CN108948142B - Fluorescent probe targeting tumor cells and new vessels and preparation method thereof - Google Patents
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Abstract
The invention provides a fluorescent probe for targeted imaging of cancer cells and new blood vessels, which solves the problem of selective imaging of cancer cells with high expression of integrin alpha v beta 3. In the design of the molecular structure of the fluorescent probe, RGD is used as a recognition group to carry out multi-site modification on triaryl fluorophores so as to construct a multivalent probe capable of being combined with integrin alphavbeta 3. The probe can selectively image cancer cells with high integrin alpha v beta 3 expression at the cellular level and can image tumor parts in a targeting manner in vivo. Meanwhile, the synthesis process of the fluorescent probe is simple and easy to implement, the raw materials are cheap and easy to obtain, the preparation cost is low, and the popularization is easy.
Description
Technical Field
The invention relates to a novel fluorescent probe for targeted imaging of cancer cells and new vessels and a preparation method thereof, belonging to the technical field of analytical chemistry.
Background
Cancer is currently a major disease worldwide, causing over 700 million deaths per year, and is predicted to become a greater problem in the next 20 years. The development of the selective visual imaging technology of cancer cells or tissues is helpful to explore the occurrence and development mechanisms of cancer, realize early diagnosis and prognosis evaluation, improve the surgical resection rate and improve the survival rate.
Molecular imaging and probes are two methods that can detect disease earlier, determine the nature of the disease, monitor the effect of therapy objectively and not invasively, and predict disease progression, primarily at the molecular and cellular level. The fluorescence imaging method using the fluorescence probe has the advantages of high selectivity, high resolution, high sensitivity, non-invasiveness and easy naked eye observation, can carry out in-situ and real-time detection on cells, tissues or organisms and the like, and can avoid radiation hazards generated by some traditional imaging technologies; thus providing a promising strategy for the selective visualization of malignant tumors. Therefore, the development of the tumor-targeted fluorescent probe can provide a powerful tool for early diagnosis of cancer, and can also visually and accurately define the tumor infiltration and metastasis range on a molecular level, realize real-time dynamic navigation in an operation, obtain safe margins and reduce the margin positive rate.
Molecular imaging studies targeting tumor stroma have mainly focused on tumor neovascularization because the occurrence, development, infiltration, metastasis and recurrence of malignant tumors are closely related to tumor neovascularization. Integrin α v β 3 is currently one of the most commonly used characteristic markers in targeted vascular imaging studies. Normally, integrin α v β 3 is expressed only at low levels in tissues such as the small intestine, blood vessels, uterus, and the like; the protein is highly expressed in tumor cells, tumor neovascular endothelial cells, wounds and inflammatory tissues, participates in a plurality of processes of activation, proliferation, apoptosis and migration of the vascular endothelial cells, and plays an important role in the processes of growth, proliferation, migration, apoptosis and the like of the tumor cells. The integrin alpha v beta 3 can specifically recognize RGD peptide, namely a short peptide containing arginine-glycine-aspartic acid (Arg-Gly-Asp). The integrin alpha v beta 3 is used as an imaging target spot, and a molecular probe targeting tumor and new blood vessels can be prepared.
By "multivalent effect" is meant that binding of multiple ligands to multiple receptors as a whole results in a synergistic effect that is much stronger than the additive effect of an equivalent number of monovalent effects, while at the same time improving the sensitivity and specificity of the ligands. Therefore, the fluorescent molecule is modified by a plurality of targeting groups, so that the 'multivalent effect' can be realized, and the sensitivity and the selectivity of the probe can be improved simultaneously.
Triaryl boron is a typical intramolecular charge transfer compound (ICT) and has strong luminescence, sensitive luminescent properties to microenvironment change, two-photon properties and the like. These properties are well suited for use in biological imaging. However, there is no report in the literature on the application of triarylboron in the design of bioluminescent probes for targeting integrin α v β 3 tumor cells.
Therefore, the development of a novel fluorescent probe which is high in sensitivity and selectivity and performs specific targeted binding on integrin alphavbeta 3 expressed by tumor neovascular endothelial cells has great significance in the exploration, diagnosis and evaluation of tumor lesion conditions and the formulation of individualized treatment schemes.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides a triarylboron-RGD fluorescent probe, which can selectively image cancer cells highly expressing integrin α v β 3.
