTA is contracted Close.The condition of carboxyl and the amino condensation for often walking be the DMF with 50mL as solvent, add 1.92g TBTU, The alkali DIPEA of 0.82g HOBt condensing agents and 5mL, 25 DEG C are reacted about 24 hours, and the specific time is with ninhydrin Colour developing is defined to judge whether carboxyl and amino condensation terminate；Condensation drains solvent after terminating, and 50mL is used respectively Methyl alcohol, is washed three times with DMF, methyl alcohol and dichloromethane.Next molecule is added to remove one before being condensed The condition of Fmoc：20% piperidines of 25mL/DMF room temperature reactions 0.5 hour, drains, and adds 25mL 20% Piperidines/DMF reacts 0.5 hour, drains, and is respectively washed three times with DMF, methyl alcohol, dichloromethane respectively.So Complete a linearly connected of radiography unit complexing agent DOTA.Repeat this process twice, complete three radiographies The linearly connected of unit complexing agent DOTA；The linearly connected of last radiography unit complexing agent DOTA with it is upper State that process is identical, but added condensation molecule only adds 1.06g Fmoc- ε-Acp-OH, 1.72g Bu^t ₃DOTA It is condensed, is not added with Mtt-Lys (Fmoc)-OH molecules.

50mL 50%TFA/ dichloromethane is all added after condensation, 25 DEG C are reacted 40 minutes.Filtering, takes filter Liquid, neutrality is transferred to triethylamine by filtrate pH, is concentrated to dryness.Ether is added, white solid is separated out, obtained slightly Product.

3rd, by the DOTA of deprotection₄-linker-TPP₂With Gd³⁺Complexing, obtains (Gd-DOTA)₄-linker-TPP₂ The contrast agent molecule of targetted mitochondria.It is specific as follows：By the GdCl containing 12.8mg₃·6H₂The aqueous solution of O is about 0.5mL is added drop-wise to the above-mentioned DOTA of 100mg₄-linker-TPP₂The aqueous solution in mix 3 hours, carefully use 1.0M Ammoniacal liquor adjusts pH value to 6 or so, and static mixer is rotated overnight, then with 1.0M ammoniacal liquor and 1.0M salt at room temperature Sour careful tune pH value obtains white powder to 7-8 after clear aqueous solution freeze-drying (Gd-DOTA)₄-linker-TPP₂The contrast agent molecule of targetted mitochondria.Detected with HPLC (Gd-DOTA)₄-linker-TPP₂Purity.HPLC conditions, Waters2535_2707_2998, Sapphire 5 μm of 4.6 × 250mm of C18, mobile phase is：Solvent orange 2 A (0.1%TFA water), solvent B (0.1%TFA, 80%CH₃CN, 20% water), solvent B rose to 44% in 20 minutes from 24%；Flow velocity 1.0mL/ minutes.It is pure Degree more than 95%.

Embodiment 9：The preparation side of the mescenchymal stem cell of the contrast agent molecule mark of triphenyl phosphonium targetted mitochondria Method

1st, people source mesenchymal stem cells MSCs (hMSCs) that will be frozen in liquid nitrogen is taken out, in 37 DEG C of water-baths In thaw rapidly.In super-clean bench, the frozen stock solution of cell after defrosting is taken out with 1mL liquid-transfering guns is placed in In 10mL sterile centrifugation tubes, while addition 2mL complete mediums (Basal mediumDMEM-F12 80%~ 90%, Australia hyclone 10%~20%, it is dual anti-1%), 1000 rpms be centrifuged 5 minutes, suck Culture medium.Plus 3mL complete mediums dispel cell precipitation in centrifuge tube, cell suspension is taken out, put In 100 × 20mm culture dishes, 5mL complete mediums are continuously added, gently rock culture dish, make cell It is dispersed in complete medium.37 DEG C are placed into, is cultivated in the incubator of 5% carbon dioxide.Cell is answered Soviet Union second day, changes culture medium, continues to cultivate.When attached cell density reaches 80%~90%, culture is sucked Base.The culture dish for covering with cell is gently washed with the aseptic PBS solutions of 2mL, 1mL PBS and 1mL is added Trypsase, timely observation of cell form, after cell dissociation is complete, sucks pancreatin under microscope, with containing 4m complete mediums blow and beat cell, are divided into two and are transferred in two 100 × 20mm culture dishes, then add respectively Enter 6mL complete mediums to continue to cultivate.The cell in 6-9 generations is taken in experiment.

2nd, cell treatment：It is light with the aseptic PBS solutions of 2ml when attached cell density reaches 80%~90% Fine laundering washs the culture dish for covering with cell, adds 1mL PBS and 1mL trypsase, is seen in time under microscope Cellular morphology is examined, after cell dissociation is complete, trypsase is sucked.Cell is blown and beaten with 4mL complete mediums, Cell suspension is transferred to 10mL sterile centrifugation tubes, 1000 rpms are centrifuged 5 minutes, suck culture medium Obtain cell precipitation.Same resuspended operation is carried out with 4mL PBS again.

3rd, electroporation mark cell

The contrast agent molecule of the triphenyl phosphonium targetted mitochondria that will be prepared according to the inventive method is dissolved in physiology salt Water, be made into 1,2,5,10,20, the concentration series of 40mM.Take 100 μ L sample solution and be placed in cell precipitation In (100 μ L containing cell concentration be 1,000,000~2,000,000), dispel cell, the cell that will be dispelled is placed in 96 orifice plates In, electrotransfection instrument and voltage 120V are reached using one, the μ s of pulsewidth 100 are spaced 1000ms, the electricity being repeated 6 times Experiment condition is hit, the contrast agent molecule of the targetted mitochondria is imported in cytoplasm, applying certain electricity When pulse is tested, need to ensure that the cell in 96 orifice plates is dispersed in physiological saline, rather than The deposited bottom in 96 orifice plates.

Embodiment 10：Triphenyl phosphonium targetted mitochondria contrast agent molecule mark mescenchymal stem cell it is external MRI image method

1st, the contrast agent molecule of the triphenyl phosphonium targetted mitochondria prepared with embodiment 1 and 2, according to embodiment 9 Method mark mescenchymal stem cell, the cell half of every kind of concentration and probe concentration mark is transferred in 10mL centrifuge tubes, And culture dish is washed with 3mL PBS, in equally moving to centrifuge tube.1200 rpms are centrifuged 5 minutes, remove PBS. Cell is transferred in the capillary of the end closure that internal diameter is 1.5mm with the capillary of external diameter 1.3mm, 1500 Rpm centrifugation 10 minutes, by cell closs packing in the bottom of capillary, for the experiment of external MRI image.

2nd, another semicell continues to cultivate, after the quantity of cell is double, then will a wherein semicell Mi Dui Product is tested in the bottom of capillary for external MRI image.Another semicell continues to cultivate, until cell Quantity is double.According to this until cell in vitro MRI image experimental result does not have difference.

3rd, the cell in capillary is carried out into T in 11.7T nuclear magnetic resonance spectrometers₁Weighted sum T₂Weighted imaging.T₁ Weighting is as using saturation recovery sequence, TE=5.2ms, TR=500ms, FOV=12 × 12mm², matrix =96 × 96, slice thickness=0.8mm, interlevel=0.2mm, accumulative frequency=4；T₂Weighting is as using Stage construction echo, TR=3000ms, TE=80ms, 20 echoes, FOV=12 × 12mm², matrix=96 × 96, Slice thickness=0.8mm, interlevel=0.2mm, accumulative frequency=1.

Figure 14 is that the present embodiment is existed using the contrast agent molecule of the targetted mitochondria containing triphenyl phosphonium of different structure Mescenchymal stem cell is marked under different probe concentration, the body obtained after being bred through different time again after cell marking Outer T₁Weighted sum T₂Weighted mri imaging results.Also include using do not have cell binding ability in embodiment Gd-DOTA mark cells are contrasted as external MRI image laboratory reference.