In order to achieve the purpose, the invention provides a fluorescent probe for targeting tumor cells and new vessels, which has a chemical structural general formula shown in the following (I):
wherein R is1、R2、R3May be the same or different, is a halogen atom or an-N-heterocyclic group substituted by a group containing the arginine-glycine-aspartic acid (Arg-Gly-Asp) (RGD) sequence, and R1、R2、R3Not all of them being halogen atoms at the same time.
The fluorescent probe takes the triarylboron compound with excellent luminescent property as a fluorescent group, the nitrogen atom on the-N-heterocyclic group is connected with the benzene ring of triaryl phenylboron to form a conjugated system with the boron atom, and the nitrogen atom can be used as an electron donor and taken as an acceptor with the boron atom, so that an Intramolecular Charge Transfer (ICT) compound is formed, and the excellent luminescent property of the triarylboron probe is ensured. The fluorescent probe uses RGD group capable of targeting tumor marker integrin alphavbeta 3 receptor as recognition group to carry out structural modification on triarylboron-N-heterocyclic compound, thereby realizing the purpose of selective imaging of cancer cells with high expression of integrin alphavbeta 3.
In addition, when the fluorescent probe is in a water environment, the fluorescent group part in the fluorescent molecular structure is surrounded by RGD, a hydrophobic environment is provided for the fluorescent group, the polarity of the microenvironment around the fluorescent group is reduced, and therefore the luminescence of the probe in PBS (phosphate buffered saline) shows cyan fluorescence under lower polarity. Thereby achieving the purpose that the fluorescent probe still has strong fluorescence property under the high-polarity water environment.
Preferably, the arginine-glycine-aspartic acid (Arg-Gly-Asp) (RGD) sequence-containing group is a cyclic arginine-glycine-aspartic acid (cRGD) group. The unique ring structure of the probe has higher binding force to cancer cells highly expressing integrin alpha v beta 3, thereby improving the selectivity of the probe.
Preferably, the-N-heterocyclic group may be a polyazacycloalkane derivative group. On one hand, the multi-nitrogen heterocyclic ring can form Intramolecular Charge Transfer (ICT) with boron by taking one N atom as an electron donating group, and on the other hand, other multiple N atoms can be used for introducing multiple recognition groups, so that the multi-valence effect is realized. Due to the existence of the multivalent effect, the probe has higher selectivity and sensitivity. More preferably, the polyazacycloalkane is any one of piperazine, triazacyclononane and tetraazacyclododecane.
In the present invention, the triarylboron compound has a structure which can be modified at multiple sites, wherein R is1、R2、R3Not all halogen atoms, which may be Cl, Br, preferably Br. When only one RGD group is substituted in the probe structure, the fluorescent probe substituted by the single RGD group has certain selectivity on cancer cells highly expressing integrin alphavbeta 3; when the RGD groups are substituted, the RGD groups are combined with integrin, so that the bonding force is stronger, and a multivalent effect is generated. In the preferred embodiment of the present invention, R is preferably1、R2、R3Are each-N-heterocyclyl substituted by a group of the RGD sequence. The 'multivalent effect' generated by a plurality of RGD groups can improve the binding capacity of the probe and the receptor.
In an embodiment of the present invention, the-N-heterocyclic group substituted with a cyclic arginine-glycine-aspartic acid (cRGD) group may be selected from any one of the following structures:
the fluorescent probe can be further used for cell imaging, and because the probe molecule can sensitively and specifically bind to the integrin alpha v beta 3 receptor, the probe can be combined with the alpha v beta 3 receptor on the surface of a corresponding cell when being cultured together with the corresponding cell. Tumor cells and neovascular cells that overexpress α v β 3 exhibit more intense fluorescence than other cells. Therefore, tumor cells and new blood vessel cells can be selectively imaged.
Preferably, the fluorescent probe is under cell imaging conditions of: excitation wavelength was 405nm, collection range was: 500-550 nm.
The fluorescent probe can be further used for in-vivo imaging, the probe can be injected into a nude mouse body and can enter a blood circulation system, and when probe molecules in blood flow through a tumor part, the probe molecules can be combined with an alpha v beta 3 receptor on the surface of a tumor cell. Thereby enriching the probe molecules at the tumor part and leading the tumor part to present stronger fluorescence. The purpose of selectively imaging the tumor site in vivo is achieved.
Meanwhile, the invention also provides the application of the fluorescent probe, which is mainly applied to cell imaging and living body imaging detection, and the preferable determination method during detection and evaluation comprises the following steps:
injecting PBS solution of the fluorescent probe into a cell culture medium for culturing, then flushing the cultured cells with PBS (phosphate buffer solution), and then carrying out fluorescence imaging; the difference in imaging was observed for different cells.