Figure 15 is the external T obtained after the Magnetic labeled cells that the present embodiment is obtained are bred through different time₁Weighting MRI image signal intensity with cell generation time change.It can be seen that：(1) when cell is just labeled, Gd-DOTA marks the T of cell₁Weighted mri signal of video signal enhancement effect is notable, through the targeting containing triphenyl phosphonium The T of the Magnetic labeled cells of mitochondrial contrast agent molecule mark₁Weighted mri signal of video signal enhancement effect is not notable, Signal is even presented and weakens effect；(2) with the breeding of Magnetic labeled cells, the T of all Magnetic labeled cells₁Plus Power MRI image signal intensity all recovers rapidly the signal strength level (within 1~2 day) to n cell, Show magnetic resonance T₁The limitation of long-time spike transplanted cells body under weighting pattern.

Figure 16 is the external T obtained after the Magnetic labeled cells that the present embodiment is obtained are bred through different time₂Weighting MRI image signal intensity with cell generation time change.It can be seen that：(1) when cell is just labeled, The T of the Magnetic labeled cells of Gd-DOTA marks₂Weighted mri signal of video signal is presented significant enhancement effect, through containing The T of the Magnetic labeled cells of the contrast agent molecule mark of the targetted mitochondria of triphenyl phosphonium₂Weighted mri signal of video signal Significant signal is presented and weakens effect, the increase of concentration and probe concentration during with cell marking, signal Weaken degree is more aobvious Write, or even can reach even below noise level；(2) with the breeding of Magnetic labeled cells, through containing triphenyl The T of the Magnetic labeled cells of the contrast agent molecule mark of the targetted mitochondria of Phosphonium₂Weighted mri signal of video signal intensity is extensive Complex velocity is considerably slower than its T₁The resume speed of weighted mri signal of video signal enhancement effect, is still within about 5 days Noise level, still significant contrast difference (dark signal) is presented, it is necessary to about in about 10 days with n cell 16 talentes reach the signal strength level of n cell, show magnetic resonance T₂Long-time spike under weighting pattern The feasibility of transplanted cells body.

Embodiment 11：The mesenchymal stem cell transplantation of the contrast agent molecule mark of triphenyl phosphonium targetted mitochondria is small 11.7T live body magnetic resonance T in mouse body₂Weighting image

1st, as described in Example 9, (Gd-DOTA) is utilized₄The contrast agent molecule of-TPP targetted mitochondrias leads to Extra pulse electroporation method marks mescenchymal stem cell, and the contrast agent molecule concentration of targetted mitochondria is 20mM；

2nd, will about 3 × 10⁵The cell of individual magnetic marker is transplanted to mouse intracranial by the method for fixed-point injection, and Different time points (D0~D10) carry out 11.7T magnetic resonance T to cell transplantation position after cell transplantation₂It is weighted to As (S is the lamella sequence number of image).Using the birdcage coil of a diameter of 38mm, joined using RARE sequences Number is set：TE=7ms, TR=125,300,500,750,1000,1500,3000,5000ms, FOV= 20×20mm², matrix=128 × 128, slice thickness=0.5mm, average=4.The image effect for obtaining is such as Shown in Figure 17：A transplanted cells part is located at encephalic, and (D0~D10) is presented significant dark letter for a long time Number (white arrow indicating positions)；A part is located in the ventricles of the brain, the part cell same day (D0) after the transfer Just fast transferring and significant dark signal is presented in the ventricles of the brain, subsequent (D1~D4) cell transplantation body starts Discharge the contrast agent molecule of targetted mitochondria and make its residing perienchyma that significant bright signal (grey arrow is presented Head indicating positions).Cell starts death after one week, the targeting line grain discharged after the membranolysis of dead cell The contrast agent molecule of body also makes its residing perienchyma that significant bright signal (black arrow indicating positions) is presented. This is contrast agent molecule and a notable spy of image method of the magnetic resonance targetted mitochondria that the present invention is provided Point, clearly can come cell transplantation body with the tissue division on its periphery in actual applications.

Embodiment 12：The mesenchymal stem cell transplantation of the contrast agent molecule mark of triphenyl phosphonium targetted mitochondria is big 3T live body magnetic resonance T in mouse body₂Weighting image

2nd, will about 1 × 10⁷Individual Magnetic labeled cells are transplanted to before mouse in leg muscle by the method for fixed-point injection, And 3T magnetic resonance T is carried out to Magnetic labeled cells implant site after Magnetic labeled cells transplanting₂Weighted imaging, obtains As shown in figure 18, cell transplantation body is presented significant dark signal to the image effect for arriving, and illustrates present invention offer The contrast agent molecule of targetted mitochondria, the Magnetic labeled cells marked by it and nuclear magnetic resonance image vivo tracking method Also there is application feasibility on clinical image documentation equipment.

Embodiment 13：The body of the mescenchymal stem cell of the contrast agent molecule mark of Shuan triphenyl phosphonium targetted mitochondrias Outer MRI image

The contrast agent molecule of the Shuan triphenyl phosphonium targetted mitochondrias prepared with embodiment 8, according to embodiment 9 Method marks mescenchymal stem cell, and the external T of Magnetic labeled cells is carried out according to the method for embodiment 10₂Weighting MRI image.

Figure 19 is the external T obtained after the Magnetic labeled cells that the present embodiment is obtained are bred through different time₂Weighting MRI image signal intensity is with the change of cell generation time and the contrast of partial results in embodiment 10.Can see Arrive：The T of the Magnetic labeled cells marked through the contrast agent molecule of the targetted mitochondria containing Shuan triphenyl phosphoniums₂Weighted mri Signal of video signal intensity resume speed is considerably slower than through containing a contrast agent molecule for the targetted mitochondria of triphenyl phosphonium The Magnetic labeled cells of mark, show the former in magnetic resonance T₂Can be in longer time range under weighting pattern Vivo tracking transplanted cells body.

Figure 20 is the external T obtained after the Magnetic labeled cells that the present embodiment is obtained are bred through different time₂Weighting MRI image signal intensity and the relation of Gd changes of contents in cell, show Magnetic labeled cells to be made in T₂Plus Minimum cell Gd contents required for power MRI image signal intensity drops to noise level can be as little as 5×10⁹Gd/ cells.

Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the scope of the present invention. All any modification, equivalent substitution and improvements made within the spirit and principles in the present invention etc., are all contained in In protection scope of the present invention.

Claims

1. a kind of contrast agent molecule of targetted mitochondria is used as T₂The purposes of contrast agent, the targetted mitochondria Contrast agent molecule includes targeting unit and radiography unit, wherein,

The targeting unit is that have-P⁺(X₁)(X₂)(X₃) general structure phosphonium cations, wherein X₁、X₂、 X₃The C that representative is unsubstituted or replaces through one or more substitution bases_1-12Alkyl, C_1-12Alkenyl or C_6-10 Aryl, the substitution base includes 1,2 or 3 halogen atoms, C_1-12Alkyl, C_6-10Aryl, hydroxyl, C_1-12 Alkoxy, halo-C_1-12Alkoxy；Wherein X₁、X₂、X₃Can be identical group, or difference Group；

The radiography unit is superparamagnetic metals complex compound.

2. purposes according to claim 1, the targeting unit is triphenyl phosphonium cation or its derivative Thing.

3. purposes according to claim 1, the superparamagnetic metals complex compound is by superparamagnetic metals and network Mixture is formed, wherein：

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

The complexing agent is selected from DOTA, HP-DO3A, DO3A-butrol, DTPA-BMA, DTPA, DTPA-BMEA, BOPTA, EOB-DTPA or derivatives thereof and its any combination.

4. purposes according to claim 1, the targeting unit is directly or by connexon and dendritic Or linear molecule is connected, the dendritic or linear molecule are connected directly or by introns with radiography unit, The construction unit of wherein described dendritic or linear molecule be it is any can homopolymerization or copolymerization form dendritic or line style The monomer of macromolecular, more preferably preferred amino acid, lysine.

5. purposes according to claim 4, described connexon is straight-chain amino acid, preferably relies ammonia Acid；The introns are straight-chain amino acid, preferably NH₂(CH₂)_pCOOH or NH₂(CH₂CH₂O)_qCH₂COOH, wherein p are 0~12 integers, and q is 0~4 integer, works as p=0 Or q=0 interval scales do not have introns.