Injecting the PBS solution of the fluorescent probe into a mouse body through intraperitoneal injection, and observing the difference of fluorescent signals of a tumor part and a non-tumor part after injection. Thereby realizing the targeted imaging of the tumor in vivo.
In another aspect, the present invention also provides a method for preparing the fluorescent probe, comprising the steps of:
under the protection of inert gas, taking 5-halogenated-2-iodometaxylene as a raw material, and adding an alkyl lithium reagent and a boron trifluoride diethyl etherate complex to obtain halogenated triaryl phenylborane;
step two, coupling reaction: under the protection of inert gas, under the action of a coupling reagent, reacting halogenated triaryl phenylborane with poly-nitrogen heterocyclic alkane with part of amino protected to obtain a carbon-nitrogen coupling product;
step three, deprotection reaction: removing the protecting group of the amino group in the carbon-nitrogen coupling product to obtain a deprotected nitrogen heterocyclic product;
step four, acylation reaction: under alkaline conditions, reacting the deprotected nitrogen heterocyclic product with halogenated acyl chloride to obtain an amide product;
step five, substitution reaction: and (3) reacting an amide product with cRGD under the action of alkali to prepare the fluorescent probe.
In the specific embodiment of the invention, in the first step, the reaction temperature is low, preferably-78 ℃; the inert gas can be selected from nitrogen or argon; the alkyllithium reagent may be selected from n-butyllithium or t-butyllithium; the 5-halogenated-2-iodine meta-xylene can be selected from 5-bromo-2-iodine meta-xylene or 5-chloro-2-iodine meta-xylene; after the reaction is finished, the product is preferably washed by water and purified by silica gel column by using petroleum ether as a developing agent.
In the second step, the reaction temperature is preferably 90 ℃; the inert gas can be selected from nitrogen or argon; the coupling reagent is a toluene solution of sodium tert-butoxide (NaOtBu), tri-tert-Butylphosphine (BINAP) and palladium acetate (Pd (AcO) 2); the amino protecting group of the polyazacycloalkane whose partial amino group is protected may be selected from a benzyloxycarbonyl (Cbz) or a tert-butoxycarbonyl (Boc) protecting group; the polyazacycloalkane can be any one of piperazine, triazacyclononane and tetraazacyclododecane; after the reaction, the reaction mixture is preferably washed with water, dried over magnesium sulfate, and purified by a silica gel column.
Removing the protecting group of amino by adding acid, wherein the reaction is preferably carried out under the ice-bath condition; wherein the acid may be selected from HCl, TFA or HBr; after the reaction is finished, purifying by using basic ion exchange resin.
In the fourth step, the reaction is preferably carried out under ice-bath conditions; the base may be triethylamine and the haloacyl chloride may be selected from chloroacetyl chloride or bromoacetyl chloride; after the reaction, the reaction mixture is preferably washed with water, dried over magnesium sulfate, and purified by a silica gel column.
In the fifth step, the reaction is preferably carried out at normal temperature; the alkali is potassium carbonate; after the reaction, the purification is preferably carried out by dialysis.
Technical effects
(1) The fluorescent probe for targeted imaging of tumor cells and neovascularization can be obtained by chemical synthesis, and has the advantages of simple and feasible synthesis process, cheap and easily-obtained raw materials, low preparation cost and easy popularization.
(2) The fluorescent probe for targeted imaging of tumor cells and neovascularization can be used for selective imaging of cancer cells with high integrin α v β 3 expression.
(3) The preferred fluorescent probe contains a plurality of RGD groups, has a multivalent effect, improves the binding capacity of the probe and a receptor, and enables the probe to have higher selectivity and sensitivity.
(4) The fluorescent probe for targeted imaging of tumor cells and neovascularization can realize the purpose of targeted imaging of tumor parts in living bodies, so that the tumor parts show stronger fluorescence.
The conception, process, specific structure and technical effects of the present invention will be further described in conjunction with the following embodiments and drawings to fully understand the objects, features and effects of the present invention.
Drawings
FIG. 1 shows the absorption spectrum and fluorescence spectrum of a TAB-3-cRGD fluorescent probe in a PBS solution according to a preferred embodiment of the present invention.
FIG. 2 is a diagram of the use of the TAB-3-cRGD fluorescent probe in NIH/3T3 cell imaging in accordance with a preferred embodiment of the present invention.
FIG. 3 is a diagram of the use of the TAB-3-cRGD fluorescent probe in HUVEC-1 cell imaging in accordance with a preferred embodiment of the present invention.
FIG. 4 is a diagram of the use of the TAB-3-cRGD fluorescent probe in U87MG cell imaging in accordance with a preferred embodiment of the present invention.
Detailed Description
The following series of specific examples are given to further illustrate the teaching of the present invention, but the present invention is not limited to these specific examples, and any modification of the present invention that would be obvious to those skilled in the art can be made to achieve similar results and is also included in the present invention.
EXAMPLE 1 Synthesis of TAB-3-cRGD
(1) Preparation of compound 2:
10g (32mmol) of Compound 1 are dissolved in 60mL of dry diethyl ether under nitrogen, cooled to-78 ℃ and 20mL of a 1.6M n-butyllithium n-hexane solution are added dropwise thereto. The reaction was warmed to room temperature and stirred for 20 min. Then, it was cooled again to-78 ℃ and 1.25mL (10mmol) of boron trifluoride diethyl etherate was added. The reaction was continued at room temperature and stirred overnight. The solvent was dried by spinning and further purified by silica gel column chromatography to obtain 4.0g of a white solid with a yield of 71%.
(2) Preparation of compound 3:
595mg (3.2mmol) of N-tert-butyl piperazine formate, 560mg (1mmol) of the compound 2, 864mg (9mmol) of sodium tert-butyl alkoxide and 27mg (0.12mmol) of palladium acetate are placed in a three-necked flask, pumped three times on a double-row pipe, 50mL of toluene are added under the protection of nitrogen, 25mg of tri-tert-butylphosphine is further added, and the mixture is stirred at normal temperature for 15 min. Then the temperature is raised to 90 ℃ and the reaction is carried out for 24 h. The reaction was then cooled to 20 ℃, washed with water, extracted with ethyl acetate, dried over anhydrous magnesium sulfate and concentrated. The crude product was further purified by silica gel column to give 334mg of compound 3 in 38% yield.
(3) Preparation of compound 4:
88mg (0.1mmol) of Compound 3 are dissolved in 5mL of dichloromethane; methanol 5: 2, and 2mL of concentrated hydrochloric acid. The reaction was stirred at room temperature for 48h (until no starting material was present), after which time the solvent was spun off to give a viscous solid which was dissolved in 10mL of methanol and poured onto a strongly basic anion exchange column (Ion exchange III; Merck). Rinsed with 100ml of methanol. The solution was spin dried to give 57mg (99%) of compound 4 as a yellow solid.
(4) Preparation of compound 5:
under nitrogen protection, dry dichloromethane and 0.5ml triethylamine were added to a round-bottom flask containing 57g (0.99mmol) of Compound 4, and 67.2mg (0.6mmol) of chloroacetyl chloride was added dropwise in a ice salt bath and stirred overnight. After the reaction, the crude product was further purified by means of a silica gel column to obtain 71.2mg of Compound 5 in 89% yield.
(5) Preparation of compound 6:
8mg (0.01mmol) of Compound 5, 17.3mg (0.03mmol) of cRGD, and 4.1mg (0.03mmol) of potassium carbonate were dissolved in 5mL of a mixed solvent of acetonitrile and water in a ratio of 4: 1, and the mixture was stirred overnight. The solution was spun dry, dissolved in 1ml of water and dialyzed with a dialysis bag having a molecular weight of 1000 for three days. Concentration gave 22.15mg of TAB-3-cRGD in 91% yield.
Example 2: detection of absorption and fluorescence spectra of TAB-3-cRGD fluorescent probes
Preparing a 10 mu M TAB-3-cRGD probe PBS solution, adding the solution into a two-way cuvette with the concentration of 3mL to 10mm x 10mm, placing the cuvette into a spectrophotometer with a well-regulated base line, and testing the data within the range of 500-300; the solution was then poured into a 10mm by 10mm four-way cuvette and the fluorescence spectrum was measured in the range of 430-700nm using 405nm as the excitation wavelength (see FIG. 1). As can be seen from FIG. 1, TAB-3-cRGD has a maximum absorption peak at 380nm and a maximum emission peak at 490 nm.