6. the purposes according to claim any one of 4-5, described dendritic or linear molecule and 1~2 Individual targeting unit and 1-8 radiography unit connection, are preferably connected with 2 targeting units and 4 radiography units.

7. the purposes according to claim any one of 4-6, described dendritic or linear molecule and 2 When targeting unit is connected, the binding site of the targeting unit can be preferably in the same of linear molecule with adjacent One end；Can also be non-conterminous, it is preferably in the two ends of linear molecule.

8. a kind of contrast agent molecule of targetted mitochondria, it includes targetting unit and radiography unit, wherein：

The radiography unit is superparamagnetic metals complex compound；

It is described targeting unit be connected with dendritic or linear molecule directly or by connexon, the dendritic or Linear molecule is connected directly or by introns with radiography unit, wherein the knot of the dendritic or linear molecule Structure unit be it is any can homopolymerization or copolymerization form the monomer of dendritic or linear macromolecule, preferred amino acid, more It is preferred that lysine.

9. the contrast agent molecule of targetted mitochondria according to claim 8, described connexon is selected from straight Chain amino acid, preferably lysine；The introns are selected from straight-chain amino acid, preferably NH₂(CH₂)_pCOOH or NH₂(CH₂CH₂O)_qCH₂COOH, wherein p are 0~12 integers, and q is 0~4 Integer, when p=0 or q=0 interval scales do not have introns.

10. the contrast agent molecule of targetted mitochondria according to claim 8 or claim 9, described dendritic Or linear molecule is connected with 1~2 targeting unit and 1-8 radiography unit, preferably with 2 targetings unit and 4 Individual radiography unit connection.

The contrast agent molecule of 11. targetted mitochondria according to claim 8-10, described dendritic or When linear molecule is connected with 2 targeting units, the binding site of the targeting unit can preferably be located with adjacent In same one end of linear molecule；Can also be non-conterminous, it is preferably in the two ends of linear molecule.

A kind of 12. methods of the contrast agent molecule for preparing the targetted mitochondria described in claim 8-11, its bag Include：

- P described in each⁺(X₁)(X₂)(X₃) cation and halogenated carboxylic acid or halogenated amine reaction generation have carboxyl Or-the P of amido functional group⁺(X₁)(X₂)(X₃) the cation ,-P⁺(X₁)(X₂)(X₃) cation by obtain carboxylic Base or amino are connected with dendritic or linear molecule；The halogenated carboxylic acid is chloro, bromo or iodo aliphatic acid Or aromatic acid；

The superparamagnetic metals complex compound is connected by its carboxyl or amino with dendritic or linear molecule；It is described The carboxyl of superparamagnetic metals complex compound is selected from second carboxyl, the third carboxyl or fourth carboxyl, the superparamagnetic metals complexing The amino of thing is selected from ethylamino, the third amino or fourth amino.

A kind of magnetic mark of the contrast agent molecule mark of 13. targetted mitochondrias with according to claim 8-11 Note cell, the Magnetic labeled cells are any can be used for by the contrast agent molecule mark of targetted mitochondria The cell of cellular transplantation therapy, does selected from mescenchymal stem cell, NSC, Cardiac Stem Cells, embryo Cell, induced multi-potent stem cell.

The combination of Magnetic labeled cells and timbering material described in a kind of 14. claims 13, the support material Material is any medical material that combination can be formed with cell, selected from collagen, various synthesis macromolecules Or inorganic timbering material；The various nutrition that the timbering material includes or survived not comprising sertoli cell and grown The factor.

A kind of 15. nuclear magnetic resonance image vivo tracking methods, it includes：

By Magnetic labeled cells and support described in the Magnetic labeled cells or claim 14 described in claim 13 The combination of material is arrived in human or animal's body by pinpointing operation transplantation/intravenous injection；

Above-mentioned human or animal is placed in nuclear magnetic resonance image equipment, in magnetic resonance T₂It is imaged under weighting pattern.