Example 3: application of TAB-3-cRGD fluorescent probe in imaging of living cells
Mouse fibroblast NIH/3T3, venous endothelial cell HUVEC-1 and glioma U87 cells were cultured on a confocal culture dish by using a culture medium (the volume ratio of DMEM culture solution to fetal bovine serum in the culture medium is 9: 1). Placing in an incubator with the conditions of 37 ℃, 5% (volume fraction) CO2 and 20% (volume fraction) O2 for 24-48 h. The culture medium was removed, washed three times with PBS, then serum-free DMEM culture solution was added, the solutions of the TAB-3-cRGD fluorescent probe of the present invention were added to each dish, and the culture was continued in the incubator for 15min, and the cells were washed with PBS (phosphate buffered saline) for 6 times. Fluorescence imaging was performed using a confocal microscope. The imaging conditions were: excitation wavelength 405nm, collection range: 500-550 nm. FIG. 2 is an image of TAB-3-cRGD in NIH/3T3, from which it can be seen that TAB-3-cRGD cannot image NIH/3T3 cells. FIG. 3 is an image of TAB-3-cRGD in HUVEC-1 cells, which shows that TAB-3-cRGD can bind to venous endothelial HUVEC-1 cells and can perform signal output through a two-channel mode. Fig. 4 is an image of TAB-3-cRGD in a nude mouse tumor model, which shows that TAB-3-cRGD can bind to glioma cell U87MG and that it binds primarily to the cell membrane surface as confirmed by co-localization experiments with cell membrane dyes.
Example 4: application of TAB-3-cRGD fluorescent probe in imaging in living body
Establishing a glioma nude mouse tumor-bearing model: adjusting glioma U87 cell to logarithmic growth phase, digesting with 0.25% trypsin, resuspending in DMEM culture solution containing 10% fetal calf serum, washing with PBS solution twice, preparing into single cell suspension with PBS solution, and diluting to obtain cell with density of 1 × 107Per mL, tumor cell suspension (2X 10) was extracted with 1mL syringe6) Inoculating to the dorsal lateral subcutaneous part of hind limb of nude mouse, injecting 0.2ml TAB-3-cRGD solution with concentration of 4mM into abdominal cavity when tumor grows to be observed, and performing optical imaging detection with small animal imager. The imaging of the TAB-3-cRGD fluorescent probe in a nude mouse tumor model shows that the TAB-3-cRGD can target the tumor site of the imaging mouse.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (5)
1. A fluorescent probe for targeting tumor cells and new blood vessels is disclosed, which has a chemical structural general formula as shown in the following (I):
wherein R is1、R2、R3Identical or different, is a halogen atom or is selected from any one of the following structures:
and R is1、R2、R3Not all of them are halogen atoms at the same time.
2. A method of preparing a fluorescent probe according to any of claim 1, comprising the steps of:
under the protection of inert gas, taking 5-halogenated-2-iodometaxylene as a raw material, and adding an alkyl lithium reagent and a boron trifluoride diethyl etherate complex to obtain halogenated triaryl phenylborane;
under the protection of inert gas, reacting halogenated triaryl phenylborane with the multi-nitrogen heterocyclic alkane with part of amino protected to obtain a carbon-nitrogen coupling product under the action of a coupling reagent;
removing the amino protecting group in the carbon-nitrogen coupling product to obtain a deprotected nitrogen heterocyclic product;
reacting the deprotected nitrogen heterocyclic product with halogenated acyl chloride under an alkaline condition to obtain an amide product;
and fifthly, reacting the amide product with cyclic arginine-glycine-aspartic acid (cRGD) under the action of alkali to prepare the fluorescent probe.
3. The method of preparing a fluorescent probe according to claim 2, wherein the reaction temperature in the first step is-78 ℃; the inert gas is selected from nitrogen or argon; the alkyl lithium reagent is selected from n-butyl lithium or tert-butyl lithium; the 5-halo-2-iodo-m-xylene may be selected from 5-bromo-2-iodo-m-xylene or 5-chloro-2-iodo-m-xylene.
4. The method for preparing a fluorescent probe according to claim 2, wherein the reaction temperature in the second step is 90 ℃; the inert gas is selected from nitrogen or argon; the coupling reagent is sodium tert-butoxide (NaOtBu), tri-tert-Butylphosphine (BINAP) and palladium acetate (Pd (AcO)2) A toluene solution of (4); the amino protecting group of the polyazacycloalkane whose partial amino group is protected may be selected from a benzyloxycarbonyl (Cbz) or a tert-butoxycarbonyl (Boc) protecting group; the polyazacycloalkane is selected from any one of piperazine, triazacyclononane and tetraazacyclododecane.
5. The method of claim 2, wherein the protecting group of the amino group is removed in step three by adding an acid selected from the group consisting of HCl, TFA and HBr; in the fourth step, the base is triethylamine, and the halogenated acyl chloride is selected from chloroacetyl chloride; and the alkali in the step five is potassium carbonate.
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| CN108948142A (en) | 2018-12-07 |
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